Embryology

i149 Abstracts Study funding/competing interest(s): This trial is supported by a grant of the Netherlands Organisation for Health Research and Development (ZonMw Clinical fellow grant 90700154). Trial registration number: ISRCTN 48210491 Trial registration number: Not Applicable P-076 Sucrose needs not to be added in vitrification solution for freezing the artificial shrunken mouse blastocysts J.K. Joo1, J.E. Jeung1, K.R. Go2, and K.S. Lee1 1 Pusan National University Hospital, OB/GY, Busan, Korea South, 2Pusan National University Hospital, infertility clinic, Busan, Korea South Embryology ACCU-VIT: a new strategy for managing poor responders G. Gandhi1, G. Allahbadia2, S. Kagalwala1, A. Allahbadia2, S. Ramesh2, K. Patel2, R. Hinduja2, V. Chipkar1, M. Madne1, and R. Ramani1 Rotunda - Centre for Human Reproduction, Assisted Reproduction Laboratory, Mumbai, India, 2Rotunda - Centre for Human Reproduction, Assisted Reproduction, Mumbai, India 1 Study question: To evaluate the efﬁcacy of serial minimal stimulation IVF cycles with vitriﬁcation of embryos for treatment of poor responders as compared to conventional IVF protocols. Summary answer: Accumulating vitriﬁed embryos in serial minimal stimulation cycles (ACCU-VIT) followed by a frozen embryo transfer is a better treatment option for poor ovarian responders as compared to conventional IVF. What is known already: Previous trials have shown that neither conventional IVF nor natural cycle IVF is an effective treatment option for poor ovarian responders. However, none of the trials have examined the efﬁcacy of accumulating embryos with serial minimal stimulation cycles and vitrifying the resulting embryos. Women with poor ovarian reserves, who commonly do not respond to conventional stimulation protocols, are left with few options when planning a family. Study design, size, duration: This is a retrospective data analysis of poor responders from February 2011 to March 2012. A total of 140 patients were included in the study. 55 patients were offered minimal stimulation cycles with vitriﬁcation and embryo banking (ACCU - VIT Group) and 85 patients underwent conventional controlled ovarian stimulation for IVF. Participants/materials, setting, methods: The inclusion criteria for ACCU-VIT group were patients with at least one previous conventional IVF cycle with poor response (deﬁned as ≤ 4 MII oocytes). Embryos were vitriﬁed using Cryotec Vitrﬁcation protocol on Day 3. Once six embryos were banked with us, a frozen embryo transfer was planned. A maximum of 3 embryos were transferred. Main outcome measure was the clinical pregnancy rate deﬁned as positive fetal heartbeat at 12 weeks of pregnancy. Main results and the role of chance: The mean age was 38.5 years in the ACCU-VIT group and 35.7 years in the conventional IVF group. In the ACC-VIT group, each patient underwent an average of 2.7 cycles of embryo accumulation before planning a frozen embryo transfer. An average of 6.2 embryos were vitriﬁed for each patient. The cycle cancellation rate was 16.6% in the ACCU-VIT group and was signiﬁcantly higher in the conventional IVF group (22.2%). The clinical pregnancy rate was higher in the ACCU-VIT group (28.5%) than the conventional IVF group (18.7%). The cumulative pregnancy rate was statistically higher in the ACCU – VIT group (41.5%) than the conventional IVF group (22.3%). Limitations, reason for caution: A limitation of our analysis is a small sample size. However, we are very encouraged by the better results observed in the ACCU-VIT group and are continuing to apply this approach to more patients. Wider implications of the findings: ACCU-VIT using minimal stimulation and reliable vitriﬁcation methods is a successful approach to treat poor responders creating a situation similar to normal responders. This approach allows the poor responder women to have consecutive cycles of embryo accumulation before the follicular reserve is depleted. It will maximize the ovaries’ already limited life span, allowing patients the opportunity to store embryos while oocyte production is still active. Study funding/competing interest(s): No funding was used. There are no competing interests to declare. P-077 ATP contents in immature oocytes obtained from graafian follicles decreased compared with those from small follicles H. Goto1, S. Hashimoto1, A. Amo1, T. Yamochi1, H. Iwata 2, and Y. Morimoto3 IVF Namba Clinic, Reserch division, Osaka, Japan, 2Tokyo University of Agriculture, Department of Animal Science, Atsugi, Japan, 3IVF Namba Clinic, Medical ofﬁce, Osaka, Japan 1 Study question: To assess the relationships among ATP contents in oocytes, ovarian stimulation procedures, donor age, oocyte cell cycle, and oocyte diameter, we measured ATP contents in immature oocytes obtained from graaﬁan and small follicles. Summary answer: ATP contents in immature oocytes obtained from small follicles (diameter: approx. 10-mm) followed by maturation culture was signiﬁcantly higher than that in immature oocytes obtained from graaﬁan follicles after ovarian stimulation and natural cycles (diameter: approx. 19-mm). What is known already: ATP contents in oocytes have been suggested to be a marker of oocyte quality including maturity and developmental competence. On the other hand, it has been also reported that oocytes containing extremely high ATP content had low developmental competence. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 P-075 Study question: Does sucrose need to be added in vitriﬁcation solution for freezing the artiﬁcial shrunke mouse blastocysts? Summary answer: Sucrose needs not to be added in vitriﬁcation solution for freezing the artiﬁcial shrunken mouse blastocysts. What is known already: Sucrose included in freezing medium causes embryos to shrink by losing intracellular water, so that intracellular ice formation is reduced and embryos can be frozen rapidly. Blastocyst consists of trophoblast and inner cell mass. It may be considered that the artiﬁcial shrunken blastocyst is not embryo but tightly packed somatic cells. Sucrose is not added to somatic cell freezing medium. Study design, size, duration: Mouse morulae were collected from superovulated, mated mice(C57BL/CBA), cultured in G2.2 to develop into expanded blastocysts. Vitriﬁcation and thawing were prepared. Two vitriﬁcation solutions with and without sucrose were prepared. Control(G25E25) and treatment(G25E25S0.5) were composed of 25%glycerol + 25%ethyleneglycol and 25%glycerol + 25%ethyleneglycero +0.5 mol/l sucrose, respectively. Participants/materials, setting, methods: Blastocoel ﬂuid was aspirated in the expanded mouse blastocysts and shrunken blastocysts were equilibrated in EBS1, EBS2. After loading capped pulled-straw blastocysts were vitriﬁed or rehydrated. Assisted hatching was performed using assisted hatching pipette after thawing procedure or rehydration. Rates of re-expanding and hatching were examined after culture for 6h. Main results and the role of chance: Re-expanding rate of mouse blastocyst exposed to VS(without and with 0.5 mol sucrose) were not different in G25E25(98%) and G25E25S0.5(92%) (P . 0.05) and hatching rate was higher in G25E25(95%) than in G25E25S0.5(88%) but did not differ in two treatments (P . 0.05). Re-expanding rate of mouse blastocyst vitriﬁed in G25E25 and G25E25S0.5 was 95% and 90%, respectively (P . 0.05), and hatching rate was higher in G25E25(90%) than in G25E25S0.5(76%) (P . 0.05). Limitations, reason for caution: This study was done with mouse blastocysts. So, we are not sure about the effect in human blastocyst. Before applying this method to human blastocyst, we have to conﬁrm the negative effect of sucrose-free vitriﬁcation solution to human blastocyst. Wider implications of the findings: It can be applied to human embryo freezing procedure. This technique make freezing process easy, convenient, so we can reduce laboratory mistakes in freezing precess. Study funding/competing interest(s): None Trial registration number: None i150 P-078 Is ICSI yielding better clinical outcome compared to IVF in poor responders with normal sperm analysis? M. Koifman, S. Lahav-Baratz, E. Blais, Z. Megnazi-Wiener, D. Ishai, R. Auslender, and M. Dirnfeld Carmel Medical Center, Gyn/Obs IVF unit, Haifa, Israel Study question: To compare the effect of insemination using standard In vitro fertilization (IVF) Vs Intra Cytoplasmic Sperm Injection (ICSI) on clinical pregnancy and delivery rates in poor responders with 4 or less available oocytes and a normal sperm count. Summary answer: Clinical pregnancy and delivery rates were higher but not statistically signiﬁcant in IVF Vs ICSI in poor responders with normospermia. Signiﬁcantly lower fertilization rates and more "NO embryo transfer " events occurred with ICSI Vs IVF. In poor responders with normal sperm the use of ICSI was not beneﬁcial. What is known already: The introduction of ICSI has led to dramatic improvement in pregnancy rates in severe male factor and cases with failed fertilization. In view of high pregnancy rates and take home baby rates and reports on the relative safety of the ICSI technique, centers have considered using ICSI for other indications than Male factor. With only few available oocytes retrieved, embryologists and clinicians face a real dilemma regarding the choice of the insemination technique. Study design, size, duration: The study reviewed 195 treatment cycles of known poor responders, with 4 or less retrieved oocytes. Only cases with normal sperm count were included. The study group included 148 cycles in which standard IVF was used and 47 cycles with ICSI. Participants/materials, setting, methods: The parameters studied were age, number of retrieved oocytes, basal day 3-5 serum FSH, number of MII oocytes, fertilization rates, cleavage rate, number of embryos transferred, the percentage of cycles with embryo transfer, clinical pregnancy and delivery rates, analyzed in the group of IVF and ICSI cycles. Main results and the role of chance: There was no difference between the groups of IVF and ICSI cycles in the parameters of age, number of oocytes retrieved, FSH, number of MII oocytes per cycle and cleavage rates. A signiﬁcantly higher fertilization rate was observed in the IVF group as compared with ICSI group (81.51% and 67.73% respectively, p ¼ 0.004). The mean number of transferred embryos after IVF was 1.54 + 0.83 Vs 1.21 + 1.10 with ICSI (p ¼ 0.019). A "No embryo transfer" event was observed in 32% of the ICSI group Vs 11.50% in the IVF group (p ¼ 0.004). Clinical pregnancy rates and delivery rates were higher but not statistically signiﬁcant in IVF Vs ICSI (24.2% Vs 15.6%, 8.1% Vs 6.4% respectively). Limitations, reason for caution: This study is retrospective. As all cases included were treatments with normal sperm count, the decision to choose the use of IVF or ICSI was based on patients’ and clinicians’ request and therefore the IVF group was larger as compared to the ICSI group. Wider implications of the findings: In view of the above results and recent reports on increased malformation rates in ICSI, it is reasonably to advise that in the presence of normal sperm counts, the technique of choice for insemination in poor responders should be IVF rather than ICSI. Study funding/competing interest(s): The researchers declare that this study is free of any ﬁnancial support or other interests. Trial registration number: N/A P-079 A modified cryotop vitrification protocol with polimer cryoprotectant agent ficoll in the cryopreservation of human oocytes V. Zaletova1, E. Zakharova 2, I. Krivokharchenko 2, and S. Zaletov1 1 MAMA Fertility Center, Medical Department, Moscow, Russia C.I.S, 2MAMA Fertility Center, Embryology and Genetic Laboratory, Moscow, Russia C.I.S Study question: We examined a modiﬁed cryotop vitriﬁcation protocol in the cryopreservation of human oocytes. Suggested modiﬁcation had an additional polimer cryoprotectant agent ﬁcoll in vitriﬁcation medium with standard cryoprotectants compound. Summary answer: Applying of modiﬁed cryotop vitriﬁcation protocol with ﬁcoll enables a high survival rate of thawed oocytes, a high rate of blastocyst formation and pregnancy rate. What is known already: Until now all standard human oocytes vitriﬁcation protocols used two- and tree-component vitriﬁcation mediums with different sets of cryoprotectants, excluding polimers. Ficoll is a neutral, highly branched, highmass, hydrophilic polysaccharide. Cryoprotectant effect of ﬁcoll is that this polimer increases viscocity of vitriﬁcation medium, and therefore hinders formation of crystals. A number of published articles on cryopreservation of animals oocytes demonstrated efﬁciency of protocols with polimer cryoprotectants, including ﬁcoll. Study design, size, duration: 388 oocytes were obtaned from 54 anonymous donors and vitriﬁed using modiﬁed cryotop vitriﬁcation protocol with ﬁcoll. Vitriﬁed oocytes were stored in Cryobank for 1 to 6 months. Thawed oocytes of good quality were donated for IVF treatment, in 30 cases. Then oocytes were fertilized by husband’s sperm. Embryos were transferred on Day 5 after fertilization (blastocyst stage, 1-2 embryos), in 28 cycles. Participants/materials, setting, methods: During ccryopreservation oocytes were successively exposed to three vitriﬁcation mediums with increased concentrations of ethylen glycol (3,75 - 15%); DMSO (3,75 - 15%); ﬁcoll (2,5 - 10%); sucrose (0,125 - 0,5 M) at 370 C. Oocyte exposure time to each solution varied from 1 to 6 min. Oocytes were vitriﬁed by cryotop method. Thawing oocytes were successively exposed to four sucrose solutions with decreased concentration (1M - 0,125 M). Then oocytes were fertilized 3 hours after thawing by ICSI method. Main results and the role of chance: In 30 IVF cycles 263 donor’s oocytes were thawed. After thawing 19 out of 263 oocytes degenerated (7,2% – 19/ 263), 244 oocytes showed no signes of degeneration (92,8% – 244/263). Normal fertilization occured in 207 oocytes (84,8% – 207/224), all zygotes started to cleave at 48 hours after fertilization. By Day 5 of their culture, we had 98 embryos of good quality (47,3% - 98/207). Embryos were transferred into the uterus in 28 IVF cycles. 10 out of 28 patients had a positive beta-hCG blood test. All 10 pregnancies were clinically recognized (35,7% for an embryo transfer - 10/28), 8 out of which resulted in the birth of healthy children an one - of healthy twins. Limitations, reason for caution: Four-component vitriﬁcation medium containing ﬁcoll was used only for human oocytes vitriﬁcation and not for cryopresevation of embryos/sperm. Wider implications of the findings: Applying modiﬁed cryotop vitriﬁcation protocol with ﬁcoll resulted in laboratory and clinical data comparable to those Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study design, size, duration: This was an experimental study using 97 immature oocytes obtained from graaﬁan follicles (diameter: approx. 19-mm) after ovarian stimulation or natural cycles and 79 immature oocytes obtained from small follicles (diameter: approx. 10-mm) followed by maturation culture between April 2012 and October 2012. The local IRB approved this study. Participants/materials, setting, methods: Donated oocytes were used after informed consent. ATP contents in oocytes were measured individually after the measurement of their diameter and the removal of their cumulus cells. The ATP assay was performed individually based on the luminescence reaction. Luminescence was measured using a luminometer. Data were compared using student t-test. Main results and the role of chance: There were no differences in the ATP contents between GV (6.7 pM, n: 110) and MI stage oocytes (6.2 pM, n: 66), and between stimulation (5.0 pM, n: 66) and natural cycles (4.4 pM, n: 32). Moreover, there were no relationships between the ATP contents and oocyte diameter (diameter: 107.5-135.0 mm, 120.5 mm, n: 147, r2 ¼ 0.01, P ¼ 0.2), and between the ATP contents and donor age (age: 25-45 years old, n: 176, r2 ¼ 0.02, P ¼ 0.06). However, the ATP content in oocytes obtained from small follicles (8.6 pM, n: 79) was signiﬁcantly higher (P , 0.05) than that in oocytes obtained from graaﬁan follicles (4.8 pM, n: 97). Limitations, reason for caution: Further studies are required to clarify the link between a decrease of ATP contents in immature oocytes obtained from large follicles compared with oocytes obtained from small follicles. Wider implications of the findings: This study provided new insights on the implications of a decrease of ATP contents during follicle growth. Study funding/competing interest(s): Part of this work was supported by a grant from the Japan Society for the Promotion of Science (JPS-RFTF 23580397 to S.H.). No other competing interests are declared. Trial registration number: None. Abstracts Abstracts published in regard to early known vitriﬁcation protocols. We demonstrated its stable efﬁciency, which enables us to create and successfully use Cryobank of donor’s oocytes. Study funding/competing interest(s): None. Trial registration number: None. P-080 Cryopreservation of cleavage stage embryo or blastocyst: which option is better L. Zhu, Y. Li, H. Zhang, J. Ai, and L. Jin Tongji Hospital Afﬁliated to Tongji Medical College, Huazhong University of Science and technology, Wuhan, China P-081 Quantitative analysis of operator-dependent variability in blastocyst vitrification with a High Security Vessel (HSV) vitrification system X. Zhang, N. Rajan, A. Kovacs, C. Foley, J. Flanagan, J. O’Callaghan, J. Waterstone, and T. Dineen Cork Fertility Centre, Embryology, Cork, Ireland Study question: This study was performed as part of process validation when embryo vitriﬁcation was introduced at the fertility center. The purpose of this study was to quantify variability in the performance of blastocyst vitriﬁcation with a closed HSV system by a number of operators with a wide-spectrum of experience. Summary answer: Survival rates after blastocyst vitriﬁcation were high and did not vary signiﬁcantly between operators both with and without previous embryo vitriﬁcation experience. Quantitative analysis of operator-dependent variability provides a practical approach for training when implementing a successful vitriﬁcation program. What is known already: A successful outcome in vitriﬁcation is highly operator dependent, and a quite different skill set is necessary compared to that required for slow rate freezing. The embryologist needs to be aware of several critical procedural details that can impact negatively on results. Skill variations for all operators must be monitored and compared in order to achieve consistent and predictable results. Study design, size, duration: 347 Blastocysts (day5/6) derived from IVF treatment were vitriﬁed and 130 were warmed between Dec 2010 and Dec 2012. Six embryologists, with experiences of vitriﬁcation ranging from 0 to 5 years, all underwent vitriﬁcation training, practicing with silicone beads and mock procedures before participating in the study. Participants/materials, setting, methods: Blastocyst survival rates post warming were used to compare for different operators. The performance of vitriﬁcation was also quantiﬁed by recording a number of key parameters: loading time, loading volume, warming speed, and speed of handling the HSV straws. Main results and the role of chance: Operators with less experience showed higher coefﬁcients of variation (CVs) in comparison with experienced operators with regard to loading volume (28.5% vs. 18.1%) and loading time (10.4% vs. 6.7%), but not warming speed (13.7% vs. 12.8%) nor straw handling (13.4% vs. 13.1%). However, all operator-dependent parameters remained within protocol limits, even though they varied among individuals. 130 vitriﬁed blastocysts were warmed. The overall survival rate post warming was 93.8% (122/130) with 93.3% (84/90) for inexperienced and 95.0% (38/40) for experienced operators. These rates were not signiﬁcantly different. Limitations, reason for caution: This audit of operator performance is being continued as vitriﬁcation is applied clinically and will be supplemented by success data. Wider implications of the findings: Quantitative analysis of operator-dependent variability is both practical and essential when implementing a successful vitriﬁcation program. Study funding/competing interest(s): N/A Trial registration number: N/A P-082 For now, I would rather have the transfer of one fresh blastocyst. The surplus, please, vitrify them! E.M. Dahdouh1, P. St-Michel2, L. Granger 1, B. Carranza-Mamane3, F. Faruqi 2, T.V. Kattygnarath2, and F.L.A. Ferreira Gomes2 1 PROCREA Clinics & University of Montreal, ART Centre, Montreal, Canada, 2 PROCREA Clinics, ART Centre, Montreal, Canada, 3PROCREA Clinics & University of Sherbrooke, ART Centre, Montreal, Canada Study question: Is a frozen elective single-embryo transfer (eSET) at the blastocyst stage effective for good prognosis patients? Summary answer: Though less effective than fresh single blastocyst transfer, frozen SET (FET) at the blastocyst stage yields adequate clinical outcomes. This will further contribute in decreasing multiple pregnancy rates, and improving the cumulative pregnancy rate in the setting of an eSET policy. What is known already: Vitriﬁed blastocysts retained good developmental competency after warming. High pregnancy and implantation rates can be expected once the blastocysts go through vitriﬁcation and warming because they have adequate developmental potential as fresh blastocysts. Study design, size, duration: This is a prospective non-randomized control study including 244 women who underwent a SET at the blastocyst stage (fresh - eSET and frozen- FET) in a 11 months period between January 9th 2012 and December 15th 2012. Participants/materials, setting, methods: Women aged 34 years or younger undergoing a fresh elective single blastocyst transfer cycle (eSET) (n ¼ 152) or a frozen-thawed single blastocyst transfer (FET) (n ¼ 92). The study was performed in a tertiary private infertility clinic. Main results and the role of chance: Patients in both groups (fresh and frozen) were homogeneous for age, duration and cause of infertility, and for levels of day 3 FSH. In the Fresh group, the biochemical pregnancy rate was 52.6% while in the Frozen group it was 33.7% (p ¼ 0.0052). Implantation rate was higher in the Fresh group compared to the Frozen group (47.4% and 31.5% respectively, p ¼ 0.0161). Ongoing pregnancy rates (41.4% and 28.3% respectively, p ¼ 0.0406) were also signiﬁcantly higher in the Fresh group. Patients who underwent a fresh eSET had a median of two surplus blastocysts cryopreserved after transfer. Multiple pregnancy rates were very low, 0.7% and 1.1% for the Fresh and the Frozen group, respectively. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: Surplus embryos available for cryopreservation in fresh cycle have been considered as a good resource for future use. However, the optimal stage of embryo cryopreservation remains unclear. Summary answer: Blastocyst cryopreservation is more time-effective and costeffective as compared to cleavage stage embryo cryopreservation though blastocyst culture will not improve the embryo developmental potential. It is a more preferable option to patients with surplus embryos destined for future use. What is known already: Blastocyst culture is an effective tool to aid embryo selection. But the beneﬁts of blastocyst transfer versus cleavage stage embryo transfer are usually evaluated in fresh cycles. Few studied have compared the results in frozen-thawed embryo transfer (FET) cycles in a prospective way. Study design, size, duration: A prospective cohort study was performed. Patients in a university-based IVF center with failed pregnancy in fresh cycle and surplus embryos available on Day 3 were enrolled during March 2011 and September 2011. Each patient was followed up at least one year. Participants/materials, setting, methods: A total of 627 patients were enrolled in the study. Patients were divided into two groups: cleavage stage embryo cryopreservation group (309 patients) and blastocyst cryopreservation group (318 patients). Clinical outcomes of the FET cycles in each patient were evaluated. Main results and the role of chance: Cycle characteristics in terms of female age, duration of infertility, basal FSH, mean number of retrieved oocytes, mean number of available embryos/top quality embryos were comparable between the two groups. Although a decreased cryopreservation rate (74.5%, 237/318), the blastocyst group achieved signiﬁcantly higher rates of pregnancy/cycle (42.8% vs 31.7%, P Limitations, reason for caution: This study was not designed for randomized fashion. Patients made the assignment decision mainly by themselves. However, the two groups had similar cycle characteristics, supporting the absence of any selection bias. Wider implications of the findings: The conclusion will be helpful in counseling patients regarding the optimal stage of cryopreservation of surplus embryos in fresh cycles and utilization of cryopreserved embryos in future FET cycles. Study funding/competing interest(s): The authors have no connection to any companies or products mentioned in this article. Trial registration number: None i151 i152 Limitations, reason for caution: This is a non-randomized study and therefore subject to bias. Primary clinical outcomes do not include live birth rates, so results must be interpreted with caution. Wider implications of the findings: Improvement in cryopreservation techniques and better synchronization between the endometrium and the embryo development, will further contribute to increase FET efﬁciency. Blastocysts can be vitriﬁed in patients for whom fresh blastocyst transfer is unsuitable, such as patients at risk of OHSS, or those in need for preimplantation genetic diagnosis. Cumulative pregnancy rate might offset the lower pregnancy rate for fresh blastocyst transfer observed in this study. Study funding/competing interest(s): This study received no funding and there are no conﬂicts of interests to be declared. Trial registration number: None. Effectiveness of vitrification in women undergoing elective freezing of oocytes and/or embryos when embryo transfer conditions are suboptimal N. Christoforidis, C. Ioakimidou, C. Papas, M. Moisidou, and A. Chatziparasidou Embryolab Assisted Reproduction Unit, IVF Unit, Thessaloniki, Greece Study question: Certain conditions that are associated with reduced ARToutcome following controlled ovarian stimulation, such as raised progesterone levels on day of hCG triggering, ovarian hyperstimulation syndrome, endometrial polyps and thin endometrium. Elective vitiriﬁcation of all oocytes/embryos and frozen embryo transfer in subsequent cycles may provide a valid alternative approach. Summary answer: Elective vitriﬁcation of oocytes/embryos after controlled ovarian stimulation in suboptimal conditions for embryo transfer, is associated with high survival rates of oocytes/embryos and high pregnancy rates in frozen embryo replacement cycles. What is known already: Successful implantation relies on both embryological features and endometrial receptivity. It is known that certain conditions have a negative impact on endometrial receptivity, in particular, raised progesterone levels on the day of hCG triggering, hyperoestrogenemia associated with ovarian hyperstimulation syndrome, as well as endometrial pathology, such as endometrial polyps and presence of endometrial ﬂuid. Vitriﬁcation is an effective procedure that allows preservation of oocytes/embryos and transfer of embryos in cycles with optimal endometrial recpetivity. Study design, size, duration: Retrospective analysis of 88 cases undergoing elective freeze-all with vitriﬁcation of oocytes/embryos following controlled ovarian stimulation, when endometrial conditions were deemed suboptimal, from March 2011 to March 2012. Participants/materials, setting, methods: Cases were categorized according to indication of elective freeze-all procedure as follows: repeated implantation failure, endometrial polyp, raised progesterone on day of hCG triggering, OHSS, embryo collection for PGD, endometrial ﬂuid, thin endometrium. Survival rate of oocytes/embryos post thawing and pregnancy rates in subsequent embryo transfers were evaluated. Main results and the role of chance: Survival rate of oocytes/embryos and CPR per category were recorded as follows: repeated implantation failure: 95% oocyte survival, 50% CPR, endometrial polyp: 96% oocyte survival, 98% embryo survival, 85% CPR, raised progesterone: 98% oocyte survival, 54.8% CPR, embryo collection for PGD: 98% embryo survival, 48% CPR, OHSS: 97% oocyte survival, 64.5% CPR, endometrial ﬂuid: 99% embryo survival, 25% CPR, thin endometrium: 96% embryo survival, 46% CPR. Limitations, reason for caution: Number of cases that underwent elective crypreservation for endometrial receptivity reasons may not be large enough. Wider implications of the findings: Oocyte and embryo survival rates after vitiriﬁcation are quite high and pregnancy rates are very satisfactory following embryo transfer in a subsequent endometrial preparation cycle. It is suggested that whenever endometrial conditions are deemed suboptimal oocyte and/or embryo vitriﬁcation may be a valid option to allow optimal endometrial preparation and hence, high pregnancy rates. Elective vitiriﬁcation may minimize the risk of developing OHSS, augment the number of available embryos for PGD. Study funding/competing interest(s): None Trial registration number: None P-084 Cleavage stage embryo versus blastocyst transfer in patients with 3 failed IVF/ ICSI cycles, a retrospective cohort study M. Klaver, K. Tilleman, and P. De Sutter University Hospital Gent, Assisted reproduction, Gent, Belgium Study question: Does day 5 embryo transfer (ET) in patients with repeated implantation failure (3 failed IVF/ICSI cycles) increase pregnancy rates in the fourth cycle if necessary after correction of thrombophilic or autoimmune pathology? Summary answer: Day 5 ET in patients with 3 failed IVF/ICSI cycles and in whom thrombophilia or autoimmune disorders are treated if present, increases the pregnancy rate per embryo transfer signiﬁcantly until 67% when compared to 37% after day 3 ET. What is known already: Pregnancy rate in the fourth cycle after 3 failed IVF/ICSI cycles is usually rather low, making this a therapeutically challenging group of patients. There is a lot of controversy about testing for auto-immunity or thrombophilia and about which is the best therapeutic strategy for this group. Study design, size, duration: This is a monocentric retrospective study. We included 72 patients with 4 consecutive IVF or ICSI treatment cycles in our centre between 2007 and 2012. The ﬁrst 3 cycles had ET at cleavage stage (day 2 or 3) and negative HCG as outcome. Patients aged above 36 years were excluded. Participants/materials, setting, methods: In the fourth cycle 51 patients were planned for ETat cleavage stage and 21 for blastocyst transfer.A repeated implantation failure protocol (RIF), including testing for thromboﬁlia, auto-immunity, karyotype disorders and hysteroscopy was performed. Depending on the test results treatment consisted of operative hysteroscopy, prednisolon, heparin or aspirinaccordingly. Main results and the role of chance: In the group scheduled for day 5 ET 81% (17 of 21 patients) were tested and 28.5% of the tests (6 patients) were abnormal and treated. In one patient (4.7%) the embryos did not reach the blastocyst stage and no transfer was performed. In the cleavage stage group 63% (32 of 51 patients) were tested and 37.5% (12 patients) were abnormal and treated. Pregnancy rate per transfer in cleavage stage transfer was 37% (19 out of 51 patients). Pregnancy rate per transfer in the blastocyst group (day 5) was 67% ( 14 out of 21 patients). This difference is statistically signiﬁcant (x2-test, P ¼ 0.023.). Limitations, reason for caution: The patient group is rather small, but still results are signiﬁcant. These are preliminary results and the cohort will be expanded to include patients with 3 IVF/ICSI cycles of which the ﬁrst 2 have failed. Results from frozen embryo’s and subsequent cryo cycles have not been included in this study. Wider implications of the findings: With blastocyst transfer chances for patients with repeated implantation failure increase signiﬁcantly. Besides correcting for implantation hindering pathology, performing day 5 embryo culture also allows to discriminate between real implantation and embryo quality problems. This provides useful information, allowing better counselling of patients regarding further steps of treatment. Study funding/competing interest(s): This study was not funded and there were no competing interests. Trial registration number: not applicable P-085 Effect of female underweight on embryo morphokinetic using time-lapse J. Lammers, T. Freour, C. Splingart, and P. Barriere CHU Nantes, 38 Bd Jean Monnet, Nantes Cedex 01, France Study question: The aim of our study was to compare early embryo morphokinetic parameters with a time-lapse monitoring system (Embryoscope) according to female BMI (Body Mass Index) category, in order to identify an eventual detrimental effect of underweight on in vitro embryo development. Summary answer: We showed that morphokinetics differs according to female BMI category, with almost all cellular events delayed in underweight women. We also found that Zona Pellucida (ZP) thickness was different according to female BMI. However, IVF cycle outcome does not appear to be affected in underweight women. What is known already: Consequences of underweight (BMI , 18.5 kg/m2) on reproductive age women’s health are numerous, including fertility. Indeed, these Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 P-083 Abstracts Abstracts P-086 Effect of oocyte activation by calcium ionophore A23187 or strontium chloride in patients with low fertilization rates T. Ikeno1, Y. Nakajyo2, Y. Sato 1, K. Hirata1, T. Kyoya 1, and K. Kyono1 Kyono ART Clinic, Department of Gynechology, Sendai, Japan, 2Kyono ART Clinic Takanawa, Department of Gynechology, Tokyo, Japan 1 Study question: To evaluate the effectiveness of oocyte activation (OA) by calcium ionophore A23187 or strontium chloride in patients with low fertilization rates. Summary answer: Artiﬁcial OA using A23187 or SrCl2 is beneﬁcial to patients with low or no fertility. Further studies are needed to conﬁrm the safety of oocyte activation. What is known already: Oocytes unfertilised after ICSI have been subsequently activated using chemical, mechanical, and electrical stimulation, and these oocytes have been able to form pronuclei. Study design, size, duration: We obtained informed consent from 144 couples who had had unsuccessful ICSI treatment at our clinic between April 2004 and August 2012. These were then divided into two groups. Participants/materials, setting, methods: Ninety two couples who had unsuccessful ICSI treatments sequentially received A23187 OA after ICSI. We compared the clinical results with and without OA. Fifty two couples who had unsuccessful previous ICSI treatments sequentially received SrCl2 OA after ICSI. We compared the clinical results with and without OA. Main results and the role of chance: Comparing the results without and with A23187, the results were as follows: two pronucleus (2PN) zygote rates, 24.3% (85/350) vs. 61.2.% (316/516): P , 0.01; top quality embryo rates, 26.1% (23/88) vs. 43.4%(115/265): P , 0.01; expanded blastocyst rates, 13.8% (9/65) vs. 15.6% (30/192): NS; clinical pregnancy rates, 2.7% (1/36) vs. 14.9% (26/ 174): NS; and miscarriage rates, 100% (1/1) vs. 38.5% (10/26): NS. Comparing the results without and with SrCl2, the results were as follows: 2PN zygote rates, 24.5% (81/330) vs. 57.5% (192/334): P , 0.01; top quality embryo rates, 27.0% (24/89) vs. 25.3% (42/166): NS; expanded blastocyst rates, 14.1% (9/64) vs. 8.4% (11/131): NS; clinical pregnancy rates, 0%(0/52) vs. 19.2% (14/ 73) :P , 0.05; and miscarriage rates, 0% (0/0) vs. 21.4% (3/14): NS. Limitations, reason for caution: None. Wider implications of the findings: In conclusion, AOA using A23187 or SrCl2 is beneﬁcial for patients with low or no fertilization by ICSI. Comparing the results for babies with A23187 and SrCl2 OA showed no signiﬁcant difference between babies born after OA and naturally conceived babies. As with all assisted reproductive technology treatment, we must inform patients of the risks and beneﬁts of undergoing fertility treatment and possible implications for the future health of their children. Study funding/competing interest(s): None. Trial registration number: This study is not RCT. P-087 Are there any differences in terms of kinetic parameters between male or female embryos - a time lapse analysis F. Bronet Campos1, M. Meseguer2, M. Nogales3, E. Martinez3, M. Ariza3, D. Agudo1, L. Rodrigo 3, and J.A. Garcia-Velasco4 1 IVI Madrid, IVF, Madrid, Spain, 2IVI Valencia, IVF, Valencia, Spain, 3IVI Madrid, PGD, Madrid, Spain, 4IVI Madrid, Gynaecology, Madrid, Spain Study question: Is it possible to estimate embryo gender according to the embryo cleavage timing? Summary answer: There is no correlation between sex ratio and developing embryo rate. What is known already: In some species male and female embryos develop at different rate during preimplantational period. It has been suggested that XY human embryos could have faster growing rate and also they could reach blastocyst stage earlier. However, no time lapse studies have been reported so far. Study design, size, duration: Retrospective, observational study including 365 embryos from our PGS (preimplantation genetic screening) program from January 2012 to December 2012. Embryos were grouped according to their sex: male (166 embryos) and female (161 embryos). Additionally another group was included with 38 embryos with Turner Syndrome (TS). Participants/materials, setting, methods: All embryos were cultured in an incubator with time lapse technology, Embryoscope. Cleavage timing from insemination to day 3 was studied and all kinetic parameters that have been described in previous studies by our group have been taken into account and compared among the three groups Main results and the role of chance: Chromosomal abnormal rate was similar in both groups (male: 61.4%; female: 57.7%, p ¼ 0.4955). We did not ﬁnd any statistical differences between male or female embryos according to the growing rate in any speciﬁc cleavage check point. Surprisingly, we found statistical differences in cleavage time from one cell to two cells (T2) in TS embryos compared with male or female embryos (TS: 27.6 + 3.7; male: 26.3 + 3.0; female: 26.3 + 2.8; p ¼ 0.032). Limitations, reason for caution: Sample size may limit our ﬁndings Wider implications of the findings: Embryo development is not affected by embryo gender and sex ratio is not affected by the embryo selected to transfer based on kinetic parameters. Study funding/competing interest(s): The authors declare no conﬂicts of interest. The study did not receive any external grant. Trial registration number: None P-088 Blastocyst survival, re-expansion, and % live cells following vitrification and warming using two commercially-available vitrification systems A.S. Lopes, V. Frederickx, G. Vankerkhoven, A. Serneels, P. Roziers, P. Puttermans, R. Campo, and S. Gordts LIFE (Leuven Institute for Fertility and Embryology), Unit for Reproductive Medicine, Leuven, Belgium Study question: The aim of this study was to evaluate and compare survival, re-expansion, and % live cells of individual Day 5-6 human blastocysts, vitriﬁed and warmed with the Vit Kit Freeze/Thaw (Irvine Scientiﬁc, CA), or with two protocols using the Global Fast Freeze/Thaw Kits (LifeGlobal, Canada). Summary answer: Survival, re-expansion, and % live cells were not different for blastocysts vitriﬁed and warmed between the two vitriﬁcation/warming kits, or between the two protocols for the Global Fast Freeze/Thaw Kits. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 women often face with infertility, anovulation, high miscarriage rate and obstetrical complications, leading to lower live birth rate after ARTcycles. Whether underweight affects preimplantatory embryo development in underweight women undergoing IVF remains to be explored. Study design, size, duration: This retrospective study was conducted on all ICSI cycles performed between 2011 and 2012 with the Embryoscopew. All clinical, ovarian reserve, ovarian stimulation and embryonic parameters (conventional morphology and kinetic events in hours post injection) were recorded. Participants/materials, setting, methods: This study was conducted at the University Hospital of Nantes, France, on all ICSI cycles. After oocyte retrieval and ICSI, all oocytes were incubated in the Embryoscopew until transfer. Clinical, ovarian reserve, ovarian stimulation and embryo morphokinetic parameters were compared according to female BMI category. Main results and the role of chance: Among the 366 couples included (2270 oocytes collected), 34 women had low BMI (,18.5 kg/m2), 224 had a normal BMI (18.5-25), 65 were overweight (25-30) and 43 were obese (.30). Patients’ clinical characteristics were not signiﬁcantly different between the 4 groups (age, smoking status, infertility, AMH, semen characteristics and stimulation parameters). Day 3 FSH, E2 and LH and peak E2 were signiﬁcantly higher, whereas total FSH dose was lower in underweight group than in others. Number of oocytes retrieved, fertilization rate, number and quality of cleaved embryos (conventional morphology) were not signiﬁcantly different between underweight and others groups. Most embryo development events (4 cell, 5 cell, 8 cell stages) occurred signiﬁcantly later in underweight than in other groups. ZP was also thicker in underweight women. Limitations, reason for caution: This retrospective study was conducted in a limited cohort of patients. Wider implications of the findings: Time-lapse analysis is a relevant method for the evaluation of female clinical characteristics’ inﬂuence on early embryo development. Not only obesity, but also female underweight, should be studied as a cofactor potentially leading to modiﬁcations in early embryo development. Study funding/competing interest(s): None Trial registration number: Local IRB approved study i153 i154 P-089 Analysis of mitochondrial DNA copy number provides an insight into the ploidy and implantation potential of human blastocysts E. Fragouli1, S. Alfarawati2, K. Spath 3, and D. Wells1 1 Reprogenetics UK/ University of Oxford, Institute of Reproductive Sciences, Oxford, United Kingdom, 2Reprogenetics UK, Institute of Reproductive Sciences, Oxford, United Kingdom, 3University of Oxford, Institute of Reproductive Sciences, Oxford, United Kingdom Abstract withdrawn by the author. P-090 Serum anti-Mullerian hormone measurements and human blastocyst development after IVF J. Liss, K. Lukaszuk, J. Glowacka, and A. Bruszczynska Invicta, Fertility and Reproductive Center, Gdansk, Poland Study question: To report on relationships among baseline AMH level, blastocyst development and other selected embryology parameters observed in non-donor oocyte IVF cycles. Summary answer: The current investigation present that baseline serum AMH level can be a useful parameter to estimate blastocyst developmental potential during IVF. What is known already: AMH was a predictive correlate for ovarian reserve and the number of oocytes retrieved, with a high positive value compared with age, early follicular FSH level and pregnancy after IVF. Its role in forecasting in vitro embryo morphology and developmental potential to the blastocyst stage is still emerging. Study design, size, duration: Between January and December 2011, 830 patients without endometriosis, who had their AMH levels analyzed were incorporated into this prospective study. Participants/materials, setting, methods: Mean age, AMH level, antral follicle count (AFC), number of follicles, oocyte retrieved, MII oocytes, early cleavage embryos, blastocyst for transfer were evaluated. Main results and the role of chance: Three groups were formed according to the AMH level: 1: ,0.6 ng/ml (n ¼ 55), 2: 0.6-1.4 ng/ml (n ¼ 134) and 3: .1.4 ng/ ml (n ¼ 641). Blastocyst formation was signiﬁcantly lower (p ¼ 0.001) in group 1 and 2 compared to group 3 (23.6%, 43.3% and 67% respectively). There were statistical differences in ﬁrst group of AMH with age (p ¼ 0.001) and early cleavage embryos (p ¼ 0.02). By multivariate analysis, only age (OR ¼ 0.81; 95% CI, 0.66-0.98) showed signiﬁcant association with the blastocyst formation in this group. All parameters were found to be associated with blastocyst formation in AMH group 2 and 3. By multivariate analysis, only early cleavage embryos (OR ¼ 2.56; 95% CI, 1.58-4.13 and OR ¼ 1.71; 95% CI, 1.52-1.93 respectively) showed signiﬁcant association with the blastocyst formation in these two groups. Limitations, reason for caution: Different types of reproductive pathology might impact serum AMH in different ways (endometriosis, PCOS). Culture to the blastocyst stage require strict environmental. This can affect the results obtained in other laboratories. Wider implications of the findings: Our study conﬁrmed correlation between AMH level and age, AFC, total number of retrieved oocytes as well as the number of MII oocytes and early cleavage embryos. The correlation between early cleavage embryos and blastocyst formation suggests that early cleavage observation (e.g. time laps procedure) will be a good prognostic factor for patient with different AMH level. Decision about embryo transfer with smaller amount of good quality embryos could be taken earlier, without long culture. Study funding/competing interest(s): There are not any commercial association of the author of any coauthors that might pose a P-091 Effect of sperm selection using hyaluronic acid binding on reproductive outcome with donated oocyte cycles S. Cortés Gallego, L. Ortega López, E. Olaya Vila, M. Gago Garcı́a, C. Luna Cañas, A. Garcı́a Segovia, A. Guijarro Ponce, R. Núñez Calonge, and P. Caballero Peregrı́n Clinica Quirurgica Tambre, Laboratorio FIV / Andrologia, Madrid, Spain Study question: Compare the efﬁciency of routine sperm selection method polyvinylpyrrolidone (PVP) with HA-selection method (Sperm-Slow) for fertilization rate (FR), cleavage rate (CR), embryo quality (EQ) and pregnancy rate (PR) as well as evaluating the relationship between HA-binding ability with sperm protamine deﬁciency and DNA fragmentation using donated oocyte cycles. Summary answer: Contrary to our expectation, selection of HA-bound spermato-zoa had no beneﬁts in terms of fertilization, em-bryo cleavage, embryo quality and pregnancy rate in ICSI cycles. In conclusion, our prospective and randomized trial suggests that both, PVP and Sperm-Slow allow a comparable clinical efﬁciency in selecting spermatozoa. What is known already: Intracytoplasmic sperm injection (ICSI) using hyaluronic acid (HA) containing media has been suggested to have higher speciﬁcity and lower biological risk than other selection methods. HA-binding ability of spermatozoa is related to sperm membrane maturity and fertilizing potential, thus it has been suggested that sperm selection using HA before ICSI might help to optimize the treatment. However, there are conﬂicting data regarding subsequent improvement of FR, CR and PR after ICSI using HA-bound spermatozoa. Study design, size, duration: This was a single-center, prospective, randomized study of 144 ICSI cycles (2012) with donated oocytes to avoid female infertility as a bias factor, randomly carried out with PVP (1119 ovocites microinjectated) or with Sperm-Slow (1104 ovocites microinjectated) for sperm selection. Participants/materials, setting, methods: Our primary outcome was to compare FR, CR, EQ and PR between two groups. A secondary outcome was to better de?ne the role of HA for selection of spermatozoa with normal chromatin content to optimize ICSI outcome. To determine the value of Sperm DNA fragmentation (SDF) levels, the Sperm Chromatin Dispersion test (SCD) was measured. Between groups differences of normally distributed continuous variables were assessed with a parametric statistic (Student’s t test).Between-group differences in no continuous variables were assessed using the c2 method. Differences were considered signiﬁcant when a P value was ,.05. Main results and the role of chance: There were no signiﬁcant differences in regard the total number of injected MII oocytes, semen quality or SDF. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 What is known already: Vitriﬁcation is a modern method for cryopreservation of human embryos, currently available in most IVF centers. Several cryoprotectants and devices are available in the market but some require excellent training skills and others led to inconsistent survival rates following thawing. Study design, size, duration: Group 1 blastocysts were vitriﬁed with the Vit Kit (n ¼ 41) and Hemi-Straw devices, Group 2 (n ¼ 50) and Group 3 (n ¼ 57) blastocysts were cryopreserved with the Global Fast Freeze Kit and 0.25 ml straws, using a direct plunge or a -1008C holding step, respectively. Group 4 (Controls, n ¼ 31) were not vitriﬁed. Participants/materials, setting, methods: Frozen/thawed Day 2-3 or discarded embryos were cultured to blastocyst (culture day 5-6). After vitriﬁcation/ warming, blastocysts were cultured for 24h. All embryos were stained individually with propidium iodide and Hoechst. Live and total cell number was assessed with ImageJ (NIH), and the percentage of live cells calculated for each blastocyst. Main results and the role of chance: Survival immediately following thawing was 90%, 94%, and 83% for Groups 1, 2 and 3, respectively, and not signiﬁcantly different (P . 0.05). Following 24h culture, survival was 72%, 74% and 77%, and not signiﬁcantly different. Re-expansion at 24h after thawing was 55%, 68% and 68%, and not signiﬁcantly different. Mean (+ SD) % live cells were 90.1 + 2.5, 92.7 + 2.5, 90.4 + 2.2, and 95.8 + 1.1, for Groups 1, 2, 3, and 4, respectively. There was no signiﬁcant effect of treatment, culture day (Day 5 or 6), or interaction between treatment and culture day. Limitations, reason for caution: The power of the comparisons of proportions is low (, 0.40) due to the relatively small sample sizes. Wider implications of the findings: The simplicity of blastocyst vitriﬁcation using the Global Fast Freeze/Thaw Kits using freezing straws may provide increased safety, and efﬁciency with lower cost, compared with vitriﬁcation using specialized embryo vitriﬁcation devices. Study funding/competing interest(s): Global Fast Freeze/Thaw Kits were provided by IVFonline. A.S. Lopes is a paid technical consultant for IVFonline. Trial registration number: Not applicable Abstracts Abstracts P-092 Does growth retardation in human blastocysts decrease implantation potential after embryo transfer through an increase of the abnormal spindles S. Hashimoto1, A. Amo1, K. Ito2, Y. Nakaoka2, and Y. Morimoto2 IVF Namba Clinic, Research division, Osaka, Japan, 2IVF Namba Clinic, Medical ofﬁce, Osaka, Japan 1 Study question: To assess the potential of growth-retarded embryos, the implantation potential and the spindle shape of vitriﬁed–warmed blastocysts were assessed among normally developing and growth-retarded blastocysts. Summary answer: The incidence of abnormal spindle morphology increased and the implantation competence decreased following vitriﬁcation in growth-retarded embryos compared with normally developing embryos, but there were no signiﬁcant differences in the chromosomal abnormalities of abortuses and the incidences of peromelus of babies between 2 groups. What is known already: There are conﬂicting data on whether the human embryo growth rate affects the outcome of vitriﬁed–warmed blastocyst transfer. Various types of spindle abnormality occur in human cleavage- and blastocyst-stage embryos. A recent systematic review and meta-analysis concluded that growth-retarded embryos that develop to the blastocyst stage by day 6 have the same implantation potential as their day 5 counterparts if the morphology of day 6 blastocyst is similar to that of day 5 blastocyst. Study design, size, duration: This was a retrospective cohort study including 878 single vitriﬁed–warmed blastocyst transfers between January 2010 and July 2012, and an experimental study using 108 vitriﬁed–warmed blastocysts donated to research. The local IRB of IVF Namba clinic approved this study. Data were compared using the Mann– Whitney nonparametric U-test. Participants/materials, setting, methods: In a clinical study, we compared the implantation rates of vitriﬁed–warmed embryos that developed to the blastocyst stage on day 5 after insemination with those that required culture to day 6. In an experimental study, vitriﬁed blastocysts were immunostained with an anti-atubulin antibody, an anti-g-tubulin antibody and DAPI. Main results and the role of chance: In the clinical study, the implantation rate of growth-retarded embryos (47%, n: 270) was signiﬁcantly lower (P n: 608). However, there were no differences in the chromosomal abnormalities of abortuses and the incidences of peromelus of babies between 2 groups. In the experimental study, a total of 533 spindles were analyzed in both day 5 and day 6 blastocysts. Confocal image analysis was accomplished by capturing a z-series stack of 0.5-mm-thick optical sections encompassing the entire blastocyst. Only spindles with fusiform poles and with chromosomes aligned at the equator were classiﬁed as normal. The incidence of abnormal spindles in growth-retarded embryos (47%, n: 274) was signiﬁcantly higher (P n: 259). Limitations, reason for caution: Further studies are required to clarify the link between an increase in abnormal spindle formation and a decrease in embryonic implantation potential. Wider implications of the findings: This study provided new insights on the implications of an increase in abnormal spindle formation in growth-retarded human blastocysts. Study funding/competing interest(s): Part of this work was supported by a grant from the Japan Society for the Promotion of Science (JPS-RFTF 23580397 to S.H.). No other competing interests are declared. Trial registration number: None. P-093 In vitro maturation of human oocytes selected by Brilliant Cresyl Blue staining D.D. Alcoba1, E.G. Valério2, M. Conzatti 1, J. Tornquist1, A.P. Kussler3, A.M. Pimentel3, H.E. Corleta3, and I.S. Brum1 1 Universidade Federal do Rio Grande do Sul, Departamento de Fisiologia, Porto Alegre, Brazil, 2Hospital de Clı́nicas de Porto Alegre, Serviço de Ginecologia e Obstetrı́cia, Porto Alegre, Brazil, 3Hospital Moinhos de Vento, Núcleo de Reprodução Humana Gerar, Porto Alegre, Brazil Study question: Evaluate the efﬁciency and safety of Brilliant Cresyl Blue (BCB) staining as a selection of developmentally competent immature human oocytes before in vitro maturation (IVM). Summary answer: BCB test can be a good marker in pre-selection procedures of developmentally competent human oocytes and, apparently, it is not toxic to the gamete. What is known already: Growing oocytes synthesize glucose-6-phosphate dehydrogenase (G6PDH), but this enzyme is inactivated in oocytes that have completed their growth phase. Once BCB can be reduced by G6PDH, the selection of developmentally competent oocytes before IVM by BCB staining is widely used for many animal species (bovine, porcine, mice, etc). Using animal models, it has been already demonstrated that this selection can improve not only nuclear and cytoplasmic oocyte maturation, but also fertilization and blastocyst rates. Study design, size, duration: Cross sectional experiment – control versus treatment groups. Three kinds of patients were included in the study: 6 hormonal stimulated patients with immature oocytes at retrieval (17 oocytes); 3 ooforectomized patients (20 oocytes recovered); and 32 pregnant patients during cesarean section (92 oocytes recovered). Participants/materials, setting, methods: Immature oocytes were divided into groups: control (disposed directly to IVM) and treated - exposed to BCB 26 mM during 60 minutes. After staining, treated group was classiﬁed as cytoplasm coloration, BCB positive or negative, and then disposed to IVM. After 24 and 48 hours of IVM nuclear status was checked. Main results and the role of chance: The IVM rate of immature oocytes recovered from stimulated and ooforectomized patients was equal among groups (control, BCB positive and BCB negative) either after 24 or 48 hours of IVM. Nevertheless, IVM was higher in BCB positive compared to BCB negative after 24 and 48 hours of IVM analyzing oocytes recovered from all patients (P ¼ 0.024 and P ¼ 0.015) and from cesarean patients (P ¼ 0.004 and P ¼ 0.032). The control group was equal to BCB positive. The meiosis resumption rate (absence of germinal vesicle) after 24 hours of IVM was equal among groups, but higher in BCB positive compared to other groups after 48 hours when all gametes were analyzed jointly (P ¼ 0.035). Degeneration rate was lower in BCB positive compared to their counterparts when oocytes were analyzed jointly (P ¼ 0.002). Limitations, reason for caution: Human oocytes that can be destined to research are limited. We have to look for effects of ovarian puncture during cesarean section after some years. As the eggs were not fertilized, embryo characteristics were not accessed; so the effects of BCB on human embryo development remain unknown. Wider implications of the findings: In human oocytes the IVM rate of BCB positive was higher than that observed for BCB negative, but equal to control group. The degeneration rate was lower in BCB positive oocytes, compared to other groups (control and BCB negative). These results were similar to that described in animals. Study funding/competing interest(s): The experiment was supported by university hospital and it has not any vested conﬂict. Trial registration number: Not applicable. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 No statistical difference was found regarding FR p: 0,961), CR (p: 0,884), EQ (0,848) and PR (0,329). Oocytes injected with HA-bound spermatozoa had not different FR (82, 33) than the conventional group (82, 46) neither CR (96, 71 vs 97, 03), or EQ (74, 23 vs 75, 6). Lower pregnancy rate was observed in oocytes injected with HA-bound spermatozoa than the conventional group, but the difference was not signiﬁcant (61, 0 vs 53, 2). When the study population was divided by the SDF level (cutoff 30 %), there were no statistical differences regarding FR, CR, EQ and PR when HA-selected spermatozoa were used. Limitations, reason for caution: We haveńt limitations in our study Wider implications of the findings: The majority of studies have consistently reported that HA-binding method is effective in selection of spermatozoa without DNA fragmentation and with normal nucleus. This is the ﬁrst report with HA-bound spermatozoa using oocytes obtained from young and fertile donors in previous cycles and this way to isolate the real inﬂuence of HA in clinical results. Study funding/competing interest(s): No study funding or competing interest Trial registration number: No trial registration numbre i155 i156 P-094 Abstracts Local evaluation and validation of open system oocyte vitrification method P. Boyer, D. Montjean, P. Tourame, and M. Gervoise-Boyer Hopital Saint Joseph, Reproductive Medicine and Embryology Department, Marseille, France P-095 Oocyte ERM proteins play a crucial role in gamete adhesion/ fusion J. Cohen, B. Lefevre, C. Ialy Radio, J.P. Wolf, and A. Ziyyat Universite Paris Descartes Institut Cochin, Inserm U1016, Paris, France Study question: Using siRNA intracytoplasmic injection in mouse oocytes, the purpose of the study was to determine the role of oocyte Ezrin, Radixin and Moesin (ERM) proteins in gamete adhesion/fusion. Summary answer: We found that fertilization index (mean number of sperm fused by zona pellucida free egg) was 57% decreased, and that oocyte microvilli were thicker after oocyte ERM inhibition. What is known already: On the mammalian oocyte, Cd9 is the only molecule known to be essential for gamete membrane adhesion/fusion. In the absence of Cd9, oocyte microvilli are thicker and adhesion exists but remains abortive. Cd9 generates adhesion sites, named pro-fusional, with strong adhesion. This adhesion is strong because of the connection between the membrane and the cytoskeleton, which is realized indirectly through other molecules, Cd9 partners, such as ERM that connect actin cytoskeleton to plasma membrane. P-096 Clinical outcome of vitrified single blastocyst transfer as related to blastocyst morphology before vitrification I. De Croo, A. Tolpe, S. Degheselle, A. Van de Velde, K. Tilleman, P. De Sutter, and E. Van den Abbeel Ghent University Hospital, Centre for Reproductive Medicine, Ghent, Belgium Study question: How does the clinical outcome of vitriﬁed single blastocyst transfer compare between poor-quality and good-quality blastocysts before vitriﬁcation? Summary answer: Vitriﬁcation and warming of poor-quality blastocysts has clinical signiﬁcance since in our hands acceptable clinical outcome is achieved as compared to vitriﬁcation and warming of good-quality blastocysts. What is known already: Results with blastocyst transfer allow the use of single embryo transfer, thereby reducing the incidence of multiple pregnancies. With successful vitriﬁcation techniques, cryopreserved embryo transfer cycles have become an integral part of IVF treatment. In fresh cycles, several reports have indicated the importance of the blastocyst morphology on clinical outcome after transfer. In most IVF centers poor-quality blastocysts are not cryopreserved and therefore little is known about their clinical outcome after vitriﬁcation. Study design, size, duration: Between February 1st 2012 to October 10th 2012, 462 vitriﬁed blastocysts were warmed in 366 warming cycles. After warming and overnight culture, we retrospectively investigated clinical pregnancy rates (CPR) as related to blastocyst morphology before vitriﬁcation in 223 single vitriﬁed blastocyst transfer (SVBT). Participants/materials, setting, methods: Blastocysts were graded according to Schoolcraft&Gardner (1999). Early, full, expanded and hatching blastocysts were vitriﬁed. On day 5, early, full and expanded/hatching blastocysts with an ICM C and/or a TE C were considered poor-quality blastocysts. Full and expanded/hatching blastocysts with ICM A/B and/or TE A/B were considered good-quality blastocysts. Main results and the role of chance: Morphological survival rate immediately after warming was 85.7% (396/462). After overnight culture, we performed Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: Is there a really need to validate oocyte vitriﬁcation technique in an IVF laboratory before establishing it in daily practice? Summary answer: Validation of micromanipulation-based technique, in this case oocyte vitriﬁcation, is essential to evaluate its efﬁciency prior to enlarging the use of the system to routine practice. What is known already: Oocyte vitriﬁcation is a new worldwide used technique and legal recently in France. This micromanipulation needs to be performed by a conﬁrmed embryologist and requires an internal assessment in each IVF unit before any wide use. Study design, size, duration: We designed a prospective study, from September 2011 to July 2012, using sibling oocytes from women who recovered more than 12 Metaphase II oocytes. A part of freshly recovered oocytes underwent immediate ICSI while the remaining oocytes were vitriﬁed. Participants/materials, setting, methods: 62 couples (872 Metaphase II oocytes) undergoing ICSI were selected based on number of oocytes available at recovery. 405 Metaphase II oocytes were microinjected after retrieval and 467 were vitriﬁed using an open system (Cryotopw). Main results and the role of chance: To date, 145 oocytes have been thawed for 24 couples. This allowed us to estimate both the mean survival rate (84.9 % +17.9) and fertilisation rate after oocytes thawing at 66.9 %+ 32.9 compared to 56.4 % +27.8 in freshly recovered oocyte sub-group No statistical difference was found. The number of embryos transferred/number of Metaphase II ratio is 38/157 for fresh oocytes and 39/145 for warmed oocytes (p . 0.05). Among the 62 included couples, 21 clinical pregnancies were recorded in the fresh ICSI oocytes and 6 were registered in the vitriﬁed/warmed oocyte cohort. Interestingly, the present evaluation led to a satisfactory cumulative pregnancy rate: 43.5%. One additional beneﬁt of such practice is the limitation of embryo freezing. Limitations, reason for caution: The study design delays oocytes warming cycles, due to pregnancies triggered by the transfer of fresh derived oocyte embryos and to the priority to transfer all the frozen embryos before starting oocytes warming. Furthermore, no data is available about children’ health. Wider implications of the findings: Oocyte vitriﬁcation represents a dramatic change in our daily practice. Indeed, it enables us to be in line with the will of local authorities to decrease the number of cryopreserved embryos while ensuring satisfactory pregnancy chances. The encouraging results suggest that oocyte vitriﬁcation may also be used for a larger panel of purposes including oocyte storing in cases of sperm retrieval failure, fertility preservation or egg donation. Study funding/competing interest(s): Kitazato BioPharma provided Cryotop safety Kits. Trial registration number: Clinical Trials Registration number: 209 R02 Study design, size, duration: We performed a cross sectional (control versus treatment) study. Number of oocytes: 149 (siRNA control injected¼ 82 / siRNA ERM injected ¼ 67). The oocytes where injected at the germinal-vesicle stage oocytes (GV). Participants/materials, setting, methods: Animals were 6 weeks-old females B6CBAF1 mice. We realized RNA silencing by oocyte intracytoplasmic siRNA microinjection under microscopic control at GV stage oocytes. Zona pellucida was removed before IVF. Efﬁciency of the protein silencing and Fertilization Index (FI) were assessed by immunoﬂuorescence (Dapi). Oocyte microvilli morphology was studied by electronic microscopy. Main results and the role of chance: Efﬁciency of the silencing was assessed by the decrease or the disappearance of oocyte membrane ERM. The ﬂuorescence intensity was quantiﬁed with specialized software. After ERM combined siRNAs injection (N ¼ 67 oocytes), Fertilization Index was 1.2 sperm per egg and 2.8 after control si RNA injection (N ¼ 82 oocytes), which means a 57% decrease (p , 0,001). After ERM siRNAs injection (N microvilli ¼ 521; N oocytes ¼ 5), oocyte microvilli radius of curvature was 37.8% increased (34.6 nm versus 47.7 nm; p , 0,001) compared to control injected siRNA (N microvilli ¼ 350; N oocytes ¼ 5). Fertilization Index was not reduced when silencing separately Ezrin, Radixin or Moesin. Limitations, reason for caution: The main limitation of this study is the partial inhibition provided by RNA silencing. It was impossible to use a triple knock-out model because of the premature lethality of Ezrin 2/- mice. Wider implications of the findings: This work provides new elements in the understanding of Cd9 mechanisms in gamete adhesion/fusion. These observations are a strong argument in favor of the cis-action of Cd9, which is the most likely mechanism: gamete fusion could succeed thanks to a strong link between plasma membrane and its underlying cytoskeleton mediated by Cd9 and ERM proteins. Study funding/competing interest(s): We declare no competing interest. Trial registration number: No registration number. i157 Abstracts P-097 A comparison of two different vitrification methods for cryopreservation of mature human oocytes S. Kagalwala1, G. Gandhi1, G. Allahbadia 2, M. Kuwayama3, A. Allahbadia2, V. Chipkar1, A. Khatoon1, R. Ramani1, M. Madne1, and S. Alsule1 1 Rotunda CHR, Assisted Reproduction Laboratory, Mumbai, India, 2Rotunda CHR, Assisted Reproduction, Mumbai, India, 3Repro-Support Medical Research Center, Assisted Reproduction, Tokyo, Japan Study question: To compare the efﬁcacy of two different methods of vitriﬁcation for cryopreservation of human oocytes: The Cryotop method and the Cryotech method. Summary answer: Cryotech vitriﬁcation method for oocyte cryopreservation gives slightly higher survival and fertilization rates and signiﬁcantly higher cleavage rate compared to the Cryotop method. What is known already: Vitriﬁcation is a highly effective method for successful cryopreservation of oocytes. Various media, methods and carrier devices are being used for optimizing the vitriﬁcation technique. Cryotop vitriﬁcation method is known to give excellent results. The Cryotech method is different from the Cryotop method in terms of the constituents of its solutions as well as the design of the plates and the carrier device. Study design, size, duration: This is a retrospective data analysis of donor egg -IVF cycles using vitriﬁed oocytes from October 2010 to August 2012. The oocytes were vitriﬁed using either the cryotop or cryotech vitriﬁcation method. A total of 611 mature oocytes were vitriﬁed and 131 embryo transfer cycles were performed using the embryos created after fertilizing the warmed oocytes with ICSI. Participants/materials, setting, methods: Donor oocytes were vitriﬁed using either cryotech or cryotop vitriﬁcation method. The frozen oocytes were then warmed using the respective warming media. The surviving oocytes were fertilized by ICSI. Embryo transfer was performed in the recipients using these embryos. The survival, fertilization, cleavage and the clinical pregnancy rates were compared in the two groups. Main results and the role of chance: A total of 275 mature oocytes were vitriﬁed using cryotech method and 336 mature oocytes using cryotop method. The mean age of the egg donors was 25.3 + 2.8 years and 24.8 + 2.4 years for Cryotech and Cryotop methods respectively. The survival rate of warmed oocytes with Cryotech media was 97.1% (n ¼ 267/275) while for Cryotop media it was 95.1% (n ¼ 319/ 336; p ¼ 0.19). The fertilization rate of Cryotech group was 90.7% (n ¼ 240/267) and Cryotop group was 86.1% (n ¼ 274/319; p ¼ 0.05). The cleavage rate for Cryotech group was 96.8% (n ¼ 232/240) and Cryotop group was 91.9% (n ¼ 251/274; p ¼ 0.01). The clinical pregnancy rate for Cryotech group was 54.8% (n ¼ 34/62) and Cryotop group was 40.6% (n¼ 28/69). Limitations, reason for caution: A limitation of this study is that it is not done using sibling oocytes. However, there was no statistically signiﬁcant difference in the age and fertility parameters of the donors of both the groups. Wider implications of the findings: Even though the survival rate of oocytes in both the groups did not show a statistically signiﬁcant difference, the embryo development was better in the Cryotech group, as reﬂected by higher cleavage rate. This may be attributed to less trauma to the oocytes vitriﬁed and warmed using Cryotech method. The Cryotech method would be very useful in building donor oocyte banks and fertility preservation of cancer patients. Study funding/competing interest(s): No funding was used. Trial registration number: Not Applicable P-098 Influence of storage period of vitrified embryos on clinical outcome Inaba1, M. A. Ohgaki1, A. Ohtani1, H. Matsumoto1, S. Mizuno1, R. Mori2, A. Fukuda2, and Y. Morimoto3 1 IVF osaka clinic, Division of reproductive technology, Osaka, Japan, 2IVF osaka clinic, Doctor, Osaka, Japan, 3IVF namba clinic, Doctor, Osaka, Japan Study question: The present study was conducted to investigate the inﬂuence of vitriﬁcation on laboratory and clinical outcomes depending on the period of storage in the stage of 2PN, cleaved embryo and blastocyst. Summary answer: The present study suggested that cryopreservation of embryos at any stages by vitriﬁcation had no detrimental effect on cryosurvival and clinical outcome. What is known already: Frozen-thawed or vitriﬁed-warmed embryo transfer (FET) in ART has been recently increasing with not only an advancement of cryopreservation technique such as vitriﬁcation, but also a trend of single embryo transfer (SET) to prevent multiple pregnancy. Cryopreservation of embryos by slow freezing method has been used more than 30 years and the inﬂuence of this method has been evaluated. However, vittriﬁcaion is a relatively new technique and inﬂuence of vitriﬁcation on clinical outcome has not been elucidated. Study design, size, duration: In 3392 FET cycles of SET (1674 patients), 8796 embryos (6069 2PNs, 1573 cleaved embryos and 1154 blastocysts) were warmed for transfer from January 2010 to December 2012. Data obtained from these embryos were analyzed retrospectively. Participants/materials, setting, methods: Those embryos were divided into 4 groups based on the storage period, A: ,1 year, B: 1-2 years, C: 2-3 years, D: .3 years. Survival after warming in each period was compared in each stage of embryos. Moreover, the inﬂuence of storage period on the rates pregnancy and live birth were compared. Main results and the role of chance: 1. Survival rates in group A, B, C, D and average were as follows. 2PN: 98.0%, 98.7%, 100.0%, 100.0% and 98.1%. Cleaved embryo: 96.3%, 95.1%, 94.9%, 100.0% and 96.1%, respectively. Blastocyst: 97.9%, 97.8%, 100.0%, 100.0% and 97.9%, respectively. There were no signiﬁcant differences among these 4 groups in each stage. 2. Pregnancy rates in group A, B, C, D and average were as follows. Cleaved embryos: 32.6%, 42.9%, 26.7 % and 25.0% and 33.0%. Blastocyst: 42.9%, 45.7%, 56.3% and 41.7%, and 43.7%, respectively. Live birth rates were as follows. Cleaved embryos: 76.5%, 83.3 %, 100.0%, 50.0%, and 77.1%. Blastocyst: 80.5%, 79.3%, 77.8%, 60.0%, and 80.0%, respectively. There were no signiﬁcant differences among 4 groups either in cleaved embryo or blastocyst. Limitations, reason for caution: None Wider implications of the findings: The present study suggested that cryopreservation of embryos at any stages by vitriﬁcation had no detrimental effect on cryosurvival and clinical outcome regardless of storage period for at least 3 years. The longest period was 5 years and 6 months with healthy baby born. Vitriﬁcation is very effective, laboratory friendly and safe cryopreservation method. Study funding/competing interest(s): None Trial registration number: None P-099 Outcome of blastocyst vitrification with cryoloops: clinical outcome of 2,924 cycles comparing with 2,003 fresh blastocyst transfer cycles Y. Umekawa, A. Yoshida, S. Tanigiwa, K. Seida, H. Suzuki, and M. Tanaka Kiba Park Clinic, Tokyo, Japan Study question: Is ultra rapid vitriﬁcation method of blastocysts using a cryoloop safe and effective? Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 278 vitriﬁed blastocyst transfers. Overall pregnancy rate (% positive HCG) was 40.3%/ET and 30.6% per warming cycle. CPR was 29.1%/ET with an implantation rate of 21.4% (99/462) per warmed blastocyst. More speciﬁcally and independent of ICM and TE quality, CPR per SVBT was 19.1% (14/ 73), 27.9% (17/61) and 27.0% (24/89) for early, full and expanded blastocysts respectively (p ¼ 0.3). In the expanded blastocyst group with ICM or TE grade C, CPR was 25.9% (7/27) compared to 28.4% (35/123) in the group with ICM and TE grade A and B. Taken together the CPR per SVTB was 21% (21/100) for poor-quality blastocysts versus 28.4% 35/ 123) for good-quality blastocysts (p ¼ 0.2). Limitations, reason for caution: Much larger study groups are required to evaluate clinical pregnancy and miscarriage rates by the speciﬁc blastocyst morphological scores especially as related to the different types of ICM and TE in full and expanded/expanding blastocysts. Wider implications of the findings: Our ﬁndings could change cryopreservation policies in IVF centres performing blastocyst vitriﬁcation because so far in most centres only good-quality blastocysts are considered eligible for vitriﬁcation. Study funding/competing interest(s): This study was not funded and there were no competing interests. Trial registration number: Inapplicable. i158 P-100 Anthocyanin improves viability and maturation of sheep oocytes cultured In the presence of cAMP modulators Z. Vahabi1, P. Eftekhari Yazdi1, A. Dalman1, B. Ebrahimi1, F. Mostafaei 2, and M. Rajabpour Niknam1 1 Department of Embryology Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine ACECR, Tehran, Iran, 2Animal Core Facility Reproductive Biomedicine Research Center, Royan Institute for Animal Biotechnology ACECR, Tehran, Iran Study question: Can high levels of cAMP alone support sheep cultured oocytes from apoptosis? Summary answer: Our ﬁndings showed that, cAMP modulators couldn’t support maturation and viability of sheep oocytes. While the Pre-IVM and IVM mediums supplemented with delphinidin chloride could signiﬁcantly Decrease apoptosis and increase maturation of sheep oocytes. What is known already: To our knowledge, no studies to data have focused on apoptosis repression in sheep oocytes cultured at high levels of cAMP. Study design, size, duration: Cumulus-oocyte complexes (COCs) were cultured in two biphasic mediums: 1) Pre-IVM and IVM mediums supplemented with delphinidin chloride and 2) Pre-IVM and IVM mediums without delphinidin chloride. Differences in maturation and viability rates between COCs cultured in two experimental groups were determined by the Chi-square test. Participants/materials, setting, methods: COCs were recovered from ovine ovaries collected at a local slaughterhouse. Good-quality COCs were randomly transferred into two biphasic IVM mediums. After maturation period, oocytes were denuded from cumulus cells and MII oocytes with ﬁrst polar body were counted. Oocyte viability was determined by means of the TUNEL technique. Main results and the role of chance: The percentage of oocytes that reached metaphase II was signiﬁcantly higher for group 1 (93.5% and 73.0%, respectively P , 0.0001) than group 2 oocytes. The percentage of viable oocytes at group 1 was higher ( 98.9% and 85.1%, respectively P , 0.001) than group 2 oocytes. Maturation rate and viability detection by TUNEL kit in two experimental groups in sheep oocytes. Limitations, reason for caution: We have any limitation. Wider implications of the findings: Some studies reported that, High levels of cAMP suppress apoptosis by inhibition of Caspase 3 activation. In addition Arnault and Colleagues (2008) showed that caspases may be dispensable for ﬁnal oocyte death in mouse. In this study we used delphinidin chloride, the activator of GSH synthesis. Based on our ﬁndings, it seems that GSH synthesis Experimental groups Oocytes Matured oocytes Oocytes for TUNEL Viable oocytes ......................................................................................... 0.1 mg/ml delphinidin chloride (group 1) 217 202 (93.5%) 90 89 without delphinidin chloride (group 2) 215 175 (73.0%) 87 74 activators more effective than inhibitors of Caspase-3 activators in oocyte death control. Study funding/competing interest(s): This work was supported by Embryology Department of Royan Institute. Trial registration number: Our study wasn’t clinical trial P-101 Correlation of the duration of the first cleavage cycle of embryos with good quality blastocyst and implantation rates in single frozen-thawed blastocyst transfer S. Watanabe1, M. Kamihata1, T. Tanaka1, R. Matsunaga1, N. Yamanaka1, C. Kani1, T. Ishikawa1, T. Wada1, H. Morita1, H. Miyamura2, E. Nishio2, M. Ito3, A. Kuwahata3, M. Ochi3, and T. Horiuchi4 1 Ochi Yume Clinic Nagoya, ART lab., Nagoya, Japan, 2Fujita Health University Hospital, Obstetrics and Gynecology, Toyoake, Japan, 3Ochi Yume Clinic Nagoya, Reproductive Medicine, Nagoya, Japan, 4Prefectural University of Hiroshima, Graduate School of Comprehensive Scientiﬁc Research, Shobara, Japan Study question: Is there any correlation of the ﬁrst cleavage duration of embryos with the success rate of obtaining good quality blastocysts and the implantation rates in single frozen-thawed blastocyst transfer cycles? Summary answer: There is a correlation between the duration of the ﬁrst cleavage cycle of embryos and good quality blastocyst and implantation rates. What is known already: It has been reported that there is a correlation between the duration of the ﬁrst cleavage cycle of an embryo and the formation of a viable blastocyst. Study design, size, duration: A retrospective study on 357 embryos from 182 cycles which were cultured to the blastocyst stage by using a time-lapse incubator between July and October 2012. Participants/materials, setting, methods: We recorded and analyzed the duration of the ﬁrst cleavage of embryos by using a time-lapse incubator (EmbryoScope, Unisense Fertilitech, Denmark), to examine the correlation of the ﬁrst cleavage duration with the success rate of obtaining good quality blastocysts and with the pregnancy rate in single frozen-thawed blastocyst transfer cycles. Main results and the role of chance: Among the 357 normal fertilized embryos cultured by using the ES, 124 (34.7%) were good quality blastocysts. The duration of the ﬁrst cleavage in the good blastocyst group and the non-good-quality blastocyst group (mean + SD) was 25.4 + 2.8 and 28.7 + 4.6 hours, respectively, which showed a signiﬁcant difference (P , 0.01). Seventy-three single frozen-thawed blastocyst transfers were performed in the good blastocyst group. The pregnancy rate of the group with a ﬁrst cleavage duration of 24–25 hours (69.2%, 9 of 13) tended to be greater than that of the 20– 24 hours group (44.4%, 12 of 27) (P ¼ 0.186), and signiﬁcantly greater than that of the 25–35 hours group (27.3%, 9 of 33) (P , 0.05). Limitations, reason for caution: None. Wider implications of the findings: We conﬁrmed that the duration of the ﬁrst cleavage cycle of embryos might be an effective parameter for ensuring viable high- quality blastocysts for transfer. Study funding/competing interest(s): No external funding was obtained for this study. Trial registration number: None. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Summary answer: Ultra rapid vitriﬁcation method of blastocysts using a cryoloop is a safe and valid method as a result of comparison between vitriﬁed-thawed blastocyst ET and fresh blastocyst ET cycles. What is known already: Blastocyst transfer has become a promising option to get the higher pregnancy rate, comparing with cleavage stage embryo transfer. Vitriﬁcation is expected to achieve a high rate of survival because of the absence of ice. Vitriﬁcation has become the method to cryopreserve blastocysts. Study design, size, duration: This is a retrospective analysis of clinical outcome in 2,924 vitriﬁed-thawed blastocyst ET cycles and 2,003 fresh blastocyst ET cycles conducted for women under the age of 40 from 2002 to 2011. Participants/materials, setting, methods: IVF and ICSI procedures were carried out with sequential media. The protocol for the cryoloop vitriﬁcation of blastocysts from IVF/ICSI patients was adopted. Embryo transfers were performed under ultrasound examination using a soft catheter. Main results and the role of chance: The average embryo transfer number of vitriﬁed-thawed blastocyst transfer was signiﬁcantly lower than that of fresh blastocyst transfer (1.4 + 0.5 vs 1.5 + 0.6: P Limitations, reason for caution: Long term follow-up of children from ultra rapid vitriﬁcation method of blastocysts using a cryoloop, including motor and psychological development, is recommended. Wider implications of the findings: Ultra rapid cryoloop vitriﬁcation method of blastocysts is easy-to use, safe and effective. Outcome of ultra rapid cryoloop vitriﬁcation method of blastocysts is comparable with that of fresh blastocyst transfer. Study funding/competing interest(s): This study was not funded and there are no conﬂicts of interest. Trial registration number: n/a Abstracts i159 Abstracts P-102 In vitro maturation does not affect the morphology of the metaphase II spindle in oocytes collected from antral follicles in IVM cycles M. Dal Canto1, M.C. Guglielmo1, R. Fadini1, M. Mignini Renzini1, D.F. Albertini2, P. Novara1, M. Lain1, F. Brambillasca1, D. Turchi1, M. Sottocornola1, and G. Coticchio1 1 Biogenesi Reproductive Medicine Centre, Istituti Clinici Zucchi, Monza, Italy, 2 Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, U.S.A P-103 Effects of sperm concentration after swim-up on conventional in vitro fertilization rates: Analysis of sperm motility using a sperm motility analysis system M. Kato 1, N. Fukunaga2, R. Nagai1, H. Kitasaka2, T. Yoshimura1, F. Tamura2, N. Hasegawa1, K. Nakayama 2, M. Takeuchi2, H. Ohno2, N. Aoyagi 1, E. Kojima1, F. Itoi3, Y. Hashiba4, and Y. Asada5 Asada Ladies Kachigawa Clinic, Laboratory, Aichi, Japan, 2Asada Ladies Nagoya Clinic, Laboratory, Aichi, Japan, 3The Asada Institute for Reproductive Medicine, Laboratory, Aichi, Japan, 4Asada Ladies Kachigawa Clinic, Doctor, Aichi, Japan, 5Asada Ladies Nagoya Clinic, Doctor, Aichi, Japan Study question: Does sperm concentration after swim-up affect fertilization rates in conventional in vitro fertilization (C-IVF)? Summary answer: It is thought that using a high concentration of spermatozoa with motility after swim-up and high curve speed and head amplitude improve the normal fertilization rate of C-IVF. What is known already: In conventional in vitro fertilization, it is important to improve the rate of normal fertilization. Our C-IVF procedure entails adjusting the count to 200,000 spermatozoa after swim-up. However, there may be differences in some cases regarding the normal fertilization rate with our procedure, and the reason for this is currently not clear. To address this problem, we used a sperm motility analysis system (SMAS) for the ﬁrst time in our clinic. Study design, size, duration: Concentration of spermatozoa with motility after swim-up was divided into two groups: not less than 1.0 × 106/ml and not more than 2.0 × 106/ml (Group A); and not less than 2.1 × 106/ml and not more than 14.9 × 106/ml (Group B). Fertilization rates were compared in this retrospective study. Participants/materials, setting, methods: From January through May 2011, we conducted a review of 524 cycles in 483 patients. In addition, we conducted a review of 42 cycles in 42 patients between May 2012 and September 2012. The motility rates of Group A and group B were analyzed via SMAS. Main results and the role of chance: The normal fertilization rate of Group Awas 48%; that of Group B was 63.0%. Thus, the normal fertilization rate was signiﬁcantly higher in Group B compared to Group A (P , /SSF . , 0.01). The rate of abnormal fertilization (1PN, ≥3PN) of Group A was 6.1%; that of Group B was 5.8%. With SMAS, the straight line speed, straight advancement, curve speed, and head amplitude were 31.5 mm/s, 0.36, 96.1 mm/s, and 2.04 mm, respectively for Group A and 34.8 mm/s, 0.34, 112.5 mm/s, 2.68 mm, respectively for Group B. Group B had a higher curve speed and head amplitude than Group A. Limitations, reason for caution: These are preliminary data from a retrospective analysis with a limited sample size. Wider implications of the findings: Not determinable until further studies using SMAS with a larger sample have been performed. Study funding/competing interest(s): None. Trial registration number: None. P-104 Study of the most effective embryo transfer with blastocyst grade H. Kikuchi, Y. Iwasa, T. Kamono, A. Suzuki, K. Yamada, H. Kanno, K. Sasaki, H. Murakawa, M. Matsubara, and H. Yoshida Yoshida Ladies Clinic, Center for Reproductive Medicine, Sendai, Japan Study question: It has reported that blastocyst transfer is able to obtain better result by synchronization with endometrium. However, depends on the situation, such as priming egg retrieval or patient’s offer, it is necessary to screen the blastocyst for fresh transfer. Summary answer: Regarding to blastocyst ICM and TE grade, grade A blastocyst resulted in signiﬁcantly high pregnancy rate in frozen-thawed cycle. It is proved that frozen-thawed cycle is susceptible to improve the pregnancy rate by classiﬁcation of grade. It is suggested that not only good embryos but inferior ones are worth cryopreservation. What is known already: Recently, the viability after thawing is getting remarkably high through the improvement of cryopreservation vitriﬁcation. It has reported that blastocyst transfer is able to obtain better result by synchronization with endometrium. Study design, size, duration: We classiﬁed 141 cases (138 patients) in fresh cycle and 673 cases (473 patients) in frozen-thawed cycle, which underwent single blastocyst transfer from January 2010 through October 2012 in our clinic. Participants/materials, setting, methods: We compared the clinical offspring and abortion rate in fresh cycle and frozen-thawed cycle with Gardner’s classiﬁcation which were Inner Cell Mass (ICM) and Trophectoderm (TE) cell numbers. Main results and the role of chance: The patient mean age was 33.8 + 3.8 years old in fresh cycle, 35.6 + 4.0 years old in frozen-thawed, so there was a signiﬁcant difference (P , 0.05). In the view of ICM grade, the pregnancy rate in frozen-thawed cycle is signiﬁcantly high in ICM grade A (P , 0.05) although Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: In vitro maturation (IVM) might affect the organization and function of the metaphase II (MII) spindle, compromising oocyte quality. The study object was to assess comparatively the morphology of the MII spindle in oocytes matured in vitro from IVM cycles and oocytes matured in vivo in stimulated cycles (IVO). Summary answer: IVM does not alter the geometry of the MII spindle in human oocytes recovered in IVM cycles matured in vitro. In addition, distribution of cortical actin appears to be polarized showing an accumulation in proximity of the spindle, perhaps to ensure localization and anchorage of this cytoskeletal component. What is known already: In the mouse, it is widely recognized that in vitro maturation can alter the structure of the oocyte MII spindle. This could have implications for chromatid segregation and embryo chromosome status. In the human, it has been repeatedly reported that IVM oocytes may be affected by spindle anomalies, but such ﬁndings are questionable because were generated using a rather inappropriate model, i.e. leftover denuded germinal vesicle (GV) oocytes obtained from conventional ovarian stimulation cycles. Study design, size, duration: Fifty-nine IVO and 10 IVM oocytes were analysed. No leftovers GV oocytes from stimulated cycles were included. Chromosomes were classiﬁed as displaced or aligned at MII plate. Their internal and external arrangement was classiﬁed as two perfect rings or partially/totally disarranged. Cortical and cytoplasmic actin was also compared. Participants/materials, setting, methods: IVO oocytes derived from women undergoing conventional ovarian stimulation. IVM oocytes matured in vitro from cumulus-oocyte complexes in the presence of FSH/HCG. Chromatin, tubulin and actin were detected by ﬂuorescence confocal microscopy. Spindles were reconstructed as 3D images. P ,0.05 was adopted as criterion to consider differences statistically signiﬁcant. Main results and the role of chance: The MII spindles of the two groups were morphologically equivalent as shown by comparing pole-to-pole axis (11.8 + 2.6 mm in IVO vs. 12.0 + 2.3 mm in IVM oocytes), equatorial axis (8.9 + 1.7 mm in IVO vs. 8.6 + 1.5 mm in IVM oocytes), and area of maximum projection (88.8 + 29.5 mm2 in IVO vs. 94.7 + 18.1 mm2 in IVM oocytes). Chromosome alignment and internal/external disposition were not related to spindle characteristics. Proportions of normal chromosome arrangement were 64.5% and 60.0% in IVO and IVM oocytes respectively. Signal intensity of cortical actin was higher near the spindle, in both oocyte types. Conversely, the mean intensity of cytoplasmic actin in IVM oocytes was higher than in IVO oocytes. (21.1 vs. 35.0). Limitations, reason for caution: Data regarding IVM oocytes are numerically limited and require further conﬁrmation. Wider implications of the findings: This is the ﬁrst study focusing on spindle characteristics in oocytes obtained from normo-ovulatory patients undergoing IVM. Data suggest that IVM conditions do not affect the structure of the MII spindle, disputing a widespread preconception. Moreover, this study introduces new criteria for the analysis of chromosome arrangement and reveals a polarization of cortical actin. Accumulation of cytoplasmic actin in immature oocytes suggests the basis for futures studies aimed at understanding spindle morphogenesis during IVM. Study funding/competing interest(s): This study was partly funded by the Italian Ministry of Health. The authors declare to have no interests conﬂicting with the study. Trial registration number: Not applicable 1 i160 no signiﬁcant difference in fresh pregnancy cycle (P , 0.005). In the view of TE grade, the pregnancy rate in frozen-thawed cycle is signiﬁcantly high in TE grade A (P , 0.025). As compared with each grade in same group, TE grade A has signiﬁcantly high rate, and grade B was the following in frozen-thawed cycle though no signiﬁcant difference was observed in fresh cycle. Limitations, reason for caution: None. Wider implications of the findings: We suggested that the best way to improve total pregnancy rate per oocyte pick-up is need to cryopreservation for effective frozen-thawed cycle in case of good quality embryo was obtained, and then perform fresh second grade embryo transfer. Study funding/competing interest(s): No competing interest is declared. Trial registration number: None. Comparison of four techniques for artificial shrinkage of the blastocoele cavity pre-vitrification C. Valdespin1, M. Elhelaly2, P. Chen3, M. Pangestu3, and S. Catt3 Nascere, Reproductive Medicine, Mexico City, Mexico, 2Al-ahram Fertility Center, Embryology, Mansoura, Egypt, 3Monash University, Obstetrics & Gynecology, Melbourne, Australia 1 Study question: Pre-vitriﬁcation artiﬁcial shrinkage: are hyperosmotic solutions as effective as laser pulse or micro-puncture techniques?. Summary answer: Collapsing expanded mouse blastocysts prior to vitriﬁcation by either laser, micro-puncture/suction or the hyperosmotic solutions: sucrose and trehalose is associated with greater survival, expansion and hatching rates when compared to non-collapsed controls. The novel use of trehalose provides an excellent low cost, low skill option with proven implantation. What is known already: Improvements in survival and implantation rates of expanded blastocysts have been demonstrated with laser pulse and micropuncture collapse, but these techniques require expensive equipment or advanced skill sets. A simple hyperosmotic pre-vitriﬁcation collapsing technique using sucrose has been tested successfully as an alternative method. We have postulated that trehalose may be a superior sugar to use due to its membrane stabilization properties. Study design, size, duration: 367 expanded blastocysts were assigned to 5 groups: laser-pulse [LP], micro-puncture/suction [MS], use of trehalose previtriﬁcation [TPV], and use of sucrose pre-vitriﬁcation [SPV] and compared to a control group (CG). 30 embryos/group were assigned for transfer (at 4h postwarm) to pseudo-pregnant female mice while the remaining blastocysts were cultured overnight. Participants/materials, setting, methods: Blastocysts were vitriﬁed, using a short protocol on Cryotops. Blastocysts were assessed prior to transfer for survival and re-expansion (classed as .70% of blastocoel re-expanded) and then, for implantation on day 12 post-transfer. The remaining cultured blastocysts were evaluated for re-expansion, and hatching rates at 24h post-warm. Main results and the role of chance: Overall 98.7% of all warmed blastocysts survived as assessed at 4h, but ,50% were classed as re-expanded at transfer. At 24h, re-expansion rates were higher for the combined collapsed groups (82%, P , 0.05, Chi Square) compared to CG (66%), with TPVand LP exhibiting the highest rates of expansion (87% and 82% respectively), and TSV and MS slightly lower (80% and 76.3% respectively). Hatching rates were also higher for the combined collapsed groups (40%, P , 0.05) compared to the control (22%), with TVP and SVP having the highest rates (56.5% and 40% respectively), compared to CG (P, 0.01 and p , 0.05 respectively). All ﬁve groups had at least one blastocyst implant and the highest rate was 6/30 for TPV. Limitations, reason for caution: Although we have demonstrated that collapsing gives better outcomes and hyperosmotic treatments was successful, further studies are required to examine these ﬁndings. Implantation was demonstrated but future transfers would beneﬁt from improved synchronization as, in this study, we transferred day 4.5 blastocysts into surrogate female mice 2.5 days after mating. Wider implications of the findings: Vitriﬁcation has become the popular choice for blastocyst preservation, and in general, studies have shown that the expanded blastocyst beneﬁts from collapse prior to vitriﬁcation. We have demonstrated that passing through two hyperosmotic solutions, just prior to the equilibration step in the vitriﬁcation process, can improve post-warm outcomes, particularly when trehalose is used. Thus, collapse can be performed cheaply and efﬁciently without the need for specialized equipment or advanced technical skill. Study funding/competing interest(s): No direct funding or competing interests. Trial registration number: NA P-106 Morphokinetics of embryos acquired from poor responders aged 40 years and above: comparison between natural and stimulated cycles N. Hojnik, B. Kovacic, P. Roglic, M. Taborin, M. Zafosnik, J. Knez, and V. Vlaisavljevic University Medical Centre Maribor, Department of Reproductive Medicine and Gynaecologic Endocrinology, Maribor, Slovenia Study question: Is there a difference in morphokinetics of embryos derived from natural cycles compared to stimulated cycles within the group of poor responders aged ≥40 years? Summary answer: There is no difference in morphokinetic parameters in group of poor responders aged ≥40 years whether embryos derive from natural or stimulated cycles. What is known already: In patients with previous unsuccessful IVF attempts with hormonal stimulation of high dosages and low number of cells retrieved, natural cycle is used as an alternative. Although the success of natural cycles in this group of patients is relatively low, controlled ovarian hyperstimulation resulting in small number of oocytes might not be better choice. Our interest is to deﬁne the quality of the embryos with the morphokinetic parameters. Study design, size, duration: Prospective study included 42 poor-responder patients, aged ≥40 years, treated in our center in year 2012. Natural cycles group: 28 patients, 27 embryos for analysis. Stimulated cycles group: 14 patients, 23 embryos for analysis Participants/materials, setting, methods During 3-day cultivation pictures of embryos were taken in 5-minute intervals. Timing of the appearance and disappearance of the pronuclei, the formation of the cleavage furrow of the ﬁrst and second mitosis and the beginning of the 2,3,4-cell stage was monitored. Time intervals (TI) were compared with Mann-Whitney test. Main results and the role of chance: TI from fertilization to disappearance of the pronuclei (p ¼ 0,42), TI from fertilization to 1st cleavage furrow (p ¼ 0,9), TI from fertilization to 2-cell stage (p ¼ 0,88), TI from 2-cell stage to 2nd cleavage furrow (p ¼ 0,33), TI from 3-cell stage to 4-cell stage (p ¼ 0,14), TI from fertilization to 4-cell stage (p ¼ 0,19). We also compared the development (TI from 3-cell stage to 4-cell stage) of the subset of embryos without the occurence of aberrant or reverse divisions (14 embryo from the natural cycle group compared to the 7 embryos from the stimulated group; p ¼ 0,09). There seems to be no signiﬁcant difference in morphokinetics of embryos from the two groups studied. Limitations, reason for caution: Selection of the treatment (natural/stimulated) was done according to previous attempts and patient preferences. Nevertheless both approaches seemed equally suitable for all patients. Small sample size poses limitations of our study. Wider implications of the findings: Our ﬁndings are in agreement with the literature that ﬁnd the two approaches for treating poor responders comparable. Study funding/competing interest(s): . Trial registration number: . P-107 Hydroxypropyl Cellulose as a novel cryoprotectant for oocyte/ embryo vitrification C. Mori, A. Yabuuchi, K. Ezoe, Y. Takayama, F. Aono, and K. Kato Kato Ladies Clinic, Advancedmedical Research Institute of Fertility, Tokyo, Japan Study question: To determine hydroxypropyl cellulose (HPC) serves as a substitute for serum substitute solution (SSS) as a novel cryoprotectant for oocyte/ embryo vitriﬁcation. Summary answer: The use of HPC as a novel cryoprotectant is advantageous with the higher embryo survival rate after vitriﬁcation. What is known already: Despite the proposal toward designing chemically deﬁned media in human ART, SSS still has been supplemented widely in the vitriﬁcation solution. HPC is an inert viscoelastic polymer, harmless and non-ionic water-soluble cellulose known as a material for food and medical-supply additive agent and also a Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 P-105 Abstracts Abstracts P-108 Morphokinetics and time-lapse technique as a predictor of embryo development to the blastocyst stage P. Radwan, R. Krasinski, K. Chorobik, and M. Radwan Gameta Szpital, Department of Repoduction, Rzgów, Poland Study question: Does the dynamic parameters of cell division assessed by timelapse analysis predicts selection of most viable embryos for embryotransfer ? Summary answer: Adapted for embryology, the time lapse analysis enables the continous monitoring of embryo development and observation of cell division abnormalities and can optimize the selection of most viable embryos for embryotransfer. What is known already: The standard assessment of embryo morphology at given time points not always allows to transfer the embryo with highest implantation potential. The effect of transfer of the improper embryo is the lack of pregnancy or miscarriage and, as a consequence, exposure of patient to unnecessary emotional stress and necessity to perform the frozen embryo transfer Study design, size, duration: Prospective cohort study. Development of 50 embryos was continuously monitored with the use of Primo Visio system (Cryo Innovation, Hungary), with image acquisition every 10 minutes. January 2012- May 2012. Participants/materials, setting, methods: The following parameters were analysed: t2 – time after ICSI to the division to two cells; t3 - time after ICSI to the division to three cells; t4 - time after ICSI to the division to four cells; t3 – t2 – time from division to two cells until division to three blastomeres; t4 – t3 – time from division to three cells until division to four blastomeres. Main results and the role of chance: The embryos developing to the blastocyst stage reached the two cell stage earlier (25,94 +2,17 h vs 28,89 +3,75 h). The time between division to 2 cells and subsequent division to three cells and the time between three- and four cell stage were shorter comparing to the arrested embryos (t3: 37,36 +2,55 h vs 42,09 +5,23 h; t4: 38,49 +2,63 h vs 42,93 +5,01 h). The time interwal between the two- and three cell stage (t3 – t2) in case of embryos developing to the blastocyst stage was shorter. (11,42 +0,80 h vs 13,20 +2,25 h). The direct transition of zygote to the three blastomere stage or too short second cell cycle (t3 – t2), ,5h, was the negative predictive factor. No difference in the duration of third cell cycle (t4 – t3) was observed (0,88 +0,80 h vs 0,84 +1,28 h). Limitations, reason for caution: The study was not powered to test differences in pregnancy rates Wider implications of the findings: Adapted for embryology, the time-lapse analysis can optimize the selection of most viable embryos for embryotransfer. Study funding/competing interest(s): No speciﬁc funding was obtained for this study; it was solely funded by Gameta Ferility Center. None of the authors have any economic afﬁliation. Trial registration number: Not applicable. P-109 Effect of early blastomere multinucleation on pre-implantation developmental timings of human embryos M. Stoppa1, R. Maggiulli1, A. Capalbo1, E. Ievoli 1, L. Dovere1, C. Scarica1, L. Albricci1, S. Romano1, F. Sanges1, N. Barnocchi2, L. Papini2, A. Vivarelli1, F.M. Ubaldi1, and L. Rienzi1 1 Genera Center for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy, 2 Genera, Center for Reproductive Medicine, Umbertide (PG), Italy Study question: The aim of the study was to evaluate whether or not the occurrence of blastomeres multinucleation may disturb the early developmental events of preimplantation embryos. Summary answer: We observed a different behavior in the early events of development of multinucleated embryos with respect to the sibling mononucleated counterpart. What is known already: The occurrence of blastomeres multinucleation may be due to nuclear replication without cytokinesis, nuclear fragmentation or defective migration at mitotic anaphase leading to errors in DNA packaging. Retrospective analyses of human embryo development have previously showed an association of multinucleation with impaired cleavage, increased incidence of aneuploidy and low implantation rates in IVF cycles. Time-lapse cinematography has been recently applied in human IVF, as useful tool to optimize embryo assessment without interfering with embryo growth. Study design, size, duration: The inﬂuence of early blastomere multinucleation on cleavage timing was retrospectively evaluated by comparing the developmental behavior of 114 sibling embryos. To avoid confounding factors, each embryo showing multinucleation at 2-cell stage (MNBs group ¼ 38) was compared with two randomly selected sibling embryos from the same cohort with mononucleated blastomeres (NBs group ¼ 76). Participants/materials, setting, methods: Microinjected oocytes were cultured in a time-lapse incubation system (Embryoscope, Unisense). Early events of development (timing of pronuclear appearance, syngamy, 1st to 7th cell division and cell cycle duration) were monitored and recorded. Multinucleation was assessed as the presence of two or more nuclei in both blastomeres, at 2-cells stage. Main results and the role of chance: Embryonic cohorts of 32 patients (mean female age ¼ 35.8 + 3.5) containing at least one MNB embryo and two randomly selected sibling NB embryos were evaluated. We observed a different behavior of multinucleated embryos with respect to the sibling mononucleated counterpart. The median time value of third (T3) cell cycle was found to be signiﬁcantly different between NBs (T3 ¼ 37.5 + 4.7, CI:36.4-38.7) and MNBs group (T3 ¼ 40.4 + 4.2, CI:39.0-41.9) (p ¼ 0.003). No signiﬁcant difference in the median time value of ﬁrst (T2), third (T4), fourth (T5), seventh (T8) cell cycle was highlighted, although the MNBs group showed consistently slower cleavage timings (T2 ¼ 28.3 + 3.3, CI:27.2-29.3, T4 ¼ 41.3 + 4.4, CI ¼ 39.8-42.8; T5 ¼ 51.4 + 8.5,CI:48.4-54.4, T8 ¼ 61.1 + 7.4,CI:58.2-63.9) compared to the NBs group (T2 ¼ 27.7 + 3.8, CI:26.8-28.6, T4 ¼ 40.0 + 4.9, CI ¼ 38.8-41.2; T5 ¼ 49.8 + 6.9,CI:48.1-51.5, T8 ¼ 59.2 + 10.7,CI:56.6-61.9). Limitations, reason for caution: The main limitation of the study is the rather low number of embryos enrolled (N ¼ 114). Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 free-water retentive polymer which may acts as cryoprotectant of the vitriﬁcation solution. Study design, size, duration: Inbred and hybrid mouse blastocysts with/without zona pellucid (ZP) with different cryoresistance (inbred: C57BL/6J, n ¼ 80, hybrid: C57BL/6JxDBA/2, n ¼ 30) were selected. Generally, hybrid embryos show better freezing tolerance than inbred. Embryos were vitriﬁed/thawed in HPC or SSS supplemented solution with Cryotop and there survival rates were compared using a cell viability assay kit. Participants/materials, setting, methods: 2-cell embryos were collected from superovulated mice and cultured in KSOM media (37C, 5%CO2, 95% Air) for 4 days to obtain blastocyts. ZP-free blastocysts were obtained by the treatment with acid tyrode’s solution. The survival rate of these blastocysts vitriﬁed/ thawed in HPC (1%/vol) or SSS (1%/vol) solutions was assessed. Main results and the role of chance: The rate of blastocyst development in inbred (C57BL/6) and hybrid (C57BL/6JxDBA/2: B6D2F1) 2-cell embryos was 85.3% and 86.8%, respectively. The survival rate of inbred blastocysts with ZP was signiﬁcantly higher in HPC 83.4% (121/145) than in SSS 50% (70/140), whereas in hybrid blastocysts, the survival rate in both HPC and SSS was 100% (32/32 in HPC, 33/33 in SSS). The survival rate of ZP free inbred blastocysts in HPC and SSS was 67.9% (38/56) and 19.6% (9/46), respectively. The survival rate was signiﬁcantly higher in HPS than SSS when embryos with less freezing tolerance (inbred blastocysts, ZP blastocysts) were selected. In addition, we observed the stickiness of different blastocysts to the Cryotop using HPC and SSS. When using SSS, the blastocyst stuck to the Cryotop, but when using HPC it didn’t stick at all. Limitations, reason for caution: The limitation of this work was the use of animal model only. Monitoring of outcomes with human embryos vitriﬁed/thawed in HPC supplemented solution is imperative to convince the safety and efﬁcacy for transferring in the clinical settings. Wider implications of the findings: We demonstrated that HPC is advantageous with higher survival rates of blastocysts even without ZP revealing that HPC is an effective cyroprotectant as a substitute for SSS. We also observed that HPC overcame the stickiness of embryos during the thawing procedure suggesting that HPC may minimize technical complications. Our study shows that the use of HPC as a cryoprotectant will be applicable and would be a standard agent in human ART. Study funding/competing interest(s): The authors declare that they have no conﬂicts of interest in the research. Trial registration number: N/A i161 i162 Wider implications of the findings: We identiﬁed distinctive early cleavage timing ranges for mononucleated and multinucleated embryos. When continuous monitoring of embryo development cannot be performed, the choice of the best embryo to transfer usually relies on the static evaluation of morphological aspect of the embryo. According to conﬁdence intervals observed in our dataset, the persistence of 3-cell stage later than 39.0 hours post insemination suggests the occurrence of blastomere multinucleation at 2-cell stage. Study funding/competing interest(s): none Trial registration number: none P-110 Human embryo aneuploidies cannot be predicted by morphokinetic assessment during the first phases of in vitro culture F. Fiorentino2 Genera c/o Clinica Valle Giulia, Reproductive Medicine, Roma, Italy, 2Genoma, Molecular biology, Roma, Italy, 3IVIOMICS, Molecular Cytogenetics Lab, Valencia, Spain 1 Study question: The aim of our study was to evaluate the reliability of morphokinetic assessments in predicting embryo aneuploidies as assessed by array comparative genomic hybridization (aCGH). Summary answer: No correlation has been found between morphokinetics variables and embryo aneuploidies. What is known already: Preimplantation genetic screening (PGS) has been introduced in IVF to avoid the transfer of aneuploid embryos. However, PGS is an invasive procedure, technically challenging that requires embryo extramanipulation and thus potentially detrimental. For this reason there is an imperative search for new non-invasive techniques able to identify healthy embryos in a reliable way. Time lapse cinematography and embryo morphokinetic assessments is a promising approach. This technology has been recently correlated with signiﬁcant improvements in pregnancy rate. Study design, size, duration: Embryos were individually monitored by time lapse cinematography (Embryoscope, Unisense). Subsequently, biopsy and chromosomal screening by aCGH (Bluegnome, UK) were performed either at day 3 (N ¼ 137) or at day 5 (N ¼ 158). Finally, embryos were categorized as euploid (group1), single aneuploid (group 2) or complex aneuploid (≥errors, group 3). Participants/materials, setting, methods: 295 embryos from 64 PGS patients were enrolled. Morphokinetic variables assessed were: timing of pronuclear formation (PN), 2-cells (T2), 3-cells (T3), 4-cells (T4), 5-cells (T5) and 8-cells (T8) divisions, length of the second cell cycle (cc2) and the synchrony in the division from two to four cells (s2). Main results and the role of chance: 83/295 (28.1%), 69/295 (23.4%), and 143/ 295 (48.5%) embryos were euploid or had single or multiple aneuploidies, respectively. No statistically signiﬁcant differences were observed for all the variables analyzed. The timings of PN formation were: 12.09 (CI 11,56-12,63) 11,50 (CI 11,00-12,01) and 11,97 (CI11,56-12,39), of T2: 26,37 (CI 25,73-27,02), 26.69 (CI 25,89-27,49) and 27,00 (CI 26,40-27,61), of T3: 37,24 (CI 35,98-38,49), 36,60 (CI 34,96-38,25) and 37,46 (CI 36,45-38,47), of T4: 39,07 (CI 38,22-39,91), 39,04 (CI 37,80-40,28) and 39,99 (CI 38,94-41,04), of T5: 49,97 (CI 47,81-52,13), 48,94 (CI 46,61-51,28) and 51,72 (50,31-52,12), of T8: 53,05 (CI 48,92-57,18), 54,68 (CI 49,67-59,69) and 56,52 (CI 53,23-59,81) in group 1, 2, and 3, respectively (NS). No signiﬁcant differences were also found for cc2 and s2 variables. Irregular cell divisions (transition from 1- to 3- and 2- to 5- cell stage) were observed in 2/83 euploid embryos (2,4%), 0/69 (0%) single aneuploid embryos and 5/143 (3,4%) multiple aneuploid ambryos (NS). Finally, direct cleavage (second cell division in less than 4 hours) was found in 3/83 (3,6%), 9/69 (13,0%) and 9/143 (6,3%) embryos in group 1, 2 and 3, respectively (NS). Limitations, reason for caution: A relatively low number of euploid embryos were transferred (52). For this reason it has been impossible to compare the behavior of implanted (22) vs non implanted euploid embryos (30). It cannot be excluded that extending the observations up to day 5 some correlations between morphokinetics behavior and aneuploidies could be found. Wider implications of the findings: Embryo behavior in the ﬁrst 3 days of in vitro culture is not useful to select embryos for chromosomal aneuploidies. What remains to be understood is if morphokinetics assessment may provide an effective tool in determining embryo developmental competence and thus be helpful in optimizing euploid embryo selection in combination to PGS approach. Study funding/competing interest(s): none Trial registration number: none P-111 Cleavage and blastocyst development rate is diminished and sex ratio is shifted by oocyte exposure to bisphenol A in Bos taurus J. Ferris, L.A. Favetta, N. MacLusky, and W.A. King University of Guelph, Biomedical Sciences, Guelph, Canada Study question: The primary objective of the current study is to evaluate the effects of bovine oocyte exposure to bisphenol A (BPA) on embryo cleavage and blastocyst development rates, as well as the sex ratio of resulting embryos surviving to blastocyst. Summary answer: In comparison to various controls, oocyte exposure to BPA during maturation resulted in a decrease in cleavage and blastocyst rates, and a higher proportion of female embryos in comparison to multiple controls. What is known already: Optimal hormone levels are important for the occurrence and maintenance of a successful pregnancy. BPA, an endocrine disrupting chemical, can have detrimental effects on fertility and reproductive success, having been linked to reproductive issues such as reduced in vitro fertilization (IVF) success in humans and recurrent miscarriage. Feminization of males and a sex skew towards females have been found in ﬁsh and amphibia in vivo as a result of early exposure to BPA. Study design, size, duration: Oocytes were matured in in vitro maturation media under various conditions. Controls included no-treatment, vehicle and estradiol controls. Treatment group media were supplemented with BPA (3 and 30 mg/ mL). Blastocysts were collected at day 8 post fertilization, treated brieﬂy with pronase to remove the zona pellucida and frozen individually. Participants/materials, setting, methods: Standard IVF was completed following maturation. Cleavage was calculated at 2 days post-fertilization (dpf) and blastocyst rate at 8dpf. PCR was used to lyse blastocysts and positive controls, and amplify TSPY and GAPDH. Embryos displaying GAPDH and TSPY bands were deemed male; GAPDH without TSPY band were deemed female. Main results and the role of chance: Cleavage rates in the 30 mg/mL BPA treatment group were signiﬁcantly lower compared to both the no-treatment and vehicle controls (p ¼ 0.0297 and p ¼ 0.0049 respectively, Fisher’s exact, twotailed). The number of cultured embryos to reach blastocyst were signiﬁcantly lower in the 30 mg/mL BPA treatment group compared to the no-treatment and vehicle controls (p ¼ 0.0087 and p ¼ 0.0035 respectively, Fisher’s exact, twotailed). There is a trend showing that oocyte treatment with high BPA results in a higher number of females surviving to blastocyst (80% females compared to 54% in the no-treatment control, p ¼ 0.0872, Fisher’s exact, two-tailed). All results are dose-dependent with the high BPA, but not low BPA, treatment showing signiﬁcant decreases in cleavage and blastocyst rates and a trend towards increased number of female embryos. Limitations, reason for caution: The sexing analysis is limited by the number of embryos analyzed; between 25 to 45 embryos per group. Additional analyses are underway to increase sample size. Analyses were conducted solely in vitro using Bos taurus. Fertility and mortality rates are underway using Onchorynchus mykiss under the same treatment conditions. Wider implications of the findings: BPA supplementation to maturation media resulted in a signiﬁcant and dose-dependent decrease in cleavage and blastocyst rates, and a trend towards a female sex skew. These results suggest that bovine oocyte exposure to BPA can compromise the resulting embryo’s early developmental competence, and inﬂuence sex ratio of surviving blastocysts. Considering the high prevalence of human exposure, these results may provide insight into oocyte viability of reproductive aged women who are regularly exposed to BPA. Study funding/competing interest(s): This research was funded by the Natural Sciences and Engineering Research Council (Canada), with no competing interests. Trial registration number: N/A Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 L. Rienzi1, S. Bono2, A. Capalbo1, L. Spizzichino2, C. Rubio3, F.M. Ubaldi1, and Abstracts Abstracts P-112 The impact of laser-assisted hatching (LAH) on clinical outcome of assisted reproduction technology and the role of maternal age T. Madani, N. Jahangiri, and R. Aﬂatoonian Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran P-113 Retrospective analysis of clinical outcome of vitrified blastocyst post thaw using time-lapse imaging and standard incubation E. Cater 1, D. Hulme1, K. Berrisford1, L. Jenner1, A. Campbell2, and S. Fishel1 1 CARE Fertility, Embryology, Nottingham, United Kingdom, 2CARE Fertility, Embryology, Manchester, United Kingdom Study question: Does a continuous incubation (ES), Embryoscope, (Unisense Fertilitech) environment and time-lapse imaging improve the clinical outcome of blastocyst transfer post thaw compared to standard incubation (SI)? Summary answer: Blastocysts incubated in a continuous environment have an improved chance of pregnancy and signiﬁcantly improved clinical outcome compared to SI. Time-lapse imaging may give more information but as yet does not appear to contribute to the increased outcome. What is known already: Many papers such as Kirkegaard K. et al (2012) and Meseguer, M. et al (2012) report a signiﬁcant increase in clinical outcome in comparison to SI. Time-lapse imaging has also been used to investigate embryo morphokinetics (I. Rubio, 2012), prognostic markers of viability (J. Herrero, 2012) and the impact of ploidy on embryo development (A Campbell 2012,2013). However, little evidence is documented regarding the use of continuous incubation and time-lapse imaging for embryos post thaw. Study design, size, duration: 96 vitriﬁed/ warmed day 5/ 6 blastocyst cycles from July to October 2012 were included in the data analysis. Group A (n ¼ 52, average maternal age 36.3 + /-3.99) were cultured in a standard incubator (HeraCell) and Group B (n ¼ 44, average maternal age 32.2 ¼ /-5.58) in a continuous time-lapse incubator. Participants/materials, setting, methods: Embryos were warmed as per protocol. Retrospective analysis examined the achievement of an embryo transfer (ET), positive bhCG, presence of a foetal heart (FH) per embryo(s) transferred, clinical pregnancy (CP), time of re-expansion and time of full recovery. Two tailed Fishers exact test will be used to assess signiﬁcance. Main results and the role of chance: 88.5% (46/52) of Group A achieved an ET. 39.1% had a positive bhCG/ET (18/46) and 21.7% (10/46) a CP/ET. Of the 56 embryos transferred, 12 (21.4%) achieved FH, with an average of 1.22 embryos/ET. 88.6% (39/44) of Group B achieved an ET. 59.0% (23/39) achieved a positive bhCG/ETand 46.2% (18/39) a CP/ET. FH/embryo transferred was 34.0% (16/47) with an average of 1.02 embryos/ET. CP rate was found to be signiﬁcantly greater in Group B (p ¼ 0.0215). All other comparisons were non signiﬁcant. In Group B, embryos that implanted were found to begin re-expansion (average 0.95hrs) and be fully recovered (average 1.35hrs) faster than those that didn’t (re-expansion begins, average 2.82 hrs, fully recovered 3.35hrs. Time lapse imaging played no role in embryo selection for transfer. Limitations, reason for caution: The decision to culture in the ES or SI was based on availability of the ES rather than randomisation. Culture dishes varied between Group A and B and there is a difference in age between the groups. These factors cannot be dismissed as contributory. Wider implications of the findings: The use of uninterrupted culture is considered likely to increase implantation in most fresh culture cycles but the data presented suggests that it also has a role in increasing the outcome of frozen embryo replacements, allowing the thaw and transfer of a single blastocyst to maximise positive outcome and minimise multiple births. To assess this further, we propose a randomised study to eliminate the limitations above. Study funding/competing interest(s): None Trial registration number: None P-114 Minimal waste of healthy human embryos due to chromosomal mosaicism X.Y. Zhang, A. Yilmaz, H. Hananel, and A. Ao McGill University Health Center, Obstetrics and Gynecology, Montreal QC, Canada Study question: What is the extent and clinical relevance of chromosomal mosaicism in the ’spare’ preimplantation embryos (i.e., embryos that are found to be unsuitable for freezing or transfer based on the results of chromosome screening performed on a single blastomere). Summary answer: Only 3.5% of the 454 embryos were mosaic which had sufﬁcient quality grade, not arrested, and thus, expected to implant if transferred. What is known already: Previous studies have shown that the presence of over 38% abnormal cells in mosaic embryos is detrimental and prevent implantation. However, the extent of mosaicism that may result in healthy births in spare preimplantation human embryos in the literature is subject to controversy. Study design, size, duration: The embryos were collected from 122 patients in 148 preimplantation genetic screening cycles performed at a hospital-based reproductive center from January 2006 to March 2011. The embryos were analyzed on day 4 or 5 post-insemination. Participants/materials, setting, methods: Fluorescent in situ hybridization (FISH) procedure for chromosomes 13, 15, 16, 17, 18, 21, 22, X and Y was used to determine chromosomal complement in each of the 5,455 nuclei obtained from 454 spare embryos. Main results and the role of chance: 165 of the 454 spare embryos (36.3%) were developmentally arrested. 26.1% (43/165) of the arrested and 24.6% of the nonarrested (70/ 284) embryos had over 38% diploid cells. However, only 16 of these 454 (3.5%) embryos were mosaic, not arrested and were of good quality, Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: This study was considered for further assessment of the LAH for promoting implantation and evaluating impact of maternal age on the embryonic hatching. Summary answer: Zona thickness appears to be impacted by a woman’s age, and LAH procedure seems to be effective in improving pregnancy and implantation rates in patients with advanced female age. What is known already: Despite great effort, there is still debate among scientists about the reported beneﬁts of AH. Study design, size, duration: This retrospective cross-sectional study was conducted from December 2010 until March 2012 at the Reproductive Biomedicine Research Center. The total number of patients entered in this study was 273 in the study group (LAH was performed) and 434 in the control group (LAH was not performed). Participants/materials, setting, methods: The participants were women aged 20-45 years undergoing IVF or ICSI using their own gametes with history of ≤ 3 ART cycles. LAH was performed on cleavage stage fresh embryos and the outcomes were compared with the intact control group. These two groups were further subdivided into women of advanced age (.36 y) and younger (≤ 36). Main results and the role of chance: The developmental rate of embryos at different stages and also rate of women with male factor infertility were comparable in both groups. With respect to age, our data showed that LAH is of no beneﬁt in women ≤ 36 years of age. As the chemical pregnancy and clinical pregnancy rates after LAH were insigniﬁcantly lower among women aged ≤ 36 years in the study group compared to control group (32.5% vs. 39.2%, P ¼ 0.131; 31.3% vs. 36.4%, P ¼ 0.249). However the only statistically signiﬁcant difference observed in implantation rate (16.5% vs. 23.2%, P ¼ 0.005). In women aged over 36 years, the chemical pregnancy, clinical pregnancy and implantation rates were higher in the study group than controls, but these differences were not statistically signiﬁcant (29% vs. 6.3%, P ¼ 0.053, 21.5% vs. 6.3%, P ¼ 0.193 and 9.9% vs. 2.8%, P¼ 0.162 respectively). Limitations, reason for caution: Wider implications of the findings: Some investigators introduce this method as a routine plan in ART, whilst others do not. No published studies recommend AH for younger women. Some researchers also claim that LAH of embryos is effective in improving the pregnancy and implantation rates of women with advanced maternal age. These disputes may be related to the type of AH and quality and stage of embryos selected for AH technique. Study funding/competing interest(s): This study has . Trial registration number: This study is a retrospective study. i163 i164 P-115 Near-equilibrium rapid freezing versus cryotop vitrification of 2-cell mouse embryos T. Vutyavanich, W. Piromlertamorn, U. Saenganan, and S. Samchimchom Chiang Mai university, Obstetrics and Gynecology Faculty of medicine, Chiang Mai, Thailand Study question: We aimed to develop a novel method of freezing that combines the advantages of both the non-equilibrium vitriﬁcation and slow equilibrium freezing. Summary answer: Our method gave comparable results to vitriﬁcation in terms of embryo survival and developmental competence. The method is user friendly, and allows fast and simple cryopreservation of embryos in a large volume of cryoprotectants in a closed container. What is known already: Slow cooling and vitriﬁcation are the most commonly used methods for cryopreservation. Vitriﬁcation can be done rapidly and achieves a high survival rate of embryos, without the need of a programmable freezing. However, it is time and operator dependent. Slow freezing, on the other hand, can be done in a closed container, and the volume and time of exposure to cryoprotectants, as well as the thawing speed, are not as critical as the vitriﬁcation method. Study design, size, duration: Mouse 2-cell embryos (n ¼ 723) were divided into controls (n ¼ 211), vitriﬁcation (n ¼ 192) and near-equilibrium rapid freezing (n ¼ 320) groups in a ratio of 1:1:1.5. After one week of storage in liquid nitrogen, embryos were warm/thawed and culture up to the blastocyst stage. Participants/materials, setting, methods: Embryos were exposed to 10% EG, 5% PROH, 0.2M trehalose in PBS for ﬁve minutes, loaded into sealed straws, and inserted into holes inside an aluminum cylinder, pre-cooled in liquid nitrogen. They were thawed at 37oC in serial dilutions of Trehalose. In another group, embryos were vitriﬁed/warmed using Cryotop kits. Main results and the role of chance: The survival, further cleavage, and blastocyst formation rate of 2-cell mouse embryos in both groups was comparable by Chi-squared tests (98.8% vs. 96.4%, P ¼ 0.06; 94.9 vs. 95.1%, P ¼ 1.00; and 86.7% vs. 88.1%, P ¼ 0.67 in the rapid freezing and vitriﬁcation groups, respectively, but further cleavage and blastocyst formation was lower than those in the controls (99.1% and 98.1%, P , 0.05). There was no signiﬁcant difference (ANOVA tests) in the average number of inner cell mass (35.5 + 7.2, 36.2 + 6.4 and 35.8 + 5.6: P ¼ 0.856), trophectoderm cells (50.9 + 9.3, 52.1 + 9.9 and 53.2 + 6.4: P ¼ 0.293), and total cells (86.4 + 15.2, 88.3 + 14.8 and 88.9 + 10.4: P ¼ 0.228) in blastocysts in the rapid freezing, vitriﬁcation and control groups, respectively. Limitations, reason for caution: Another study is now underway to transfer frozen/thawed embryos into pseudo-pregnant mice to evaluate the implantation and pregnancy rates. We also plan to employ this freezing protocol on discarded human embryos, as data obtained from the mouse might not be directly applicable to the human. Wider implications of the findings: We postulated that this novel method created a “hybrid” condition, i.e. the co-existence of small ice crystals interspersed among the vitriﬁed extracellular ﬂuid, in contrast to the presence of large ice crystals in slow freezing or the total absence of any ice crystal in vitriﬁcation. The method requires lower cooling and warming rates, and the volume is not critical. It is equally effective, but less demanding and easier to perform than the current vitriﬁcation techniques. Study funding/competing interest(s): The funding was provided by the Faculty of Medicine Endowment Fund for Medical Research, Chiang Mai University. The authors declared s that could inﬂuence the study results. Trial registration number: (Not applicable) P-116 Aseptic oocyte vitrification to circumvent oocyte aging as strategy when semen sample production is delayed B. Wirleitner1, B. Lejeune2, N.H. Zech1, and P. Vanderzwalmen2 IVF-Centers Prof. Zech-Bregenz GmbH, Bregenz, Austria, 2Centre Hospitalier Inter Regional Cavell, (CHIREC), Braine-l’Alleud, Belgium 1 Study question: When production of the semen sample on the day of oocyte pick-up (OPU) is delayed or impossible, e.g. due to erectile dysfunction or azoospermia, aging of oocytes becomes a matter. The efﬁciency of a rescue strategy where oocytes are vitriﬁed and warmed when sperm collection is possible was tested. Summary answer: Our results show that in all centers with a good, standardized protocol, aseptic oocyte cryopreservation can safely be applied in cases of unexpended failure of sperm production. What is known already: Although several articles deal with the efﬁciency of oocyte vitriﬁcation using open vitriﬁcation devices in donor cycles, still little is known about vitriﬁcation in closed aseptic conditions and its application in standard IVF-patients. To apply the oocyte vitriﬁcation technique on a larger scale and lift its experimental label, it is important also to test its efﬁciency, especially of the aseptic closed devices, on our daily clientele in routine IVF-work. Study design, size, duration: This retrospective study included 56 IVF-cycles where aseptic oocyte vitriﬁcation was performed due to absence of sperm production at least 3 hours after OPU during January - December 2011. Participants/materials, setting, methods: In all cycles no sperm was retrieved until 3 hours after the OPU. Oocytes were vitriﬁed in a closed vitriﬁcation device (VitriSafe) ensuring aseptic conditions during vitriﬁcation and storage. After sperm retrieval in a subsequent cryo-cylce, oocytes were warmed and fertilized. As main outcome live-birth rate was evaluated. Main results and the role of chance: For 56 patients 455 oocytes were vitriﬁed of which 416 were fertilized after warming and ICSI/IMSI. The cleavage rate on day 3 was 95.3%. Embryo transfer was performed on day 5 (blastocyst selection). An ongoing pregnancy rate of 43.8% and a birth rate of 35.7% was obtained and 26 healthy babies were born. These results are encouraging, especially as mean age of women in this study was 34.5 years and sperm parameter were very poor. The additionally safety and efﬁciency of aseptic vitriﬁcation (i.e. reducing the risks of contamination and increasing the viability after warming) during cooling and storage turn this strategy in to the focus not only when its application is inevitable but also when sperm parameters are very poor (e.g. after recent infections). Limitations, reason for caution: Although baby-take-home rates and health of babies born do not give any reason for concern, a long-time follow-up will be necessary to exclude any later appearing health problems originating from the applied techniques. Wider implications of the findings: In contrast to earlier ﬁndings, we report very high efﬁciency of aseptic vitriﬁcation in closed carriers. The reduced cooling rates in closed carriers as compared to open devices can be overcome by a slightly modiﬁed vitriﬁcation protocol. Aseptic vitriﬁcation achieves the same warming rates as open devices and thereby ensures high survival rates. Study funding/competing interest(s): None Trial registration number: None P-117 Sequential versus single step media in a time lapse system: is there a difference in pregnancy rate? E. Albani, V. Parini, A. Smeraldi, F. Menduni, R. Antonacci, A. Marras, S. Levi, G. Morreale, B. Pisano, A. Di Biase, A. Di Rosa, and P.E. Levi Setti Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 suggesting that they had the potential to implant. Percentage of diploid cells in the non-arrested spare embryos were not associated with changes in maternal age, number of cumulus oocyte complexes (COC) collected, or the number of chromosomally normal embryos obtained per cycle. Pearson’s coefﬁcients for the correlations of percent diploid cells with the maternal age (r ¼ -0.10), number of COC collected (r ¼ 0.10), and number of normal embryos obtained per cycle (r ¼ 0.07) were all small. Limitations, reason for caution: Only nine pairs of chromosomes were analyzed in this study. Screening of a larger number of chromosomes may detect mosaicism rates higher than those found in our study. FISH has an error rate of 5 to 7% resulting from inaccuracy of probe hybridization. Wider implications of the findings: Only 3.5% of the non-arrested mosaic spare embryos analyzed in this study had over 38% diploid cells, were of sufﬁcient quality, and thus, had the potential to implant if transferred. These results are in agreement with some of the previous publications reporting less than 5% of misdiagnosis due to mosaicism in spare embryos using FISH. These results suggest that waste of healthy human embryos due to mosaicism after chromosome screening is minimal. Study funding/competing interest(s): N/ATrial registration number N/A Abstracts Abstracts IRCCS Istituto Clinico Humanitas, Department of Gynecology Division of Gynecology and Reproductive Medicine, Rozzano, Italy P-118 Reverse phase protein array (RPPA): a new approach for the identification of cumulus cells biomarkers of oocyte quality V. Puard1, V. Cadoret2, T. Tranchant2, C. Gauthier2, E. Reiter 2, F. Guérif1, and D. Royère1 1 CHRU Tours - Hôpital Bretonneau, GYN-OBS 1 REPRODUCTION, Tours Cedex 1, France, 2INRA, UMR85 Physiologie de la Reproduction et des Comportements, Nouzilly, France Study question: We proposed to evaluate the Reverse Phase Protein Array (RPPA) to detect and analyze potential biomarkers related to oocyte developmental competence through a non-invasive procedure involving each individual cumulus (CCs) in human. Summary answer: Reverse Phase Protein Array allowed us to detect speciﬁc proteins at a level as low as the equivalent of one cell of one human cumulus. What is known already: Morphological criteria are mostly used in ART laboratories but remain poorly predictive of embryo potential to develop. Various approaches involving transcriptomics, proteomics, and metabolomics on the embryo or its cellular or non cellular environment have been proposed. RPPA is a sensitive and quantitative technique which allowed the detection of speciﬁc proteins in very low quantities of biological samples to help clinical management of cancer. Study design, size, duration: Six patients undergoing intracytoplasmic sperm injection (ICSI) for male infertility from January to May 2012 were included in this study. Thirteen human individual cumulus from 4 patients and a pool of 16 cumulus from 2 patients were collected during ICSI procedure. Participants/materials, setting, methods: The expression of the targeted proteins was suppressed by siRNA in HEK293 and the antibodies used for RPPA were validated by correlating the signal observed by Western Blot (WB) and RPPA. Proteins were detected in a pool of cells of 16 cumulus, then in 13 individual cumulus. Main results and the role of chance: Speciﬁcity of antibodies targeting the proteins of interest was assessed by WB. The expression of 4 proteins was partly suppressed in HEK293 cells by siRNA. The correlation of knockdown efﬁciencies of the VCL and SRC proteins between WB and RPPA validated the antibodies for RPPA used. We detected in cells of a pool of 16 cumulus the proteins VCL, SRC and ERK2 at the level of less than one equivalent cell of one cumulus. Then, these proteins were detected at the same sensibility in individual cumulus. No correlation was observed for the antibody targeting RGS2 and led us to reject it for RPPA used. Therefore, validation the antibodies is a crucial step for RPPA used and its application for biomarkers identiﬁcation. Limitations, reason for caution: Antibody validation remains a challenging problem for RPPA. While suppressing the expression of the proteins by siRNA approach is efﬁciency, this technique required heavy development. Overexpression the proteins in HEK293 cells might be an interesting alternative for high throughput validation. Wider implications of the findings: Once validated antibodies targeting potential biomarkers of oocyte quality at protein level and assessed their robustness by taking into account the patient variability, RPPA might be used in ART laboratory. Indeed RPPA is a sensitive, high throughput, cost and material effective technique which might be used to predict the developmental ability and implantation of the embryos in ART laboratory to increase single embryo transfer and limit multiple pregnancies. Study funding/competing interest(s): This work was supported by Merck Serono Grant for Fertility Innovation Award 2010. Trial registration number: - P-119 Abnormal distribution of type 1 Inositol 1,4,5 tri-phosphate receptor in fertilization failed human eggs S.Y. Yoon, J.H. Eum, E.A. Park, T.Y. Kim, T.K. Yoon, D.R. Lee, and W.S. Lee CHA University, Department of Biomedical Science, Seoul, Korea South Study question: IP3R1 is a key protein to induce intracellular Ca2+-oscillation during fertilization. Does fertilization failed human eggs have normal IP3R1 distribution? Summary answer: Fertilization failed human eggs showed abnormal IP3R1 distribution including inconsistent clusters in cytoplasm instead of cortical clusters. What is known already: The capacity of [Ca2+]i oscillation is acquired during egg maturation and coincides with an increase in the sensitivity of the IP3R1 and their localization in cytoplasm. Cluster formation of IP3R1 in the egg cortex is important to initiation of [Ca2+]I oscillations during egg and sperm fusion. Study design, size, duration: Immunoﬂuorescence analysis of IP3R1 in in vitro matured egg, fertilization failed egg and tripronuclear human eggs. Participants/materials, setting, methods: Patients enrolled in the assisted reproduction program at the Fertility Center of CHA Gangnam Medical Center were participated with written consent in this study. Total 62 eggs (GV, 8 eggs; NF 42 eggs, 3PN; 12 eggs) were immunostained with anti IP3R1 antibody, and examined with a confocal laser-scanning microscope. Main results and the role of chance: Distribution of IP3R1 in human eggs change gradually from GV stage to MII stage. In GV stage, most of the eggs shows progressively disperse from GV to cortex. In MII stage, 6 out of 8 eggs have that IP3R1 distributed in whole cytoplasm including clusters which is more 3 ≏ 6um in diameter on the cortex. After fertilization, in PN stage with 3PN, most of the eggs have IP3R1distribution with gradually disperse from PN to cortex, but they do not have cortical clusters. In fertilization failed MII eggs on the next morning post fertilization, more than 50% of eggs have several IP3R1 aggregates in the middle of the cytoplasm, which is more than 10um in diameter instead of cortical clusters as in MII. Limitations, reason for caution: The low numbers of human eggs analysed may not be ideal for conclusive statistical analysis. Also, evaluation of other IP3R1 biochemical changes would be need to conclusion. Wider implications of the findings: The present ﬁndings provide new understanding the reason of the fertilization failure during ART program, and suggest further evidence for the clinical application as maker of egg quality. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: Is the pregnancy rate, in infertile patients, inﬂuenced by the type of culture media used in association with time lapse analysis? Summary answer: According to our experience, the use of universal media instead of sequential media could improve the pregnancy rate. What is known already: Recent studies of different culture media on human embryo development show that there are no differences in implantation rate and clinical pregnancy rate. Furthermore embryo evaluation is often based on a static observation. Study design, size, duration: In a retrospective study, from June 2012 to December 2012, 141 infertile patients/cycles underwent Intracytoplasmic Sperm Injection (ICSI) cycles with embryo transfer. After which ICSI oocytes were incubated in a time lapse system (Unisense FertiliTech A/S Embryoscope). Participants/materials, setting, methods: The 141 patients were divided in two groups, group A and B. In group A (n ¼ 90) we used a sequential media (Sagew) and B (n ¼ 51) a single step media (Irvine Scientiﬁcw). The variables studied were the fertilisation, implantation, pregnancy, and blastocyst cryopreservation rate. Main results and the role of chance: In Group A 1217 oocytes were retrieved, 722 injected (8.02 + 1.42), 511 fertilised (FR ¼ 70.8) and 201 embryos were transferred; in B 618 retrieved, 383 injected (7.5 + 1.5), 269 fertilised (FR ¼ 70.2) and 120 transferred.No differences in mean age (A¼ 36.6 + 3.8; B ¼ 36.6 + 3.8), fertilisation rate (A ¼ 70.8%; B ¼ 70.2%) and transferred embryos (A ¼ 2.2 + 0.54; B ¼ 2.3 + 0.5). Furthermore there are no differences in time lapse analysis at two, four, eight, morula and blastocycsts development between two groups. A pregnancy rate (A ¼ 34.4%; B ¼ 47%) and percentage of cryopreserved sovrannumerary blastocysts (A ¼ 25.5%; B ¼ 33.3%) positive trend was observed, although not statistically signiﬁcantly different. Limitations, reason for caution: The use of time lapse systems without comparison with 3 gas incubators normally used in clinical practice. The sample considered had not sufﬁcient statistical power to detect the difference observed in this study. Wider implications of the findings: Although not statistically different, our results show a clinical interesting increase in pregnancy rate between the two studied groups, to be conﬁrmed in a larger sample comparing 3 gas incubators and time lapse systems. Study funding/competing interest(s): None Trial registration number: Not applicable i165 i166 Abstracts Study funding/competing interest(s): This work was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family affairs, Republic of Korea (A084923). Trial registration number: Basic research P-120 The shipment of oocytes or embryos vitrified in minimum volume by means of dry shipper containers does not impair the clinical outcome A. Cobo Cabal1, B. Vallejo1, P. Campos1, E. Sánchez1, J. Serrano1, and J. Remohi2 Instituto Valenciano de Infertilidad, IVF laboratory, Valencia, Spain, 2Instituto Valenciano de Infertilidad, Ob. Gyn., Valencia, Spain 1 P-121 Cleavage timing of euploid embryo development is age-related V. Nagornyy1, P. Mazur1, D. Mykytenko2, L. Semeniuk1, and V. Zukin1 1 Clinic of Reproductive Medicine ’NADIYA’, Embryology department, Kiev, Ukraine, 2Clinic of Reproductive Medicine ’NADIYA’, Department of molecular diagnostics, Kiev, Ukraine Study question: Can age-related differences in timing of embryo development be indentiﬁed and do they correlate with embryo ploidy? P-122 Does the type of culture media or oxygen tension affect embryo fertilization and development - analysis of sibling o ocytes P. Guilherme1, C. Madaschi1, T.C.S. Bonetti2, G. Fassolas3, and C.R. Izzo4 1 Originare - Centro de Reprodução Humana, IVF Laboratory, São Paulo SP, Brazil, 2Universidade Federal de São Paulo, Laboratory of Molecular Gynecology and Proteomics, São Paulo SP, Brazil, 3Originare - Centro de Reprodução Humana, Laboratory director, São Paulo SP, Brazil, 4Originare Centro de Reprodução Humana, Clinical director, São Paulo SP, Brazil Study question: Are the morphokinetics of growing embryos affected by the type of culture media utilized and does the oxygen tension of the incubator may inﬂuence sequencial or single step media? Summary answer: The oxygen tension does not inﬂuence the fertilization, but the GlobalTM (single-step) media presents better fertilization rate. On the other hand, embryo development was not affected by the type of culture media, but for the oxygen tension. Lower oxygen tension gives rise to better quality embryo, and subsequently better blastocysts. What is known already: Optimal culture conditions and media composition are critical for the development of embryos in vitro. It is commercially available single step and sequential media for embryo culture; however the best type of media for embryo development is not deﬁned yet. Besides the type of media, the atmosphere culture conditions are equally important. It is known that laboratory culture of embryos with oxygen at atmospheric tension impairs embryo metabolism and blastocyst development in several species Study design, size, duration: Retrospective cohort study carried out in private infertility clinic. We analyzed 253 ICSI cycles with ejaculated Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: Is shipping impairing survival or clinical outcome of vitriﬁed oocytes or embryos? Summary answer: Oocytes or embryos vitriﬁed in minimum volume with an open system can be safely transported by means of dry shippers equipped with temperature monitoring devices without impairment of survival or clinical outcome. What is known already: Vitriﬁcation has revealed as most efﬁcient method for oocyte cryopreservation, especially by means of open systems using minimum volume for loading samples. Shipping vitriﬁed material could be of great utility for donor egg-banks as well as for patients needing the transportation of their oocytes or embryos due to change of residence or clinic. Usually cryopreserved samples are transported in vapor dry shippers. Serious concerns have been raised about the shipping of micro-drop- vitriﬁed samples in this type of containers, due to possible deleterious changes in temperature caused by the manipulation needed to load the containers or during transportation. Study design, size, duration: Retrospective cohorts study, January 2007- April 2011. Participants/materials, setting, methods: University afﬁliated infertility center. 674 oocytes (N¼ 72 cycles) and 304 embryos (N ¼ 132 cycles) were shipped to different Spanish cities. Matched controls consisted of IVF and warming cycles conducted in situ including 1436 non-shipped vitriﬁed oocytes (N ¼ 144 cycles) and 524 non-shipped vitriﬁed embryos (N ¼ 272 cycles). All samples were vitriﬁed using Cryotop method. MEV vapor dry shipperw equipped with a temperature tracking system (ShipslogTM) was employed for monitoring the temperature of the device throughout the trip. Any air-contact was avoided when loading the dry shipper. Full history of temperature throughout the journey was downloaded. Survival and ongoing pregnancy rate (OPR) were analyzed as main outcome measurements. x2 test was used for statistical analysis and odds ratios (OR) with CI 95% were calculated with signiﬁcance under 0.05. Main results and the role of chance: The shipment of oocytes orembryos does not impair survival and clinical outcome. 89.8 % vs. 91% (NS) and 94.9% vs. 97.8% (NS) embryo transfers were performed when oocytes and embryos were shipped compared to controls respectively. The odds ratio (OR) for survival and OPR/ cycle in the case of shipped oocytes was 1.134 (CI95% 0.838-1.534; P ¼ 0.433) and 0.912 (CI95% 0.498-1.670; P ¼ 0.877). Similarly, no statistical differences were found for shipped embryos vs. controls: OR ¼ 0.751 (CI95% 0.325-1.738; P ¼ 0.548) and 1.051(CI95% 0.682-1.622; P ¼ 0.912) for survival and OPR/cycle. Limitations, reason for caution: Heterogeneity of study sample including ovum donation and autologous oocytes cycles and natural cycles or hormonal replacement therapy for vitriﬁed embryo transfers, although matched controls have been included. Wider implications of the findings: The safe shipment of vitriﬁed oocytes and embryos has great implications for the ﬂexibility of ovum donation and IVF programs. Study funding/competing interest(s): None of the authors are afﬁliated to the brands that commercialize the devices employed in the study. Trial registration number: N/A Summary answer: According to our investigation, transfer quality embryos of women of advanced age show delayed cleavage times, especially those with correct chromosomal constitution conﬁrmed by array comparative genome hybridization (aCGH) analysis. What is known already: Time-lapse analysis of human embryo morphokinetics had already produced sufﬁcient amount of data to help improve selection of embryos with higher implantation potential. However, there is little evidence for connection of morphokinetic parameters with embryo ploidy status and womens age Study design, size, duration: This cross-sectional study included 878 embryos of 133 patients, cultured in time-lapse imaging system during one year period. From these, 113 embryos of 24 patients were analysed for ploidy by aCGH. Of aCGH embryos 45 originated from 10 women over 34 years, other 68 embryos were from 14 younger women. Participants/materials, setting, methods: Embryos were cultured in EmbryoScopeTM (UnisenseFertiliTech A/S, Denmark). Array CGH, if applied, was performed after trophectoderm biopsy, using 24surew kit (BlueGnome, United Kingdom). After 5 or 6 days of embryo culture, time-lapse image data were analysed and event times determined. Main results and the role of chance: In general group, embryos were compared by timing of ﬁrst divisions. No differences between mean values for young and advanced age women were found. Embryos analysed by aCGH have developed to blastocyst at day 5 or day 6, allowing trophectoderm biopsy. Thus, timing was also established for later embryo development events, e.g. morula formation, cavitation and expansion start. Morphokinetic data were compared by women’s age and embryo ploidy. Euploid embryos of women aged 35 years or more, showed signiﬁcantly slower times of early development when compared to euploid embryos of younger patients. First cleavage time was 29.10h (CI95% 26.97 to 31.33h) compared to 24.57h (CI95% 23.92 to 25.22h), second cleavage was at 41.93h (CI95% 39.60 to 44.26) compared to 35.91h (CI95% 34.94 to 36.88h) Limitations, reason for caution: Presented data shows only limited possibility of noninvasive embryo ploidy assessment. Wider implications of the findings: Our ﬁndings suggest that currently used time-lapse imaging analysis criteria should be adjusted when applied to embryos of women of advanced age. In this group of patients optimal time points of cytokinesis are delayed. Study funding/competing interest(s): Authors have nothing to disclose.No funding was received for this research. Trial registration number: None i167 Abstracts P-123 Is intrafollicular retinoic acid associate with nuclear oocyte maturation M.J. De Los Santos1, D. Beltrán1, V. Garcı́a-Láez1, M.J. Escribá1, N. Grau 1, L. Escrich1, C. Albert1, J.L. Zuzuarregui2, and A. Pellicer1 1 IVI Valencia, Clinical embriology, Valencia, Spain, 2IVI Valencia, Clinical analysis, Valencia, Spain Study question: Does intrafollicular concentration of retinoic acid (RA) correlate with nuclear maturity of human oocytes? Summary answer: Despite the presence of RA in follicular ﬂuids (FF), it seems that the concentration of RA is not directly associated with the resume of meoisis. What is known already: RA synthesis is a requirement to sustain meiosis in human ovary. ALDH1A2 gene expression which converts Vitamine A to retinoic acid have been found in human cumulus cells (CC) from both unstimulated and stimulated cycles and it seems that, at least in the bovine, is associated with the acquisition of oocyte cytoplasmic competence through the promoting growth factors, COX2 expression suppression. Study design, size, duration: This sampling procedure study was performed from 2007 to 2009. A total of 39 FF were analysed; 13 FF from immature oocytes and 26 FF from mature oocytes. Oocyte retrieval was schedule 36 hours after hCG administration. Participants/materials, setting, methods: All FF were measured and centrifuged, aliquoted and stored at -80C for subsequent analysis. All the oocytes associated to the aspirated follicles were kept in culture individually. FF were analyzed for RA by means of liquid chromatography. Student T-test was used for statistical comparisons. Main results and the role of chance: We observed no differences between the groups of immature and mature oocytes in terms of age of the patients (35.5 + 3.6 vs. 34.5 + 6.5 years), size of the follicle (4.1 + 2.1 ml vs. 4.2 + 1.6 ml ) and RA concentration (4.3 + 2.3 ug/ml vs. 4.1 + 1.2 ug/ml) respectively. Limitations, reason for caution: Sample size may be a limitation of the study, however since the RA concentration frame was very narrow among samples we believe results will not vary that much. Wider implications of the findings: Despite meiosis initiation in the human ovary relies partially on RA, it seem that is not important in later steps of meiosis since no differences were found in FF from preovulatory follicles containing metaphase II oocytes an immature ones. Study funding/competing interest(s): The present project was supported by the R + D programme from the Generalitat Valenciana (Regional Valencian Goverment).Project identiﬁcation number: IMPIVA IMDTG/2008/26 and IMIDTF/ 2009/142 Trial registration number: This is not a clinical trial P-124 Evaluation of calcium machinery in a meiosis defect mouse model Y. LU 1, D. Nikiforaki1, F. Vanden Meerschaut 1, J. Neupane1, W.H. De Vos2, S. Lierman1, T. Deroo 1, B. Heindryckx1, and P. De Sutter 1 1 University Hospital Ghent, Department for Reproductive Medicine, Gent, Belgium, 2Ghent University, Department of Molecular Biotechnology, Gent, Belgium Study question: Is nuclear and cytoplasmic maturation impaired in in vivo and in vitro matured oocytes from LT/Sv mice, a mouse model showing oocyte meiotic arrest? Summary answer: In addition to abnormal spindle-chromosome complexes, defective intracellular calcium (Ca2+) signalling during in vitro maturation (IVM) points to both a nuclear and cytoplasmic maturation defect in LT/Sv oocytes. What is known already: Acquisition of oocyte meiotic competence coincides with spontaneous Ca2+- oscillations during germinal vesicle breakdown (GVBD) mediated by the type 1 inositol 1, 4, 5-triphosphate receptor (IP3R1). In addition, the occurrence of Ca2+-oscillations during fertilization serves as a marker of efﬁcient cytoplasmic maturation. LT/Sv mice show a signiﬁcant proportion of arrested metaphase I (MI) oocytes. Furthermore, LT/Sv GVoocytes that are in vitro matured to the MI and MII stage show aberrant Ca2+-responses at fertilization. Study design, size, duration: Spontaneous Ca2+-oscillations were analyzed in GV oocytes following 16h IVM from LT/Sv (n ¼ 38) and B6D2F1 mice (n ¼ 40). Strontium-induced Ca2+-oscillations were measured for 2h in in vivo and IVM LT/Sv (n ¼ 91) and B6D2F1 oocytes (n ¼ 94). IP3R1 localization and spindlechromosome complexes were assessed in in vivo and IVM LT/Sv oocytes (n ¼ 72). Participants/materials, setting, methods: Oocytes were collected from 6- to 10week-old LT/Sv and control B6D2F1 mice 48h after FSH (GV) and 14h after hCG injection (MI and MII). Spontaneous and strontium-induced Ca2+-responses were measured by ﬂuorescence time lapse imaging. IP3R1 localization and spindlechromosome complexes were analysed by immunostaining and confocal microscopy. Main results and the role of chance: During maturation none of the IVM MI and 25% of the IVM MII oocytes from LT/Sv mice showed spontaneous Ca2+oscillations compared to 60% (P , 0.01) and 64% of B6D2F1 oocytes, respectively. After strontium activation, the number of Ca2+-rises was signiﬁcantly decreased in IVM LT/Sv-MI (5.50 + 3.73) and IVM LT/Sv-MII oocytes (4.59 + 2.34) compared to in vivo LT/Sv-MI (14.28 + 5.83) and in vivo LT/ Sv-MII oocytes (11.25 + 4.23) (P , 0.01) respectively. Furthermore, in vivo LT/Sv-MI oocytes showed more Ca2+-oscillations than in vivo LT/Sv-MII (P , 0.05) and B6D2F1 in vivo MII oocytes (P , 0.05). IP3R1 localization was similar in both in vivo and IVM LT/Sv-MI and LT/Sv-MII oocytes. However, a signiﬁcantly lower number of normal spindle-chromosome complexes was observed in IVM LT/Sv-MI and LT/Sv-MII oocytes compared to in vivo matured LT/Sv-MI and LT/Sv-MII oocytes (P , 0.05). Limitations, reason for caution: LT/Sv mice are a good model for human clinical mixed MI arrest cases, yet the ﬁndings are species-speciﬁc and cannot be fully extended to the human. The mechanism and pattern of strontium-induced Ca2+oscillations may differ from those of fertilization. Wider implications of the findings: Defective Ca2+ signalling during oocyte maturation might underlie certain types of oocyte maturation arrest. Nuclear transfer may be a way to overcome this, but given the high rate of spindle abnormalities in in vitro matured oocytes, this should be applied to collected in vivo matured oocytes. Further studies are needed to determine the contribution of the IP3R1 and other signalling pathways to impaired cytoplasmic maturation. Study funding/competing interest(s): This study was supported by the China Scholarship Council and Special Research Fund from Ghent University (Bijzonder Onderzoeksfonds, BOF). No competing interest declared. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 sperm between January,2011 and December,2012. Embryos were cultured with two different types of media, single-step ( GlobalTM ) or sequential (VitrolifeTM ), in tri-gas (5% CO2, 378C, 6% O2) or conventional incubator (5% CO2, 378C, 20% O2). Participants/materials, setting, methods: Oocytes were split according to media and incubator: A: atmospheric O2 tension/single-step media; B: atmospheric O2 tension/sequential media; C: 6% O2 tension/single-step media; D: 6% O2 tension/sequential media. Variables studied included normal fertilization, embryo grade on day 3, and blastocyst expansion on day 5. p ≤ 0.05 was considered statistically signiﬁcant. Main results and the role of chance: When normal fertilization rate was evaluated, it seems the single step media result in higher rates as groupA (77.6%) versus groupB (68.5%) (p ¼ 0.286) and groupC (80.9%) versus groupD (67.5%)(p , 0.001) showed numerical and statistical signiﬁcant differences, respectively. No differences were observed in relation to the oxygen tension. When we analyzed the embryo morphology on day 3, the low oxygen tension was relevant, as we observed statistically signiﬁcant difference between groupA (67.8%) and groupC (80.3%), p ¼ 0.020. No difference was seen between groupB and groupD, showing that for sequencial media, the oxygen tension does not matter. In spite of seem that group A had lower blastocyst rate (39.1%) than other groups (B: 51.6%, C:49.3% and D: 44.2%), there is no statistical difference among them. Limitations, reason for caution: The study was not powered to test differences in pregnancy rates between the two culture media, as embryos from different groups were transferred for the most of patients. Wider implications of the findings: The absence of differences in the development of blastocyst between two different media concepts validates the algorithm for embryo selection in diverse culture conditions. On the other hand, the low oxygen tension seems to lead to improved embryo quality on day 3, and should be adopted specially in short time cultures. Study funding/competing interest(s): No speciﬁc funding was obtained for this study; it was solely funded by ORIGINARE. None of the authors have any economic afﬁliation any culture media company. Trial registration number: Not applicable. i168 Abstracts Trial registration number: No trial registration number. P-125 Embryonic human chorionic gonadotropin (hCG) in spent culture medium may tell embryo viability in IVF-ET program: a multi-center study Study question: Could embryonic beta-hCG be as a biomarker for embryo selection in IVF-ET procedure? Summary answer: A higher embryonic beta-hCG level was found in embryos with higher morphologic grading or implantation potential and beta-hCG might be a predictor for embryo viability, especially in blastocyst transfer program. What is known already: hCG was one of the ﬁrst found embryonic secretions and detected at variable levels by different methods in spent embryo culture medium since 1984. A highly sensitive electrochemiluminescence immunoassay (ECLIA) with strong repeatability, efﬁciency and stability was performed to detect beta-hCG in culture medium in our previous study. Study design, size, duration: It’s a cross sectional experiment study. Total 719 samples were individually collected at day3 (n ¼ 300) and day5 (n ¼ 419) which included fresh (n ¼ 161), frozen-thawed (n ¼ 55) and further culture to blastocyst after thawing (n ¼ 203) from 382 women in 6 IVF centers from Nov. 2011 to Oct. 2012 in China. Participants/materials, setting, methods: A sequential culture system was performed from 2PN to blastocyst stage. Samples were collected individually and stored at -808C until beta-hCG detection by ECLIA. Clinical data of these participants were collected at the same time. Main results and the role of chance: 1) Beta-hCG was found in culture media at fresh day3 (87.7%, 263/300), fresh day5 (98.1%, 158/161), further culture to day5 after thawing (96.6%, 196/203) and thawed day5 (100%, 55/55). 2) There was no difference among conventional IVF group, ICSI group and PGD group ( p ¼ 0.067). 3) A higher beta-hCG concentration appeared in subgroups of fresh blastocysts with expanded cavity or high trophectoderm grading (A OR B) ( p ¼ 0.042) and day5 (fresh/thawed and single/double embryos transferred). 5) The concentration of embryonic beta-hCG correlated positively with implantation rate (r ¼ 0.56, p , 0.001). Limitations, reason for caution: 1) Embryo transferred was still dependent on the morphology grading rather than beta-hCG concentration in spent culture medium in this study. 2) The sample size of single embryo transfer (n ¼ 88) was relatively small to describe the predictive power of beta-hCG for embryo viability assessment. Wider implications of the findings: Selecting the embryo with highest competence by embryonic beta-hCG detection in spent culture media with ECLIA alone and/or combined with morphology grading may reduce the number of embryos transferred, resulting in a decrease of multiple pregnancies rate in clinical application. Study funding/competing interest(s): This study was supported by Nature Science Foundation of China (2008; no.30872762) and the Science Foundation of Guangdong Province (2009B030801022). Trial registration number: None. P-126 Viability markers: optimal dynamic range for implantation in competent blastocyst L. Muela1, M. Roldan1, B. Gadea1, M. Martinez1, I. Perez1, M. Meseguer2, and M. Muñoz3 1 IVI Alicante, IVF Laboratory, Alicante, Spain, 2IVI Valencia, IVF Laboratory, Alicante, Spain, 3IVI Alicante, Medical Director, Alicante, Spain Probability to implant. .............................................................. Outside optimum kinetic range Inside optimum kinetic range ......................................................................................... t5 (47- 60 h) 28.8 % (n ¼ 73) 42.9% (n ¼ 92)* t5t2 (22-33h) 26.4% (n ¼ 14) 42.6% (n ¼ 78)* t8 (49-62h) 32.5 % (n ¼ 38) 45.4% (n ¼ 54)* tm (82-92h) 32.2% (n ¼ 58) 60.7% (n ¼ 34)* tb (92-105h ) 30.3% (n ¼ 46) 54.8% (n ¼ 92)* * p values , 0.05. Study question: Is there an optimal range in cleavage kinetics that increases implantation potential? Summary answer: For ﬁve of the morphokinetic parameters studied, we have found a higher signiﬁcant implantation probability for those embryos within an optimal timing range. What is known already: Evaluation of embryos outside the incubator enables the assessment of timing of events, but also exposes embryos to undesirable changes in temperature, humidity and gas composition (Zhang et al., 2010). Culture of embryos in a time-lapse monitoring system improves pregnancy outcome compared with a standard incubator. (Meseguer et al 2012). Addition of kinetics to conventional morphological assessment can be used as predictor of embryo implantation. (Meseguer et al 2011) Study design, size, duration: In a retrospective cohort study, we analyzed 236 embryos with known implantation from 240 cycles included in our egg donation program. Embryos were culture in a time-lapse incubator after intracytoplasmic injection or conventional in vitro fertilization and monitored until transfer on day5. It was conducted from November-2009 till December-2012. Participants/materials, setting, methods: The current study took place in a private IVF clinic, on embryos with Known Implantation Data (KID). We included in the study embryos with 100% implantation (KIDpositive) and embryos failing to implant 0% (KIDnegative). For each embryo, the timing of each cellular event was annotated: pronuclear formation and fading, cleavage to 2 cells (t2), t3,t4,t5,t6,t7,t8,t9, Morula(tM), early blastocyst(tB), and expanded blastocyst. We also evaluated the duration of the second cycle cc2 (t3-t2), time between 2 and 5 cells(t5-t2), and the blastomere synchrony s2 (t4-t3) Selection of embryos for transfer was exclusively based on morphological criteria. The optimum timing range is established according to embryo distribution for each parameter studied depending on implantation status. Pearson’s Chi-square was performed, p values , 0.05 were considered signiﬁcant. Main results and the role of chance The implantation rate (IR) of the study was 40.4%, KID IR was 39.0%, pregnancy rate was 64.8%. These results showed how the probability to implant rises when a blastocyst is selected inside the optimal timing range. Limitations, reason for caution These are data from a retrospective analysis, although sample size is considerably high. Results are based on observations with embryos from oocyte donors and need to be repeated with embryos from infertile patients of different ages. Wider implications of the findings: Inclusion of kinetics parameters within embryo morphology classiﬁcation may increase pregnancy rates. The ﬁnding of the optimal kinetic range is the ﬁrst step in this study. From now on, we will include this optimal kinetic range in our embryo selection criteria to demonstrate an improvement on pregnancy rates, although previous studies already did it. The present analysis will be evaluated by logistic regression in order to develop an algorithm for blastocyst selection. Study funding/competing interest(s): Not applicable Trial registration number: Not applicable Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 J. Li1, X.Y. Chen1, G. Lin2, G.N. Huang3, Z.Y. Sun4, Y. Zhong5, B. Zhang6, T. Li 1, S.P. Zhang2, H. Ye 3, S.B. Han3, S.Y. Liu5, J. Zhou4, G.X. Lu2, and G.L. Zhuang1 1 The First Afﬁliated Hospital Sun Yat-Sen University, IVF Center-Dept. of Ob/ Gyn, Guang Zhou, China, 2Central South University, Institute of Reproduction and Stem Cell, Changsha, China, 3Chongqing Obstetrics and Gynecology Hospital, Chongqing Reproductive and Genetics Institute, Chongqing, China, 4 Peking Union Medical College Hospital, Department of Obstetrics and Gynecology, Beijing, China, 5Chengdu Xinan Gynecological Hospital, Reproductive Center, Chengdu, China, 6Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Obstetrics and Gynecology, Nanning, China Timing of cellular event Abstracts P-127 Impact of exposure to music during in vitro culture on embryo development C. Castelló1, M. Asensio1, P. Fernández1, A. Farreras1, S. Rovira1, J.M. Capdevila 1, E. Velilla 1, and M. López-Teijón 2 1 Instituto Marques, Reproductive Biology, Barcelona, Spain, 2Instituto Marques, Reproduction Medicine Service, Barcelona, Spain P-128 Can a composite score based on time lapse observation aid embryo selection for single embryo transfer ñ an interim report P. Kovács1, S.Z. Mátyás1, V. Forgács2, A. Reichart2, F. Rárosi3, A. Bernárd1, A. Török 4, S.G. Kaáli1, A. Sajgó1, and C.S. Pribenszky 5 1 Kaáli Institute, IVF Center, Budapest, Hungary, 2Forgács Intézet, IVF Center, Budapest, Hungary, 3Department of Medical Physics and Informatics Bolyai Institute, University of Szeged Hungary, Szeged, Hungary, 4Pannon Reprodukciós Intézet, IVF Center, Tapolca, Hungary, 5St. Istvan University Faculty of Veterinary Science, Department of Animal Breeding and Genetics, Budapest, Hungary Study question: The aim of this study is to compare pregnancy rates in human in-vitro fertilization treatments when embryos are monitored and evaluated using Primo Vision time-lapse system (Vitrolife Ltd., Hungary) (TL) or cultured and evaluated in the traditional way to support the selection of a single blastocyst for transfer (ET). Summary answer: Based on the interim results of this ongoing study TL monitoring may assist embryo selection for single blastocyst transfer. The 32% increase in pregnancy rate (PR) in the TL group is encouraging. What is known already: A multiple pregnancy is an undesired outcome of assisted reproduction. Current embryo classiﬁcation is inefﬁcient in identifying the embryo with the highest implantation potential. Time-lapse embryo monitoring provides additional information about embryo development and therefore may aid embryo selection. It appears that the kinetics and dynamics (fragmentation, blastocyst formation) of embryo development predict embryo viability. Study design, size, duration: Ongoing, prospective, randomized, multicenter trial started in Jan/2012. All patients use the long agonist protocol with recombinant-FSH stimulation. The single blastocyst for ET is selected based on day-5 morphology (Control) or on composite score based on TL observations. Fifty patients are planned to be included per protocol. Participants/materials, setting, methods: Eligible patients are randomized to TL vs. standard daily embryo monitoring. For the TL monitoring a scoring system was developed including: timing of 1st division, duration of 2-cell cytokonesis, timing of 2-3 and 3-4 cell divisions, time to reach the 5-cell stage, fragmentation, blastocyst morphology). Patient/ cycle, parameters were compared. Main results and the role of chance: This report is based on the ﬁrst 59 randomized patients (28 TL vs 31 control). 12 patients dropped out (5 TL, 7 control) for various reasons. Patient and stimulation parameters are comparable between the groups. The per randomization pregnancy rates (PR): 14/28 (50%, TL) vs 13/31 (41.9% control) and ongoing PR: 13/28 (46.4% TL) vs 12/31 (38.7% control) were not signiﬁcantly different (p ¼ 0.5). The per protocol PR: 14/23 (60.8% TL) vs 11/24 (45.8% control) and ongoing PR: 13/23 (56.52% TL) vs 10/24 (41.6% control) were not signiﬁcantly different (p ¼ 0.3). The mean time lapse score was higher among those who achieved pregnancy in the TL group: (14.5 + /2 1.8 vs 13.1 + /2 2.0; p ¼ 0.09). Limitations, reason for caution: The sample size is relatively small to allow ﬁrm conclusions but we observed a favorable trend with TL monitoring. The scoring system that was developed based on currently available time-lapse ﬁndings and own experience also needs to be validated on a larger dataset. Wider implications of the findings: The development of effective embryo selection tools could lead to further reductions in the number of embryos transferred for an even wider patient population without affecting pregnancy rates in the fresh cycle. TL monitoring provides immediate information, is non-invasive and allows us to keep the embryos under ideal culturing conditions throughout the observation. Study funding/competing interest(s): None Trial registration number: NCT01694641 P-129 p38 MAPK signal activity is critical for GLUT1 and GLUT4 expressions, maintaining pluripotency and survival of early mouse embryos B. Sozen1, S. Ozturk1, A. Yaba-Ucar2, and N. Demir1 1 Akdeniz University Faculty of Medicine, Histology and Embryology, Antalya, Turkey, 2Istanbul Bilim University Faculty of Medicine, Histology and Embryology, Istanbul, Turkey Study question: Does p38 MAPK signaling pathway has any role on the regulation of GLUT1 (glucose transporter 1) and GLUT4 (glucose transporter 4) expressions, cell death, pluripotency and, thus cell fate in the preimplantation embryo development? Summary answer: The suppression of p38 MAPK activity affected the expression of GLUT1 and GLUT4 proteins and mRNA levels; increased cell death and altered expression of the pluripotency markers in preimplantation embryos. What is known already: Cleavage divisions after 8-cell stage generate differentiation within the preimplantation embryos, namely the segregation of primitive endoderm, epiblast, trophectoderm lineages. Nanog, Sox2, Oct4/Pou5f are known as cell fate and pluripotency regulators during this period. On the other hand, to accomplish proper preimplantation development, glucose transport in early embryo achieved by facilitative glucose transporters, GLUTs. However, although some signaling pathways associated with these processes have been identiﬁed, the role of p38 MAPK signaling is remained elusive. Study design, size, duration: Two cell stage embryos were cultured up to 8-cell, compact morula and blastocyst stages in three microdrop culture treatments; Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: The aim of this work is to determine whether or not exposure to music during in vitro culture conditions could improve outcomes in terms of oocyte fertilization and embryo quality. Summary answer: Our preliminary results show a statistically signiﬁcant increase in fertilization rate in oocytes exposed to music, but no statistically signiﬁcant differences were found regarding embryo quality in terms of cleavage stage and multinucleation. What is known already: Mechanical micro-vibration can alter both the expression of some molecules and neurogenesis itself (Alladi 2005). It has been reported that exposure of in vitro cultured human embryos to 5s intervals 44hz/h of microvibrations can improve embryo development and quality (Heo 2010; Matsura 2010). Nothing is known about music as a source of mechanical vibrations and its effect on human embryos whilst undergoing in vitro development prior to implantation. Study design, size, duration: 985 oocytes from 114 patients were analyzed (01-08/2012). Inseminated oocytes from each patient were divided in groups: A (culture with music (n ¼ 497)) and B (culture without music (n ¼ 488)). Fertilization rates and embryo quality were compared. We tested 3 types of music (A1:Pop,A2:Heavymetal,A3:Classical) by placing speakers inside standard incubators (Labotec C200). Participants/materials, setting, methods: Sibling embryos were randomly assigned to groups A/B. Morphological quality was established (scale 1-10, quality 7-10 ¼ ﬁrst-choice embryos). Generalised Linear-Mixed model. Oocyte fertilization rates, embryo quality (10-7 score), cleavage and multinucleation were analysed. Two (patient/cycle) or three levels (patient/cycle/day of transfer) were considered. Bayesian inferences were made using Integrated Nested Laplace(INLA). Main results and the role of chance: Fertilisation rates in group A (music) were 4.82% higher than in Group B (no music). Regarding the parameters used to assess embryo quality, no statistically signiﬁcant difference was found in embryo cleavage rates or the percentage of multinucleated embryos created. In group A there was no statistical difference in the percentage of ﬁrst choice transfer embryos (score 10-7) created, compared with those in group B. However, no statistically signiﬁcant difference was observed between the different types of music used (pop, heavy metal and classical). Limitations, reason for caution: Bayesian inferences were made using integrated Nested Laplace (INLA). Wider implications of the findings: Limited number of Publications . The routine use of music inside the incubators during in vitro culture would appear to be a useful tool to improve fertilisation rates. It is important to evaluate the effect of music on embryo development to day 5. We would recommend conﬁrming these results in a larger series of cases. Study funding/competing interest(s): Not applicable. Trial registration number: Not applicable. i169 i170 P-130 A prospective study evaluating the effect of high (21%) and low (5%) oxygen levels on the human embryo development N. Gelo, P. Stanic, V. Hlavati, S. ogoric, D. Pavicic-Baldani, M. prem-Goldtajn, B. Radakovic, M. Kasum, M. Strelec, T. Canic, V. imunic, and H. Vrcic University Hospital Centre Zagreb, Department of Gynecology and Obstetrics, Zagreb, Croatia Study question: The purpose of this study was to examine effect of low O2 (5%) levels in order to obtain more embryos with excellent morphology (grade Z1,A,AA) as well as higher clinical pregnancy rates (CPR). Summary answer: Cultivation of embryos in incubators with low oxygen (5%) levels contributes their better development on day 2, enhances the rate of blastocysts and those blastocysts results with higher number of CPR. What is known already: Oxygen is the major factor in embryo development in vitro along with cultivation medium. Excess of reactive oxygen species (ROS) results with damage known as oxidative stress (OS) which can cause DNA fragmentation, apoptosis, slowing or even stopping of embryo development. Excess of ROS is more common in cultures with high (21%) oxygen levels. Study design, size, duration: Between March 2012 and May 2012, 70 IVF and ICSI stimulated cycles were prospectively randomized for cultivation with high (21%) or low (5%) oxygen level. Until the time of fertilization all oocytes were cultivated with high (21%) oxygen level and after that cultivated according to the prior randomization. Participants/materials, setting, methods: Embryo development was monitored every 24 hours (except on day 4) for 3 - 5 days according to the day of transfer. Statistical analysis was performed with StatSoft program and results were compared with chi-square test with Yates correction with df ¼ 1. A P value ≤0. 05 was considered statistically signiﬁcant. Main results and the role of chance: Statistically signiﬁcant difference was observed with embryos on day 2 – 52.2% (47/90) grade A embryos developed with 5% O2 and 37.4% (34/91) with 21% O2, number of embryos that stopped in development – 5.6% (5/90) with low oxygen and 15.4% (14/91) with high oxygen levels and number of embryos that reached blastocyst stadium – 21.1% (19/90) with 5% O2 versus 6.6% (6/91) with 21% O2.Number of transfers on day 3 showed statistically signiﬁcant difference for cultivation with high (21%) oxygen levels while number of transfers on day 5 showed statistically signiﬁcant difference for cultivation with low (5%) oxygen levels-88% versus 71.2% and 26% versus 8.9%.Number of CPR after transfer of blastocyst was statistically signiﬁcant after cultivation in incubator with 5% O2 –46.6% versus 10%. Limitations, reason for caution: Results are speciﬁc and limited due to our restrictive law in that period - maximum of 3 oocytes per couple were fertilized. Wider implications of the findings: The most interesting result is statistically signiﬁcant difference observed with more grade A embryos on day 2 in favor of low oxygen cultivation. These results are in contrast with those of some previous studies that found no beneﬁt of culturing human embryos at lower O2 concentrations at this stage of development (Dumoulin et al.,1999; Bahceci et al., 2005). Other results are in correlation with previous ﬁndings (Kovacic et al.,2010; Meintjes et al.,2009; Waldenstrom et al.,2009). Study funding/competing interest(s): We have no relevant interests to declare. Trial registration number: None P-131 Growth factors status in follicular fluid of patients undergoing ICSI M. Ajina1, D. Negra 1, H. Ben-Ali2, S. Jallad1, I. Zidi1, S. Meddeb3, M. Bibi 3, H. Khairi3, and A. Saad3 1 Unit of Reproductive Medicine, University hospital F.Hached, Sousse, Tunisia, 2 Laboratories of Cytogenetic Molecular Biology and Human Biology of Reproduction, University hospital F.Hached, Sousse, Tunisia, 3Department of Obstetrics and Gynaecology, University hospital F.Hached, Sousse, Tunisia Study question: we try to ﬁnd any association between follicular ﬂuid (FF) levels of TGFb1, IGF 1 and EGF from individual follicles and the subsequent fertilization results after ICSI of the oocytes derived from the same follicles. Summary answer: The intrafollicular concentrations of growth factors were not correlated with ICSI outcomes but TGF b1 expression in the ovary seems to be regulated differentially by FSH and LH since recombinant- FSH group produced higher levels of TGF b1 compared to HMG one.TGF b1 production appears to decrease with age. What is known already: Although the exact role of each growth factor is not entirely known, a large body of evidence suggests that their harmonic cooperation is of crucial importance for the development of mature and competent oocyte. By using ICSI as the fertilization technique, the fertilization or even pregnancy outcome seems to be mainly dependant on the quality of the oocytes, and consequently, the relationship between oocyte and its environment and fertilization outcome could be investigate. Study design, size, duration: A prospective study during one year , including 100 women aged under than 38 years old undergoing ovarian stimulation following standard long agonist protocol, divided in two groups for analysis according to the treatment modalities, HMG(n¼ 35) or recombinant FSH(n¼ 65) for ICSI treatment with an indication of male factor infertility. Participants/materials, setting, methods: Follicular Fluid and its matched oocyte from each single follicle were collected individually during oocyte retrieval and stored at -208c until subsequent assays for TGF b1, IGF1 and EGF by ELISA using commercially available kits. After ICSI the levels of these factors and the subsequent fertilization results were analyzed. Main results and the role of chance: total retrieve and metaphase II oocyte numbers were signiﬁcantly higher in the r-FSH group (p ¼ 0.0001), cleavage and pregnancy rates were higher in this group (p ¼ 0.0001,), in contrast with lower rates of fertilization and top embryos. r-FSH group produced higher levels of TGF b1 (p , 0.05) and IGF 1(p . 0.05). The intrafollicular concentrations of the above factors were not signiﬁcantly associated with the ICSI outcomes. However, a strong positive correlation was found between TGF b1 levels and IGF1 (r ¼ 0.467; p ¼ 0.0001), but a negative one between TGF b1 or IGF1 and EGF(r ¼ -0.222, p ¼ 0.029; r ¼ -0.237, p ¼ 0.028. TGF b1 levels appeared signiﬁcantly higher in patients aged less than 30 years old (p ¼ 0.04) in whose pregnancy rate was higher and correlated negatively with age after 30 years (r ¼ -0.262, p ¼ 0.03). Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 control (n ¼ 195), vehicle (%0.1 DMSO-treated, n ¼ 195), SB203580-treated (p38 MAPK speciﬁc inhibitor, CSAIDTM, n ¼ 223). Statistical analysis were performed by one-way ANOVA test. Participants/materials, setting, methods: 6-week-old Balb/C female mice superovulated with 5 IU/mouse PMSG/hCG. Cell death was assayed by TUNEL and whole mount immunofuorescence staining used for p-MK2, p-hsp27, GLUT1, GLUT4. The relative Glut1, Glut4 mRNA levels were determined by quantitative RT-PCR. Expression of Oct4/Pou5f, Nanog, Sox2 determined by RT-PCR , /SSF . and analysed by Image-J software. Main results and the role of chance: With the inhibition of p38 MAPK activity, embryos displayed morphological abnormalities, developmental blockade at 8-16 cells onwards (P , 0.05). In the presence of SB203580, blastocysts contained fewer cells (mean 62 + 2.35) and an increased percentage of TUNEL positive nuclei (8.25 + 1.32%) compared to control (1.11 + 0.33%) and vehicle groups (1.08 + 0,34%) (P , 0.05). Expectedly, SB-treated embryos displayed a complete loss of p-MK2 and p-hsp27 expressions in all groups conﬁrming the effectiveness of inhibitor. Besides, dramatically decreased both GLUT1 and GLUT4 protein expressions and increased mRNA levels in SB-treated blastocysts were observed, indicating an altered glucose metabolism. Pluripotency related genes Nanog, Sox2, and Oct4/Pou5f showed altered expressions in SB-treated embryos. Nanog, a transcription factor key for epiblast differentiation, mRNA levels signiﬁcantly decreased at 8-cell stage onwards in the presence of SB203580 (P , 0.05). Limitations, reason for caution: This study was performed in vitro conditions and shown only in mice. In vivo applications of CSAIDTM molecules is not appropriate to determine the effects of p38 MAPK inhibition on preimplantation embryos. Wider implications of the findings: These results demonstrated how the inhibition of p38 MAPK activity could affect the preimplantation development. As clearly shown in the present study, the expression of glucose transporters and pluripotency genes to ensure lineage allocation are most likely regulated by p38 MAPK activity in early mouse embryos. Therefore, our study might provide new insights into the mechanism of regulation of preimplantation development and new approaches for idiopatic infertility problems. Study funding/competing interest(s): This study was supported by Akdeniz University Research Project Coordination Unit (Project no: 2011.02.0122.005). Trial registration number: None. Abstracts i171 Abstracts P-132 Detailed kinetics and morphology analyis of human triplonucleated embryos: a comparison with correctly fertilized transferred embryos L. Escrich, N. Grau, M. Meseguer, P. Gámiz, T. Viloria, and M.J. Escribá IVI Valencia, FIV, Valencia, Spain Study question: Is the in vitro dynamics of tripronucleated embryos (TPN) comparable to the morphokinetics of correctly fertilized transferred embryos (BPN)? Summary answer: TPN and BPN embryos showed different dynamic features which became reﬂected into a different embryo morphokinetic quality distribution. What is known already: Classical assessment have shown that ICSI-TPN cleave into two cells as BPN embryos do, but on day 3, the number of cells and ability to progress in vitro to the blastocyst stage are inferior to that observed in TPN embryos. Using time lapse technology, six discriminative morphokinetic parameters (t2-t3-t4-t5-cc2-s2) were deﬁned for implantation of BPN. Using these parameters and a hierarchical classiﬁcation procedure (A-E) we identiﬁed embryos with high implantation potential (Meseguer et al 2011). Study design, size, duration: Retrospective study of the morphokinetic parameters of 378 BPN transferred embryos and 163 TPN embryos. Participants/materials, setting, methods: Embryos were evaluated by detailed time-lapse analysis, which measured the exact timing of t2, t3, t4, t5, cc2, s2 (in hours post-ICSI). They were then classiﬁed according to a hierarchy based on these parameters. These parameters were compared in the two embryo groups (BPN and TPN). Main results and the role of chance: We observed signiﬁcant differences between BPN and TPN embryos in t2(25.9hrs vs 31.1hrs), t3(36.9hrs vs 39.7hrs), t4(38.4 vs 44.2), cc2(11.0 vs 9.4hrs), cc3(14.7hrs vs 13.1hrs), s2(1.5hrs vs 4.6hrs), s3(8.2hrs vs 10.4hr) and t5-t2(25.6hrs vs 23.4hrs). Concerning t5, timings were comparable in both groups. Differences in the hierarchical embryoclassiﬁcation were evident. Signiﬁcantly more BPN embryos were classiﬁed as “A” and “B” than TPN embryos (49.5% vs 22.2%), whereas more embryos were classiﬁed as “E” in the TPN than in the BPN group (44.4% vs 6.9%, respectively). Comparable percentages of BPN and TPN embryos were classiﬁed as “D” (16.1% vs 11.1%, respectively). Limitations, reason for caution: Although none of our TPN embryos were eventually transferred, the results reported here suggest that embryo quality is impaired in these abnormally fertilized embryos. Wider implications of the findings: Normally and abnormally fertilized embryos seem to be distinguished by the morphokinetics of in vitro embryo development. The present results reinforce the relevance of morphokinetics as a reliable marker of embryo quality. Study funding/competing interest(s): None Trial registration number: Not Applicable P-133 Embryo morphokinetic after artificial oocyte activation by using calcium ionophore E. Taboas Lima1, M. Pérez Fernández1, J.A. Aguilar Prieto 1, M. Ojeda Varela 1, D. Kassa1, and E. Muñoz Muñoz2 1 IVI Vigo, FIV laboratory, Vigo, Spain, 2IVI Vigo, Gynelogy, Vigo, Spain Control iCa Sig ......................................................................................... Age 39.35 + 2.7 38 + 2.9 .0.05 Fertilization rate 66.28 + 19.63 58.88 + 21.77 .0.05 % TQE 41.01 + 29.84 47.18 + 33.66 .0.05 .0.05 1.74 + 0.53 1.80 + 0.52 Pn Fading 26.15 + 3.40 23.50 +3.65 0.03 T4 41.70 + 5.57 38.24 + 3.47 ,0.01 T5 54.92 +5.60 50.17 + 7.38 ,0.01 Number ET Study question: Is embryo morphokinetic affected after artiﬁcial oocyte activation (AOA) with Calcium ionophore? Summary answer: Pronucleus fading(PN fading), The time from 3 to 4 cells (T4) and the time from 4 to 5 cells (T5) occurred earlier in the Calcium ionophore (iCa) group than in the control group, although this may not affect the pregnancy and live birth rates. What is known already: Total fertilization failure occurs in almost 10% of ICSI cycles. Artiﬁcial oocyte activation (AOA) with calcium ionophore (iCa) showed acceptable fertilization rates and successful pregnancies and deliveries of a healthy infant however, previous animal studies have demostrated that the use of AOA may inﬂuence the embryonic development to blastocyst stage. Study design, size, duration: Between January 2011 and January 2013, 13 couples who had experienced previous total failed fertilization after ICSI cycles with oligoasthenozoospermic sperm characteristics (iCa group) were compared with 12 couples with similar sperm quality (control group). Written informed consent to share the outcomes for research purposes was obtained from all them. Participants/materials, setting, methods: The AOA consisted of sperm injection with buffered medium containing iCa (Ionomicym from Streptomyces conglobatus). After sperm injection, oocytes were incubated for 20 minutes in culture medium with iCa. In the control group conventional ICSI was performed. To evaluate the embryo morphokinetic, oocytes were cultured in a tri-gas timelapse incubator. Main results and the role of chance: No signiﬁcant differences were observed between groups for age, fertilization rate, proportion of TQE and number of embryos transferred.Signiﬁcant differences were shown in embryo morphokinetics. The implantation rate was 29.41% in control versus 30% in ICa group and ongoing pregnancy rate was 80% and 83.33%, respectively. To date 4 live births were achieved in the control group and 5 in iCa group. Limitations, reason for caution: Total fertilization failure occurs in almost 10% of ICSI cycles. The AOA with Ionomicym from Streptomyces conglobatus is an experimental technique, at this moment. Wider implications of the findings: AOA induced by microinjecting sperm and calcium ionophore is an acceptable technique in couples who faced previous failed fertilization cycles. Pronucleus fading, T4 and T5 occurred earlier in the studied group than in control group, although this not affect the pregnancy and live birth rates. Study funding/competing interest(s): None Trial registration number: no RCT trial P-134 Improvement of blastocyst culture by a time-lapse incubator Morita 1, H. S. Watanabe1, M. Kamihata1, R. Matsunaga1, T. Wada1, K. Kani1, T. Ishikawa1, H. Miyamura2, M. Ito2, A. Kuwahata3, M. Ochi3, and T. Horiuchi4 1 Ochi Yume Clinic Nagoya, ART laboratory, Nagoya, Japan, 2Fujita Health University Hospital, Obstetrics and Gynecology, Nagoya, Japan, 3Ochi Yume Clinic Nagoya, Reproductive medicine, Nagoya, Japan, 4Prefectural University of Hiroshima Graduate School, Comprehensive Scientiﬁc Research, Hiroshima, Japan Study question: We compared the results of embryos incubated in the conventional incubator and those cultured in the time-lapse incubator. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Limitations, reason for caution: IGF 1 tented to be higher in r-FSH group although the difference was not signiﬁcant (p . 0.05) and that was correlated positively with TGF b1, so this result could be explained by our small sample size. EGF levels were very low and slightly detected coming probably through blood passive diffusion. Wider implications of the findings: The intrafollicular concentrations of growth factors can not predict ICSI outcomes but TGF b1 expression in the ovary seems to be regulated differentially by FSH and LH; it is stimulated by FSH and inhibited by LH. The lower IGFI levels in HMG group may suggest that LH inhibits IGF1 and/ or stimulates IGF binding protein 1. We suggest that TGFb1 together with IGF I, may regulate ovarian follicle growth in response to gonadotropin stimulation. Study funding/competing interest(s): TGF b1 production is stimulated par FSH and inhibited by LH and appears to decrease with age; So Studies on the follicular features of the aging ovary are much needed. Trial registration number: BASIC SCIENCE i172 P-135 The effect of piezo-assisted biopsy on preimplantation mouse embryo development and gene expression M.N.K. Nor-Ashikin1, J.M.Y. Norhazlin 1, S. Norita1, W.J. Wan-Haﬁzah1, M. Mohd-Fazirul1, D. Razif2, and B.P. Hoh1 1 Institute of Medical Molecular Biotechnology, Faculty of Medicine Universiti Teknologi MARA, Sungai Buloh Selangor, Malaysia, 2Faculty of Health Science, Universiti Teknologi MARA, Puncak Alam Selangor, Malaysia Study question: Are the development and genes expressed by piezo-biopsied embryos signiﬁcantly different from non-biopsied embryos, at morula and blastocyst stages? Summary answer: A signiﬁcant difference ( p , 0.05) was observed between the development of piezo-biopsied and non-biopsied embryos at the blastocyst stage, and a total of 75 and 66 genes of piezo-biopsied embryos were found signiﬁcantly different in expression ( p , 0.05) compared to non-biopsied controls, at morula and blastocyst stages respectively. What is known already: Zona breaching by chemical and laser drilling are implicated in delayed development and abnormal hatching of blastocysts. Although it has been reported that acid Tyrodes biopsy did not signiﬁcantly affect development and gene expression of blastocysts, the effect of piezo-assisted biopsy on gene expression has not been examined. Study design, size, duration: A 2 x 2 factorial study design was used to compare development and gene expression proﬁles of non-biopsied with piezo-biopsied embryos at morula and blastocyst stages. The development of 291 embryos were observed. Thirty embryos were pooled from each treatment group for RNA extraction, with three replicates per group. Participants/materials, setting, methods: Piezo-assisted (Eppendorf PiezoXpertw) single-blastomere biopsies were performed on 8-cell ICR mouse embryos (68 h post-human Chorionic Gonadotropin injection) in Ca2+/ Mg2+-free M2 medium. Biopsied embryos were cultured in M16 medium until RNA extraction. cDNAwas ampliﬁed, labelled and hybridized on the AffymetrixGeneChipw. Microarray was analysed using Gene Spring GX 12. Main results and the role of chance: Signiﬁcant difference ( p , 0.05) between the development of non-biopsied and piezo-biopsied embryos was observed only at the blastocyst stage (96.8% versus 89.0%, respectively). Gene expression of non-biopsied and piezo-biopsied mouse embryos were compared at morula and blastocyst stages, using the unpaired t-test ( p , 0.05), including fold change of ≥ 1.5 for each gene. At the morula stage, 32 genes were found upregulated and 43 genes downregulated. At the blastocyst stage, 43 genes were upregulated and 23 genes downregulated. DAVID pathway analysis software was used for annotation and visualization of statistically signiﬁcant genes. KEGG database annotated 14 genes for morula and 11 genes for blastocyst into different categories of pathways. Both stages showed regulation of genes involved in oxidative phosphorylation and fatty acid metabolism. Limitations, reason for caution: The use of in vivo instead of in vitro fertilized embryos may have reduced the impact on gene expression proﬁles. Critical changes in gene expression proﬁles may be diluted because of the pooling of embryos for RNA extraction. Wider implications of the findings: Elucidation of gene expression of preimplantation embryos after biopsy will add to a better understanding of the effect of piezo-assisted blastomere biopsy on early development. This knowledge can subsequently contribute towards the improvement of Assisted Reproductive Technology (ART) outcomes. Study funding/competing interest(s): This research was supported by our institutional grant (600-RMI/ST/DANA5/3/Dst(337/2011) and national Fundamental Research Grant Scheme (600-RMI/ST/FRGS5/3/FST(71/210). Animal procedures were approved by institutional Animal Care and Use Committee (ACUC-7/11). Trial registration number: Not applicable P-136 Retrospective analysis of sperm incubation time pre IVF insemination S. Dale, E. Cater, G. Woodhead, L. Jenner, and S. Fishel CARE Fertility, Embryology, Nottingham, United Kingdom Study question: Does sperm incubation pre IVF effect fertilisation, embryo transfer and clinical outcome? Summary answer: From the data presented in the abstract, clinical outcome was not signiﬁcantly affected by duration of sperm incubation pre IVF fertilisation. What is known already: Hyperactivation and capacitation of sperm is known to be induced by warming to body temperature, as would occur naturally in the female genital tract, and this is a prerequisite for fertilisation. Seminal plasma inhibits this hyperactivation (Mortimer et al 1998). Ragaa et al (2008) reported the rate of acrosomally reacted sperm was greatest after incubation for 5 hours but that a better fertilisation rate was achieved with 3 hours incubation pre-ICSI. Study design, size, duration: Retrospective analysis, non randomised analysis of IVF cycles from a single independent UK clinic. 153 continuous IVF cycles included in analysis from January to October 2012, regardless of age, previous history, sperm or oocyte source. Results to be statistically analysed using unpaired t-test and Fisher’s exact test. Participants/materials, setting, methods: Cycle preparation and stimulation varied as per consultant recommendation based on previous history. Oocyte recovery, insemination, culture and embryo transfer were performed as per protocol. Transfer day varied between day 3 and day 5 based on embryo development, and number of embryos transferred varied based on age and previous history. Main results and the role of chance: 222 patients were included in the study. Of these 201 achieved embryo transfer. These patients were divided into 2 groups depending on pre IVF sperm incubation times (Group 1 60-89 mins and Group 2 90-119 mins). Fertilisation rates were comparable (71.9%, 72.9%, p ¼ 0.8308 respectively) as were the abnormal fertilisation rates (3pn) (4.3%, 4.4%, p ¼ 0.9222 respectively). Both were analysed using unpaired t tests. Patients that didn’t achieve embryo transfer due to failed fertilisation, embryo development or freeze all due to OHSS risk were analysed but showed no correlation. Positive bhCG (55.4%, 60.6%), clinical pregnancy (47.5%, 47.9%) and biochemical loss rate (14.3%, 20.9%) were analysed using Fisher’s exact test but showed no signiﬁcance (p ¼ 0.5339, p ¼ 1.000 & p ¼ 0.4286 respectively). Limitations, reason for caution: The study was limited due to a change in laboratory protocol in January 2012. Sperm was incubated for 1-2 hours prior to insemination, therefore data pre 1 hour and post 2 hours is limited. The majority of cases performed at the clinic are ICSI, giving low numbers for analysis. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Summary answer: Culture using the time-lapse incubator largely reduced the frequency of removal and the time required to remove embryos from the culture chamber, and improved the development rate of blastocysts. Furthermore, frozen-thawed embryo transfer had high success rates. What is known already: A time-lapse imaging incubator for embryos facilitates the observation of embryos within the culture chamber. This can minimize stress factors such as decrease in temperature, change in culture medium pH, and light exposure, and may allow ’gentle culturing of ovum Study design, size, duration: The time-lapse imaging incubator culture group comprised 428 procedures (350 individuals; average age, 40.1 years) from whom oocytes were collected from August to December 2012. The conventional culture group comprised 1273 procedures (757 individuals; average age, 38.5 years) from whom oocytes were collected in 2011. Participants/materials, setting, methods: Embryos were placed in the timelapse imaging incubators (TLI) or the conventional culture incubators (CCI) for up to 7 days. We removed embryos form culture chamber twice in TLI group and up to 17 times in CCI group for inspection and changing the medium. Good quality blastocysts were cryopreserved. Main results and the role of chance: The rate of obtaining a good quality blastocyst was 43.5% (486 of 1116) in the ES culture group and 33.1% (1032 of 3120) in the conventional culture group, and the difference was signiﬁcant (P , 0.05). The pregnancy rate from frozen-thawed blastocyst transfer in the the time-lapse imaging incubators culture group and the conventional culture group was 48.9% (65 of 133) and 52.7% (371 of 704), respectively, and there was no signiﬁcant difference. Limitations, reason for caution: none Wider implications of the findings: The improvement of the culture environment by using the time-lapse imaging incubators increased the success rate of obtaining good quality blastocysts. Study funding/competing interest(s): No ezternal funding was obtained for this study. Trial registration number: Nothing Abstracts i173 Abstracts Wider implications of the findings: There is little literature available regarding length of time sperm should be incubated for IVF inseminations. There are many studies discussing sperm activity and capacitation upon which laboratories must base their protocols for incubation pre insemination. Many of these studies assess acrosome activity but do not relate to IVF outcome. This study, although limited, provides evidence that for the speciﬁc times of incubation included there is no statistical difference in outcome. Study funding/competing interest(s): None Trial registration number: None damage. A larger sample in a prospective study will be required to conﬁrm these ﬁndings are robust, and whether there is an effect on embryos of younger women. Study funding/competing interest(s): None Trial registration number: N/A P-138 Cyclin E1 as a new ICM marker identifies a discrete lineage in epiblast cells of the human blastocyst M. Krivega1 and H. Van de Velde2 Vrije Universiteit Brussel, Reproduction and Genetics - REGE, Brussels, Belgium, 2Vrije Universiteit Brussel and UZ Brussel, Reproduction and Genetics (REGE) and Centre for Reproductive Medicine (CRG), Brussels, Belgium 1 P-137 The effect of CO2 levels in embryo culture on clinical outcome Study question: We sought to determine whether a change in CO2 level supplied to IVF incubators from 6.0% to 5.5% had an effect on fertilization rate (FR) and clinical pregnancy rate (CPR). This retrospective analysis of women ≤34 and ≥35 years of age was conducted in the context of the recommendations of the culture media manufacturer. Summary answer: Although not statistically signiﬁcant, overall FR was higher with a CO2 supply of 5.5% compared to 6.0%. In contrast to younger women, a statistically signiﬁcant decrease in CPR was observed in the older group of patients when embryos were cultured under 5.5% rather than 6% CO2 level. There was no statistical difference in FR for either of the groups under the different CO2 conditions. What is known already: Most culture media utilize a bicarbonate/CO2 buffer system to keep pH in the range of 7.2-7.4. Media manufacturing companies recommend a value of CO2, having taken into consideration the Henderson-Hasselbach equation, under which their media will achieve the desired pH. In theory 1% difference in CO2 should only alter the pH by 0.1. However, as pH is a fundamental factor impacting oocyte viability and subsequent embryo development, any slight ﬂuctuation may affect the resulting embryo’s developmental potential. Study design, size, duration: A retrospective analysis to examine the effects of changes in CO2 levels on FR and CPR in 384 ART cycles over a 12 month period from January -December 2012. Results were compared among two different age groups (≤34 and ≥35 years old). A proprietary commercially available medium was used for gamete/embryo culture. Participants/materials, setting, methods: In 209 cycles the gametes/embryos were cultured under 6.0% CO2 supply (Condition A) and in the remaining 175 cycles under 5.5% CO2 supply (Condition B). These conditions were compared among two different age groups. 72 cycles of Condition A were compared to 54 cycles of Condition B in patients ≤34 years (Group I). 137 cycles of Condition A were compared to 121 cycles of Condition B in patients ≥35 years (Group II). T-test was employed to examine the difference in the mean maternal age among the groups. One sample Z-test with signiﬁcance level of 0.05 was used for statistical analysis of FR and CPR. Main results and the role of chance: The mean maternal age of Group I under Condition Awas comparable to the mean maternal age of Group I under Condition B (30.6 years + 3.0 vs. 31.4 years + 2.5, NS). Similar results were observed for Group II (Condition A: 38.7 years + 2.5 vs. Condition B: 38.4 years + 2.3, NS). FR in Group I was slightly higher under Condition B than in cycles under Condition A (63.2% vs. 58.4%, NS). Almost identical results were observed when FR of Group II was compared under different CO2 conditions (Condition B: 60.8% vs. Condition A: 60.0%; NS). CPR in Group I under Condition Awas comparable to CPR under Condition B (41.7% vs. 40.7%, NS). However, a statistically significant decrease in CPR was observed in Group II under the different CO2 conditions (Condition A: 28.5 % vs. Condition B: 17.4%; p ≤ 0.05). Limitations, reason for caution: Though the ﬁndings are intriguing, more data is required to determine whether these ﬁndings are valid. Ideally, the pH of the media should be measured as well as the CO2 levels. Wider implications of the findings: The data demonstrated that slight difference in CO2 levels in the IVF culture had signiﬁcant impact on clinical outcome among older women. One possible explanation is that when handling gametes/embryos outside their normal environment, replacing them into an incubator with a higher CO2 level allows for a faster recovery to physiological pH levels; older patients’ embryos due to poorer quality may be more prone to pH ﬂuctuation induced Study question: This study was aimed to describe new molecular players in human embryonic cells. In particular, we investigated Cyclin E1 (CCNE1) as a potential regulator of inner cell mass (ICM) in human embryos. Summary answer: The new marker of ICM in expanding blastocysts, CCNE1, is restricted to a deﬁned cell type in human blastocysts at the moment of implantation. What is known already: Cyclin E1 is one of the major regulators of the cell cycle. It forms a complex with CDK2 (cyclin-dependent kinase 2) and together they promote transition from G1 to S phases of the cell cycle. CCNE1 protein is known to be sustained to high levels in pluripotent cells. CCNE1 is necessary for re-entry of G0 cells into the cell cycle and for oncogenic transformation. Knock-out studies in mice proved redundancy for CCNE1 and CDK2. Study design, size, duration: We analyzed the expression pattern of CCNE1 in human preimplantation embryos and hESC in combination with pluripotency and early differentiation markers. Day 3 frozen-thawed embryos were cultured in presence of Roscovitine and harvested on day 6 for gene expression analysis. HESC were cultured on matrigel and transfected with FUGENE. Participants/materials, setting, methods: The study was approved by the Local and Federal Ethical Committees for research on human embryos. Human embryos were obtained from patients treated for infertility at our IVF Centre. Human embryonic stem cell (hESC) lines had been derived by our group. Samples were analyzed by qRT-PCR and immunocytochemistry. Main results and the role of chance: CCNE1 protein is exclusively expressed in nuclei of all ICM cells in expanded blastocysts. Later at the moment of implantation (day 6-7) CCNE1-positive cells only mark NANOG-negative cells of the epiblast. Inhibition of CCNE1 function during the cell cycle using Roscovitine did not affect preimplantation embryo development and expression of general embryonic markers for pluripotency and differentiation. Moreover, CCNE1 was found in hESC, particularly in VIMENTIN-positive cells acquiring epithelialmesenchymal transition and VIMENTIN-negative cells, but not in NANOG expressing cells. Overexpression of CCNE1 in hESC strongly upregulated SOX17 and mildly induced GATA4 and GATA6. This effect implies speciﬁcation into the endodermal lineage, although SOX17 marks both endoderm progenitors and PGCs. We currently investigate this hypothesis to ﬁnd out the nature of CCNE1-positive epiblast cells. Limitations, reason for caution: There are limited numbers of good quality human embryos donated for research. Wider implications of the findings: The improved knowledge on human preimplantation development will contribute to reﬁne artiﬁcial reproductive techniques and consequently increase live birth rates. Study funding/competing interest(s): Our research is supported by grants from the Fund for Scientiﬁc Research - Flanders (FWO-Vlaanderen) and the Methusalem (METH) of the VUB. Trial registration number: none P-139 Correlation of PRSS35 and SERPINE2 gene expression levels in cumulus cells with oocyte maturation and the potential as a biomarker to predict embryo quality R.K. Lee 1, Y.M. Hwu1, C.H. Lu2, and S.H. Li2 Mackay Memorial Hospital, Department of Obstetrics and Gyneocology, Taipei, Taiwan R.O.C, 2Mackay Memorial Hospital, Department of Medical Research, New Taipei, Taiwan R.O.C 1 Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 S. Andronikou, G. Francis, S. Tailor, M. Vourliotis, and P.A. Almeida Chelsea & Westminster Hospital, ACU, London, United Kingdom i174 P-140 Biologic efficiency of fresh oocytes in a large Italian ART program in relation to International benchmarks A. Vaiarelli, R. Antonacci, A. Smeraldi, M. Desgro, E. Albani, A. Baggiani, E. Zannoni, and P.E. Levi Setti IRCCS Istituto Clinico Humanitas, Department of Gynecology Division of Gynecology and Reproductive Medicine, Rozzano, Italy Study question: Is the ratio of babies delivered in relation to number of utilized oocytes a constant element in different infertile populations and countries? Aim of the present study was to compare retrospectively the biological efﬁciency of our ART program in relation to USA and European benchmark published data. Summary answer: Although great differences in term of delivery rates have been reported between European and USA results, representing still a matter of debate. The mean number of oocytes utilized to obtain a single baby delivered seems not signiﬁcantly different among our results and European and USA benchmarks. What is known already: A large and constant number of oocytes is needed to reach a delivered baby. This number seems a constant biological limitation, although improvements in ART technology during the last 30 years have been implemented. According to the American data “live birth” per oocyte retrieved was 4.6 %. European data shows that 4.4% to 3.8% (according to the age of patients) oocytes are needed to deliver a baby. Study design, size, duration: Retrospective analysis of clinical and embryological data of cycles performed at the Humanitas Clinical and Research Institute (Italy) in the period from 1 January 2011 – to 31 December 2011. Participants/materials, setting, methods: 1739 patient’s cycles with 9194 oocytes inseminated (7989 ICSI and 1205 IVF) were analyzed. Mean female age was 37 + 3.9 years. Data were divided by age into two groups (≤38 A and ≥39 B). Live born rate was calculated from the total number of oocytes including only fresh embryo transfer. Main results and the role of chance: The live baby born rate per inseminated oocytes was 3.5%. In group A and B the live baby born rate was respectively 4.4 % and 2.1% per oocyte used. The number of eggs needed to have a baby was 22.6 in group A while 48.4 in group B. American and European data show no increase in babies born if . 15-20 oocytes were collected. This range of oocytes could be the optimal number to maximise delivery rate, minimizing the risk of OHSS in fresh ART cycles. Our results even without considering pregnancies obtained from oocytes and embryos cryopreservation are not so far from benchmark results. Limitations, reason for caution: Our results do not consider cumulative delivery rate, because most of patients have still cryopreserved oocytes and embryos. Our data consider only used oocytes and not all mature retrieved oocytes. Wider implications of the findings: A strict correlation exists between the number of the eggs collected and babies born in ART cycles. This enormous biological wastage is probably due to the great number of intrinsically abnormal oocytes. This knowledge could be important in couple’s counseling. Our efforts should be addressed to modify earlier stages of folliculogenesis, to improve oocyte intrinsic quality, before ovulation induction, were little could be done. Study funding/competing interest(s): None Trial registration number: Not applicable P-141 Factors affecting the twin-delivery rate in sperm donation programme L. Bacer Kermavner1, I. Virant Klun1, B. Pinter2, and E. Vrtacnik-Bokal2 University Medical Centre Ljubljana, Dep. of Obstetrics and Gynaecology - IVF Laboratory, Ljubljana, Slovenia, 2University Medical Centre Ljubljana, Dep. of Obstetrics and Gynaecology Reproductive unit, Ljubljana, Slovenia 1 Study question: In our sperm donation programme there is still twin-pregnancy rate 20%, therefore the aim of this study was to elucidate if the female age and the number of retrieved or transferred blastocysts affect the twin-delivery rate and the potential role of elective single embryo transfer (eSET) in these couples. Summary answer: eSET is recommended in sperm donation programme when at least 4 good quality blastocysts are developed after prolonged embryo culture regardless the female age. What is known already: eSET has already became a practice in the in vitro fertilization programme using autologous fresh partner’s semen to prevent the twinpregnancy and to avoid the risk for both the woman and the baby. It is less known about the factors affecting the twin-pregnancy in the sperm donation programme using frozen-thawed sperm. Study design, size, duration: The data on 497 in vitro fertilization cycles using frozen-thawed donated sperm were performed at our department during the time period from 2001 to 2011 and were retrospectively analyzed to elucidate the effect of female age and number of retrieved or transferred blastocysts on twin-delivery rate in these couples. Participants/materials, setting, methods: One or two blastocysts were transferred. The couples were divided into three groups: 1.) non-pregnant, 2.) with single-delivery or 3.) with twin-delivery. All groups were divided according to the female age (≤36 or .36 years) and compared in terms of the number of retrieved and transferred blastocysts by the Chi-Square test. Statistical signiﬁcance was set up at P , 0.05. Main results and the role of chance: In couples with transferred one blastocyst the mean number of blastocysts per cycle was lower than in couples with a transfer of two blastocysts; after the transfer of two blastocyts there was a higher number of blastocyts per cycle in couples with pregnancy/delivery than in couples with no pregnancy regardless the female age. In couples with twin-delivery there was a higher number of blastocysts per cycle (younger: 3.8, older: 4.0) than in Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: Are PRSS35 and SERPINE2 gene expression levels in cumulus cells correlated with oocyte maturation, fertilization, and embryo quality? Summary answer: PRSS35 and SERPINE2 gene expression levels in cumulus cells were correlated with oocyte maturation and fertilization as well as good embryo quality. What is known already: The oocyte is surrounded by several layers of cumulus cells and that bi-directional communication between the oocyte and the cumulus cells is crucial for oocyte developmental competence and embryo development. PRSS35, SERPINE2, PTX3, and GJA1 are expressed in human cumulus cells. PTX3 and GJA1 mRNA levels are known to be associated with embryo quality. However, the correlation of PRSS35 and SERPINE2 expression levels with oocyte maturation and embryo developmental potential remains to be clariﬁed. Study design, size, duration: Total 211 cumulus cell samples of individual oocytes were obtained from 30 patients in the IVF laboratory. Patients undergoing intracytoplasmic sperm injection treatment program were recruited into the study. Participants/materials, setting, methods: PRSS35, SERPINE2, PTX3, and GJA1 mRNA levels were assessed in cumulus cells of individual oocytes using quantitative RT-PCR. PTX3 and GJA1 expression levels were also assayed for reference. The housekeeping gene RPL19, ribosomal protein L19, was used as the internal loading control to normalize the relative gene expression levels. Differences were analyzed by one-way analysis of variance followed by the Bonferroni post hoc test using GraphPad software. P , 0.05 was considered signiﬁcant. Main results and the role of chance: Cumulus cells from mature oocytes expressed signiﬁcantly lower PRSS35 and SERPINE2 mRNA levels than those from immature oocytes (P , 0.05 and P , 0.0001, respectively). Conversely, Cumulus cells surrounding mature oocytes expressed signiﬁcantly higher PTX3 mRNA levels than those encircling immature oocytes (P , 0.05). GJA1 mRNA levels also showed the tendency, although there was no signiﬁcant differences. PRSS35 mRNA levels were 5-fold lower in cumulus cells from mature and fertilized oocytes than those from mature but non-fertilized oocytes. GJA1, PRSS35, and SERPINE2 mRNA levels in cumulus cells from mature oocytes that developed to high quality embryos (grades 1 and 2) were also signiﬁcantly lower than those developed to poor quality embryos (grades 3 and 4) (P , 0.05). However, PTX3 mRNA levels exhibited the reverse tendency, which is similar to the previously published results. Limitations, reason for caution: A small sample size may cause insufﬁcient power. Thus, further large cohort studies are required. Wider implications of the findings: PRSS35 and SERPINE2 mRNA levels in cumulus cells correlate with oocyte maturation, fertilization, and good-embryo quality. This may provide novel biomarkers to predict oocyte developmental competence and embryo development. Study funding/competing interest(s): This work was funded by a grant, NSC 101-2314-B-195-009-MY3, from the National Science Council of Taiwan. is declared. Trial registration number: none Abstracts i175 Abstracts couples with single delivery (younger: 2.9, older: 2.1). This indicated that the number of at least 4 blastocysts retrieved per cycle is an important tool to introduce the eSET into the embryo transfer strategy regardless the female age. In all couples with twin-delivery number of blastocyst per cycle was signiﬁcantly higher than in couples with single delivery. Limitations, reason for caution: The number of cycles was realtively low. Wider implications of the findings: These results indicated that developmental potential of embryos and a number of blastocysts per cycle may be a good prognostic factor for twin-pregnancy/delivery and an important tool to introduce the eSET in the sperm donation programme regardless the female age. Study funding/competing interest(s): The study was performed in the frame of our clinical programme. There are no competing interests to be declared. Trial registration number: 0 Differentiation during early human embryogenesis C. De Paepe1, G. Cauffman2, G. Verheyen2, D. Stoop2, I. Liebaers3, and H. Van de Velde2 1 Vrije Universiteit Brussel, Department of Reproduction and Genetics (REGE), Jette, Belgium, 2Universitair Ziekenhuis Brussel, Centre for Reproductive Medicine (CRM), Jette, Belgium, 3Universitair Ziekenhuis Brussel, Centre for Medical Genetics (CMG), Jette, Belgium Study question: How are the trophectoderm (TE) key regulators CDX2, TEAD4 and YAP expressed during early human embryogenesis? Summary answer: In human embryos, the restriction of TE-speciﬁc components to the TE occurs after expansion of the embryo, when the two cell lineages (TE versus inner cells mass (ICM)) are morphologically distinguishable, suggesting a role for other molecules in the initial segregation between the ICM and TE lineages. What is known already: In mice, the Hippo Signaling pathway has been described in the ﬁrst lineage segregation (Nishioka et al. 2009). Differences in cell-cell contacts through differences in cell position lead to activation or inactivation of YAP, which subsequently can activate or inactivate TEAD4 in the nucleus and consequently regulate CDX2 expression in the inner versus outer cells. In the human it is not known how these Hippo signaling pathway components regulate TE speciﬁcation. Study design, size, duration: In this study, human preimplantation embryos were analysed for the expression of the transcription factors CDX2 and TEAD4, and the co-activator protein YAP. Both localization within the embryo and the timespeciﬁc expression were analysed. Participants/materials, setting, methods: The study was approved by the Local and Federal Ethical Committees for research on human embryos. Good quality embryos were obtained at our IVF Laboratory after informed consent of the patients. They were ﬁxed in 4% paraformaldehyde, permeabilized with 0,1% triton and stained for the respective proteins using immunocytochemistry. Main results and the role of chance: In cleavage-stage embryos, compaction and early blastocyst stages CDX2 was found weakly cytoplasmic. From the full blastocyst stage onwards, some cells showed nuclear CDX2 expression. In expanded blastocysts CDX2 was found either in the TE nuclei or in the ICM and TE nuclei. In hatching/hatched blastocysts CDX2 was found in the ICM and TE nuclei except for one big hatched blastocyst that started to downregulate CDX2 expression in ICM nuclei. YAP and TEAD4 started to be expressed in the nuclei of some cells of late cleavage-stage and compacted embryos. In expanded, hatching and hatched blastocysts TEAD4 and YAP were expressed in ICM and TE nuclei, except for one big hatched blastocyst in which YAP started to become downregulated in the nuclei of ICM cells. Limitations, reason for caution: Due to ethical concerns the amount of material is limited. This work is rather descriptive, but functional studies will be performed in the future. Wider implications of the findings: Information about the key players leading to the ﬁrst lineage segregation in the human embryo and the mechanisms underlying differentiation will contribute to our basic knowledge on human embryogenesis. This fundamental research is unique and crucial to the ﬁeld of reproductive medicine. Even though a lot is known about this subject in mice, results obtained in the human are different. This study shows that data obtained in mice cannot always be extrapolated to humans. Study funding/competing interest(s): This research is supported by grants from the Scientiﬁc Research Foundation- Flanders (FWO-Vlaanderen) and the Research Council (OZR) of the VUB. Trial registration number: None A. Stecher, B. Wirleitner, P. Vanderzwalmen, M. Zintz, A. Neyer, M. Bach, B. Baramsai, D. Schwerda, and N.H. Zech IVF-Centers Prof. Zech Bregenz GmbH, Bregenz, Austria Study question: Low fertilization rates (FR) -predominantly observed in severe male factor patients- negatively affects IVF outcome. We evaluated whether activation with calcium ionophore after sperm injection increases FR in these patients and studied the impact on blastocyst development, implantation rates (IR) and pregnancy rates (PR) in a sibling study. Summary answer: Oocyte activation signiﬁcantly increased FR in male factor infertility (e.g. severe OAT, testicular sperm extraction) and unexplained fertilization failure. Activation did not compromise blastocyst development but led to a higher number of fertilized embryos. Thus, overall more blastocysts with similar potential to implant are achieved per cycle. What is known already: To release the meiotic arrest in the oocyte and initiate the fertilization process, repeated calcium oscillations are essential. One main trigger for this intracellular signal was identiﬁed as the sperm-speciﬁc phospholipase C zeta I. Therefore failed fertilization is thought to be mainly a sperm factor. FR was shown to be increased with oocyte activation by calcium ionophore in patients with previous implantation failure and case series described no impact on health of babies born. Study design, size, duration: This prospective study included 62 IVF-cycles between November 2009 and December 2012. Inclusion criteria were severe male factor infertility and/or unexplained fertilization failure (,30%) in previous cycles. Sibling analysis was performed. Injected oocytes were randomly grouped in half and allocated either to the active or non-activated group for direct comparison. Participants/materials, setting, methods: ICSI or IMSI was performed 2-3 hours post-OPU. Half of the injected oocytes of each patient were activated with calcium ionophore. FR was checked 17 hours post injection, embryo quality on day 3 and blastocyst rate on day 5 before embryo transfer. IR was calculated by embryonic heart beat/embryo transferred. Main results and the role of chance: In 62 OPUs 966 oocytes were retrieved and 798 MII oocytes were injected. Of 397 non-activated oocytes 256 (64.5%) were fertilized on day 1 as compared to 302/401 in activated (75.3%; p , 0.001). In the further development the blastocyst rate/ MII oocyte was 31.8% without activation as compared to 36.6% with calcium ionophore. On day 5 the best blastocysts were chosen for transfer by morphological criteria. In the male-factor group the number of transferred blastocysts was the same in both groups, but in the non-male factor patients more embryos from the non-activated group were chosen. Similar pregnancy rates (54.5% vs. 52.9%) were obtained with nonactivated and activated, the IR was 35.3% vs. 29.6% and the ongoing PR 45.5% vs. 41.2%. Limitations, reason for caution: A higher number of cases as well as a long-term follow-up of the children’s health will be necessary to prove the safety of this procedure. Wider implications of the findings: Our results are in line with the literature. Additionally, we show for the ﬁrst time, that oocyte activation does not negatively impact on blastocyst development. Similar IR and ongoing PR for activated and non-activated sibling embryos were observed. By applying this technique the total number of developing blastocysts/ cycle is increased. This may lead to a higher cumulative PR. Study funding/competing interest(s): None Trial registration number: Informed consent was signed by all patients. P-144 Synchronized blastomere cleavage at cryopreservation: Effect on subsequent embryo survival, pregnancy and live birth rates Z. Wiener-Megnazi, M. Fridman, M. Koifman, S. Lahav-Baratz, I. Blais, R. Auslender, and M. Dirnfeld Carmel Medical Hospital, IVF Unit Obstetrics and Gynecology, Haifa, Israel Study question: To evaluate the effect of blastomere synchronicity of frozen embryos on post - thaw survival, morphological grading and outcome parameters in frozen embryo transfer (FET) cycles. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 P-142 P-143 Oocyte activation increases fertilization rate resulting in a higher final number of blastocysts without compromising implantation potential per transferred embryo: a sibling prospective study i176 Abstracts P-145 Histidine-rich glycoprotein improves embryo development H. Åkerud, K. Lindgren, K. Kårehed, K. Wånggren, and J. Hreinsson Department of Women’s and Children’s Health, Uppsala University, Uppsala, Sweden Study question: Does a Histidine-rich glycoprotein (HRG) peptide improve human embryo development and is the Embryoscope useful for evaluation of embryo development? Summary answer: A 35 amino-acid long HRG peptide seems to improve human embryo development and maturation when added to culture media. An Embryoscope is useful for monitoring embryo development. What is known already: Histidine-rich glycoprotein (HRG) is a protein known to interact with a number of different biological pathways. We have previously shown that a single nucleotide polymorphism (SNP) in the HRG gene (HRG C633T) is associated with recurrent miscarriages and pregnancy success rate after IVF treatment. In addition, infertility seems to associate with the HRG genotype. The different pathways through which HRG affects embryo development, implantation and placentation are not yet well established. Study design, size, duration: The study was designed as a case-control study and was approved by the regional ethics committe. Fourty-eight couples donated their surplus embryos which were cultured for four days and continoulsy monitored by a time-lapse methodology using an Embryoscope (UniSense Fertilitech, Denmark). Participants/materials, setting, methods: The study was carried out at the Reproductive Center, Akademiska sjukhuset, Uppsala, Sweden. Embryos frozen on day two after fertilization were thawed and cultured with or without HRG peptide added to culture media. Time-lapse sequences were used for timing of developmental events and to set ﬁnal blastocyst scores. Main results and the role of chance: After four days of culture 71% of the embryos in the peptide group (n ¼ 24) and 58% of the embryos in the control group (n ¼ 24) had developed to blastocysts. 46% of the embryos in the peptide group and 25% of the embryos in the control group were given the highest ﬁnal blastocyst scores. The time needed from ﬁrst cleavage to development of morula differed signiﬁcantly between the groups (43.1 + 9.8 h peptide group vs 34.6 + 14.3 h control group, p ¼ 0.049). In conclusion, the embryos cultured with the HRG peptide added developed slower and the difference in time needed between ﬁrst cleavage and development of morula might reﬂect a better timing of events of relevance for adequate maturation of an embryo. Limitations, reason for caution: This is a relatively small study and the results need to be conﬁrmed in a larger study population. Also, the protocol for scoring the embryos is new and needs to be evaluated further. The results are although signiﬁcant and the protocol was easy to use and reproducible. Wider implications of the findings: The results indicate that HRG is a protein of importance in human embryo development and that the HRG peptide might be of interest to add to culture media to optimize maturation of embryos and also to improve IVF treatment results. The Embryoscope is useful for evaluation of human embryo development. Study funding/competing interest(s): This study was funded by Swedish Society of Medicine and the Family Planning Foundation in Uppsala, Sweden. The authors declare no competing interests. Trial registration number: Not applicable P-146 The efficacy of hyaluronate (HA): a comparative study to evaluate the selection of mature sperm in ICSI-IMSI cycles S. Rovira1, J.M. Capdevila1, B. Freijomil1, C. Castelló 1, A. Farreras1, P. Fernández1, M. Asensio1, M. López-Teijón2, and E. Velilla 1 1 Instituto Marques, Reproductive Biology, Barcelona, Spain, 2Instituto Marques, Reproduction Medicine Service, Barcelona, Spain Study question: The objective of the study is to determine whether or not sperm selection by means of hyaluronate selection by SpermSlow is better than polyvinylpyrrolidone (PVP) in terms of the subsequent embryo fertilisation rates, viability, and pregnancy rates, in patients with teratozoospermia who undergo ICSI or ICSI-IMSI. Summary answer: There is a statistically signiﬁcant increase in the number of viable embryos created when hyaluronate rather than PVP is used to select sperm prior to ICSI. This difference was not noted if IMSI is used. No statistically signiﬁcant difference was noted in fertilisation rates or pregnancy rates with hyaluronate. What is known already: Mature sperm have receptors for hyaluronic acid, a protein found on the outer membrane of the oocyte. These sperm can then remodel the protein on the oocyte. Hyaluronidate in the microinjection media improves the selection of sperm with greater DNA integrity (Gabor Huszar, 2003), improves fertilisation rates (Nasr-Esfahani, 2008) and improves embryo quality, development and implantation (Parmegiani, 2009; 2010). However, these improvements have not been noted in all groups (Van der Bergh, 2009). Study design, size, duration: A prospective year-long study was set up in 2012 to compare hyaluronate (HA) by SpermSlow media (Medicult) and polyvinylpyrrolidone (PVP) (Vitrolife). The comparison was made both in patients who used ICSI and those who used IMSI-ICSI, resulting in the creation of 4 groups:G1: PVP-ICSI, G2: HA-ICSI, G3: PVP-IMSI-ICSI; G4: HA-IMSI-ICSI. Participants/materials, setting, methods: Inclusion criteria included all pacients who required egg donation, and whose partner’s sperm showed a severe teratozoospermia (normal morphology ,4%, Kruger et al, 1988). Main results and the role of chance: 537 oocytes were fertilised, 323 using PVP-ICSI (G1) and 214 using HA-ICSI (G2). No signiﬁcant differences were noted between the two groups in terms of fertilisation rates (76,47% vs 77,1% p ¼ 0,8652), nor pregnancy rates (41,46% vs 46,43 p ¼ 0,6829). There was Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Summary answer: Embryos with both synchronous and asynchronous blastomere cleavage can be safely cryopreserved. Embryos for cryopreservation should be selected by classical embryo grading and not by the criteria of synchronicity of blastomere cleavage. What is known already: While morphologic scoring of embryos in IVF is estimated subjectively, number of blastomeres and the distinction between synchronous and asynchronous cleavage are objective parameters. Embryos with synchronized blastomere cleavage are usually preferentially selected for cryopreservation. Time-lapse analysis techniques implicated synchronicity of blastomere cleavage as a possible predictor of treatment success. Study design, size, duration: Retrospective analysis of 1060 FET cycles performed from 2004 - 2006. Cycles were divided into 3 groups: 1: cycles with only embryos with synchronous blastomere cleavage were frozen; 2: cycles with only embryos with asynchronous blastomere cleavage were frozen; 3: cycles with both, synchronous and asynchronous blastomere cleavage embryos frozen. Participants/materials, setting, methods: One thousand and sixty FET cycles were analyzed. Clinical and laboratory data was recorded and analyzed. Main outcome parameters were: Post - thaw embryo survival, morphologic grading, pregnancy and live birth rates. Main results and the role of chance: Out of 1863 embryos, embryo survival rate was 68%; in 90%, ≥1 embryo survived. This was similar among all groups. Full blastomere survival rate was higher among the synchronous group (62.7%, 43.3%, 38.3% respectively, P ¼ 0.0001). Mean and maximal embryo grading at cryopreservation and at thawing were higher among the synchronous group (P ¼ 0.0001, P ¼ 0.037, P ¼ 0.004, P ¼ 0.03, respectively). When controlled for number of thawed embryos, differences remained signiﬁcant for 2 embryos (P ¼ 0.028). Mean and maximal thawing parameters were in association with conception. (P ¼ 0.056, P ¼ 0.023, respectively). Groups were similar regarding pregnancy and live birth rates. In a multivariate regression analysis, number of transferred embryos and maximal embryo grading at thawing affected the occurrence of conception (P ¼ 0.021, P ¼ 0.027, respectively). Limitations, reason for caution: Validity of results may be limited due to the retrospective nature of the study. Wider implications of the findings: Our study suggests that synchronicity of blastomere cleavage at cryopreservation should not serve as a criteria to select embryos for freezing. Although some of the embryo survival parameters were better among synchronous embryos, this was not associated with the chances of implantation. Therefore, the implantation potential of asynchronous and synchronous thawed embryos should be regarded as equal, in the same manner as fresh embryos, and not be discarded from being candidates for cryopreservation as such. Study funding/competing interest(s): The study was not funded by any commercial company. There were no competing interests in the study. Trial registration number: The study is not an RCT, so there is no need for a study registration number. i177 Abstracts P-147 Oocyte maturity rate as function of time from ovulation trigger to oocyte aspiration in IVF-ICSI cycles A. Weiss 1, R. Neril2, J. Geslevich 1, R. Beck-Fruchter1, M. Lavee1, J. Golan1, A. Ermoshkin1, and E. Shalev2 1 Emek Medical Center, Obstetrics and Gynecology, Afula, Israel, 2TechnionIsrael Institute of Technology, Rappaport Faculty of Medicine, Haifa, Israel Study question: Is the rate of oocyte maturity dependent on the time from ovulation trigger to oocyte aspiration within a four hour time frame during which the pickup procedures take place and does variation in this time interval warrant individualized scheduling of ovulation induction injection and oocyte aspiration? Summary answer: Thirty four hours is the minimum time required from oocyte maturation injection to ovum pickup. Variation in time between 34 and 37 hours did not affect the maturity rate of oocytes. Within this time frame there is no need for indivualized scheduling of ovulation induction injection and oocyte aspiration. What is known already: In spite of the prevalence of IVF, few studies have addressed the exact timing of oocyte aspiration after ovulation trigger. While 36 hours is an accepted time interval, the outpatient nature of the procedure may make meticulous timing of oocyte pickup difﬁcult. Furthermore, advances in IVF, such as the emergence of antagonist protocols and the introduction of GnRH agonist for ovulation trigger in high responders, may make older studies obsolete. Study design, size, duration: A retrospective study analyzing 526 IVF/ICSI cycles from January 2010 to May 2012. Participants/materials, setting, methods: Patients were instructed to inject HCG (or GNRH agonist for high responders) at 23:00 two days prior to ovum pickup. Patients were aspirated in the order they arrived at the clinic. The time of ovum pickup, laboratory and patient records were analyzed retrospectively. Main results and the role of chance: A total of 526 ICSI cycles were analyzed. On average 8.38 + 6.22 oocytes were aspirated per cycle. Overall, 64 + 28% of aspirated oocytes were MII oocytes. A one-way ANOVA demonstrated that oocyte maturity differed signiﬁcantly among four different time groups: 33.45 to 34 hours (n ¼ 94), 34 to 35 hours (n ¼ 256), 35 to 36 hours (n ¼ 125) and 36 to 37 hours (n ¼ 34); [F (3,506) ¼ 5.244, P , .001]. Tukey post hoc comparison of the four groups, showed that only the ﬁrst group differed signiﬁcantly from each of the other three groups (54 + 31%, 66 + 27%, 66 + 27% and 72 + 25% respectively). The time groups did not differ regarding other clinical and treatment parameters. Limitations, reason for caution: The study is limited by its retrospective nature. The sample size was sufﬁcient to reach signiﬁcance, though the fourth time group is smaller than the other three time groups. Wider implications of the findings: Simply changing the time patients are instructed to administer the ovulation triggering agent from 23:00 to 22:00 should increase the rate of MII oocytes in our unit. There is no need to individualize and meticulously schedule injections and ovum pickups as long as between 34 and 37 hours transpire. Further studies should reveal if an even longer delay will increase or decrease MII oocyte maturity rates. Study funding/competing interest(s): We declare no outside funding or competing interest. Trial registration number: The study was approved by the local ethics committee. Due to its retrospective non-interventional nature, trial registration is not required. P-148 Factors related to clinical pregnancy of vitrified-warmed embryo transfer: a retrospective and multivariate logistic regression analysis of 2313 transfer cycles W. Shi, S. Zhang, W. Zhao, X.I.A. Xue, M.I.N. Wang, H. Bai, and J. Shi Maternal & Child Health Care Hospital, Assisted Reproduction Center, Xi’anShaan’xi, China Study question: what factors affect clinical pregnancy in vitriﬁed-warmed embryo transfer (VET)? Summary answer: Assisted hatching (AH) had a positive effect on clinical pregnancy and the reason of freezing was one of the most signiﬁcant factors related to the clinical pregnancy of VET.No. of embryos transferred showed the lowest relative importance in 8 variables on the effect of successful clinical pregnancy. What is known already: Most previous studies have analyzed some of the factors related to the outcome of VET using one-factor analysis. To take account of confounding factors such as patient age, embryo quality, number of embryos to transfer, different laboratory procedure and clinical alternations, it is important to perform a multivariate logistic regression analysis to determine which one or a group of factors are most related to clinical pregnancy of VET. Study design, size, duration: The study was a retrospective analysis of 2313 VET cycles from 1481 patients performed between January 2008 and April 2012. A multivariate logistic regression analysis was performed to identify the factors to affect clinical pregnancy outcome of VET. Participants/materials, setting, methods: There were 22 candidate variables identiﬁed from clinical experiences and the literature. Eleven variables were chosen by a bootstrapping stepwise variable selection algorithm (n ¼ 1000) with the thresholds of aentry ¼ aremoval ¼ 0.05 for both variable entry and variable removal.3 factors were excluded and 8 factors were chosen to contribute the model. Main results and the role of chance: For the reason of freezing, OHSS group showed a high OR than the other groups (OHSS vs Other, OR: 2.145 CI: 1.4-3.286; Supplement vs Other, OR: 1.152 CI: 0.761-1.743). Assisted hatching also showed a high OR (OR: 2.105, CI: 1.554-2.85). The 3 variables (age of COH, damaged blastomere, and presence of blood on catheter) had an adverse effect on the outcome of VET (b , 0). The number of good-quality embryos showed the highest marginal PEV and partial PEV (marginal PEV 3.91%, partial PEV 2.28%). The reason of freezing showed the second highest marginal PEV (2.77%). However, No. of embryos transferred showed the lowest marginal PEV and partial PEV (marginal PEV 0.21%, partial PEV 0.46%). Limitations, reason for caution: This was a retrospective multivariate analysis of the data obtained in 5 years from a single IVF center. Prospective analysis of large data sites from a multicentre study is necessary to conﬁrm this ﬁnding . Wider implications of the findings: Except for the quality of embryos and the number of good embryos, assisted hatching and the reason for freezing were two important variables on clcinical pregnancy of VET.No. of embryos transferred showed less relative importance on the effect of successful clinical pregnancy Study funding/competing interest(s): This study was ﬁnancially supported by the Scientiﬁc and Technological Research Projects of Shaanxi Province (project number: 2011k15-02-01). All the authors have to declare. Trial registration number: N/A P-149 Understanding the impact of Assisted Reproductive Technologies (ART) on embryo health, child health and disease and longevity in later life H.L. Smith1, L. Shaw1, S. Kimber1, and D. Brison2 1 University of Manchester, reproductive medicine, Manchester, United Kingdom, 2 St. Mary’s Hospital, reproductive medicine, Manchester, United Kingdom Study question: To use existing microarray data and ampliﬁed cDNAs from different stages of human embryo development to investigate the expression of Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 however a signiﬁcant difference in the number of embryos that continued to evolve – i.e. the number of viable embryos created (G1: 112: 45,34% - vs G2: 102: 61,82%, p ¼ 0,001). 516 oocytes were fertilised, 361 using PVP-IMSI-ICSI (G3) and 155 using HA-IMSI-ICSI (G4). No signiﬁcant differences were noted between the two groups in terms of fertilisation rates (70,91% vs 69,03% p ¼ 0,6679), pregnancy rates (47,73% vs 35,29 p ¼ 0,3807), or in the number of viable embryos created (G3: 179: 69,92% - vs G4: 69: 64,49%, p ¼ 0,3101). Limitations, reason for caution: The study was not randomised. Wider implications of the findings: The use of hyaluronate in order to select mature sperm prior to ICSI would appear to be a good technique to improve embryo viability in patients with teratozoospermia, and therefore could be used as an alternative to IMSI. Study funding/competing interest(s): Not applicable. Trial registration number: Not applicable. i178 P-150 Developmental regulation of apoptosis related-genes during early embryonic development: a comparison between human and mouse species I. Boumela1, S. Assou1, D. Haouzi1, O. Ait Ahmed1, H. Dechaud2, and S. Hamamah3 1 CHU Montpellier Institute for Research in Biotherapy Hôpital Saint-Eloi, INSERM U1040, Montpellier Cedex5, France, 2CHU Montpellier Institute for Research in Biotherapy Hôpital Saint-Eloi, Hôpital Arnaud de Villeneuve Dept. of Ob/Gyn, Montpellier Cedex5, France, 3CHU Montpellier Institute for Research in Biotherapy Hôpital Saint-Eloi, INSERM U1040 Hôpital Arnaud de Villeneuve Dept. of ART/PGD, Montpellier Cedex5, France Study question: What are the apoptotic-regulatory genes expressed during early embryonic development in humans and mice and are they comparable between the two species? Summary answer: The apoptotic machinery is expressed during human and mouse early embryonic development. Although some differences may exist, most apoptosis-related genes exhibit similar expression patterns in both species. What is known already: Apoptotic cell death has been reported in human and mouse oocytes and pre-implantation embryos in vivo and in vitro, but its regulation and the conservation of the molecular factors between human and mice are not well known. Study design, size, duration: This experimental study included 15 patients referred to our ART department for IVF treatment and 25 mice (B6CBAF1) between 2010 and 2012. Both patients and female mice underwent controlled ovarian stimulation. Participants/materials, setting, methods: Human mature MII oocytes, 8-cell stage embryos and blastocysts were included after informed consent of the patients and IRB approval. Mouse MII oocytes, 2-cell stage embryos and balstocysts were collected from mice in vivo. Using DNA microarray, the expression proﬁles of apoptosis-related genes were determined before and after embryonic genome activation (EGA) in human and mouse. Main results and the role of chance: In human, genes involved in the extrinsic pathway of apoptosis are overexpressed in MII oocytes (Edar, ×11, p ¼ 0.008) and day5 blastocysts (Tnfrsf10b, ×10, p ¼ 0.03; Tnfrsf21, ×115, p ¼ 0.04) while in mouse Tnfrsf25 and Tnfsf12 are constitutively expressed. Several members of the Bcl2 family, that regulate the intrinsic pathway, show similar expression patterns in human and mouse such as for Bcl2l10 which followed a maternal proﬁle (×10, p ¼ 0.01; ×3.5, p ¼ 0.03) and Mcl1 which was overexpressed after EGA at the 8- and 2-cell stages in human and mouse respectively (×8, p ¼ 0.008; ×6, p ¼ 0.002). Among the numerous caspase genes expressed during preimplantation development, Caspase6 showed differential expression patterns between the two species with abundant levels in human MII oocytes (×7, p ¼ 0.01) and a speciﬁc increase in mouse blastocysts (×12, p ¼ 0.008). Limitations, reason for caution: Human and mouse oocytes and embryos were obtained after controlled ovarian stimulation, and thus the gene expression proﬁles of apoptosis-related genes could have been inﬂuenced. In addition, the in vitro culture conditions of human embryos may also have an impact. Wider implications of the findings: This study opens new perspectives for understanding the molecular regulation of oocyte and pre-implantation embryo survival and death. Study funding/competing interest(s): This work was partially supported by a grant from Ferring Pharmaceuticals company. The authors of the study have no competing interests to report. Trial registration number: Not applicable P-151 Ultrastructural alterations in different developmental stages of in vivo and in vitro preimplantation murine embryos exposed to cryopreservation R. Dasiman1, A.R. Nor-Shahida2, W.J. Wan-Haﬁzah2, J.M.Y. Norhazlin 2, M. Mohd-Fazirul2, O. Salina2, R.A.F. Gabriele2, and M.N.K. Nor-Ashikin2 1 Universiti Teknologi Mara, Faculty Of Health Science, Puncak Alam Selangor, Malaysia, 2Universiti Teknologi Mara, Faculty Of Medicine, Sungai Buloh Selangor, Malaysia Study question: This study was designed to address the issues listed below: 1) Which stage of development is best for cryopreservation in vivo and in vitro embryos? 2) What are the effects of cryopreservation using vitriﬁcation and slow freezing methods on ultrastructural organizations of the in vivo and in vitro embryos? Summary answer: The ﬁndings indicated that 8-cell stage of in vivo and in vitro embryos is more suitable for cryopreservation in terms of embryonic cryotolerance and ultrastructural damages. Highly signiﬁcant changes were observed in the ultrastructural organizations of both in vivo and in vitro cryopreserved embryos (p , 0.001). What is known already: Ultrastructural organizations of the preimplantation embryos are highly unique and consist of a fragile intracellular network of microﬁlaments and microtubules that is essential for embryonic development. The organizations undergo dramatic architectural changes which show variable physiological necessities and requirements in order to survive. Cryopreservation reduced embryonic survivability and caused deleterious changes on the Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 metabolic and epigenetic pathways identiﬁed as important in embryonic health, disease and longevity, e.g. mTOR, IGF, AMPK and zfp57. Summary answer: Genetic variability between embryos results in inconsistent expression of many of the key genes. However, several alternative genetic and epigenetic pathway members appear to be differentially expressed between different stages of development, indicating potential points of control in embryo development. What is known already: Many studies have shown the relationship between the maternal environment and offspring health, using animal models and epidemiological data. Children with diabetic mothers/ pre-blastocyst stage embryos removed from a diabetic environment have an increased risk of developing obesity, diabetes and metabolic syndrome in later life. Mouse studies demonstrate the importance of epigenetic methylation stability during embryonic development. However few studies have attempted to clarify the link between the environment/ART technology and human embryo health. Study design, size, duration: To use existing microarray data and ampliﬁed cDNAs from different stages of human embryo development. Initial analysis conducted on oocytes, 4cell and blastocyst stage embryos, 3 samples per developmental stage. Participants/materials, setting, methods: Embryos were donated by patients undergoing IVF at St. Mary’s hospital, Manchester, UK. cDNA was ampliﬁed via polyAPCR and analysed using the Affymetrix microarray HG U133 plus 2 chip, at the Paterson cancer Institute, Manchester. CEL. ﬁles are downloaded and statistical analysis performed within Partek and Metacore. Main results and the role of chance: The epigenetic regulator Trim28 was shown to be expressed during early human embryonic development. Although Trim28 was not differentially expressed between stages, its co-repressing binding partner MM-1 was 98 fold over-expressed in oocytes/4cell stage, but not at the blastocyst stage. MM-1 over-expression may lead to increased methylated regions, transcription factor inhibition and a point of gene silencing within oocyte/embryo development. IGF-related genes are also suggested as key points of metabolic control, relating the maternal nutrient availability to downstream pathways in the embryo. IGFBP1 was heavily (373 fold) over-expressed in oocytes compared to blastocysts. IGFBPs bind and inhibit IGF’s therefore the differential regulation is an apparent point of control in oocyte and embryo development. A number of other genes were upregulated by the blastocyst stage. Limitations, reason for caution: The number of samples was kept low in order to focus on genetic variability, however; utilisation of other microarray samples within the database will help clarify/verify ﬁndings. Embryos are inherently different to one another and therefore less stringent settings need to be applied to identify trends in gene expression. Wider implications of the findings: Understanding developmentally important pathways which may be perturbed by ARTaids our understanding of potential risk factors to which human embryos are subject in vitro. Assessing the perturbation of such genes in response to ART is an essential part of a fully-informed risk assessment. This will ultimately lead to increased understanding of long term health outcomes for ART children and will help to improve ART conditions to avoid unwanted impacts. Study funding/competing interest(s): This research is funded by the European Commission under the FP7 Health programme, as part of the EpiHealth consortium (co-ordinator Professor Andras Dinnyes) Trial registration number: no trial registration number Abstracts Abstracts P-152 Time-lapse microscopic analysis for exploring the dynamics of embryonic cell cycles following blastomere biopsy D. Ben-Yosef, T. Shwartz, T. Cohen, A. Carmon, N. Mey Raz, M. Malcov, T. Frumkin, B. Almog, I. Vagman, R. Kapustiansky, A. Reches, F. Azem, and A. Amit Tel Aviv Sourasky Medical Center, Racine IVF Unit, Tel Aviv, Israel Study question: Preimplantation genetic diagnosis (PGD) involves the aspiration of single blastomeres from a 6-8 cell embryo at day 3 of development. This study explored the dynamics of the developmental events following blastomere biopsy by determining the timing of subsequent cleavages and compaction following culture in the EmbryoScopeTM concomitantly with time-lapse analysis. Summary answer: Analysis of morphokinetic parameters enables unraveling of developmental events associated with blastomere biopsy. Synchronized cleaving embryos biopsied at the 8-cell stage enter the next cell cycle later than nonsynchronized ones. The latter are biopsied during the M phase of the cycle, thus resuming cleavage or entering compaction shortly after biopsy. What is known already: Morphokenetics evaluated by time-lapse analysis was recently added to the embryo scoring system to assist in embryo selection. To date, selection of healthy embryos following PGD was based mainly on their dynamic development following biopsy as observed by static evaluation, i.e., addition of cells following cleavage and/or compaction. There is only one publication (Kirkegaard et al., 2012) in which time-lapse analysis was performed following blastomere biopsy in order to study its effect on embryo development. Study design, size, duration: Embryos from all PGD treatments from September, 2012 to date were cultured in a time-lapse incubator (EmbryoScopeTM) until days 4-5 after fertilization. Images of 178 embryos from 24 couples carriers of genetic disorders were acquired every 20 min. Time-points of key embryonic events before and after embryo biopsy were registered. Participants/materials, setting, methods: Participants included 13 couple carriers of monogenic disorders and 11 couple carriers of chromosomal translocations. The acquired images of each embryo were retrospectively analyzed with the EmbryoViewer by image analysis software. All the selected embryo developmental events were annotated together with their corresponding time points (in hours) after insemination. Main results and the role of chance: Blastomere biopsy was performed at 71.2 + 2.9h post ICSI on embryos with 8.6 + 1.4 cells. A dynamic in embryo development was observed within 7.8 + 4.7h following biopsy: most embryos (77.5%) initially cleaved, as evidenced by the addition of blastomeres, followed by compaction. Interestingly, embryos biopsied at the 8-cell stage took longer to progress into subsequent developmental stages compared to embryos which underwent biopsy at a stage 8 cells (median 8.8h vs. 4.4h until cleavage and 22h vs. 10h until compaction, respectively). Fifty of the 178 cultured embryos were diagnosed as inheriting the normal allele (41 embryos transferred; 9 frozen). In 21 cycles 41 healthy embryos were transferred resulting in 8 pregnancies and 9 beating hearts (38% ongoing pregnancy rate; 22% implantation rate). Limitations, reason for caution: These new ﬁndings warrant validation in a larger cohort of embryos. Wider implications of the findings: Blastomere biopsy at ≏70 h postfertilization probably does not interfere with the embryonic cell cycle. This supports our previous observations that non-synchronized cleaving embryos with a good cleavage pattern probably have the same potential for further development as synchronized cleaving embryos. These ﬁndings make it possible to explore the timing of developmental events characterizing human preimplantation embryos, and they may have broader implications in choosing the optimal timing for blastomere biopsy for PGD. Study funding/competing interest(s): No funding, Trial registration number: 06/214 P-153 An additive model using morphokinetics to predict embryos of highest implantation potential M. Cetinkaya, C. Pirkevi, H. Yelke, Y. Kumtepe, Z. Atayurt, and S. Kahraman Istanbul Memorial Hospital, Assisted Reproductive Technologies and Reproductive Genetics Center, Istanbul, Turkey Study question: Can we predict embryos with highest implantation potential from morphokinetic parameters obtained until day3? Summary answer: The ﬁve formulas cited below were used to identify cleavage synchronicity: (F1): (t8-t2)/((t3-t2) + (t5-t4)); (F2): (t8-t5)/(t8-t4); (F3): (t4-t3)/ (t4-t2); (F4): (t4-t2)/(t8-t4); (F5): (t3-t2), additionally two criteria (irregular divisions and evenness of blastomeres at 2 and 4-cell stages) were applied to build an additive embryo scoring model predicting implantation. What is known already: A hierarchical model, predicting embryo implantation, was built through the retrospective morphokinetic analysis of 247 embryos (Meseguer et al., 2011). The most predictive parameters were described as being; ﬁrst, the time of division to 5 cells (t5), followed by, the time between division to 3 cells and subsequent division to 4 cells (s2) and the time between division to 2 cells and division to 3 cells (cc2). Study design, size, duration: This retrospective cohort study was conducted between October 2011 and October 2012 in a private ART Center and approved by the institutional review board. The study included 454 embryo having a (t8) with known implantation data (KID ¼ 0 or1) from 257 infertile patients (Female age: 33.1 + 5.1; BMI: 24.4 + 6.8; MII: 5.3 + 4.1). Participants/materials, setting, methods: Embryos were cultured in a single step culture medium, which was refreshed on day3. Incubation was performed under oil at 378C, 5%O2 and 6%CO2. Each time of cleavage was recorded. Clinical pregnancy was deﬁned as the presence of a gestational sac with fetal heartbeat detected on ultrasound on week7. Main results and the role of chance: Embryos should mainly stay in even cell stages, which should be balanced when compared to each other, ideally reﬂecting cleavage synchronicity. Each formula was applied to KID embryos, which were classiﬁed in 5%groups and implantation rates (IR) were computed for the 20intervals. In order to follow mathematically the natural trends obtained in IR, the average of ≥3consecutive 5%groups was subtracted from the average of the two following groups, and divided to the initial IR until the theoretical threshold set at 0.3 was reached. Each class had a mean IR, which was subtracted from Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 ultrastructural elements. However, to date, limited information is available about the ultracellular alterations in in vivo and in vitro cryopreserved embryos. Study design, size, duration: This study is carried out using a 2 x 3 x 2 factorial design. The sample size is 50 embryos for each treatment arm. The cryopreserved in vivo and in vitro produces embryos at 2-, 4-, and 8-cell stages were used in this study. Participants/materials, setting, methods: The ultrastructural elements of the embryos namely tubulin, actin and nucleus were labelled by immunoﬂuorescent staining, after being cryopreserved by either vitriﬁcation or slow freezing method. The embryos were then viewed under Confocal Laser Scanning Microscope and ﬁnally the intensities of the labelled elements were analyzed using QWin Software V.3. Main results and the role of chance: The results clearly showed that both cryopreservation methods caused highly signiﬁcant changes in ultrastructural elements of in vivo and in vitro embryos as compared to non-cryopreserved embryos (p , 0.001). In all cryopreserved group, as the number of cells increased, ﬂuorescent intensities of tubulin, actin and nucleus were decreased slightly as compared to non-cryopreserved groups. Vitriﬁcation and slow freezing caused highly signiﬁcant changes in tubulin intensities (p , 0.001), but the intensities of actin and nucleus were not signiﬁcant between 4- and 8-cell in in vivo embryos. In the in vitro embryos, the differences between intensities of tubulin, actin and nucleus were highly signiﬁcantly between 2-, 4- and 8-cell (p , 0.001). Limitations, reason for caution: The use laser with high energy and wavelengths during confocal microscopy observations is absorbed by the pigment granules that can cause intense local heating and contribute to ultrastructural alterations. Other than that, the ﬂuorescent signals may be overlapped during the immunoﬂuorescent staining and confocal analysis. Wider implications of the findings: Information obtained from this study will provide vital information needed in the selection of the best stage and cryotolerant embryo candidates for cryopreservation and directly reduce the cost of long term storage by eliminating unnecessary storage of non-viable embryos. Study funding/competing interest(s): This research was funded by Dana Kecemerlangan Universiti Teknologi MARA (DANA-600-RMI/ST/DANA5/3/Dst/ 37/2009) and Fundamental Research Grant Scheme (FRGS-600-RMI/ST/ FRGS/5/3/SFT/71/2010), Ministry of Science, Technology and Innovation (MOSTI), Malaysia. Trial registration number: None i179 i180 P-154 Safespeed technology - the myth of ultrahigh cooling rates: a close device and a serum-free media for the vitrification human oocytes/embryos with the highest recovery rates R. Risco1, M. Hebles2, A.M. Saa3, M.A. Vilches-Ferron 4, P. Sanchez-Martin5, E. Lucena3, and M. Lucena6 1 Engineering School, University of Seville, Seville, Spain, 2Clinica Ginemed, IVF Laboratory, Seville, Spain, 3Cecolfes, Fertility Laboratory, Bogota, Colombia, 4 Hospital Torrecardenas, Ginecology Department, Almeria, Spain, 5Clinica Ginemed, IVF Laboratory, Sevilla, Spain, 6Nidacon International, Product Development, Mölndal, Sweden Study question: While no studies have demonstrated unintentional uptake by a human or mammalian oocyte or embryo of any pathogen during vitriﬁcation or storage, under experimental conditions such contamination may occur (Bielanski et al., 2000); Also the presence in the vitriﬁcationsolutions of substances of human or animal origin is a drawback Summary answer: SafeSpeed is brand new technology that accomplishes the optimal requirements unveiled in recent studies for oocyte vitriﬁcation (Mazur et al., 2011), although with evidences since 1990 (Steponkus et. al, 1990). We have reached the highest recovery rates of human oocytes by optimizing the cooling and warming rates What is known already: Various techniques have been developed in the past to overcome this difﬁculty by improving the heat transfer, such as Open Pulled Straws, ‘‘solid-surface vitriﬁcation’’, the use of electron microscope copper grids, cryoloops, and more recently cryotop and cryotip (Kuwayama, 2007). However, in each of those systems, the philosophy underneath is increasing, but not optimizing, the cooling/warming rate (Mazur, 2011). SafeSpeed has born as the best optimization of those parameters in a close system. Study design, size, duration: Experimental trials have been performed with mouse and human oocytes. Out of 1700 samples, ninety ﬁve percent of vitriﬁed mouse oocytes survived after warming, and ninety three percent of human oocytes (out of 94) were recovered (Ginemed, Torrecardenas Hospital, Spain) . Fertilization rate observed after ICSI was 70 %. Participants/materials, setting, methods: Currently transfer trials are carried out in Cecolfes (Colombia). Images of ﬂuorescence immunochemistry with tubulin-speciﬁc antibodies to visualize microtubular structures of human oocytes vitriﬁed with SafePreservation technology were excellent, showing the good behavior of the cryoprotectant as well (Torrecardenas). Main results and the role of chance: This work has been possible thanks to an international collaboration of several partners: Nidacon, SafePreservation, Cecolfes, Clinica Ginemed, Hospital Torrecardenas and the Engineering School of the University of Seville. Limitations, reason for caution: By optimizing the cooling/warming rates, we have been able to maintain the use of conventional liquid nitrogen for our close device. Wider implications of the findings: In SafeSpeed, the container is heat-sealed at both ends, and consequently the solution containing the oocyte or embryo and the liquid nitrogen is hermetical isolated during cooling and storage. The technique is simple and robust, without sophisticated devices and easy to learn. Our system does not require special cryogenic agents, like slush nitrogen or other special mixtures (Risco et al., 2007), what would make the everyday practice very cumbersome, and would elevate the costs Study funding/competing interest(s): This study is relevant in the ﬁeld of oocyte and ambryo vitriﬁcation Trial registration number: 1 P-155 Vitrification: a useful tool in IVF lab when fresh embryo transfer can not be performed M. De Las Heras1, J.A. Agirregoikoa2, E. Martinez1, G. Barrenetxea2, and J.L. De Pablo 1 1 Quirón Bilbao, IVF Lab, Bilbao, Spain, 2Quirón Bilbao, Gynaecology, Bilbao, Spain Study question: The aim of this retrospective observational study is to assess the effectiveness of embryo vitriﬁcation on day 3 of embryo development comparing pregnancy rate, implantation rate and ongoing pregnancy rate between fresh embryo transfer and warmed embryo transfer. Summary answer: Vitriﬁcation could be not only a good cryopreservation method for good quality embryos that are not transferred in the fresh cycle but it is also a good strategy when the fresh embryo transfer must be postponed What is known already: Cryopreservation allows improving the costeffectiveness of IVF and expanding the options available to infertile couples. Many studies have shown that vitriﬁcation in contrast to slow freezing offers a higher survival rate, minimal deleterious effects on post-warming embryo morphology and it can improve clinical outcomes. Other studies show a better embryo-endometrial synchrony in frozen-thawed embryo transfer cycles than fresh embryo transfer with higher pregnancy and implantation rates after the transfer of warmed embryos. Study design, size, duration: This retrospective observational study includes 887 fresh and 690 warmed embryo transfers performed on day 3 between 2009 and 2012. Only transfers in which the two embryos transferred had the same score or single embryo transfers were included. A total number of 1456 fresh and 1153 warmed embryos were transferred. Participants/materials, setting, methods: Patients were ≤ 39 years old and their own oocytes were used. Embryos were scored according to the Spanish Association for Reproductive Biology. Vitriﬁcation and warming was performed using cryotop method. Clinical pregnancy was determined 7 weeks after embryo transfer. Statistical analysis was done using the Fisher test (p , 0,05). Main results and the role of chance: The pregnancy rate after warmed embryo transfer of two very good quality embryos was 43,7%, 29,9% when only one very good quality embryo was transferred, 32,3% when the transfer included two good quality embryos and 26,6% when two medium quality embryos were transferred. The pregnancy rates after fresh cycles were 42,1%, 22,9%, 24,1% and 20,2% respectively not showing signiﬁcant differences with those from warmed cycles (p . 0,05). The implantation rates did not show signiﬁcant differences between the two groups either (29,4% vs. 27,7%; 29,9% vs. 22,9%; 18,2% vs. 24,1% and 14,4% vs. 10,6%) (p . 0,05). There were not signiﬁcant differences between ongoing pregnancy rate (12 weeks after embryo transfer) (20,7% vs. 23,6%; 22,4% vs. 18,3%; 14,6% vs. 18,2% and 13,3% vs. 9,6%) (p . 0,05). Limitations, reason for caution: Although the study included many patients, a prospective randomized study should be carried out in order to conﬁrm our data. Wider implications of the findings: It is neccesary an efﬁcient cryopreservation method that allows the storage of the remaining embryos maximizing the cumulative effectiveness of an in vitro fertilization cycle. Our data show that vitriﬁcation offers the patient the same pregnancy rate as fresh cycles so it could be a good strategy when transfer must be postponed for personal or medical reasons such as in the ovarian hyperstimulation syndrome or when the endometrium is not prepared. Study funding/competing interest(s): no Trial registration number: no P-156 Embryo density in group culture of human embryos may influence embryo quality A. Lehner1, C. Pribenszky2, A. Murber1, J. Rigó Jr. 1, J. Urbancsek1, and P. Fancsovits 1 Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 the initial IR, giving a positive or negative score. Obtained scores were added for the ﬁnal prediction of the implantation potential. The logistic regression model built gave an AUC ¼ 0.684 (95% CI 0.628-0.740). Limitations, reason for caution: Embryos having a (t8) were exclusively included in the additive model, leaving out evaluation of day2 embryo transfers. The cohort studied involved only infertile patients (female, male or combined) and is in this respect a heterogeneous population. Wider implications of the findings: Time points deﬁning precise embryo cleavage events may not be generalized to infertile patients with different etiologies. However, using ratios based on selected cleavage cycles deﬁning synchronicity of embryos, allows in this retrospective cohort study an individualized analysis giving high predictivity of implantation. The proposed additive model has to be further tested in a randomized prospective trial to evaluate its efﬁcacy, and may in the future improve embryo selection in IVF treatments. Study funding/competing interest(s): Authors have nothing to disclose. Trial registration number: The study was approved by the institutional review board. Abstracts i181 Abstracts 1 Semmelweis University, First Department of Obstetrics and Gynaecology Division of Assisted Reproduction, Budapest, Hungary, 2Szent István University, Department of Animal Breeding and Genetics Faculty of Veterinary Sciences, Budapest, Hungary Trial registration number: This investigation is a retrospective analysis of a subgroup of data obtained from a larger prospective randomized study (ClinicalTrials.gov: NCT01774006). Study question: The aim of this study was to analyze the effect of embryo density on embryo quality and development when culturing in Well-of-the-Well (WOW) Petri dishes (Primo Dish, Vitrolife Hungary) in human IVF treatments. Summary answer: According to our results 3.5-5 ml/embryo density seems to have a beneﬁcial effect on the embryo quality compared to lower and higher densities. Embryo density can affect embryo development when culturing embryos in groups. What is known already: Group culture of human embryos is a widely used culture method, which may have a beneﬁcial effect via autocrine and paracrine mechanisms. However an optimal embryo density (volume of culture media divided by the number of embryos ¼ ml/embryo) in human in vitro culture has not been deﬁned yet. The WOW dish, which contains 9 + 1 microwells in the center of the dish, allows us to identify individual embryos cultured together in the same microdrop. Study design, size, duration: Data of 675 normally fertilized oocytes from 119 IVF cycles were retrospectively analyzed. Embryos were classiﬁed into four groups: Group A (n ¼ 336): individual culture, Group B (n ¼ 62): 2-4 embryos, Group C (n ¼ 152): 5-7 embryos, Group D (n ¼ 125): 8-10 embryos cultured in a single droplet of 25 ml media in WOW dishes. Participants/materials, setting, methods: Embryo densities were the following: Group A: 25 ml/embryo, Group B: 6-12.5 ml/embryo, Group C: 3.5-5 ml/embryo, Group D: 2.5-3 ml/embryo. Number of blastomeres, fragmentation and morphology score on Day 2 and Day 3 were analyzed with one-way ANOVA and Tukey HSD test. Frequency of top quality embryos were compared with Chi-squared test. Main results and the role of chance: Number of blastomeres was similar in the four groups (P ¼ 0.36) on Day 2. However cell number in Group C was signiﬁcantly higher than in Group A (7.5 + 2.1 vs. 6.4 + 2.2; P , 0.001) and in Group D (7.5 + 2.1 vs. 6.6 + 2.0; P ¼ 0.007) on Day 3. Fragmentation rate was signiﬁcantly lower in Group C compared to Group A (11.5 + 8.3% vs. 15.1 + 11.9%; P ¼ 0.01) on Day 2. The ratio of top quality embryos were higher in Group C compared to Group A (29.3% vs. 17.2%; P ¼ 0.004) and to Group B (29.3% vs. 8.3%; P ¼ 0.003), as well as in Group D compared to Group B (22.6% vs. 8.3%; P ¼ 0.03) on Day 3. Limitations, reason for caution: This study presents preliminary data. A larger study is needed to have more exact information about the effect of embryo density on embryo quality. Wider implications of the findings: Embryo density of 3.5-5 ml/embryo seems to have the most beneﬁcial effect on embryo development. Culturing embryos in a WOW group culture dish may increase embryo quality and allows assessment and tracing of individual embryos. Study funding/competing interest(s): - P-157 Effect of oil density in donor oocyte IVF cycles D. Gumbao Baño1, A. Sánchez-León 1, J. Marcos1, M. Mollá1, B. Amorocho1, M. Nicolás2, L. Fernández2, and J. Landeras2 1 IVI Murcia, FIV Laboratory, Murcia, Spain, 2IVI Murcia, Gynecologist, Murcia, Spain High-density oil Low-density oil p Nº of treatments 34 57 Nº of transfers 32 55 Age 40.6 39.6 Average nº embryo transfers 1.5 1.7 Fertilisation rate 76.8 %(298/388) 77.9 %(514/660) 0.69 Clinical pregnancy rate 65.6 %(21/32) 69.1 %(38/55) 0.74 ............................................................................................................................................................................................... Implantation rate 50 %(24/48) 55.3 %(52/94) 0.55 Biochemical pregnancy rate 4.5 %(1/22) 11.6 %(5/43) 0.27 Clinical miscarriage rate 33 %(7/21)* 5.3 %(2/38)* 0.02 Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: The aim of this study was to evaluate the effect of two different culture oils with different densities on the donor oocyte clinical outcome rates at day ﬁve of embryo development using a time-lapse culture system. Summary answer: The density of the oils used in a time-lapse culture system at day ﬁve of embryo development may have some impact on ART outcomes. What is known already: The quality of oil used in an embryo culture is one of the most critical issues for successful IVF treatment. In fact, different quality oils may affect embryonic development in vitro by containing inappropriate or toxic organic substances. Study design, size, duration: To evaluate the oil density inﬂuence in the clinical outcomes on our embryo culture system, two different densities of the same oil were evaluated retrospectively. 91 FIV-ICSI treatments were carried out between 2011 and 2012 with patients who were included in our oocyte donor program. Participants/materials, setting, methods: Embryos were divided into two groups and cultured to day 5 in a time-lapse system using culture plates with 12 wells including 24 mL of medium and overlaid with 1400 mL of the two different culture oils. The statistical t-student software analysis signiﬁcance was deﬁned when P , 0.05. Main results and the role of chance: No signiﬁcant differences were found except in clinical miscarriage rate which was signiﬁcantly lower in the group of embryos cultured in light oil. The results are shown in Table 1. Table 1. Clinical outcome rates. * statistical signiﬁcance p ,0.05. Limitations, reason for caution: Although culture oil density did not affect pregnancy and implantation rates, statistic differences in the miscarriage rates were observed, so further prospective and randomised studies are underway in order to discover something else related with the oil density including fertilization to day 5 time-lapse division parameters analysis. Wider implications of the findings: The results appear to point towards the importance of the oil using in culture. These results would be in agreement with the literature and the results obtained by other groups that also defend the role played by oil and the idea that further studies will be necessary in order to provide new proofs. Study funding/competing interest(s): The present study was supported by IVI Murcia SL. Murcia. Spain Trial registration number: No trial registration number. i182 P-158 A comparative gene expression analysis of slow frozen and vitrified metaphase II human oocytes O.A. Adeniyi, S.M. Ehbish, and D.R. Brison Central Manchester University Hospitals, NHS Foundation Trust, Manchester Academic Health Science Centre P-159 Comparison of two different cryo-devices for vitrified-warmed single blastocyst transfer using chemically defined vitrification/warming solutions supplemented with recombinant human albumin A. Egashira1, M. Murakami2, E. Nagafuchi1, K. Tanaka1, A. Tomohara1, C. Mine H. Otsubo1, A. Nakashima 3, M. Otsuka3, N. Yoshioka3, and T. Kuramoto4 1, 1 Kuramoto Women’s Clinic, IVF Laboratory, Hakata-ku Fukuoka City, Japan, Kuramoto Women’s Clinic, Reseach Laboratory, Hakata-ku Fukuoka City, Japan, 3Kuramoto Women’s Clinic, Reproductive Medicine, Hakata-ku Fukuoka City, Japan, 4Kuramoto Women’s Clinic, Clinical Department, Hakata-ku Fukuoka City, Japan 2 Study question: To investigate whether a closed devise (Rapid-i) is useful for human blastocyst vitriﬁcation compared with an open vitriﬁcation devises (Cryotop) Summary answer: Closed vitriﬁcation system using recHAwere effective for the cryopreservation of human blastocysts to obtain good clinical outcome and to avoid the possibility of disease transmission and potential risk of crosscontamination. What is known already: The vitriﬁcation/warning solutions supplemented with recHA used for human blastocysts vitriﬁcation with cryotop were ﬁrst validated in a preclinical study (Murakami et al. 2012, abstract O-183 ESHRE). However, there are no proven viral transmissions between embryos or to the patient at embryo transfer, the use of an open system for vitriﬁcation potentially introduces a risk of cross-contamination during liquid nitrogen storage. Study design, size, duration: From August 2012 to December 2012, 79 patients forvitriﬁed-warmed single blastocyst transfer under a hormone-replacement cycle were divided into two groups (group A: Cryotop, n ¼ 41; group B: Rapid-i, n ¼ 38). On day 5 and day 6, blastocysts with ICM and trophectoderm type A or B (Gardner’s scoring system) were vitriﬁed. Participants/materials, setting, methods: The base solution was PBS with 2.5 mg/ml recHA. Blastocysts were exposed to two solutions: equilibration (10% EG) and vitriﬁcation (15% EG + 15% DMSO + 0.5 mol/l sucrose). For thawing, embryos were placed into four sucrose solutions (0.5M, 0.25M, 0.1M, and 0M respectively). Data were analysed by x2 test. Main results and the role of chance: There were 41 warmed cycles in group A and 38 warmed cycles in group B. Mean female age was 35.8 + 3.9 years in group A and 35.1+ 2.5 years in group B.There were no signiﬁcant different in embryo survival rates (100% [41/41] vs. 100% [38/38]) and the implantation rates (53.7% [22/41] vs. 63.2% [24/38]) between groups A and B. The ongoing pregnancy rates per embryo transfer were no signiﬁcant different between group A and group B (43.9% [18/41] vs. 52.6% [20/38]). Limitations, reason for caution: In our study, study population and number of vitriﬁed blastocysts were small. Therefore, large-scale investigation including follow-up of children born after embryos vitriﬁcation using a Rapid-i are required. Wider implications of the findings: Closed vitriﬁcation method used in this study useable each blastocyst to be vitriﬁed in super-cooled air and loaded in a closed straw. As a result, the blastocyst has supposed to have low risk of disease transmission and cross-contamination from liquid nitrogen, without impairing developmental competence. This method will be useful vitriﬁed-warmed single blastocyst transfer in the future. Study funding/competing interest(s): None Trial registration number: None P-160 Fresh embryo transfer versus frozen embryo transfer with GnRH-antagonist protocol in ART D. Choi, H. Yang, J.H. Park, J.H. Jung, H.G. Hwang, J.H. Lee, J.E. Lee, A.S. Kang , J.H. Yoo, H.C. Kwon, and S.J. Lee Mirae and Heemang Ob/Gyn Clinic, Obstetrics and Gynecology, Seoul, Korea South Study question: In ART of high ovarian responders undergoing a GnRH-antagonist protocol, which has beneﬁt for patients between fresh embryo transfer (FT) and frozen-thawed embryo transfer (FTT)? Summary answer: If fresh embryo transfer(FT) could be replace by frozen-thawed embryo transfer(FTT) in high ovarian responder undergoing ART with GnRH-antagonist protocol, the patients will have some advantages in both higher pregnancy rate and avoiding OHSS What is known already: Some studies reported that in ART, the pregnancy rate of FTTare higher than FT. Also several meta-analyses mentioned similar results. The endometrial receptivity during ART is very important in implantation of the transferred embryo. The endometrial receptivity can be controlled more precisely in FTT than in FT. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: To investigate the impact of metaphase II (MII) oocyte cryopreservation by slow freezing and by vitriﬁcation on the expression of genes involved in oocyte cryo-survival and in-vitro developmental competence. Summary answer: Oocytes showed a higher survival rate (86.7%) from vitriﬁcation compared to slow freezing (76.2%). This was not signiﬁcantly associated with maternal age. Gene expression data for BRCA 1 and 2, OCT4, TUBB4Q and a number of other candidate genes show differences between the vitriﬁed and slow frozen oocytes. What is known already: Routine application of oocyte cryopreservation procedures for both long and short-term fertility preservation and treatment has been widely accepted. Several modiﬁcations in the processes of vitriﬁcation and slow oocyte freezing, have signiﬁcantly improved the survival rates, pregnancy and live-birth rates obtained from both procedures. However, vitriﬁcation seems to produce better results than slow oocyte freezing. Unfortunately, the safety of these procedures and their impact on oocytes at the molecular level is not completely understood. Study design, size, duration: A prospective study of 15 consenting patients in the vitriﬁcation group had controlled ovarian stimulation between May and October 2012. Oocytes were vitriﬁed using a closed system. Two patients in the slow freezing group had oocytes cryopreserved between 2002 and 2003. A minimum of 10 oocytes were analysed per group. Participants/materials, setting, methods: Fifteen pairs of unfertilised MII oocytes were obtained from fresh ICSI cycles in a standard NHS public sector hospital, of which 15 were vitriﬁed and 15 sham treated as paired controls. Fertility preservation patients donated 21 slow frozen MII oocytes. Survival rates were compared and cDNA was analysed by polyAPCR and qPCR Main results and the role of chance: The average age of patients and oocyte survival rates from slow freezing, vitriﬁcation and control groups were 31.4yrs and 76.2%, 32.8yrs and 85.7% and 32.8yrs respectively. The survival rate in the vitriﬁcation group was not statistically signiﬁcant, when compared with the slow freezing group (p ¼ 0.6897). Ongoing gene expression analysis of genes including TUBB4Q, OCT 4, BRCA 1, BRCA 2 and some speciﬁc genes associated with oocyte cryo-survival, show different gene expression patterns in both groups compared with the study control group. Limitations, reason for caution: The use of unfertilised MII oocytes from ICSI cycles in the vitriﬁcation and control groups can be argued to be a sub-optimal source of MII oocytes, pre-exposed to micromanipulation and overnight culture. However, this did not appear to compromise the higher survival rate obtained, compared with slow freezing group. Moreover we have made genetically normal embryonic stem cell lines from this source of oocytes demonstrating that they retain developmental capacity. Wider implications of the findings: Our preliminary results show that MII oocyte vitriﬁcation using a closed system produces better survival outcome compared with slow freezing. Vitriﬁcation can be argued to be essential in optimising outcomes involving fertility preservation and treatment. However, further investigations involving sibling MII oocytes to compare cryopreservation techniques is warranted. In particular, gene expression patterns in pre and post-cryopreserved MII oocytes can allow us to fully evaluate the association between oocytes cryopreservation techniques and molecular gene expression. Study funding/competing interest(s): Adeniyi, OA and Brison, DR are funded by Central Manchester NHS Trust; Ehbish, SM is funded by the Libyan Government. Trial registration number: None Abstracts i183 Abstracts P-161 Induction of autophagy in the vitrified-warmed mouse oocytes S. Bang, H. Shin, and H.J. Lim Konkuk University, Biomedical Science & Technology, Seoul, Korea South Study question: Is autophagy induced during vitriﬁcation-warming process in mouse oocytes? Summary answer: Autophagy is induced in mouse oocytes during vitriﬁcationwarming process. What is known already: Vitriﬁcation uses cryoprotectants and liquid nitrogen, which may cause osmotic stress and cryodamage to oocytes. Autophagy is widely considered as a survival or responsive mechanism to various environmental and cellular stresses. However, the status of autophagy in vitriﬁed-warmed oocytes has not been studied. In this work, we investigated if vitriﬁcation-warming process induces autophagy in mouse oocytes. Study design, size, duration: Oocytes obtained from several mice were pooled and divided into three groups. Group1: fresh oocytes. Group2: oocytes treated with vitiﬁcation solutions (1.3M EG + 1.1M DMSO and 2.7M EG + 2.1M DMSO + 0.5M sucrose) and warming solutions. Group3: vitriﬁed-warmed oocytes (loaded onto an EM copper grid, and were stored in LN2 for 2weeks). Participants/materials, setting, methods: Four-week-old female ICR mice and GFP-LC3 transgenic mice were used. The mice were superovulated with 5IU PMSG and 5IU hCG and ovulated MII oocytes were collected from oviducts. RT-PCR and confocal live imaging of GFP-LC3 were performed to examine the effects of vitriﬁcation-warming process on autophagy in oocytes. Main results and the role of chance: In RT-PCR analyses, the expression of autophagy related (Atg) genes, such as Atg5, Atg7, Atg12, LC3a, LC3b, and Beclin1 were examined. In Group 2, the expression of Beclin1 was increased in comparison to fresh oocytes (Group 1), and it was recovered in Group 3. Thus, induction of Beclin1 mRNA is associated with the exposure to the vitriﬁcation solution. The expression of other Atg genes did not change. Confocal live imaging analysis using oocytes from GFP-LC3 transgenic mice revealed that some vitriﬁed-warmed oocytes showed green puncta which indicate autophagic activation. All oocytes of Group 1 and Group 2 show no puncta formation. Each experiment was performed three times. Limitations, reason for caution: Increased autophagy was only conﬁrmed by live imaging of GFP-LC3 as a qualitative analysis, thus quantitative analysis such as Western blotting needs to be performed. Further investigation is warranted to address the role for autophagic activation in vitriﬁed-warmed oocytes. Wider implications of the findings: Induction of autophagy may serve as an indicator of conditions of vitriﬁcation-warming process. Moreover, it offers the possibility that development of methods to modulate autophagic response during cryopreservation could improve efﬁcacy of oocyte cryopreservation. Study funding/competing interest(s): This study was supported by a grant of the Korea Health technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A120080). Trial registration number: N/A P-162 Forced blastocoele collapse of the blastocoel improves developmental potential in cryopreserved bovine blastocysts by vitrification and slow-rate freezing methods S.H. Min, J.Y. Yeon, and D.B. Koo Daegu University, Department of Biotechnology, Daegu, Korea South Study question: The purpose of this study was to evaluate the effectiveness of forced blastocoele collapse of the blastocoel before vitriﬁcation and slow-rate freezing of bovine blastocysts. Summary answer: Cryopreservation of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production by using embryo transfer technique. What is known already: In general, the slow-rate freezing using a programmed cryo-machine are traditionally employed for the cryopreservation of embryos. Vitriﬁcation with higher concentrations of cryoprotectants and a fast cooling rate, it transforms cells into a glassy state without ice crystal formation. Study design, size, duration: Bovine in vivo and in vitro blastocysts were slowrate frozen and vitriﬁed after forced blastocoele collapse of the blastocoel by puncturing the blastocoele with a pulled pasteur pipet. Participants/materials, setting, methods: The differences in survival and pregnancy rates of blastocysts derived from forced blastocoele collapse and non-forced blastocoele collapse groups were found in both slow-rate freezing and vitriﬁcation. Furthermore, we examined the total cell numbers and apoptosis index of blastocysts. Main results and the role of chance: We found that the total cell numbers of blastocysts in forced blastocoele collapse groups were increased and the numbers of blastomeres undergoing apoptosis in forced blastocoele collapse groups were decreased. Consistent with the results, qPCR data showed that the expression of anti-apoptotic gene (Bcl-xL) was signiﬁcantly increased by forced blastocoele collapse groups, whereas the expression of pro-apoptotic gene (Bax) was signiﬁcantly decreased. Furthermore, pregnancy rates in the both slow-rate frozen and vitriﬁed bovine in vivo blastocysts could be improved by reducing the ﬂuid content after forced blastocoele collapse of the blastocoel. Limitations, reason for caution: However, the low efﬁciency of frozen-thawed embryos survival and further development is a crucial problem. Wider implications of the findings: The forced blastocoele collapse of the blastocoel method suggesting that it could be improving the developmental competence and qualities of frozen-thowed bovine embryos that may be used for mammalian embryo preservation. Study funding/competing interest(s): We have no conpeting interest. Trial registration number: We have no trial registration number. P-163 Effect of Hydroxypropyl cellulose on survival of bovine and human oocytes after vitrification M. Kuwayama1, S. Higo1, and L. Ruvalcaba 2 1 Repro-Support Medical Research Center, Research & Development, Tokyo, Japan, 2Infertility Institute of Mexico, Research & Development, Guadalajara, Mexico Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study design, size, duration: All ART cycles were performed from January 1, 2012 to December 31, 2012. FT were divided into three sub-groups: FT-LR: low-ovarian-responder (344 cycles), FT-NR : normal-ovarian-responder (227-cycles), FT-HR : high-ovarian-responder (104 cycles). Also, FTT were divided into three sub-groups: FTT-LR: low-ovarian-responder (16 cycles), FTT-NR; normal-ovarian-responder (78 cycles), FTT-HR: high-ovarian-responder (104 cycles). Participants/materials, setting, methods: All patients had GnRH-antagonist protocol. The embryos used in FTT were cryopreserved by vitriﬁcation method. We classiﬁed the patients in low, normal and high ovarian responder, according to the number of retrieval oocytes: low-ovarian-responder: from 1 oocyte to 9 oocyte, normal-ovarian-responder: from 10 to 19 oocytes, high-ovarian-responder: over 20. Main results and the role of chance By analyzing the results, we investigated the clinical-pregnancy rate, the implantation rate and the ongoingpregnancy of all groups and compared them. However, we failed to ﬁnd statistically signiﬁcant difference in comparison of FT-LR and FTT-LR [the clinical pregnancy rate: 26.8% vs. 25%, the implantation rate: 9.8% vs. 13.5%, the on-going pregnancy rate: 16.8% vs. 18.8%] or in FT-NR and FTT-NR [the clinical pregnancy rate: 39.6% vs. 38.5%; the implantation rate: 12.4% vs. 15.4%; the on-going pregnancy rate: 28.2% vs. 29.5%.]. However, in comparison with FT-HR and FTT-HR, we could finally find significant difference (FT-HR vs. FTT-HR: the clinical pregnancy rate; 42.3% vs. 50.9%, p < 0.01; the implantation rate: 10.5% vs. 18.9%, p < 0.01, ongoing-pregnancy rate: 25.9% vs. 30.7%, p < 0.05) Limitations, reason for caution There were some variation in this study, including GnRH dosage, the day of antagonist treatment, and classiﬁcation of patients. Wider implications of the findings: The High ovarian responder undergoing GnRH-antigonist treatment can choose the strategy of cryopreservation of all embryos and transferring them subsequently in optimal conditions and this protocol increases the synchrony between embryo and endometrial development, and therefore, improves the ongoing pregnancy rate and outcome of ART cycles Study funding/competing interest(s): none Trial registration number: none i184 P-164 Restoration of developmental competence of oxidatively damaged oocytes by meiotic spindle transfer M. Kobayashi1, T. Takeuchi2, and A. Yoshida1 Kiba Park Clinic, Tokyo, Japan, 2Kyono ART Clinic Takanaw, Tokyo, Japan 1 Study question: Whether induced oxidative damage compromise the maturational and developmental capabilities of maturing oocytes as well as such insult can be reversed by spindle transfer (ST). Summary answer: Oxidative stress impaired meiotic and mitotic development of mammalian oocytes with reduced mitochondrial activity and aberrant spindle formation. Ooplasmic replenishment by STwas able to conquer such injuries on maturing oocytes. What is known already: Oxidative stress during the in vitro maturation (IVM) process compromises the oocyte maturation. In addition, it is suggested that oxidative stress confronted during oocyte maturation might contribute to the age-related oocyte aneuploidy and consequent infertility in the human. However, the ability of ooplasmic replenishment by ST at the metaphase I (MI) stage to treat oxidatively damaged oocytes has not yet been assessed. Study design, size, duration: The spindle morphology, mitochondrial membrane potential (MMP), maturation rate and developmental ability were compared between intact MI oocytes and those treated with H2O2. In order to assess the ability of ST to treat oxidatively damaged oocytes, spindles isolated from oocytes exposed to H2O2 were transferred to intact ooplasts. Participants/materials, setting, methods: Mouse MI oocytes were exposed to 50 mM H2O2 for 15 min. H2O2 treated MI spindle was transferred into an enucleated intact oocyte with inactivated Sendai virus. After IVM, morphology of MII spindle was examined immunohistochemically, MMP was evaluated with JC-1 dye, and matured oocytes were fertilized by ICSI. Main results and the role of chance: Maturation rate of the H2O2 treated MI oocytes was signiﬁcantly lower than the control (51.7% vs. 79.4%, p , 0.05). In oocytes reached MII stage after H2O2 treatment, length of the spindle was remarkably shorter, and MMP was lower than that of simply in vitro matured oocytes. After ICSI, only 34.1% of the H2O2 treated oocytes developed to the 2-cell stage, and none reached to the blastocyst stage. However, following ST, 73.5% of the H2O2 treated oocytes matured normally exhibiting MII spindle with normal morphology. MMP in the ST oocytes was higher than the H2O2 treated oocytes. In addition, all of the ST oocytes were fertilized normally after ICSI, and 20.5% developed to the blastocyst stage. Limitations, reason for caution: This study was conducted using a mouse model with artiﬁcially induced oxidative stress. This ﬁnding does not directly represent human infertility. Wider implications of the findings: This study shows clearly that the oxidative damage induced in cytoplasm/mitochondria of mammalian oocytes impairs their developmental competence. Such ooplasmic damage can be reversed by replenishment of ooplasm, indicating that STcan play an important role in understanding age-related oocyte pathology. Study funding/competing interest(s): There are no conﬂicts of interest to declare. Trial registration number: n/a P-165 Avoiding the occurrence of smooth endoplasmic reticulum clusters in oocytes improves ART outcomes A. Miwa, Y. Nagai, Y. Momma, K. Takahashi, M. Chuko, A. Nagai, and J. Otsuki Nagai Clinic, Obstetrics and Gynecology, Saitama, Japan Study question: Is it possible to prevent the presence of smooth endoplasmic reticulum clusters (sERC) in human oocytes and improve the take home baby rate? Summary answer: In this study, it was found that presence of sERC in oocytes could be avoided in most patients. About 30% of patients who previously had sERC positive oocytes and had failed to become pregnant were able to have healthy babies in their sERC negative cycles. What is known already: The presence of sERC in MII human oocytes indicates cytoplasmic damage. We have previously reported that the presence of sERC decreased pregnancy rates and increased the miscarriage rate (ESHRE 2002, Hum Reprod 2004) and that elevated oestradiol and progesterone concentrations and larger follicles induced sERC formation (ESHRE 2003, Hum Reprod 2004). Study design, size, duration: Consecutive ICSI and combined IVF and ICSI attempts from April 2002 to December 2012 in our clinic are included in this study. Based on our previous ﬁndings three procedures described below were carried out for patients previously positive for sERC. Participants/materials, setting, methods: The following three procedures, i) earlier administration of hCG than in previous sERC positive cycles, ii) alternative ovarian stimulation methods, iii) exclusion of even smaller sized sERC, were carried out and pregnancy outcomes were subsequently investigated. sERC positive oocytes rates were also compared in seven different stimulation procedures. Main results and the role of chance: Unfavorable sERCs were present in 12.1% (68/564) of patients representing 7.3% (86/1174) of cycles. Among the 68 patients who had sERC positive cycles, 14 (20.6%) patients delivered healthy babies. Among the 54 couples who failed to deliver, 46 couples had further ART treatment using the three procedures described above and 41 out of 46 (89.1%) patients were found to have sERC negative cycles. In their sERC negative cycles 12 (29.3%) patients delivered healthy babies. The following are the sERC positive rates for each stimulation protocol: long, 8.9% (31/349); short, 25.0% (2/8); FSH-antagonist, 6.3% (32/512); CC-antagonist, 6.7% (11/164); CC, 6.9% (2/ 29); FSH, 9.1% (1/11) and letrozole-antagonist, 0% (0/29). The short protocol had statistically lower sERC rates than the letrozole-antagonist (p , 0.01) and the FSH-antagonist protocols (p , 0.05). Limitations, reason for caution: Since we previously proposed that the transfer of embryos derived from sERC positive oocytes should be avoided (Hum Reprod 2004), sERC positive oocytes in this study were not fertilized in all sERC positive cycles. Thus, the pregnancy outcomes derived from sERC positive oocytes cannot be investigated in this study. Wider implications of the findings: As reports show that babies derived from sERC positive oocytes had multiple abnormalities, it is important to avoid the occurrence of sERC formation. Since there were no sERC positive oocytes in the Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: Is Hydroxypropyl cellulose in vitriﬁcation solution effective on post-warm survival of bovine oocytes and blastocysts, and human oocytes after vitriﬁcation ? Summary answer: Hydroxypropyl cellulose was very effective as a macromolecule supplement in vitriﬁcation solution on post-warm survival of bovine oocytes and blastocysts, and human oocytes after vitriﬁcation. It is more effective than conventional synthetic serm substitute which has been normaly used in human IVF . What is known already: Supplementation of synthetic macromolecule to the cryopreservation solution is necessary to obtain stable and high post-warm survival for human and animal oocytes and embryos vitriﬁcation. Study design, size, duration: Perspective study. Comparison of in vitro survival and growth of oocytes after treatment. Total number of oocytes and embryos was 874, the duration is 20 months from 04/2011. Participants/materials, setting, methods: Four experiments were conducted to assess the efﬁcacy of HPC for use in vitriﬁcation solutions for bovine oocytes and blastocysts and for human oocytes. Four-hundred and twenty bovine oocytes and 300 blastocysts and 154 human oocytes were vitriﬁed by the Cryotop method (Kuwayama, 2005). The solutions for vitriﬁcation were supplemented with 0.06 mg/ml HPC or with 10% commercial Synthetic Serum Substitute (SSS) or with no added macromolecule (NA). Some HPC solutions and SSS solutions were stored at 48C or -808C for 12months before vitriﬁcation. Main results and the role of chance: The survival rates of bovine oocytes vitriﬁed in HPC, SSS and NAwere 100%, 100% and 86% and the respective blastocyst rates were 22%, 18% and 8%, respectively. The survival rates of bovine blastocysts vitriﬁed in HPC, SSS and NA were 99%, 93% and 88%, respectively.The survival rate of bovine oocytes vitriﬁed in HPC solutions stored for 12 months at 48C or -808C was 100%; the rates for those vitriﬁed in SSS solutions were somewhat lower than HPC. The survival rates of human oocytes vitriﬁed in HPC, SSS and NA were 100%, 98% and 92%, respectively. Limitations, reason for caution: This study is in vitro only. Wider implications of the findings: Found macromolecule hydroxypropyl cellulose may be used effectively for embryo culture media and freezing solutions for sperm. Study funding/competing interest(s): The study was performed by an ordinary research expenses in the clinic Trial registration number: The study does’t contain clinical trials. Abstracts Abstracts letrozole protocol, it could be used as a stimulation method for patients who have recurrent sERC positive cycles. Study funding/competing interest(s): The authors received no funding for this study and there is . Trial registration number: 0022654 P-166 Thawing in the day of embryo transfer had improved pregnancy rate in HRT cycles, but not in the natural cycles S.G. Kim1, J.H. Lee1, Y.Y. Kim1, H.J. Kim1, I.H. Park 1, H.G. Sun1, K.H. Lee1, and H.J. Song2 1 Mamapapa&baby Obstetrics Gynecology Clinic, Infertility Lab., Ulsan city, Korea South 2Bucheon Seoul Women’s Hospital, BuCheon City, Korea South P-167 Single medium culture in a time-lapse incubator until the blastocyst stage with or without medium renewal on Day-3: a prospective randomised study with donor oocytes N. Costa-Borges1, M. Belles1, J. Herreros1, J. Teruel1, A. Ballesteros2, A. Pellicer3, and G. Calderón1 1 IVI Barcelona, IVF Laboratory, Barcelona, Spain, 2IVI Barcelona, Clinical Department, Barcelona, Spain, 3IVI, Clinical Department, Valencia, Spain Study question: Is it really necessary to renew the medium on Day-3 when using a single medium for embryo culture to the blastocyst stage? Summary answer: Our preliminary data suggest that embryo quality, morphokinetics, and clinical performance are not affected by single-step (uninterrupted) embryo culture in a single medium. The same medium can be used throughout the 5 days of culture with no replacement on Day-3. Moreover, this strategy does not affect embryo kinetics. What is known already: Embryo culture to blastocyst stage can be accomplished using either a single medium or sequential media. Traditionally, culture in a single medium includes medium renewal on Day-3. There are insufﬁcient data to conclude that this step can be avoided without affecting embryo quality and clinical performance. In this study, we wanted to clarify this question and evaluate whether embryo culture until blastocyst stage in single medium can be performed safely without medium replacement on Day-3. Study design, size, duration: We compared embryo development and quality, morphokinetics, and clinical outcomes (implantation and clinical and ongoing pregnancy rates) between single-step culture (without renewal) and two-step culture (Day 3 renewal) culture. This prospective, randomised study comprised 302 embryos (from donor oocytes) from 29 patients. ICSI was performed on all oocytes. Participants/materials, setting, methods: Injected oocytes were evenly distributed in EmbryoSlidesw of a time-lapse incubator (EmbryoScope TM, Unisense, FertiliTech). Slides were prepared with Global Totalw medium (LifeGlobal), equilibrated overnight, and randomised for medium renewal or no renewal on Day-3. Results were compared by Student’s t-test or X-test, P , 0.05 was considered statistically signiﬁcant. Main results and the role of chance Blastocyst rates (67.5 vs 64.9%, P ¼ 0.751), percentages of frozen (49.0 vs 58.2%, p ¼ 0.248) and discarded embryos (22.5 vs 25.5%, P ¼ 0.745) were similar between two-step and singlestep culture groups. The proportion of good-quality blastocysts (transferred plus frozen) was also identical for both groups (77.5 vs 74.5%, P ¼ 0.745). Morphokinetic variables from early cleavage until late blastocyst stage were not different between culture treatment groups. No differences (P . 0.05) were found in clinical and ongoing pregnancy rates between transfers performed with embryos from two-step culture (69.2 and 77.8%), single-step culture (75.0 and 100%) and mixed transfers (42.9 and 66.7%). A total of 24 of 45 transferred embryos implanted and no signiﬁcant differences were found among pure and mixed transfers. Limitations, reason for caution: These are preliminary results of an ongoing study and a larger sample size is required to increase statistical power. Results are based on observations with embryos from oocyte donors and need to be conﬁrmed with embryos from infertile patients of different ages. Wider implications of the findings: Simpliﬁed laboratory protocols, lower costs and fewer human errors are associated with the use of a single step culture system without medium renewal on Day-3. The absence of differences in morphokinetics between both groups demonstrates that medium renewal does not affect timings of embryo development events. Study funding/competing interest(s): This study was funded by IVI-Barcelona. The authors do not have any P-168 Regulation of inositol 1,4,5-trisphosphate receptor during human oocyte maturation and fertilization D. Nikiforaki1, L. Vossaert2, F. Vanden Meerschaut1, C. Qian 1, Y. Lu1, J.B. Parys3, W.H. De Vos4, D. Deforce2, T. Deroo 1, E. Van den Abbeel1, L. Leybaert5, B. Heindryckx1, and P. De Sutter 1 1 University Hospital Ghent, Department for Reproductive Medicine, Ghent, Belgium, 2Ghent University, Laboratory for Pharmaceutical Biotechnology, Ghent, Belgium, 3KU Leuven, Department of Cellular and Molecular Medicine, Leuven, Belgium, 4Ghent University, Department of Molecular Biotechnology, Ghent, Belgium, 5Ghent University, Department of Basic Medical Sciences, Ghent, Belgium Study question: What are the modiﬁcations occurring to the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) during human oocyte maturation that enhance its sensitivity for intracellular Ca2+ release during fertilization? Summary answer: During human oocyte maturation, the IP3R1 level increases with a concomitant rise of its phosphorylation. Simultaneously, the IP3R1, which is located in the endoplasmic reticulum (ER), relocalizes and forms dense clusters that decrease after fertilization. The amount of Ca2+ stored in the ER ([Ca2+]ER) increases signiﬁcantly during in vitro maturation (IVM). What is known already: Intracellular [Ca2+] oscillations mediated by the IP3R1 regulate oocyte activation and embryo development. Prior to fertilization, the Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: What is the optimal thawing time in day 3 frozen-thawed embryo transfer(FET) during natural and hormone replacement therapy(HRT) cycles? Summary answer: Immediate embryo transfer after thawing in the morning had improved pregnancy rate compared with thawing in the evening 1 day before embryo transfer in HRT cycles. What is known already: Embryo-endometrial synchronization seems to be improved by control of thawing day in FET cycles. However, the optimal timing for thawing still remains uncertain. Study design, size, duration: In a retrospective study, the relationship between thawing time and pregnancy rate has been studied in 678 FET cycles between Jan 2008 and Dec 2011. FET cycles were divided into four groups according to thawing time and endometrium preparation methods. Participants/materials, setting, methods: Embryos which were thawed evening 1day before ET were cultured overnight and transferred the next day morning (group A: in natural cycle(n ¼ 160), group B: in HRT cycle (n ¼ 277)), while embryos from thawed in the morning were transferred within 3h (group C: natural cycle (n ¼ 138), group D: in HRT cycle (n ¼ 103)). Main results and the role of chance: There were no differences among four groups in patient age(group A: 34.7 + 4.0, group B: 34.7 + 4.5, group C: 33.9 + 3.5 and group D: 34.2 + 4.2), the mean number of transferred embryos(2.2 + 0.4, 2.1 + 0.4, 2.5 + 0.5 and 2.1 + 0.3), the mean number of cryopreserved embryos(9.4 + 4.8, 10.9 + 6.2, 10.9 + 5.9 and 11.3 + 5.6), endometrial thickness(10.4 + 1.9, 10.3 + 2.0, 9.9 + 1.5 and 10.1 + 1.6) and embryo quality. The mean embryo survival index before transfer was comparable in the four groups(72.4%, 71.7%, 70.6% and 71.4% p ¼ 0.30). The clinical pregnancy rates in natural cycles were no signiﬁcant difference between group A(28.1%) and group C(37.7%, p ¼ 0.19). However, the clinical pregnancy rates in HRT cycles were signiﬁcantly higher in group D(50.5%) than group B(31.0%, p , 0.01). Limitations, reason for caution: The number of cycles between each group was different. In particular, the number of HRT cycles was small. Wider implications of the findings: In our results, the overall pregnancy rate in FET cycles had improved in immediate embryo transfer after thawing in the morning. Especially, pregnancy rate was signiﬁcantly higher in the HRT cycles. We do not know the exact reason for the high pregnancy rate in the HRT cycles. Probably, it seems that better embryo-endometrial synchronization was achieved in HRT cycles. Study funding/competing interest(s): None Trial registration number: None i185 i186 multivesicular bodies, and are frequently fused with the neighboring cells. Various cell types are carrying exosomes. To date, their characterization, based on morphology and, more importantly, biochemical and functional activity has not been provided. Study design, size, duration: Total of 35 embryos, having 6C to 8C, with various degrees of fragmentation, are stopped at Day 3 and examined. Overall cohort of embryos were developed under standardized IVF clinical programme and donated for science with written informed consent provided from couples. Participants/materials, setting, methods: Using transmission electron microscopy (TEM) on human preimplantation embryos, we observe presence, distribution and ultrastructural details of various membranous extracellular structures. A prospective observational ongoing study was initiated at ObGin Clinic - Clinical Centre of Serbia and Faculty of Biology – Belgrade University. Main results and the role of chance: The blastomeres certainly do shed exosomes and show their agglomerations in sub-plasmalemal area. Released exosomes are observed in inter-blastomeric space and not clearly conﬁrmed in perivitelline space. Release zone was characterized with sER vesicles and mitochondria presence. TEM shows characteristic cup-shaped exosomes with their mean size of 165 nm + 0.5 nm. Standard bell-shaped curve indicates homogeneous population. We observed exosome production in A-class embryos and slightly negative correlation between their number and embryo quality. Presence of various multivesicular bodies and structures resembling apoptotic bodies increases with lower embryo score. There are clear signs of exosome uptake by blastomeres, and it correlates with fragmentation degree. Fragments showed retained capability to endocytose exosomes as well. Rather excessive exosome uptake was found in embryos showing apoptotic blastomeres. Limitations, reason for caution: Embryo-derived exosomes are mostly located in the central inter-blastomere environment. Their position, small quantity and highly dynamic uptake make their isolation, puriﬁcation and content depiction very challenging, when compared with other cell systems. Revealing their spatial dynamics is the ﬁrst step for their future molecular and physiological mapping. Wider implications of the findings: To our knowledge, this is the ﬁrst report that embryos release exosomes. Studies on various cell types showed immunomodulatory, antigen and transcriptomic activity. Excessive exosome uptake by dying blastomeres suggests their involvement in activation-induced cell death. Their presence in good embryos implies their role in normogenesis and deserves great attention. In future, improved puriﬁcation and nomenclature strategies will help to illuminate their place in complex and fragile biological system - early human embryo. Study funding/competing interest(s): Study has institutional review board approval and no conﬂicts of interest exist. Trial registration number: This work was supported by Serbian Ministry of Education, Science and Technological Development, Grant #173054. P-169 Exosomes as bioactive messengers in a critical time for preimplantation embryos ñ an ultrastructural study L. Surlan1, V. Otasevic2, K. Velickovic3, I. Golic3, M. Vucetic3, V. Stankovic 1, J. Stojnic4, N. Radunovic4, I. Tulic4, B. Korac 2, and A. Korac3 1 Clinic for Obstetrics and Gynaecology Clinical Centre of Serbia, ART Department, Belgrade, Serbia, 2University of Belgrade Institute for Biological Research “Sinisa Stankovic”, Physiology Department, Belgrade, Serbia, 3 University of Belgrade Faculty of Biology, Cell Biology, Belgrade, Serbia, 4 Clinic for Obstetrics and Gynaecology Clinical Centre of Serbia, Faculty of Medicine, Belgrade, Serbia Study question: Among the embryo fragments and blastomeres, various populations of smaller but distinctive membranous extracellular organelles: exosomes, shedding microvesicles and apoptotic blebs deserve particular attention. The goal of this study was to provide an ultrastructural characterization of blastomerederived exosomes in a critical period of embryo blastulation and fragmentation. Summary answer: Based on morphology, size and origin we deﬁned the release of exosomes from both intact and fragmented blastomeres, their uptake by fragments and other blastomeres, and we provide their ﬁrst extensive characterization in human IVF embryos. What is known already: IVF embryos show rather crafty developmental capacity and various phenotype markers. Recent works conﬁrmed exosomes as important intercellular communication mediators – they carry proteins, miRNAs and menu of other molecules. They are formed by the inward budding and releasing of P-170 Prospective randomized study comparing human embryo culture in group in the Well-of-the-Well dish or individually in droplets. Results from an intermediate analysis P. Fancsovits 1, C. Pribenszky 2, A. Lehner1, A. Murber1, J. Rigo Jr.1, and J. Urbancsek1 1 Semmelweis University, Division of Assisted Reproduction First Department of Obstetrics and Gynaecology, Budapest, Hungary, 2Szent István University, Department of Animal Breeding and Genetics Faculty of Veterinary Sciences, Budapest, Hungary Study question: The aim of this study is to compare embryo development and pregnancy rate in human in vitro fertilization treatments when embryos are cultured individually or in group culture in Well-of-the-Well (WOW) Petri dish (Primo Dish, Vitrolife Hungary). Summary answer: Group culture in WOW Petri dish results in a higher cell number and better embryo morphology than individual culture on Day 3 of embryo development. A non-signiﬁcant increased clinical pregnancy rate is observed after group culture. What is known already: Embryos in human IVF treatment are frequently cultured individually in microdrops allowing individual assessment and tracing of embryo quality. Culturing more than one embryo in the same microdrop may result in accumulation of autocrine and paracrine factors in culture media Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 oocyte’s Ca2+ machinery is optimized. In animal models, multiple IP3R1 modiﬁcations occur during maturation from the germinal vesicle (GV) to metaphase II (MII) stage, affecting its sensitivity and ensuring that the optimum Ca2+ response concurs with fertilization. These IP3R1 modiﬁcations include its redistribution, elevated level, enhanced Ca2+-releasing ability and relative IP3R1 phosphorylation (p-IP3R1) potentially by M-phase kinases. Study design, size, duration: Immature, in vivo-matured and failed-fertilized oocytes were obtained with informed consent (2010/182) from 98 women (32.2 + 4.6 years old) undergoing infertility treatment. Fifty oocytes were used for immunoblotting to assess the p-IP3R1 status, 35 for [Ca2+]ER evaluation and 37 for IP3R1 immunostaining and confocal microscopy. Participants/materials, setting, methods: GV and MI oocytes were matured in the presence of inhibitors of Polo-like kinase 1 (Plk1) (BI 2536) and cyclindependent kinase 1(CDK1) (roscovitine, RO-3306) respectively. MPM-2 and Rbt03-IP3R1 antibodies were used for immunoblotting and CT1-IP3R1 for immunostaining. [Ca2+]ER was assessed after thapsigargin treatment in Ca2+-free medium. Results are expressed as median (interquartile range). Main results and the role of chance: Immunoblotting analysis indicated a 2.2-fold maturation-associated increase in IP3R1 level between the GV and MII stage. The p-IP3R1 increased by 75% from the GV to GV breakdown (GVBD) stage and reached a maximum level in MII oocytes. Speciﬁc inhibition of Plk1 (until GVBD stage) and CDK1 (until MII stage) decreased the p-IP3R1 by 30% and 50% respectively. During maturation, IP3R1 redistributed from a diffuse pattern to an increasingly localized pattern in both in vitro- and in vivo-matured oocytes with prominent clusters that decreased in size after fertilization and in vitro aging. [Ca2+]ER increased from 0.4 (0.3-0.5) AU at the GV stage to 1 (0.8-1.4) AU at the GVBD, 1.4 (1.2-1.7) AU at the MI and 1.8 (1.7-2.1) AU at the MII stage (P , 0.05). Limitations, reason for caution: It is currently unclear which is the critical IP3R1 modiﬁcation underlying its increased sensitivity. Further analysis is required to clarify the effect of M-phase kinases on IP3R1 phosphorylation and IP3R1 Ca2+releasing ability in human oocytes. Wider implications of the findings: This is the ﬁrst report showing that various components of cytoplasmic maturation can render human oocytes competent to initiate [Ca2+] oscillations at fertilization. These not only include the increase in [Ca2+]ER and IP3R1 redistribution but also the rise in IP3R1 level and phosphorylation, which in animal models lead to increased sensitivity for Ca2+ release. Any perturbations of these processes could potentially hinder normal human fertilization and embryo development. Study funding/competing interest(s): This work was supported by a Ghent University grant to B.H. The authors have no competing interests to declare. Trial registration number: Not applicable. Abstracts Abstracts P-171 Origin and role of transient DNA strand breaks during spermiogenesis R. Elias 1, Q.V. Neri1, T. Fields1, P.N. Schlegel2, Z. Rosenwaks1, and G.D. Palermo 1 1 Weill Cornell Medical College, Reproductive Medicine, New York, U.S.A, 2Weill Cornell Medical College, Urology Department, New York, U.S.A Study question: To assess the etiology, localization, and meaning of DNA strand breaks occurring during sperm production versus those generated in the male genital tract. Summary answer: From this preliminary data, it appears that the origin of DNA fragmentation in these infertile men is caused by a post-spermatogenic insult. Therefore, the utilization of testicular specimen may enhance pregnancy outcome. What is known already: Tests for sperm DNA integrity have been proposed to assess male gamete competence. They are currently gaining popularity and are more often used as a supplemental assay to traditional semen analysis. Sperm DNA nicks and breaks are considered the culprits for the impaired reproductive capacity in natural and assisted reproduction, however, it remains to be elucidated when and at what level during sperm production the mishap occurs. Study design, size, duration: In patients with recurrent ICSI failure in ejaculated spermatozoa, DNA fragmentation was assessed. To determine whether a testicular biopsy would yield spermatozoa with a healthier chromatin, men underwent ICSI with this specimen. Embryo developmental capacity and pregnancy outcome were compared among the two semen source. Participants/materials, setting, methods: We identiﬁed infertile men (n ¼ 9) with high DNA fragmentation in their ejaculates that agreed to undergo testicular biopsy for this indication. Chromatin fragmentation index was assessed by SCSA or TUNEL on both types of sperm sources. Reproductive outcomes of ICSI cycles utilizing the two sperm sources were analyzed and compared. Main results and the role of chance: In 9 men with recurrent ICSI failure (3.2 cycles) their sperm yielded 60.9 + 29% fragmentation (up to 96%). After counseling, eight underwent TESE with 6.5 + 5% DFI. In a paired analysis the ejaculated cycle closest to the TESE cycle, gave a fertilization of 55.9% and 50.0%, respectively. The embryo cleavage was lower in the ejaculate at 63.6% with a pregnancy rate of 12.5%, while in the TESE cohort all embryos cleaved resulting in a 25.0% clinical pregnancy. When the overall ejaculated cycles (n ¼ 28) and TESE (n ¼ 13) were analyzed, fertilization was higher in the former (60.0 vs 46.9%;P , 0.01) with a comparable embryo cleavage rate. Following the replacement of an average of 2.8 conceptuses, the clinical pregnancy was 10.7% (3/28) in the ejaculate and 30.8% (4/13) for the TESE. Limitations, reason for caution: Patients need to be informed of the risks with regards to the surgery, anesthesia, and the possibility that even with TESE a pregnancy may not occur. Therefore, appropriate counseling should be conducted since these men have spermatozoa in their ejaculate. Wider implications of the findings: When couples have recurrent pregnancy failures after ICSI, associated with a high sperm DNA fragmentation, they should be offered the option to undergo testicular biopsy. Study funding/competing interest(s): Institutional Trial registration number: Not applicable P-172 Clinical outcomes following the transfer of vitrified blastocysts derived from a single or sequential culture medium A. Gilson, N. Piront, B. Heens, C. Vastersaegher, A. Vansteenbrugge, and P.C.P. Pauwels Centre Hospitalier Régional de Namur, Procréation Médicalement Assistée, Namur, Belgium Study question: The purpose of this study was to examine the effect of single versus sequential culture medium on the survival of post-thawed blastocysts and their related chances of implantation and pregnancy rates. Summary answer: No statistical differences in terms of survival, implantation and pregnancy rates of post-thawed blastocysts were observed between the two culture media studied. What is known already: Both single and sequential culture media contribute to the development of zygote to the blastocyst stage. A large number of clinical studies have shown that blastocyst transfer results in higher implantation and pregnancy rates than cleavage-stage embryo transfer. Obtaining good quality blastocysts before a vitriﬁcation procedure is a crucial step in predicting successful pregnancy rates. However, the impact of culture medium on blastocyst cryotolerance remains poorly studied. Study design, size, duration: The current study includes a total of 177 warming cycles of blastocysts from infertile women with a mean age of 32. Among these, 101 were derived from culture in the single medium and 76 from culture in the sequential medium. Data were retrospectively analysed for blastocyst survival, implantation and pregnancy rates. Chi-square test was used for statistical analysis. Participants/materials, setting, methods: Embryos arising from IVF cycles were randomly cultured in four-well dishes containing either Global (single) or Sage (sequential) medium covered with oil. Surplus embryos meeting our laboratory standards for cryopreservation were vitriﬁed in closed condition. Following warming, blastocysts were incubated for 3 hours prior to their transfer. Morphological assessment was used to evaluate embryo viability/survival before and after vitriﬁcation. Only blastocysts that show sign of reexpansion and with a cell viability above 50% were considered for transfer. Main results and the role of chance: There were 142 thawed embryos vitriﬁed after a 5 day culture in Global medium and 96 thawed embryos vitriﬁed after a 5 days culture in Sage medium. The survival of post-warmed blastocysts was similar for both Global and Sage culture medium (78.9% vs. 82.3% respectively). Similarly, no signiﬁcant differences were observed between the two culture medium studied with respect to implantation rate (35.7% vs. 43.0%), clinical pregnancy rate (38.4% vs. 46.6%) and ongoing pregnancy rate (32.6% vs. 34.6%). Limitations, reason for caution: N/A Wider implications of the findings: This study shows that the use of single or sequential culture medium does not affect the recovery of vitriﬁed blastocysts and that both media ensure similar chances of implantation and pregnancy following frozen blastocyst transfer. Study funding/competing interest(s): N/A Trial registration number: N/A Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 having a beneﬁcial effect on embryo development. The WOW dish contains 9 + 1 microwells in the center of the dish allowing identiﬁcation of individual embryos cultured together in the same microdrop. Study design, size, duration: One hundred nineteen consecutive IVF-ET cycles were enrolled in this prospective randomized study between September and December 2012. Cycles were randomized using a computer generated table. Five hundred thirty-ﬁve embryos from 57 cycles were cultured in WOW dish and 581 embryos from 62 cycles were cultured individually (Control group). Participants/materials, setting, methods: IVF cycles were randomized into WOW group or individual culture. In WOW group up to 10 embryos were cultured together in 25 ml culture media. In Control group embryos were placed into a 25 ml droplet individually. Pregnancy rate and embryo morphology were compared. Main results and the role of chance: Fertilization rate in ICSI cycles -where oocytes were placed into culture dish immediately after sperm injection- were signiﬁcantly higher in WOW than in Control group (74.5% vs. 66.7 %, P ¼ 0.022). Number of blastomeres was similar in the two groups on Day 2. However, degree of fragmentation was signiﬁcantly lower in WOW than in Control group (12.7 + 8.4% vs. 15.0 + 11.8; P ¼ 0.005). We observed a higher number of blastomeres (7.0 + 2.0 vs. 6.5 + 2.1; P ¼ 0.002) and a lower degree of fragmentation (13.5 + 10.6% vs. 15.5 + 12.8; P ¼ 0.045) in WOW group on day 3. A higher proportion of embryos were available for cryopreservation in WOW group (41.3% vs. 32.9%; P ¼ 0.024). Clinical pregnancy rate was 50.9% in WOW and 38.7% in Control group but the difference did not reach signiﬁcance (P ¼ 0.182). Limitations, reason for caution: These are preliminary data of a larger study. There is more than 10% difference in clinical pregnancy rate which is not signiﬁcant due to limited case numbers. Wider implications of the findings: Our intermediate ﬁndings suggest that using WOW group culture dish may have a beneﬁcial effect on IVF outcome. This is in agreement with previous ﬁndings on the superiority of group culture, however, WOW system allows for individual assessment and tracing of individual embryo quality. A larger study is currently ongoing. Study funding/competing interest(s): Trial registration number: ClinicalTrials.gov: NCT01774006 i187 i188 P-173 Embryo culture under two different incubator temperature: a double blind randomized clinical trial M.F. Abdel-Raheem1, M.Y. Abdel-Rahman2, H.M. Abdel-Gaffar2, M. Sabry2, H. Kasem3, S.M. Rasheed 2, M. Amin2, A. Abdelmonem2, and A.S. Ait-Allah2 1 IBN-SINA IVF/ICSI Center, IVF Lab, Sohag, Egypt, 2Sohag University, OB/ GYN, Sohag, Egypt, 3Akhmim General Hospital, OB/GYN, Sohag, Egypt P-174 An uninterrupted culture medium protocol in conjunction with a non-humidified incubation environment can provide more patient treatment options for IVF programs M. VerMilyea, J. Anthony, J. Bucci, S. Croly, and C. Coutifaris University of Pennsylvania, Penn Fertility Care, Philadelphia, U.S.A Study question: The purpose of this study was to investigate the effects of embryo development and alternative patient treatment options when using a renewed two-step sequential media protocol and traditional “big-box” incubators compared to a continuous uninterrupted single-medium protocol and benchtop incubators with no humidity. Summary answer: Embryos grown in a non-invasive single culture medium under oil and retained in humidity-free benchtop incubators are three-times more likely to become usable blastocysts thereby doubling the number of day-5 embryo transfers and providing supernumerary blastocysts for vitriﬁcation for use in subsequent frozen embryo transfer cycles. What is known already: Culture media is based on either a sequential (two/threestep) media protocol, which requires the use of one media followed by a different media, or a single step culture medium. Previous reports have often suggested that a sequential series of media are necessary to accommodate changes in the physiology and metabolism of embryos but recent studies have shown comparable pregnancy outcomes but improved blastocyst development when embryos are cultured in an uninterrupted media and humid environment. Study design, size, duration: This retrospective study consisted of data from 956 zygotes, which resulted in 179 embryo transfers over the course of nine months. 304 embryos were cultured in a sequential culture media system in standard size incubators while 652 embryos were place in a single-step medium in nonhumidiﬁed benchtop incubators. Participants/materials, setting, methods: Irvine Scientiﬁc’s Continuous Single Culture (CSC) was compared with LifeGlobal’s Fertilization and Global sequential renewed-medium with respect to human embryo development, blastocyst utilization rates and patient treatment options. Embryos cultured in CSC and K-Systems G-185 incubator were compared to GF and G media in Forma 3130 incubators. Main results and the role of chance: Overall, signiﬁcantly more embryos cultured in CSC and non-humidiﬁed benchtop incubators were transferred on day-5, compared to those cultured in Global sequential medium in humidity controlled “big-box” incubators (57% vs. 29%, P ¼ 0.0013). Autologous patients in different age groups (,35, 35-37, 38-40, .40) had more blastocyst transfers as a result of CSC compared to Global (74% vs. 47%, 60% vs. 28%, 27% vs. 21%, 33% vs. 0%, respectively). In addition, surplus blastocysts available for cryopreservation, which were cultured in CSC compared to LifeGlobal medium, were signiﬁcantly greater (30% vs. 6%, P ¼ 0.0001). Ultimately, patients across all age groups had a signiﬁcantly higher blastocyst utilization rate when cultured in a noninvasive one-step embryo culture protocol (45% vs. 16%, P ¼ 0.0001 respectively). Data were analyzed by Chi-square analysis or Fisher’s exact test with P ≤ 0.005 regarded as statically signiﬁcant. Limitations, reason for caution: Although the data set is not extremely large, this observation clearly shows an upward trend supporting the beneﬁt of a noninvasive culture system, which incorporates an uninterrupted single culture medium (from day-0 to -6) and benchtop incubators. Further investigation into pregnancy outcomes will also soon be reviewed. Wider implications of the findings: The interest in single step culture medium has grown due to several practical and theoretical advantages over a sequential media protocol. A non-invasive static approach to embryo culture, where unwarranted embryonic stresses from varying composition of media, potential osmotic shock and deprivation of any paracrine or autocrine factors has recently been shown to be beneﬁcial for embryo development. Practical advantages of using a single step protocol include a substantial reduction in labor, error and cost. Study funding/competing interest(s): None Trial registration number: None P-175 Efficiency of biopsy strategy and vitrification procedure in PGS cycles R. Maggiulli1, L. Rienzi1, D. Cimadomo1, A. Capalbo1, L. Dusi2, S. Colamaria1, E. Baroni1, M. Giuliani1, A. Vaiarelli1, F. Sapienza1, L. Buffo2, and F.M. Ubaldi1 1 Genera Center for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy, 2 Genera Center for Reproductive Medicine, Clinica Salus, Marostica, Italy Study question: We aim to evaluate the efﬁciency of biopsy strategy and vitriﬁcation procedure, assessed as impact on implantation potential of euploid embryos and on laboratory workload, in PGS cycles. Summary answer: Embryo implantation potential of euploid embryos seems not to be affected by the biopsy day and/or the vitriﬁcation procedure at blastocyst stage. Laboratory workload is higher for day 3 biopsy, without any advantage on the clinical outcome. What is known already: The prevalence of aneuploidy in human embryos provides a likely explanation for the relatively low success observed during IVF cycles, especially in advanced maternal age patients. Unfortunately, a poor correlation between conventional embryo morphological evaluation and chromosomal complement has been shown. This observation led to the introduction of PGS to avoid the transfer of aneuploid embryos. However, there is not yet a clear consensus about the impact of embryo biopsy strategy on implantation potential. Study design, size, duration: We compared day 3 biopsy (of one blastomere) and transfer on day 5 (strategy 1) (36 cycles); day 5 biopsy (of 5-10 trofoectoderm cells) and transfer on day 6 (strategy 2) (11 cycles) and day 5 biopsy, vitriﬁcation and transfer in a subsequent prepared cycle (strategy 3) (10 cycles). Participants/materials, setting, methods: The patients enrolled in this study underwent ICSI/PGS cycles due to advanced maternal age (N ¼ 57). The mean female age was 38.1 + 2,9, 37,5 + 3,4 and 37,9 + 3,5 years for strategy 1, 2, and 3 respectively. No differences were observed in the basic patients characteristics between the 3 groups. Aneuploidies were assessed by whole genome Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: Does embryo culture at 36.5 degree Celsius result in better pregnancy rate compared with embryo culture at the traditional 37 degree Celsius incubator temperature? Summary answer: There is a statistically signiﬁcant increase (*P ¼ 0.0196) in the pregnancy rate at the group cultured at incubators with lower temperature 36.5 degree Celsius (65.79%) compared to the pregnancy rate at the group cultured at incubators with traditional 37 degree Celsius (44.44%). What is known already: To date all IVF/ICSI procedures are conducted at 37 degree Celsius, yet little studies had discussed the effect of temperature on development of embryos. It was principally accepted that 37 degree Celsius is the best temperature for all aspects of human in vitro procedure. The ‘quiet embryo hypothesis’ proposed that viable embryos have a ‘quiet’ metabolism. Others proposed that gametes and early embryos function in vivo at a lower temperature than core body temperature. Study design, size, duration: This is a double blind randomized controlled clinical trial conducted at a private IVF/ICSI center comprising 130 participant at the period between October 2012 to January 2012. Participants/materials, setting, methods: One hundred thirty women undergoing fresh IVF/ICSI cycles in a private center were randomly assigned either to 36.5 degree Celsius incubator temperature (Group I, N¼ 76) or 37 degree Celsius incubator temperature (Group II, N¼ 54). Main results and the role of chance: There was a clear statistical signiﬁcant increase (*P ¼ 0.0196) in the pregnancy rate at (group I) which cultured at incubators with lower temperature 36.5 degree Celsius 50/76 (65.79%) compared to the pregnancy rate at (group II) which cultured at incubators with traditional 37 degree Celsius 30/54 (44.44%). Limitations, reason for caution: Lack of clear molecular background that can explain the interesting increase of the pregnancy rate in the group cultured at lower temperature. Wider implications of the findings: To change the traditional incubator temperature to 36.5 degree Celsius. Study funding/competing interest(s): There was no study funding or competing interests. Trial registration number: ClinicalTrials.gov identiﬁer: NCT01706900 Abstracts i189 Abstracts P-176 Use of culture media amino acid mass spectrometry/liquid chromotography (LCMS) measurments in identifing the embryo with the best implantation potential E. Zivi1, E. Aizenman 1, D. Barash2, D. Gibson2, and Y. Shufaro 1 Hadassah Ein Kerem, IVF unit, Jerusalem, Israel, 2Hebrew university, Institute for Drug Research School of Pharmacy, Jerusalem, Israel 1 Study question: Is there an assocoation between culture media amino acids’ (serine, proline, taurine, aspartic acid) alterations, as detected in mass spectrometry (MS), with reproductive outcome? Summary answer: Serine and proline excretion into the fertilization media was found to be in association with pregnancy. What is known already: Several studies investigated the correlation between pyruvate and glucose uptake with embryo viability and pregnancy. Others found that an increased uptake is correlative with embryo development and implantation. Amino-acids metabolism has also been investigated. Studies have found an association between low concentrations of glycine and leucine and high concentration of aspargine in the culture media, with high pregnancy rate. High glutamate levels also correlate with pregnancy and live birth. Study design, size, duration: The study is observational.45 IVF patients of various ages, infertility etiologies and different embryo qualities undergoing treatment, are included. Exclusion criteria: patients with a signiﬁcant male factor abnormality, structural anomaly of the uterus, endometriosis and pregestational diagnosis embryos. Spent culture media samples are collected and analyzed on MS. Participants/materials, setting, methods: Day 1 and day 3 samples of spent media are collected from each embryo, and analyzed on MS, in comparison to control empty medium droplets incubated identically. The samples preparation includes adding distilled water and hexane in order to remove the oil phase that coats the incubated embryos. Main results and the role of chance: Spent medium samples from each oocyte/ embryo were obtained in 45 IVF cycles, performed in different patients, were analyzed by LCMS. 22 patients conceived after fresh embryo transfer (all singletons). Day 1 spent fertilization media of 219 oocytes was analyzed, of which 84 embryos were transferred fresh. On day 1, excretion of serine and / or proline were found highly predictive for pregnancy; for proline excretion the PR’s were 40% compared to 18.5% (OR2.9, 95% CI 1.1-8, p ¼ 0.04). For serine excretion the PR’s were 50% compared to 17% (OR 5, 95% CI 1.75-14.28, p ¼ 0.003). No signiﬁcant differences in PRs were found in regard to aspartic acid and taurine excretion or uptake. The results of day 3 embryo culture media are currently analyzed. Limitations, reason for caution: Thechnical limitations due to difﬁculties in sample preparations regarded to the oil phase in the incubation. Wider implications of the findings: Serine and proline excretion into the fertilization media was found to be in association with pregnancy. The biological signiﬁcance of this observation is that an embryo’s quality and implantation potential can be determined even immediately after fertilization. Accurate measurements of media minute amino acid concentration changes are feasible even at this early stage. Study funding/competing interest(s): research fund of the hospital. there is no competing interests. Trial registration number: the study is observational - not a clinical trial P-177 Pronuclear score does not predict embryo implantaion. Retrospective study on different checkpoint times M. Pérez 1, J. Aguilar 1, E. Táboas1, M. Ojeda1, L. Suárez 1, and E. Muñoz2 IVI VIGO, IVF Lab, Vigo (Pontevedra), Spain, 2IVI VIGO, IVF Unit, Vigo (Pontevedra), Spain 1 Study question: Can pronuclear score predict the embryo implantation? Summary answer: The aim of this study is to compare the pronuclear score (Z-score) at 17 hours post-insemination and at the frame before both pronucleus (PN) disappear to correlate them with implantation rate by using a time-lapse system. What is known already: A major objective of IVF laboratories is the identiﬁcation and selection of embryos with the highest implantation potential after transfer, avoiding multiple pregnancies. Pronuclear evaluation under light microscopy has been employed as a determining method for embryo fertilization assessment, and also for embryo selection. Previous studies endorse the prognostic effect of pronuclear evaluation for embryo viability and chromosomal abnormalities, while other authors have not been able to ﬁnd any correlation. Study design, size, duration: Retrospective study of 2697 microinyected oocytes from 420 ICSI patients between January 2011 and November 2012, all of them incubated in an incubator with time-lapse technology. A x2-test was performed as the statistical analysis to assess the inﬂuence of the multinucleation in the implantation rate, and Yates correction when applicable. Participants/materials, setting, methods: The embryos which either failed to implant or fully implanted, with full implantation meaning that all transferred embryos in a treatment implanted, were deﬁned as KID (Known Implantation Data) positive (+) and negative (-) respectively. Only they were included in the study. The distribution of nuclear precursor bodies (NPB) is classiﬁed into four groups given Z-scores 1, 2, 3 and 4. Score group 1 includes zygotes with three to seven polarized NPB; score group 2 consists of zygotes with homogeneously dispersed NPB; score group 3 covers all the zygotes with a NPB distribution not included in the other three groups, i.e., more than seven polarized NPB, one PN polarized and the other dispersed or both dispersed but not homogeneously; and score group 4 is formed of zygotes with only one or two NPB as described by Gamiz et al (2003). Z-score was registered at 17 hours post-insemination and the frame before the pronuclear disappearance (BPD). Embryo transfer was performed on day 2-3 of development and embryo implantation was conﬁrmed at an ultrasound scanning for gestational sacs. Main results and the role of chance: 268 KID embryos wereanalyzed. 33.5% (n ¼ 90) KID + , and 66.5% (n ¼ 178) KID-6. At 17h post-insemination,the Z-score was Z1 ¼ 10% (n ¼ 27); Z2 ¼ 7.8 (n ¼ 21); Z3 ¼ 15,6% (n ¼ 42) and Z4 ¼ 0% for theKID+ embryos, and Z1 ¼ 17.1% (n ¼ 46); Z2 ¼ 14.2% (n ¼ 38); Z3 ¼ 32.8% (n ¼ 88); andZ4 ¼ 2.2% (n ¼ 6) for the KID- embryos. At BPF, the Z-score was Z1 ¼ 14.9% (n ¼ 40);Z2 ¼ 3.7% (n ¼ 10); Z3 ¼ 13.8% (n ¼ 37) and Z4 ¼ 1,1% (n ¼ 3) for the KID + , and Z1 ¼ 23.13%(n ¼ 62); Z2 ¼ 7.5% (n ¼ 20); Z3 ¼ 30.9% (n ¼ 83) and Z4 ¼ 4.8% (n ¼ 13) for KID-. Statistical analysisshowed no signiﬁcant differences in implantation rate and Z-score between groups,(p ¼ 0.30) and p ¼ (0.32) respectively. It showed an increase in the percentage ofZ1, Z3 and Z4 and a decrease in Z2 which could be observed comparing 17h vs.BPD (p , 0,05). Limitations, reason for caution: Limitations due to the number of embryos analysed. Wider implications of the findings: The two PN show a dynamic pattern and Z-score depends on when it is performed. As closer to BPD, NPB tend to be Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 ampliﬁcation and array CGH (Bluegnome, UK). The primary outcome measure was ongoing implantation rate (.12 weeks of gestation), while secondary outcomes measures were related to number of biopsied embryos and euploid blastocyts obtained. Main results and the role of chance: A signiﬁcant higher mean number of embryos per cycle were biopsied on day 3 vs day 5 (8,3 + 2,5 vs 4,9 + 2,2 and 4,8 + 1,3 respectively, p , 0.001). A similar percentage of embryos were diagnosed as euploid when strategy 1 was applied: 81/300 (27,0%), vs 20/54 (37,0%) strategy 2 and 18/48 (37,5%) strategy 3 (NS). Moreover, the mean number of euploid embryos that reached the blastocyst stage (thus available for transfer and/or vitriﬁcation) per cycle was also comparable (1,9 + 0,7 (70/36), 1,8 + 0.9 (20/11), 1,8 + 0,6 (18/10), for strategy 1,2,3 respectively). In warming cycles, blastocyst survival rate was 100% (16/16). Ongoing implantation rate was similar in the 3 groups [40,0% (28/70), 44,4% (8/18) and 43,8% (7/16)] for strategy 1,2,3 respectively). Limitations, reason for caution: The main limitation of this study is the relatively low number of embryos included (402 biopsied, 104 transferred) and the retrospective approach. Wider implications of the findings: Since embryo biopsy strategy and/or cryopreservation procedure do not seem to have any impact on embryo developmental potential, the most appropriate PGS strategy (day 3 or day 5 biopsy) can be adopted according to the required genetic information and the laboratory workload. Study funding/competing interest(s): none Trial registration number: none i190 polarized. According to our data, there is not any prognostic effect of pronuclear evaluation at 17h or at BPD on implantation rate. Further studies with a greater number of embryos analyzed are necessary to conﬁrm these preliminary results. Study funding/competing interest(s): No commercial interest Trial registration number: Trial none Abstracts Study funding/competing interest(s): No external funding was obtained for the present study; has to be declared with any ﬁnancial organization regarding the material discussed in the manuscript. Trial registration number: Not applicable P-179 Time affects time: increased maternal age delays embryo morpho- kinetics P-178 Time laps monitoring of embryo development in patients with testicular or ejaculated sperm: a comparative study Study question: Our purpose was to compare, with the use of time-lapse technology, the developmental stages of embryos obtained with intracytoplasmic injection of spermatozoa retrieved either by ejaculation or by the testicles, in order to ﬁnd out possible differences in the developmental kinetics between the two groups. Summary answer: No difference appeared in the fertilization timing between testicular and normal ejaculated sperm, except a very slight anticipation of the second polar body (IIPB) extrusion. In the following cellular divisions, it was possible to observe a signiﬁcant retardation in the 4-cells stage in embryos deriving from testicular sperm. What is known already: It is well known that ejaculated and testicular sperm differ in the degree of nuclear maturation. During spermiogenesis, the transit of spermatozoa in the epididymal tract favors DNA packaging by stabilizing the chromatin structure through protamine dephosphorilation and the formation of molecular bridges. Additional difference in sperm maturity may involve the centriols as structures implicated in embryo cleavage. Based on these observations, sperm maturity can affect the timing of fertilization and later cellular divisions. Study design, size, duration: In this retrospective observational study (September 2012-January 2013), we analyzed the developmental kinetics of embryos obtained either with testicular sperm (N ¼ 40 embryos; TS-group) or with normal (WHO, 2010) ejaculated sperm (N ¼ 101 embryos; ES-group). The developmental markers observed were: IIPB extrusion, 2PN appearance, 2PN disappearance, divisions at 2 to 8-cells. Participants/materials, setting, methods: Development timings were recorded for all embryos (TS-group: 9 ICSI cycles, female mean age ¼ 33.78 + 4.58; ES-group: 25 cycles, mean age ¼ 35.57 + 4.09). Only correctly fertilized oocytes were observed (40/50 ¼ 80% in TS; 101/117 ¼ 86.3% in ES, NS). Student’s t-test was used for statistical analyses. Average hour + SD from ICSI insemination are reported for all stages. Main results and the role of chance: In TS versus ES groups respectively, IIPB was extruded at 3.86 + 1.48h (N ¼ 38) and 4.03 + 1.39h (N ¼ 88) (p ¼ 0.035); 2PN appeared at 10.05 + 1.77h (N ¼ 40) and 10.33 + 2.78h (N¼ 101); 2PN disappeared at 24.93 + 3.59h (N ¼ 32) and 23.91 + 2.48h (N ¼ 100); cleavage at 2-cells was at 27.94 + 3.75h (N ¼ 25) and 26.66 + 3.00h (N¼ 92); 3-cells at 39.07 + 6.27h (N ¼ 18) and 38.65 + 4.68h (N ¼ 53); 4-cells at 42.08 + 4.62h (N ¼ 23) and 39.70 + 3.65h N ¼ 88) (p ¼ 0.019); 5-cells at 52.23 + 9.45h (N ¼ 16) and 51.08 + 7.87h (N ¼ 70); 6-cells at 54.09 + 10.08h (N ¼ 17) and 53.18 + 5.05h (N ¼ 53); 7-cells 52.65 + 9.21h (N ¼ 16) and 52.26 + 3.95h (N ¼ 58); 8-cells 54.99 + 9.73h (N ¼ 12) and 57.56 + 6.63h (N ¼ 60). In brackets are reported numbers of embryos at a speciﬁc developmental stage. Data on embryos are relative to cultures up to day-2 or day-3. Limitations, reason for caution: A limitation of the present study is the small size of our population due to the fact that we have started using the time-lapse technology only in September 2012. An additional limitation may relates to the imaging system of such technology that allows the visualization of only 7 focal layers. Wider implications of the findings: Differences in maturity of ejaculated and testicular sperm seem not to interfere with fertilization when ICSI is performed. Sperm factors involved in oocyte activation are likely present in testicular spermatozoa in similar amount and with similar biological activity as in normal ejaculated sperm. The retardation in the 4-cell stage observed with testicular sperm could be due to immaturity at different levels which may involve the sperm head as well as other structures participating in cellular divisions. Study question: To determine whether maternal age affects embryo morphokinetic parameters. Summary answer: Embryos derived from older mothers have a slower development to the morula and blastocyst stage compared to embryos derived from younger mothers. What is known already: Maternal age is the most signiﬁcant factor to affect embryo implantation potential due to the increased incidence of aneuploidy in embryos derived from older patients. Aneuploid embryos have been shown to have delayed embryo morphokinetics. Therefore, it was hypothesized that increased maternal age delays embryo development. Study design, size, duration: This blind retrospective study in an IVF clinic assessed morphokinetic parameters of 348 human embryos cultured using a timelapse imaging system (EmbryoscopeTM, Unisense-Fertilitech, Denmark) from April to October 2012. Embryos were categorized as derived from older (age greater or equal to 37 years) versus younger (36 years and under) patients. Participants/materials, setting, methods: Morphokinetic data was collected from 24 ICSI patients cultured in the EmbryoScopeTM (sensitivity: 15 minutes). Following ICSI, hours for embryos to reach the 2-cell, 3-cell, 4-cell, 5-cell, 6-cell, 7-cell, 8-cell, 9 + cell, morula, expanded and hatched blastocyst were recorded and compared between 10 older and 14 younger patients. Main results and the role of chance: Embryo morphokinetic parameters were compared between embryos derived from older and younger patients and differences tested for signiﬁcance using a two-tailed two sample t-test. Differences were found to be signiﬁcant at p , 0.05. Data are presented as mean+ standard deviation. Patient age did not signiﬁcantly affect the time for embryos to reach the 2-cell, 3-cell, 4-cell, 5-cell, 6-cell, 7-cell, 8-cell or 9 + cell stages. Younger patients reached the morula (92.9 + 11 vs 98.8 + 14 hours post insemination, hpi, p ¼ 0.03), expanded blastocyst (111.7 + 8.4 vs 118 + 8.8hpi, p ¼ 0.008) and hatched blastocyst (115.1 + 7.5 vs 129 + 10.7hpi, p , 0.001) stages faster than older patients (younger vs older patients respectively). Limitations, reason for caution: As expected, the mean FSH dose for older patients (3069 + 1531,n ¼ 144) was signiﬁcantly greater (p , 0.001) than for younger patients (2148 + 1052, n ¼ 204). Therefore, further studies are required to determine whether the differences between the two treatments were due to maternal age or differences in dose of FSH. Wider implications of the findings: Time-lapse technology has the potential to improve embryo selection if parameters are identiﬁed as accurate predictors of implantation potential. Improving our understanding of how these parameters interact with current established predictors of implantation, such as age, may aid in the formation of models to predict implantation. Such models would have considerable clinical value, with the potential to improve IVF success rates. Study funding/competing interest(s): None Trial registration number: None The effect of Sildenafil Citrate (Viagraw) in vivo on endometrial receptivity, ovulation and embryo development in mice P-180 L. Asgari, D. Paouneskou, K. Jayaprakasan, W. Maalouf, and B.K. Campbell University of Nottingham, School of Clinical Sciences, Nottingham, United Kingdom Study question: What are the effects of pre-conception sildenaﬁl treatment on: subsequent embryo development through culturing of embryos until the blastocyst stage, endometrial receptivity through assessment of the thickness of the epithelial cell layer and oocyte yield through the estimation of the number of corpora lutea in mice. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 V. Casciani1, M.G. Minasi1, F. Scarselli1, M. Terribile1, D. Zavaglia1, A. Colasante1, G. Franco 2, and E. Greco 1 1 European Hospital, Centre for Reproductive Medicine, Rome, Italy, 2Sapienza University, Gynaecological-Obstetrical and Urological Sciences, Rome, Italy C. Hickman, C. Cook, D. Gwinnett, G. Trew, A. Carby, and S. Lavery Boston Place Clinic, Embryology, London, United Kingdom i191 Abstracts P-181 Binucleation versus multinucleation in 2-cells embryos how do they affect to implantation rate? J. Aguilar1, E. Taboas1, M. Perez1, E. Muñoz2, M. Ojeda1, and J. Remohi3 IVI Vigo, FIV, Vigo, Spain, 2IVI Vigo, Gynecology, Vigo, Spain, 3IVI Valencia, Gynecology, Valencia, Spain 1 Study question: How do binucleation and multinucleation in 2-cells embryo affect the implantation rate Summary answer: Embryos with one blastomere multinucleated (more than two nuclei per cell) have a lower implantation rate than those with one binucleated blastomere and those with both blastomeres multinucleated (binucleated or multinucleated) What is known already: Multinucleation has been related with an increase in the aneuplody rate, in chromosomal anomalies and a lower blastocyst rate, although the impact of multinucleation on live birth rate remains elusive. Nucleation errors are relatively frequent and, can be transitory phenomenon. They are classiﬁed either as binucleation (2 nuclei per cell), multinucleation (3 or more nuclei per cell). Study design, size, duration: Cross-sectional study of 2697 microinjected oocytes from 420 ICSI patients between January 2011 and November 2012, all of them incubated in an incubator with time-lapse technology. Participants/materials, setting, methods: Only KID (Known Implantation Data) embryos were analized.They were classiﬁed in 6 groups according to implantation (positive/negative),and the number of multinucleated blastomeres on 2-cells stage, (group 0¼ none; group 1 ¼ one; group 2 ¼ two) Multinucleation on 4-cells embryo was employed as exclusion criteria. x2-test and a Fisher exact t-test were performed Main results and the role of chance: 245 out 475 embryos transferred provided known implantation information and were analyzed, whereas 32.24% (n ¼ 79) fully implanted and 67.75% (n ¼ 166) failed to implant. Multinucleation was present in 46.17% of the embryos. 17.1% (n ¼ 42) fully implanted embryos were group 0, 8.97% embryos (n ¼ 22) were group 1, and 6.1% (n ¼ 15) group 2. The distribution for the not-implanted embryos was (n ¼ 90) 36.7 % of embryos group 0, 17.5%, one cell multinucleated (group 1) (n ¼ 43), and 13.46% both cells multinucleated (group 2) (n ¼ 33). In group 1, those embryos binucleated (n ¼ 17) showed greater implantation rate than those multinucleated (n ¼ 5), 26.15% v.s 7.69% (p ¼ 0.02) In group 2, three subcategories were analyzed, 2 binucleated cells, 2 multinucleated cells, 1 binucleated + 1 multinucleated. No signiﬁcant differences were found among them. Limitations, reason for caution: Studies with a greater number of embryos analyzed are necessary to conﬁrm these preliminary results Wider implications of the findings: Multinucleation in 2 cells embryos is relatively frequent during the embryo development, even in those transferred embryos selected by morphology and could disappear when embryos develop into 4-cells.The early cleavage checkpoint helps to improve embryo selection criteria. Study funding/competing interest(s): This work has not received any ﬁnancial support from any commercial entity. None of the authors have any particular beneﬁt to run this project. Trial registration number: none P-182 Improvement in blastocysts formation by selecting hyaluronic acid binding sperm E. Rega, A. Alteri, R.P. Cotarelo, P. Rubino, A. Colicchia, and P. Giannini Ferticlinic-Villa Margherita, Laboratorio PMA, Roma, Italy Study question: To evaluate the ability of hyaluronic acid (HA) binding sperm to increase the efﬁciency of intracytoplamic sperm injection (ICSI) in terms of blastocysts formation. Summary answer: The selection of normal sperm for ICSI with hyaluronic acid binding assay (HA-ICSI) led to increase of the blastocyst formation rate, in comparison with the conventionally selected spermatozoa. What is known already: The majority of studies are focused on the use of HA-binding as a method to select spermatozoa with normal nucleus and intact DNA. These parameters play a critical role in the paternal contribution to successful preimplantation embryogenesis; however, there are conﬂicting data about the positive correlation between HA-ICSI and improvement of fertilization rate and embryo development. Study design, size, duration: In this cohort study, we retrospectively analyzed the percentage of blastocysts obtained, between July and December 2012, from 67 sibling oocytes injected with either HA-bound (n ¼ 33) or conventionally selected spermatozoa (n ¼ 34) in a randomized way. Participants/materials, setting, methods: Patients using testicular or criopreserved sperm were excluded from the study. Half of the oocytes of women retrieving more than 4 metaphase II oocytes were injected by the HA procedures. Semen samples were treated via swim-u and placed in two different ICSI dishes with HA and PVP drops. Main results and the role of chance: A clear trend towards a higher blastocyst formation rate in oocytes injected with HA-bound spermatozoa has been observed: the frequency of blastocyst formation in HA-ICSI was 62.0%, while for PVP-ICSI was 40.0% (p ¼ 0,0598). These data suggest that the embryos developed from HA-ICSI seem to have a greater chance of developing to the blastocyst stage. Limitations, reason for caution: Low number of oocytes available for the study. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Summary answer: Pre-conception administration of sildenaﬁl at both the lower and higher doses tested impaired embryonic development relative to vehicle treated controls and the median dose of sildenaﬁl. Further, the higher dose increased endometrial thickness but none of the doses had any effect on ovulation rate or follicle number. What is known already: Sildenaﬁl has been suggested as a useful adjuvant therapy in women undergoing ART to increase the blood ﬂow to both the developing endometrium and the ovary. Previous studies from our laboratory have shown that while addition of sildenaﬁl to mouse embryos cultured in vitro had no effect on embryo development at doses in the normal physiological range, higher concentrations had a detrimental effect. This study therefore tested whether preconception sildenaﬁl administration had a similar negative effect. Study design, size, duration: Mice were randomly assigned to either vehicle controls (n ¼ 16) or three different doses of sildenaﬁl (S1:1.25 mg/kg n ¼ 16, S2:2.5 mg/kg n ¼ 16, S3:5 mg/kg n ¼ 16) administered by oral gavage twice daily over 2 days before mating on the morning of Day 3 before sacriﬁce for tissue recovery on Day 4. Participants/materials, setting, methods: Day 1 zygotes were collected and cultured in vitro to the blastocyst stage before being scored and the total cell number (TCN) counted. Ovaries were analysed histologically for number of corpora lutea (CL) and follicles whereas in the uterus endometrial thickness and number of blood vessels (neo-vascularisation) were counted. Main results and the role of chance: Relative to vehicle treated controls, cleavage rates, speed of cleavage and blastocyst rates were signiﬁcantly lower (P , 0.05) in embryos from mice treated with the lowest and highest doses of sildenaﬁl (S1 and S3, P , 0.05), while there was no signiﬁcant difference with the middle S2 dose. No difference was detected in the TCN of blastocysts formed (P . 0.05). Sildenaﬁl at the highest concentration (S3: 5 mg/kg) also caused a signiﬁcant increase in the uterine thickness (P , 0.05), while ovulation rate and follicular development remained unaffected (P . 0.05). These results suggest therefore that sildenaﬁl may be acting via different mechanisms at different concentrations. Limitations, reason for caution: This work has been carried out in mice so caution is needed when translating to the human situation as it is known that the pharmacokinetics of sildenaﬁl differ between species. Wider implications of the findings Although our study supports the possible use of sildenaﬁl as adjuvant therapy to improve endometrial receptivity it also raises concerns over the possible safety of this drug in terms of possible detrimental effects on embryo development. Further animal studies on the potentially deleterious effects of pre-conception sildenaﬁl treatment of females on subsequent embryo and fetal development are required before the introduction of sildenaﬁl as an adjuvant therapy could be adopted. Study funding/competing interest(s): Study funding came from my beloved father, Mohandes Ehsan Asgari and the University of Nottingham. Trial registration number: NA i192 Wider implications of the findings: This study suggest that the selection of normal sperm with HA might overcome potential embryo arresting due to pathogenesis inherent in the paternal genome. Study funding/competing interest(s): This study received no funding and there are no conﬂicts of interests to be declared. Trial registration number: This was a coohort study and a trial registration does not apply P-183 Embryo quality predictive models based on cumulus cells gene expression for clinical application Study question: The aim of the study was to use cumulus cells (CC) gene expression differences of leukemia inhibitory factor (LIF), anti – Mullerian horomone receptor, type II (AMHR2), follicle stimulating hormone receptor (FSHR), vascular endothelial growth factor C (VEGFC) and serpin E2 (SERPINE2) to differentiate high grade and low grade embryos. Summary answer: As signiﬁcant resulted AMHR2 (p ¼ 0.03) and LIF showed a tendency to signiﬁcance (p ¼ 0.11) between high grade and low grade embryos where their combination resulted in the area under the curve (AUC) 0.72 + 0.08 and 0.73 + 0.03 for binary logistic or decision tree prediction model, respectively. What is known already: Cumulus cells (CC) have a speciﬁc gene expression proﬁle according to the developmental potential of the oocyte they are surrounding and therefore speciﬁc gene expression could be used as a biomarkers. So far no single uniform biomarker has been found to be accurate enough for clinical application. The combination of several biomarkers and various statistical models might improve the usefulness of CC biomarkers in the clinics. Study design, size, duration: The CC samples from oocytes developed in high grade embryos (n ¼ 26) and low grade embryos (n ¼ 32) from 21 patients were analyzed. The CC expressions of LIF, AMHR2, FSHR, VEGFC and SERPINE2 were compared between high grade and low grade embryos and two models were used to test their prediction value. Participants/materials, setting, methods: Infertile women with tubal, unexplained infertility, aged less than 35 years and normal partners’ spermiograms were included in the study. Quantitative PCR was used to analyze CC expression. A Mann – Whitney test was used for testing differences and a binary logistic model and data a decision tree model were used. Main results and the role of chance: The CC gene expression between high grade and low grade embryos was signiﬁcantly different at AMHR2 (p ¼ 0.03) and among other genes only LIF showed a tendency to signiﬁcance (p ¼ 0.11). The CC expression values of AMHR2 and LIF were used in two models for prediction of high grade embryos. The ﬁrst, binary logistic model yielded in AUC 0.69 + 0.08 for AMHR2 and AUC 0.63 + 0.08 for LIF alone. Combining both genes the binary logistic model yielded in AUC 0.72 + 0.08. The second, decision tree model yielded in AUC 0.67 + 0.01 for AMHR2 and AUC 0.57 + 0.02 for LIF alone. Combining both genes in the decision tree model yielded an AUC 0.73 + 0.03. Limitations, reason for caution: Only two genes were signiﬁcant enough for implementation in models. With more genes in the model the predictive power would improve. Wider implications of the findings: The present study indicates that prediction of high grade embryos with CC expression is dependent on a type and number of CC biomarkers used. Even though LIF is not signiﬁcantly different between high grade and low grade embryos it improves prediction value of AMHR2 in both tested models. In term of eventual use in clinical practice the decision tree model has easy rules which can be applicable when making clinical decisions. Study funding/competing interest(s): This work was supported by Slovenian Research Agency (www.arrs.gov.si) grants P1-0104 and L3-4162. Trial registration number: Not a clinical trial. P-184 Pronuclear behavior and timing of embryo development: a time-lapse point of view C. Scarica1, F.M. Ubaldi1, M. Stoppa1, R. Maggiulli1, A. Capalbo1, E. Ievoli1, L. Dovere1, L. Albricci1, S. Romano1, F. Sanges1, A. Vaiarelli1, and B. Iussig2 1 GENERA, Center for Reproductive Medicine Clinica Valle Giulia, Rome, Italy, 2 GENERA, Center for Reproductive Medicine Clinica Salus, Marostica (VI), Italy Study question: Evaluate whether speciﬁc morphodynamic events during pronuclear (PN) formation are correlated with the early stages of embryo development, investigated with time-lapse cinematography. Summary answer: Through the use of time-lapse cinematography is possible to highlight that a delay in the PN appearance is predictive for abnormal PN formation. Moreover, PN eccentricity, asymmetry in size and failure of juxtaposition are related to a delay in timing of cleavage. What is known already: It was hypothesized that the PN evaluation at 16-18h post-ICSI is correlated with embryo development and chromosomal abnormalities and proposed as an important embryo selection parameter. However, conﬂicting data are available in literature. Parameters analyzed are normally related to the number and distribution of nuclear precursors bodies in PNs, the size and the position of the PN. Due to the dynamicity of PN formation events, static evaluation is probably insufﬁcient evaluating this stage of embryo development. Study design, size, duration: We retrospectively evaluated the impact of speciﬁc PN abnormal characteristics (eccentricity, asymmetry in size and failure of juxtaposition) on timing of embryo development by comparing 86 sibling embryos, cultured in a time-lapse incubator. To avoid confounding factor, each embryo with abnormal PN dynamics was compared with two sibling embryos with normal PN dynamics of the same cohort. Participants/materials, setting, methods: All the embryos analyzed derived from oocytes microinjected and cultured in a time-lapse incubator system (Embryoscope, Unisense). Events of development, such as timing of PN appearance, syngamy, timing of 1st to 7th cell division and cell cycle duration were evaluated by time-lapse cinematography. Main results and the role of chance: The morphokinetic parameters analyzed show that the timing of appearance of the PNs is signiﬁcantly lower in zygotes with normal PN dynamics (10,6 + 1,75 hours, CI 10,10-11,12), than those with abnormal PN dynamics (12,01 + 1,96 hours, CI 11,27-12,45) p ¼ 0,002. This delay is reﬂected in the subsequent development and, in particular, in the time division into 4 cells (39,56 + 5,01 hours CI38,15-40,57, vs 42,83 + 9,7 hours CI38,43-46,52 respectively) p ¼ 0,05 Limitations, reason for caution: Further analysis is necessary to conﬁrm this data on a larger population and to understand the impact of abnormal PN behavior on implantation potential and chromosomal abnormality of the deriving embryos. Wider implications of the findings: Due to the high dynamicity of pronuclear formation, different events can be missed during static evaluation normally performed at 16-18 hours post ICSI procedure. Our results suggest the predictive value of pronuclear appearance on successive abnormal PN behavior. An evaluation around half past 11 hours could help to avoid the selection of embryos that have the risk to display PN eccentricity, abnormal size and/or failure of juxtaposition during early formation. Study funding/competing interest(s): none Trial registration number: none P-185 Artificial collapse of blastocoelic cavity improves clinical outcome of closed blastocyst vitrification: a randomized controlled trial A. Gala1, A. Ferrières1, S. Assou1, C. Vincens2, S. Bringer-Deutsch2, C. Brunet2, and S. Hamamah1 1 Université Montpellier 1 Inserm U1040 CHU Montpellier, ART/PGD Department Institut de Recherche en Biothérapie, Montpellier Cédex 5, France, 2 CHU Montpellier, ART/PGD Department, Montpellier Cédex 5, France Study question: Does blastocyst artiﬁcial shrinkage (AS) prior to vitriﬁcation in a closed carrier device improve clinical outcomes? Summary answer: Artiﬁcial shrinkage of blastocoelic cavity signiﬁcantly improves clinical pregnancy rate after closed vitriﬁed blastocysts transfer. What is known already: Expanded blastocysts on day 5/6 are more sensitive to vitriﬁcation than early blastocysts because of an insufﬁcient dehydration of the blastocoelic cavity. Based on this hypothesis, several authors have described techniques to reduce the blastocoele and prevent the formation of damaging ice crystals. However, all those studies have been realized with opened devices. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 R. Devjak1, T. Burnik Papler1, K. Fon Tacer 2, and I. Verdenik1 University Medical Centre Ljubljana, Departmen of Obstetrics and Gynecology, Ljubljana, Slovenia, 2University of Ljubljana Veterinary Faculty, Institute for Hygiene and Pathology of Animal Nutrition, Ljubljana, Slovenia 1 Abstracts Abstracts P-186 Dynamic assessment of early embryo fragmentation by time-lapse analysis may improve cell cycle timing-based embryo selection J. Conaghan1, L. Tan2, M. Gvakharia3, K. Ivani4, A. Chen2, and R. Reijo Pera 5 1 Paciﬁc Fertility Center, IVF Lab, San Fransisco CA, U.S.A, 2Auxogyn Inc., Clinical R&D, Menlo Park CA, U.S.A, 3Fertility and Reproductive Health, Reproductive Laboratories, San Jose CA, U.S.A, 4RSC of the SF Bay Area, IVF Lab, San Ramon CA, U.S.A, 5Stanford University, Stem Cell Biology and Regenerative Medicine, Stanford CA, U.S.A Study question: What are the fragmentation dynamics in human embryos from 1to -4-cell stages, and could time-lapse assessment of fragmentation improve embryo selection when used together with early cell cycle timings? Summary answer: For most embryos, fragmentation occurs between the 2- and 4-cell stages; time-lapse analysis of early embryo fragmentation can augment the predictive power of cell cycle timing parameters for selecting the embryo with the highest chance of implantation. What is known already: Fragmentation is commonly associated with decreased embryo viability, and most clinics assess fragmentation on Day 2 and/or 3 (4- to 8-cell stage). However, a recent paper by Chavez et al. (Nat Comm, 2012) suggests that dynamic assessment of embryo fragmentation, together with cell cycle timing parameters, is indicative of human embryo ploidy at the 4-cell stage. The current study examined the incidence and outcomes for embryos with early fragmentation in a clinical IVF setting. Study design, size, duration: Retrospective analysis of 850 embryos from 95 patients (6/2011-10/2012) with embryos imaged using the EevaTM Test, a platform that makes blastocyst predictions by integrating time-lapse imaging and automated analysis of cell cycle timing parameters P2 (time between cytokinesis 1 and 2) and P3 (time between cytokinesis 2 and 3). Participants/materials, setting, methods: Embryo videos were analyzed between the 1- to 4-cell stages for fragmentation appearance, volume and pattern over time. This data was used to determine if fragmentation analysis could augment P2 and P3 to better predict implantation. Statistical signiﬁcance was determined by Fischer’s Exact or Chi-Square test. Main results and the role of chance: Dynamic assessment offragmentation by timelapse imaging revealed that fragmentation was present in 94% (795/850) of embryos, and a majority of fragments ﬁrst appeared at the 2-cell stage (90%, 720/795). At Day 3, conventional fragmentation reported that only 77% (653/850) of embryos had fragmentation, suggesting that time-lapse may be more sensitive in detecting fragments. Time-lapse assessment also showed that the percentage of fragmentation was persistent to the 4-cell stage for 89% (707/795) of embryos. Without the fragmentation assessment, speciﬁc ‘in-window’ cell cycle timings (P2, P3) were predictive of embryo implantation (implantation 39% vs. 6%, in window vs. out of window, p , 0.0001). The addition of the early fragmentation assessment to ‘in-window’ cell cycle timings (P2, P3) resulted in a further improvement to implantation (46%). Limitations, reason for caution: The dynamic fragmentation assessment was done manually and thus subject to inter-observer variances. Automated and more quantitative fragmentation assessment based on state-of-the-art computer vision technologies is currently underway to address this limitation. Wider implications of the findings: Our results support previous ﬁndings suggesting that early fragmentation assessment from 1 to 4-cell stages, together with cell cycle timings P2 and P3, is predictive of embryo developmental competence, including aneuploidy status. Furthermore, our ﬁndings provided two additional insights: 1) time-lapse assessment of fragmentation is more sensitive than traditional Day 3 evaluation; 2) adding early fragmentation assessment may further augment cell cycle timing-based embryo selection and improve implantation rates. Study funding/competing interest(s): This work was supported by Auxogyn, Inc. Trial registration number: ClinicalTrials.gov # NCT01369446 and NCT01617993 P-187 Comparison of clinical results, between standard and uninterrupted embryo culture, when there are no embryos to select between N. Bowman1, S. Montgomery 1, L. Best1, A. Campbell2, S. Duffy 1, and S. Fishel2 1 CARE Manchester, Embryology, Manchester, United Kingdom, 2CARE Fertility, CARE Fertility Group, Nottingham, United Kingdom Study question: Is there a beneﬁt of uninterrupted (time-lapse) incubation over standard incubation for ICSI patients who only have the number of embryos available that they require for transfer? Summary answer: When there is no selection of embryos required, as all created are transferred and where female age is ≥38 years, there appears to be a signiﬁcant beneﬁt of uninterrupted embryo culture compared to standard (interrupted) methods. What is known already: Meseguer et al (2012) reported a signiﬁcant improvement in the relative probability of clinical pregnancy (CP) when the EmbryoScopeTM (ES)(Unisense Fertilitech, Denmark) was used compared to standard incubation methods for ICSI patients. This uplift was reported likely to be due to a combination of uninterrupted culture and the enhanced embryo selection, made possible by the time-lapse imaging. Study design, size, duration: Retrospective analysis of CP and implantation rates in a private IVF centre between May 2011 and October 2012. 85 embryos were incubated in the ES’s uninterrupted conditions and 591 in Galaxy incubators (SI), both 5%O2, 5.5%CO2, 89.5%N2. Participants/materials, setting, methods: All patients undergoing ICSI treatment under either incubation condition were included, when they only had the number of embryos they required for transfer. IVF patients and oocyte recipients were excluded. 53 patients underwent ES culture and 391 SI. Main results and the role of chance: There were no signiﬁcant differences between the CP rates and implantation rates when embryos were cultured in SI and ES culture when patients were aged ≤35 (n ¼ 244 patients SI, n ¼ 19ES). When two embryos were transferred, the CP rate was 27.3% SI vs 33.3% ES (p ¼ 0.7381 not signiﬁcant (NS)) and the implantation rate, per embryo was 16.4% SI vs 20.8% ES (p ¼ 0.5703 NS). For patients aged ≥36 there was a trend towards an increased CP and implantation rate in ES. For patients ≥38, there was a signiﬁcant increase in implantation rate under ES incubation when two embryos were transferred (n ¼ 51 patients SI, n ¼ 16 ES, 3.9% SI vs 15.6% ES p ¼ 0.035) and a trend towards an increased CP rate (7.8% SI vs 25% ES p ¼ 0.085). Limitations, reason for caution: Embryos were cultured under different incubation conditions at patient request. Therefore, other than age, patient history, has not been taken into account. The number of ‘interruptions’ during SI was variable when door openings of the 48L incubators and dish removal for developmental progress checks are included. Wider implications of the findings: This relatively new technology is not widely available. Clinics commonly run both ES and SI and may prioritise which embryo cohorts are placed into ES culture. Our previous data showed overall increased CP rates for patients over 36(Best 2013, ACE) but these ﬁndings suggest an Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Concerning the income of artiﬁcial shrinkage (AS) prior to vitriﬁcation in closed systems, data remain unclear. Study design, size, duration: Prior to vitriﬁcation with closed device, blastocysts were either collapsed by laser pulse or non treated at all according to a randomized procedure. Clinical pregnancy rate was assessed in 147 warming cycles from April 2011 to December 2012. Participants/materials, setting, methods: On day 5/6, full (3), expanded (4) or hatching (5-6) blastocysts with at least a trophectoderm quality type B according to Gardner classiﬁcation were cryopreserved. Clinical pregnancy rate was compared between blastocysts with AS (n¼ 35 cycles) and blastocysts without AS (n ¼ 112 cycles). The mean women age was 33.2 + 4.9 years. Main results and the role of chance: The overall clinical pregnancy rate after transfer was 41.8% (59/141). The clinical pregnancy rate was signiﬁcantly higher in the AS group (19/35; 54.2%) compared with the control group (40/ 106; 37.7%) (1 ¼2.414, p , 0.02). The survival rate was higher in the AS group but no signiﬁcantly (52/53, 98.1% Vs. 157/170, 92.3%; 1 ¼1.468, p . 0.09). The two groups were similar concerning women age, endometrial preparation and vitriﬁcation day Limitations, reason for caution: This results need to be completed with the living birth rates to ensure the efﬁciency of our treatment. Wider implications of the findings: To our knowledge, this is the ﬁrst randomized controlled trial assessing the impact of AS prior to vitriﬁcation in a closed device. This study reveals that blastocyst AS with laser pulse is a successful technique to improve clinical pregnancy rate after closed vitriﬁcation. Our results are in line with previous publications on the beneﬁt of AS. Study funding/competing interest(s): This work was partially supported by a grant from Ferring Pharmaceuticals company. The authors declare that there is . Trial registration number: Not applicable i193 i194 implantation rate beneﬁt to ES incubation for patients ≥38 even without embryo selection. Therefore, this may encourage units to culture embryos for this patient group in ES culture. Study funding/competing interest(s): None. Trial registration number: None. P-188 The zona pellucida thickness of a human embryo is associated with implantation potential in fresh blastocyst transfer but not in day 3 transfer Study question: Is the zona pellucida thickness (ZPT) of cleavage embryo associated with implantation potential in a fresh cycle? Summary answer: The day 5 embryos with thickened zona pellucida (ZP) on day 3 had a lower implantation rate compared with normal embryos. Assisted hatching (AH) could be beneﬁcial for fresh day 5 transfer. However, the day 3 embryo had no difference in implantation rate regardless of ZPT. What is known already: The ZPT is considered to be a good indicator for embryo potential. However, several studies have reported that the notion of a relationship between ZPT and implantation has not been supported. Study design, size, duration: This study was a retrospective analysis of implantation rate after fresh elective single-embryo transfer cycles. A total of 376 embryos were included between 2010 and 2012. Participants/materials, setting, methods: The AH procedure was performed on patients who had ≥2 failed IVF cycles, or were ≥38 years of age, by laser, regardless of ZPT. The measurement of ZPT was performed by Zilos imaging software on day 3 embryo images. Main results and the role of chance: The overall mean of ZPT was 18.44 + 2.4 mm. In day 3 transfer, ZPT did not correlate with implantation rate whether AH was performed or not. In day 5 transfer, unhatched embryos whose zona were thicker than 20 mm on day 3 had a signiﬁcantly lower implantation rate compared with the embryos with a normal ZP of 15-19 mm (30.8% vs. 54.8% respectively, P , 0.05). However, in day 5 transfer with AH treatment, no signiﬁcant differences were observed between the thick ZP group and normal ZP group in the rates of implantation (42.9% vs. 36.7% respectively). Limitations, reason for caution: The characteristics of the two groups; the AH-treated group and non-treated group, are not comparable because the AH was performed on patients with a poor prognosis; those with ≥2 failed IVF cycles and older women (≥38 years of age). Wider implications of the findings: Our results suggest that the AH procedure is useful for embryos with a thick ZP in day 5 transfer but not in day 3 transfer. Extended embryo culture may affect the hatching of embryos, especially in embryos that have a thick ZP. Study funding/competing interest(s): This study received no funding and there are no conﬂicts of interests to be declared. Trial registration number: Not applicabe. P-189 A novel biological role of embryo derived oxytocin in early stages of embryo development in mice V. Dinopoulou, G.A. Partsinevelos, R. Bletsa, D. Mavrogianni, E. Anagnostou, K. Stefanidis, P. Drakakis, and D. Loutradis Athens University Medical School, Division of Human Reproduction IVF Unit 1st Department of Obstetrics and Gynaecology Alexandra Hospital, Athens, Greece Study question: What is questioned in this study is whether oxytocin levels measured in the culture medium post-fertilization are correlated with the stage of embryo development (2-cell, 4-cell, morula/blastocyst and blastocyst stage) and whether these levels reﬂect embryo implantation potential in mice. Summary answer: Oxytocin was secreted at all stages of early embryo development, although a gradual decrease until morula-early blastocyst stage was found, which was followed by an increase at the blastocyst stage. These ﬁndings suggest a role of oxytocin in early embryo development as well as in the implantation process in mice. What is known already: Oxytocin receptor (OTR) is downregulated at the endometrial areas surrounding the attaching embryo, but is upregulated at the periimplantation areas, throughout the later stages of implantation. OTR mRNA is detected in mouse oocytes and embryos up to the blastocyst stage. The increase in OTR expression immediately after fertilization suggests a possible role of oxytocin in this process, whereas the gradual decrease after the 4-cell stage implies a possible role in implantation. Study design, size, duration: This prospective observational animal study was performed on a study population consisted of 10 female mice and 10 male mice (C57BL/6 x CBA) F1 hybrids, which were allocated in two sequential experiments of 5 female and 5 male mice each. Participants/materials, setting, methods: Female mice, superovulated and paired with male, were sacriﬁced and zygotes were ﬂushed from the fallopian tubes. Two-cell embryos were cultured in groups of 20. The culture medium was sampled and stored at -20o C at 2-cell, 8-to-16-cell, morula-blastocyst and blastocyst stage for oxytocin determination using an enzyme immunoassay kit. Main results and the role of chance: Baseline oxytocin levels in the medium used to culture mouse embryos was 0.03 + 0.000 ng/ml (mean + SEM) on Day 0. On day 2, 56 hours post-hCG injection, at the 4- to 8-cell embryo stage, oxytocin levels were 0.070 + 0.009 ng/ml (mean + SEM). On day 3, 76 hours post-hCG injection, at the 8- to 16-cell embryo stage, oxytocin levels were 0.068 + 0.014 ng/ml (mean + SEM). On day 4, 109 hours post-hCG injection, at the morula-blastocyst embryo stage, oxytocin levels were 0.054 + 0.055 ng/ ml (mean + SEM). On day 5, 131 hours post-hCG injection, at the blastocyst embryo stage, oxytocin levels were 0.079 + 0.007 ng/ml (mean + SEM). Limitations, reason for caution: A possible limitation of our study is the use of a restricted number (20) of mouse embryos for 5-day culture. We believe that a greater difference in oxytocin levels would have probably been seen between stages of embryo development in case a higher number of mouse embryos had been utilized. Wider implications of the findings: Taking into account that oxytocin might have some biological role at the early stages of development of fertilized oocytes and the fact that embryos and their secreted oxytocin may exert local effects, we could implement measurement of oxytocin levels in the culture medium as a biological marker in the selection of the best embryos, in terms of implantation potential, for transfer in IVF protocols. Study funding/competing interest(s): No funding supported this study and no competing interest is declared. Trial registration number: The study was registered to Local University Hospital Ethics Committee (registration number: 15/26-01-2009). P-190 Incidence of live birth using hyaluronic acid (HA) for sperm selection J. Hernandez 1, C. Lorenzo León 1, M. Puopolo2, and A. Palumbo1 Centro de Asistencia a la Reproducción Humana de Canarias, La Laguna, Spain, 2Istituto Superiore di Sanitá, Department of Cell Biology and Neurosciences, Rome, Italy 1 Study question: PICSI has been proposed as a sperm selection technique capable of improving pregnancy rates in couples with male factor infertility. Since 2009 we have introduced this technique in our IVF laboratory for selected couples to test its effect on live birth rates. Summary answer: Our data suggest that use of hyaluronic acid for sperm selection by PICSI may have a beneﬁcial effect in patients with male factor by reducing the miscarriage rate and improving the live birth rate. What is known already: PICSI is a technique of sperm selection based on the ability of mature sperm to bind to hyaluronic acid in vitro. Although some reports suggest a beneﬁcial effect of PISCI in male factor cases, current literature is controversial. Study design, size, duration: This is a retrospective observational study on 44 patients recruited from 11/2009 to 12/2012 who underwent a treatment cycle using HA for sperm selection (PICSI). We selected an historical control group recruited from 1/2008 to 10/2009 composed of 104 ICSI cycles in couples with sperm parameters comparable to the study population. Participants/materials, setting, methods: Patients in the study group had a sperm count in the range 5.000.000-20.000.000/ml. Variables analyzed included patientś and partnerś age, fertilization rate, viable, transferred and top quality Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 R. Hirata1,2, Y. Aoi1, T. Habara1, and N. Hayashi1 Okayama Couplès Clinic, Gynecology, Okayama, Japan, 2Laboratory of Reproductive Physiology, Graduate School of Environmental and Life Science, Okayama University 1 Abstracts i195 Abstracts P-191 Impact of seminal trace elements and glutathione levels on sperm DNA fragmentation and assisted reproductive techniques outcome Atig1, Kerkeni2, Saad3, Ajina 3 A. A. and M. F. University Farhat Hached Hospital, Unit of reproductive medicine, Soussa, Tunisia, 2University of Monastir, Research Laboratory of “Trace elements free radicals and antioxidants” Biophysical Department Faculty of Medicine, Monastir, Tunisia, 3University Farhat Hached Hospital, Unit of reproductive medicine, Monastir, Tunisia 1 Study question: Can seminal non-enzymatic antioxidants and sperm DNA fragmentation help to predict the in vitro fertilization (IVF) outcome of Tunisian infertile couples? Summary answer: Standard semen parameters were poor predictors of the IVF outcome. Besides; sperm genome quality, seminal trace elements “Zinc and Selenium” and glutathione levels were strongly inter-related and demonstrated signiﬁcant and close relationships with the in vitro early embryogenesis, thus with the success of IVF attempt. What is known already: Valuable evidence has emerged about the crucial role of seminal trace elements and glutathione as non-enzymatic antioxidants that protect spermatozoa against lipid peroxidation and oxidative damages. Nevertheless, contrary to the seminal reactive oxygen species, there is no clear consensus about the involvement of these antioxidants to protect sperm from oxidative DNA fragmentation and to predict the success of the early embryonic development after IVF. Study design, size, duration: This is a prospective controlled study carried out on Tunisian infertile men participating in the conventional IVF program at our Unit of Reproductive Biology, Farhat Hached University Hospital (Tunisia). A total of 200 consecutive males (24-50 years) were recruited in our investigation between June 2011 and July 2012. Participants/materials, setting, methods: Obtained semen samples were analyzed according to WHO criteria (1999) and divided into four groups: nomozoospermics (controls, n ¼ 70), oligozoospermics (n ¼ 40), asthenozoospemics (n ¼ 45) and teratozoospermics (n ¼ 45). Seminal zinc and selenium concentrations were determined using ﬂame and furnace atomic absorption. The different forms of glutathione and sperm DNA fragmentation levels were measured spectrophotometrically and by TUNEL assay, respectively. IVF outcome was presented as follows: fertilization, cleavage and embryo quality (Grade I, II and III). Main results and the role of chance: The key results in this study were: (1) increased seminal trace element amounts were observed in normozoospermics when compared with abnormal groups mainly with Asthenozoodpermics and Teratozoospermics. Total and reduced forms of glutathione (GSHt and GSHr) were also signiﬁcantly elevated in seminal plasma of controls than asthenozoospermics (P ¼ 0.002, P ¼ 0.003; respectively). (2) Controls established highly signiﬁcant decline of sperm DNA fragmentation levels compared to the three abnormal groups (P , 0.001). (3) Negative correlations were found between enhanced seminal antioxidant proﬁle and sperm DNA fragmentation (zinc [r ¼ -0.71**, P , 0.001], selenium [r ¼ 0.63**, P , 0.001], GSHt [r ¼ -0.21*, P ¼ 0.01], GSHr [r ¼ -0.72**, P , 0.001]). (4) Inversely to the non-enzymatic antioxidants, sperm DNA fragmentation was strongly correlated to poor fertilization rate (r ¼ -0.53*, P ¼ 0.02), cleavage (r ¼ -0.71**, P , 0.001) rate and embryo quality (Grade III) (r ¼ 0.62*, P ¼ 0.01). Limitations, reason for caution: The results may be biased by the determination of sperm DNA fragmentation on the same day as the IVF procedure. It would be interesting to measure the sperm DNA fragmentation and antioxidant levels before IVF, in order to predict their role in the procedure. Wider implications of the findings: We identiﬁed that the routine determination of non-enzymatic antioxidants and sperm DNA fragmentation levels is a more reliable prognostic tool for male infertility assessment which can help selection of semen samples with the least amount of sperm DNA fragmentation and thus reduce the risks associated with the use of DNA-fragmented sperm for fertilization and avoid ﬁnancial, social and emotional problems associated with failed IVF attempts. Study funding/competing interest(s): None of the authors have any competing interest. This work is part of a scientiﬁc project (Thesis). Trial registration number: None. P-192 Matured human oocytes with cumulus cells and assisted reproduction techniques G. D’Ommar, A.K. Herrera, L. Lozano, and M. Majerfeld Centro Medico Docente La Trinidad, Fertility Clinic, Caracas, Venezuela Study question: Determine effects in fertilization rate and quality of embryos obtained from oocytes remnants of assisted reproduction techniques (ART), matured with or without their cumulus cells (CC). Summary answer: Mature oocytes with CC, equates the fertilization rate and embryo quality to levels similar to those obtained with mature oocytes collected. What is known already: The oocyte plays an important role regulating its own development, by the production of paracrine growth factors that affect the surrounding granulosa cells (Gilchrist et al., 2008). These cells represent an essential component in the maturation and fertilization of oocytes. Because oocytes undergo intracytoplasmic sperm injection (ICSI) are denuded, the potential positive effects of cumulus cells in future development cannot be observed (Ebner et al., 2006). Study design, size, duration: Retrospective analysis of the effect of CC on completion of oocyte maturation. Population: 124 patients (n ¼ 1239 oocytes), age: 33.0 + 3.9 years, performing ART and ICSI, is compared fertilization and embryo quality. Participants/materials, setting, methods: Oocytes classiﬁcation: Denuded: Appeared to be mature, were immature when denuded. (n¼ 198) Not denuded: Appeared to be immature, were not denuded. (n¼ 360) Control group: Were mature by crown classiﬁcation, resulting MII when denuded (n ¼ 681). Results were analyzed by the chi-square test. Main results and the role of chance: In collected mature oocyte, fertilization rate was 69.6 %; in Denuded group 35.2 %; in Not denuded group 68.3 %. With values of P¼ 0.000001 between mature oocytes and Denuded group, P¼ 0.000002 between Denuded group and Not denuded group, but with P¼ 0.8426 between mature oocytes and Not denuded group. In relation to embryos quality, in mature oocytes 43.7 % corresponds to good quality embryos; 24 % of good quality embryos in Denuded group; in Not denuded group 43.3 %. With values of P¼ 0.003271 between mature oocytes and Denuded group, P¼ 0.003874 between Denuded group and Not denuded group, but with P¼ 0.9586 between mature oocytes and Not denuded group. For values ??of P ,0.05 there is no association between the variables. Limitations, reason for caution: The main limitation in this study because it is human oocytes was obtaining informed consent, which was reﬂected in the sample size. Wider implications of the findings: Was observed that coculture with cumulus cells promotes maturation and meiotic progression, which is reﬂected in the high rates of fertilization in vitro matured oocytes with cumulus cells, which is consistent with other studies. Study funding/competing interest(s): Clı́nica de Fertilidad, Centro Médico Docente La Trinidad, Caracas, Venezuela. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 embryos, clinical pregnancy and miscarriage rate. Statistical analysis was performed using t-test for continuous variables and Fisher’s exact probability test for categorical variables. [Maria1]era nei parametri ﬁssati per la selezione dei controlli? Main results and the role of chance: The study group and the control group did not differ in terms of mean patientś age and partners’ age (35.8 + 1 vs 36.4 + 0.4 and 40.76 + 0.9 vs 39.1 + 0.6 respectively); fertilization rate (64.4% vs 65.9%), and pregnancy rate [50% (22/44) vs 40% (42/104)]. There was a trend towards a lower miscarriage rate in the study group [9% (2/22)] vs 21% (9/42) in controls, however this difference did not reach statistical signiﬁcance, which may be due to the small sample analyzed. Limitations, reason for caution: The main limitation is the retrospective design and the small number of cases in the PICSI group. A further weakness is the use of an historical control group which might be biased due to improvements in clinical practice, however no major changes occurred in our center during this time frame. Wider implications of the findings: The possibility that the PICSI technique reduces the miscarriage rate increasing live birth rates is attractive and further prospective randomized studies are warranted. Study funding/competing interest(s): None Trial registration number: Not applicable i196 Trial registration number: No P-193 Direct unequal cleavages: Cell stage onsets, embryo developmental potential and chromosomal abnormalities Z. Ye, N. Zaninovic, R. Clarke, R. Bodine, and Z. Rosenwaks Center for Reproductive Medicine, Reproductive Medicine, New York, U.S.A P-194 Can timing of post-cleavage embryo development stages reveal ploidy or implantation potential? P. Mazur1, V. Nagorny1, D. Mykytenko2, L. Semeniuk1, and V. Zukin1 Clinic Of Reproductive Medicine "Nadiya", Embryology, Kyiv, Ukraine, 2Clinic Of Reproductive Medicine "Nadiya", Department of Molecular Diagnostics, Kyiv, Ukraine 1 Study question: Can detailed time-lapse observation of embryo post-cleavage development stages suggest embryo ploidy or its enhanced implantation? Summary answer: Although ploidy is not related with timing of compaction, cavitation and expansion, thorough analysis of blastocyst expansion may help in assessment of embryo implantation potential. What is known already: Limited data connects timing of embryo development with its ploidy. Human embryo genome activation is known to start at day 3 of cultivation in-vitro, so analysis of late stages of in-vitro culture seems promising. Study design, size, duration: This cross-sectional study includes 84 patients whose embryos were cultured in time-lapse imaging system. Of those, 106 blastocysts of 60 patients were transferred in fresh cycles and have known pregnancy outcome. Second group consisted of 24 infertility treatment patients where embryo ploidy was established by array comparative genome hybridization (aCGH). Participants/materials, setting, methods: Embryos were cultured in EmbryoScopeTM (Unisense FertiliTech, Denmark). After 5 or 6 days of embryo culture, time-lapse image data were analysed. Array CGH, if applied, was performed after trophectoderm biopsy, using 24surew kit (BlueGnome, United Kingdom). In aCGH group of 113 analysed embryos 58 were euploid and 56 were aneuploid. Main results and the role of chance: Using time-lapse observation, development time points like morula formation, cavity appearance and expansion time were deﬁned. Efﬁcient expansion was presumed when expanding blastocyst started to stretch zona pellucida (ZP). To locate this moment, inner ZP diameter was calculated from image data with 1h interval and subsequent linear regression analysis performed. In aCGH group assisted hatching performed at day 3 of culture caused expansion time to match with moment of trophectoderm protrusion trough ZP opening. No correlation was seen between timing of late in-vitro culture events when embryos were compared by ploidy or implantation fate. However graphic interpretation of blastocyst volume expansion speed in group of embryos with known implantation showed that implanted embryos usually had uniform midspeed expansion without deep collapsing or oversized growth. Limitations, reason for caution: Comparison of timing of late in-vitro development events is complicated by lags often observed in pre compaction and morula stages. It is possible that in future investigations these difﬁculties will be overcomed. Wider implications of the findings: To the date, assuming that each embryo is unique, we cannot recommend to select embryos for transfer relying on their timing of post-cleavage stages. Blastulation dynamics, after development of labour-saving software, can be applied for embryo scoring. Study funding/competing interest(s): The authors have nothing to disclose. No external funding was received for this work. Trial registration number: This study was not an RCT. P-195 Prospective trial of vitrification of all embryos in cycles of ART: implantation and pregnancy rate A. Zabala1, T. Pessino2, S. Outeda2, L. Blanco2, F. Leocata 2, and R. Asch3 1 Hospital Interzonal de Agudos Jose de San Martin, Ginecology, La Plata, Argentina, 2PROCREARTE, Ginecology, C.A.B.A, Argentina, 3Ministerio de Salud de la Provincia de Buenos Aires, Ginecology, La Plata, Argentina Study question: To determine the pregnancy outcome after vitriﬁcation of all fresh embryos produced in stimulated assisted reproduction technique cycles (ART) and replacing them in subseuqemt non-gonadotropin stimulated cycle Summary answer: Vitriﬁcation of all fresh embryos produced in stimulated ART cycles and replacing them in subsequent non-gonadotropin stimulated cycles resulted in highly successful implantation rate and cumulative pregnancy rates. What is known already: It has bee propoced that supraphysiological hormone levels during ovarian stimulation may adversely affect embryo implantation in assisted reproductive treatments. Very few studies have advocated the elective cryopreservation of all embryos as a methos to avert the undesirable effect of gonadotropin ovarian stimulation on implantation and pregnancy rate, as well as to prevent the occurrence of iatrogenic events such as ovarian hyperstimulation syndrome (OHSS). Study design, size, duration: A prospective trial of series of cases in a private fertility center-ART program. 122 patients (average age 35 years) with risk to develop OHSS or with uterin factor (poor endometrium or difﬁcult transfer) who underment vitriﬁcaion of all fresh embryos from Junuary 2011 to December 2012 Participants/materials, setting, methods: 1000 embryos were vitriﬁed at 8 blastomeres or blastocyst stage. Embryo survival rate (at last 80% of blastomeres intact post-thawing) was 97%. After thawing, 527 embryos were transferred into hormone replacement cycles in a total of 215 cycles. the average number of embryos transferred per cycle was 2,4. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: To investigate cell stage occurrence, developmental potential, and chromosomal abnormalities in embryos displaying direct unequal cleavages (DUC). Summary answer: When identiﬁed correctly, direct unequal cleavages at early cell stages have a greater negative impact on subsequent embryo development than later stage onsets, and are correlated with a high incidence of chromosomal abnormalities, but not triploidy. What is known already: The occurrence of DUC (1 to 3 cells) has been reported in many IVF labs, a phenomenon unrelated to speciﬁc patient population, culture media or fertilization technique. A recent multicenter study showed limited implantation potential of embryos with direct unequal cleavages at ﬁrst mitosis. Triploidy has been suggested as a cause; however developmental potential and chromosomal composition of these embryos in relation to the cell stage onset was not reported. Study design, size, duration: Fertilized oocytes (n ¼ 5604 from 408 patients) were cultured in time-lapse EmbryoScope incubators (Unisense Fertilitech) and annotated for patterns and times of cleavage. PGS analysis was performed on the subset of patients (n ¼ 26, avg. age 37.7) that demonstrated DUC. Participants/materials, setting, methods: Direct unequal cleavages were deﬁned as cleavage of one cell directly to three or more cells which were annotated during the ﬁrst, second or third cleavage division. PGS analysis (biopsy day 3 or 5) was performed on 26 patients exhibiting DUC using FISH, CGH or SNP array. Main results and the role of chance: DUC were observed in 12% of the embryos: 58% at 1st cleavage, 25% at 2nd cleavage and 6.7% at 3rd cleavage division. DUC embryos were developmentally compromised, and only 9% reached blastocyst stage and were frozen. The developmental potential of DUC embryos was associated with cell stage onset, where DUC occurrence at ﬁrst cleavage is more critical to development than DUC at second or third division. 255 PGS embryos were analyzed; 74 (29%) annotated as DUC. Chromosome abnormalities were detected in 89% of DUC embryos, which included different patterns of chromosomal aberrations (but not triploidy), and were not of speciﬁc maternal or paternal origin. The detailed analysis of PGS normal embryos, originally annotated as DUC, revealed ambiguous cell or fragment identiﬁcation, and/or cell fusion. Limitations, reason for caution: N/A Wider implications of the findings: When identiﬁed correctly with time-lapse microscopy, DUC at various cleavage stages can be a useful tool for selecting embryos for transfer. Embryos demonstrating DUC have limited developmental potential and are associated with high level of chromosomal abnormalities; therefore they should be avoided for ET or freezing. Study funding/competing interest(s): Institutional Trial registration number: N/A Abstracts i197 Abstracts P-196 Early cleaving mouse embryos are better candidates for vitrification W.J. Wan-Haﬁzah1, M.H. Rajikin2, A.S. Nuraliza2, M. Mohd-Fazirul 3, J.M.Y. Norhazlin 3, D. Razif4, and M.N.K. Nor-Ashikin3 1 Faculty of Medicine, Universiti Kuala Lumpur-Royal College of Medicine Perak, Ipoh, Malaysia, 2Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Malaysia, 3Institute of Medical Molecular Biotechnology Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Malaysia, 4Faculty of Health Sciences Puncak Alam, Universiti Teknologi MARA, Sungai Buloh, Malaysia Study question: Do early cleaving (EC) embryos make better candidates for vitriﬁcation compared to late-cleaving (LC) embryos? Summary answer: EC embryos make better vitriﬁcation candidates compared to LC embryos, because they exhibit signiﬁcantly higher post-vitriﬁcation survival rate and viability. What is known already: The use of EC embryos has been suggested for embryo transfer in Assisted Reproductive Technology (ART) because of their good quality and high viability. However, no study has been conducted to investigate whether these EC embryos also make better vitriﬁcation candidates Study design, size, duration: In a prospective study, post vitriﬁcation survivability and viability were compared between EC embryos and LC embryos. A total of 124 EC embryos and 156 LC embryos were vitriﬁed in liquid nitrogen for 1 hour. Participants/materials, setting, methods: Embryos were retrieved from in vivofertilized ICR mice, 28 hours after human Chorionic Gonadotrophin injection. Two-cell embryos were categorized as EC embryos and two-pronuclear zygotes as LC embryos. Vitriﬁcation using EFS20/40 method was performed at 2-cell stage. Post-vitriﬁcation viability of EC and LC embryos was compared using chi-square test. Main results and the role of chance: The survival rate was signiﬁcantly higher in vitriﬁed EC embryos (96.8%) compared to vitriﬁed LC embryos (89.2%) (p , 0.05). A signiﬁcantly higher proportions of vitriﬁed EC embryos developed to the blastocyst stage, compared with vitriﬁed LC embryos (90% versus 57.1%, p , 0.0001). This may be related to the ability of early cleaving embryos to survive the high cooling rates, high osmotic changes and toxicity of cryoprotectant during the vitriﬁcation process. Limitations, reason for caution: The use of in vivo- instead of in vitro-derived embryos may have resulted in greater cryotolerance, as in vivo-derived embryos exhibit reduced sensitivity to chilling and freezing due to their lower lipid to protein ratio compared with in vitro-derived embryos. Wider implications of the findings: The selection of early cleaving embryos as vitriﬁcation candidates could provide better cryopreservation outcomes. This ﬁnding can subsequently contribute towards the improvement of ART outcomes and reduce the cost related to procedures in ART. Study funding/competing interest(s): This research was supported by institutional grant [600-RMI/ST/DANA5/3/Dst(335/2011)] and national grant [600-RMI/ST/FRGS5/3/FST(71/2010)]. There were no competing interests. Trial registration number: Not applicable P-197 A novel method for human oocyte vitrification with a closed device using super-cooled air S. Machac1, V. Hubinka2, M. Larman3, and M. Koudelka1 1 Reproﬁt International s.r.o., Gyneacology, Brno, Czech Republic, 2Reproﬁt International s.r.o., Embryology, Brno, Czech Republic, 3Vitrolife, Research Department, Englewood, U.S.A Abstract withdrawn by the author P-198 Is embryo quality affected by different approaches to stimulation in IVF? T. Pehlivan Budak1, O. Oliana Membrado1, E. Sevillano Martinez1, P. Wilson1, A. McClure1, and G. Nargund2 1 Create Health Clinics, IVF Laboratory, London, United Kingdom, 2Create Health Clinics, Obstetrics and Gynecology, London, United Kingdom Study question: Does stimulation protocol affect embryo quality on day 2 in different age groups? Summary answer: In patients ,40 years and ≥40 years of age, higher percentage of Grade A and B embryos were obtained from patients submitted to natural cycle IVF compared to stimulated cycles. The differences were statistically signiﬁcant in the group ≥40 years of age. What is known already: In previous studies, natural cycle approaches to stimulation in IVF have been associated with better embryo quality and IVF outcome. Study design, size, duration: This retrospective observational data included 3044 embryos assessed on day 2 of embryo development during 2011 and 2012. Embryos scored based on ASEBIR criteria were compared in patients ,40 years and ≥40 years of age whom underwent following treatment protocols: Natural cycles(NC), Modiﬁed Natural cycles(MN) and stimulated cycles (SC). Participants/materials, setting, methods: Three main groups were made: NC, MN and SC (Group 1, 2 and 3 respectively). Group 1a(,40 years, 73 embryos) and Group 1b(≥40 years, 104 embryos). Group 2a(,40 years, 167 embryos) and Group 2b(≥40 years, 392 embryos). Group 3a(,40 years, 1644 embryos ) and Group 3b (≥40 years, 743 embryos). Main results and the role of chance: In Group 1a, 65.8% were grade A and B, 34.2% were grade C and D. In Group 1b, 73.1% were grade A and B, 26.9% were grade C and D. In Group 2a, 64.1% were grade A and B, 35.9 % were grade C and D. In Group 2b, 65% were grade A and B, 35% were grade C and D. In Group 3a, 58.9% were grade A and B, 41.1% were grade C and D. In Group 3b, 62.2% were grade A and B, 37.8 were grade C and D. In general, independent of age, there was a tendency for better embryo quality in NC, and MN compared to SC. The difference was statistically signiﬁcant between groups 1b and 3b (p ¼ 0.04). Limitations, reason for caution: Due to the study design, selection bias and confounding factors may have affected the results. Wider implications of the findings: In women undergoing IVF, natural cycle approach to IVF seems to yield better quality embryos on Day 2 compared to stimulated cycles. This ﬁnding is more signiﬁcant in women with advanced maternal age . Study funding/competing interest(s): None Trial registration number: None P-199 Accumulation of vitrified oocyte: a successful strategy to achieve an elective embryo transfer in young low responders patients D. Raso1, M.F. Insua2, B. Lotti1, S. Giordana2, C. Baldi2, J. Barattini1, M. Cogorno 1, N. Fernandez Peri 1, and F. Neuspiller 1 1 Instituto Valenciano de Infertilidad, Ginecology, C.A.B.A., Argentina, 2Instituto Valenciano de Infertilidad, FIV Laboratory, C.A.B.A., Argentina Study question: To describe the results of vitriﬁed oocytes accumulation (VOA) in low responder (LR) patients as a way of achieving an elective embryo transfer. Summary answer: The pregnancy rate in low responder patients is usually suboptimal due to the low number of embryos. Vitriﬁcation techniques allow oocytes to accumulate several cycles of ovarian stimulation, increasing the possibilities of elective embryo transfer. What is known already: The low response represents a difﬁculty to the management within women who must undergo assisted reproductive treatment. Reduced Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Main results and the role of chance: 103 pregnancies were achieved for a cumulative clinical Pregnancy Rate (CCPR) of 84% per patient and 48% per cycle. These results are higher than fresh embryos transfer cycles for the center and study period (48% vs 31%). Implantation Rate (IR) was 22%. Pregnancies were achieved 57% in the ﬁrst, 29% in the second, 11% in the third and 3% in the fourth cycle of thawing per patient, respectively. Of those patients that did not achieved successful clinical pregnancies, 57 % sill have embryos vitriﬁed (4,5 embryos/ patient). Limitations, reason for caution: Descriptive measure of prospective trial. Wider implications of the findings: These results reassure the role of embryo vitriﬁcation in an IVF program, and could also be a possible approach to prevent the alleged adverse effects of ovarian hyperstimulacion on the implantation process, and it is tempting to propose its use routinely in ART cycles in the future. Study funding/competing interest(s): no Trial registration number: no i198 P-200 Cryopreserved versus fresh blastocyst transfer: a way to improve implantation rate S. Resta, A. Filannino, E. Maggi, G. Cafueri, A.P. Ferraretti, M.C. Magli, and L. Gianaroli S.I.S.M.E.R. s.r.l., Reproductive Medicine Unit, Bologna, Italy Study question: Do blastocysts belonging to the same cohort have different chances of implantation when transferred in fresh or a frozen cycles? Summary answer: Blastocysts transferred in frozen cycles seem to have higher implantation rates compared to the sibling blastocysts transferred in the fresh cycle. What is known already: Traditionally, the tendency in ART is to give priority to the fresh cycle by selecting the best quality embryos for transfer. Several reports support the culture to the blastocyst stage with the aim of improving the selection criteria. More recently, in view of the high efﬁciency of vitriﬁcation, the advantages of transferring warmed blastocysts have been underlined. Actually, the rates of pregnancy and implantation seem to be increased compared with fresh blastocyst transfer cycles. Study design, size, duration: Between 2008 and 2012, 437 blastocysts transfer were performed (309 fresh, 128 frozen). A subset of 36 patients who did not achieve term pregnancy in fresh cycles and had spare cryopreserved blastocysts were included in the study for a total of 36 fresh transfers and 38 cryopreserved transfers. Participants/materials, setting, methods: Private center. Blastocyst development and quality, and implantation were evaluated in 36 patients that transferred at the blastocyst stage in the fresh cycle and cryopreserved one or more spare blastocysts for further attempts. The studied parameters were compared between fresh and cryopreserved cycles. Main results and the role of chance: In all, 136 blastocysts were obtained (73% blastocyst rate over 185 fertilized oocytes). As priority was given to fresh transfers, 35 patients (97%) transferred their best quality blastocysts (all day-5 blastocysts) in the fresh cycle (1.7 + 0.5 mean embryos transferred) with a pregnancy rate of 14% (5/36) and an implantation rate of 6% (4/62). After the transfer of the cryopreserved blastocysts in subsequent 38 cycles (1.3 + 0.4 mean embryos transferred), the pregnancy rate was 40% per transfer (15/38, P , 0.05) and 42% per patient (15/36, P , 0.025). The implantation rate of 31% (15/48) was signiﬁcantly higher when compared to fresh cycles (P , 0.005). In the group of frozen cycles, 29 transferred day-5 blastocysts and 9 day-6 blastocysts with implantation rates of 29.7% and 36.4% respectively. Limitations, reason for caution: An extremely efﬁcient cryopreservation program is necessary to minimize the possible damage to blastocysts during the procedure. The results reported here regard a special group of patients that did not achieve a term pregnancy in the fresh cycle, and should be veriﬁed on a larger set of data. Wider implications of the findings: The high implantation rate after the transfer of spare blastocysts in frozen cycles suggests an improved endometrial receptivity, especially when considering that the best quality blastocysts were selected for fresh transfers. Transfers in frozen cycles have the additional advantages of 1) reducing the risk of OHSS, 2) increasing the cumulative pregnancy rate due to the higher implantation, and 3) providing the better obstetric and perinatal outcome that are associated with the transfer of thawed embryos. Study funding/competing interest(s): None. Trial registration number: Not applicable. P-201 Vitrification of cleavage and morula stage mouse embryos with DMSO/ EG and PROH/ EG: effects on blastomere viability, cytoskeleton and development A. Sioga1, Z. Oikonomou1, K. Chatzimeletiou2, L. Oikonomou1, E. Kolibianakis2, and B.C. Tarlatzis2 1 Aristotle University Medical School, Lab. of Histology - Embryology, Thessaloniki, Greece, 2Aristotle University Medical School, 1st Department of Obstetrics and Gynaecology Papageorgiou Hospital, Thessaloniki, Greece Study question: Is there a difference in blastomere viability, cytoskeletal abnormalities and development of cleavage and morula stage mouse embryos vitriﬁed with a DMSO/EG kit compared with those vitriﬁed with a PROH/EG kit and fresh embryos? Summary answer: There was no signiﬁcant difference in blastomere viability, cytoskeletal abnormalities and development between the DMSO/EG and the PROH/EG groups but fresh embryos showed a lower incidence of spindle abnormalities compared to the vitriﬁed groups. What is known already: Morula and blastocyst stage vitriﬁcation has successfully been applied in human and mouse embryos. Most studies assess feasibility of the methodology based on survival rates and development and only limited studies in human blastocysts report on the incidence of cytoskeletal abnormalities. Here, we investigate in the mouse immediate effects on blastomere viability following staining with viability markers and any potential effects on the cytoskeleton, by analysing spindle and chromosome conﬁgurations by confocal scanning microscopy. Study design, size, duration: This study was performed between January 2011-January 2013 in an Embryology Laboratory. 280 embryos were ﬂushed out of BalbC female mice oviducts. 215 were vitriﬁed with DMSO/EG (n¼ 110) and PROH/EG (n¼ 105) and 65 were used fresh for comparison of Immediate effects (n ¼ 15(4-6cell),n ¼ 15(8-cell),n ¼ 15morulae) and cytoskeleta analysis, n ¼ 20 blastocysts). Participants/materials, setting, methods: 50 DMSO/EG (n ¼ 15(4-6 cell), n ¼ 18(8-cell), n ¼ 17morulae) and 45 PROH/EG (n ¼ 15(4-6 cell), n ¼ 15(8-cell), n ¼ 15morulae) vitriﬁed embryos were subjected to viability CFSE/ PI staining. 60 DMSO/EG (n ¼ 20(4-6 cell), n ¼ 20(8-cell), n ¼ 20morulae) and 60 PROH/ EG (n ¼ 20(4-6 cell), n ¼ 20(8-cell), and n ¼ 20morulae) vitriﬁed embryos were cultured to the blastocyst stage and stained with a-tubulin/DAPI. Main results and the role of chance: This is the ﬁrst study to examine in detail using viability markers the immediate effects of DMSO/EG and PROH/EG vitriﬁcation on blastomere survival and compares the type and incidence of cytoskeletal abnormalities in vitriﬁed cleavage and morula stage embryos with fresh Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 number of oocytes obtained in these patients is usually associated to poor egg quality, low number of embryos and high rate of cancellation of their cycles. The pregnancy rate in this group of patients is usually suboptimal due to the inability to perform an elective embryo transfer. Study design, size, duration: We carried out an observational, retrospective, comparative and descriptive study. During the period from January 1st 2011 to July 31st 2012, 705 assisted reproduction cycles were performed, 20% (140) out of which behaved as low responders. LR was deﬁned as those patients who obtained ≤4 oocytes after puncture. Participants/materials, setting, methods: VOA was offered to 87 patients (22 accepted). Patients were divided in two groups: ≤39 years and ≥40 years. Each group was also divided in those that did not accumulate oocytes (A), and those who did (B). Stimulation was performed with clomiphene citrate and hMG. Oocytes were cryopreserved (Cryotop method). Main results and the role of chance: Average age was 36.4 [31-43]. Average cycles/ patient were 2.2 + 0.4. Main retrieved oocytes/aspiration were 4.1 + 2.1; average MII was 2.9 + 1.3; main retrieved oocytes/patient were 9.1 + 4.3 and main retrieved MII 6.4 + 2.1. Oocytes survival rate was 81.8%. Global fertilization rate (FR) was 62.1%. Pregnancy rate (PR)/transfer was 34.7% and PR/ patient was 36.3%. Women ,40 years of GroupB exhibit signiﬁcantly better results than their peers in GroupA. Elective embryo transfer was 50% vs. 25%, PR 33.3% vs 15% and the implantation rate (IR) 25% vs 8.3%, respectively. The main age was 37.2 + 1.74 vs 35.81 + 2.46. In patients ≥40 years, results for GroupA and GroupB elective embryo transfer were 28.6% vs 23.5%, PR 14.3% vs 18.8%, IR 14.3% vs 11.4%. The main age for these sub groups was 41.9 + 1.5 vs 41.9 + 1.4. Limitations, reason for caution: None Wider implications of the findings: Oocyte vitriﬁcation is a useful technique to accumulate oocytes of different stimulation cycles, increasing the initial pool. According to our results, in women ,40 years, this procedure signiﬁcantly improves the possibility of an elective transfer, impacting positively on pregnancy rates. For ≥40 years, this strategy would not provide signiﬁcant beneﬁts when compared with fresh cycles. This could be due to oocyte ageing, which in turn impacts negatively on reproductive outcomes. Study funding/competing interest(s): None Trial registration number: None Abstracts i199 Abstracts embryos. There was no signiﬁcant difference in the survival rates following warming between the two vitriﬁed groups at all stages (p . 0.05). Cytoskeletal analysis revealed that fresh embryos that had developed to the blastocyst stage had a signiﬁcantly higher incidence of normal spindles (72.2%) and lower incidence of abnormally shaped (22.2%) or multipolar spindles (2.8%) compared to both DMSO/EG and PROH/EG vitriﬁed embryos (p,0.05). The level of abnormal spindle/chromosome conﬁgurations including abnormal shape, and multipolarity, was similar between the two vitriﬁed groups (p.0.05). Limitations, reason for caution: Mouse embryos may differ from human embryos in the way they survive post-warming. Wider implications of the findings: The results presented in this study document high survival rates post warming and no major immediate effects following staining with viability markers. This suggests that vitriﬁcation with both the DMSO/EG and PROH/EG kits at cleavage and morula stages is a safe procedure that can be applied in routine clinical IVF practice. Study funding/competing interest(s): No external funding was raised for this study. Trial registration number: Not applicable Impact of oxygen concentration on early human embryo development in vitro M. Roychoudhury Sarkar1, D. Ray 1, and J. Bhattacharya2 1 A.H IVF & Infertility Research Centre Pvt Ltd, Embryology, Kolkata, India, 2A.H IVF & Infertility Research Centre Pvt Ltd, IVF Specialist, Kolkata, India Study question: How do the oxygen concentration affect early human embryo development in - vitro from fertilized oocytes to blastocyst? Summary answer: Result in this study demonstrated that embryo culture in higher oxygen concentration reduce the human embryo developmental rate in vitro. What is known already: Early embryo development in-vitro is not only dependent on the culture media but also upon the physical environment of incubator such as concentration of CO2 and O2 gas.Studies on mammalian embryo culture have demonstrated that culture at reduced O2 results in better embryo development. However, it has also been observed that embryo can also grow in elevated O2 level, this has led to some confusion regarding the role of oxygen in human embryo culture. Study design, size, duration: Retrospective study. Embryos cultured in 3 different groups:- Group-I: Fertilisation upto blastocyst in 20% Oxygen, Group-II: Fertilisation(day O) in 20% Oxygen, followed by (day 1 to day 5)of embryo culture in 5% Oxygen and Group-III: Fertilisation upto blastocyst in 5% Oxygen. Only (PN2) oocytes were scored from (day 1-5). Participants/materials, setting, methods: Patients selected with a criteria of ≥ 35 years of age, devoid of endometriosis and PID and ≥ 8 oocytes after IVF / ICSI. Group-I: 60 patients with 421 normally fertilized oocytes. Group-II: 40 patients with 289 normally fertilized oocytes and Group-III: 45 patients with 311 normally fertilized oocytes. Main results and the role of chance: Result in this study shows that proportion of development of 2-celled, 4-celled embryo are almost similar in the three groups and from this stage, embryo development is signiﬁcantly reduced in group-I compared to that of group-II and group-III. Therefore, percentages of compact embryo and blastocyst development in the 3 groups are as follows: Group-I: 45.1% and 39.9% Group-II: 63.4% and 57.09% and Group-III: 65.8% and 59.16% respectively.We observed that there was no signiﬁcant differences in any parameters in group-II and group-III. Limitations, reason for caution: Care was taken to ensure that other parameters such as culture time and CO2 (6%) were kept constant. Wider implications of the findings: Signiﬁcant dip in the growth rate is observed after day-3. The delay in the late cleavage stage is probably due to maturation arrest of embryos cultured at 20% O2 before reaching the blastocyst stage. Study funding/competing interest(s): NA Trial registration number: NA P-203 Randomised assisted localised hatching in cryopreserved blastocyst transfers J. Marcos Alises1, D. Gumbao1, A. Sánchez-León1, B. Amorocho1, M. Mollá1, M. Nicolás2, L. Fernández2, and J. Landeras2 1 IVI Murcia, Infertility, Murcia, Spain, 2IVI Murcia, Gynecologist, Murcia, Spain AH TRP N 72 76 Nº transfer 68 76 Age 37 38 p Average nº embryo transfers 1.6 1.5 Pregnancy % 51.4 (35/68) 42.1 (32/76) 0.26 Implantation % 36.4 (39/107) 34.5 (39/113) 0.77 Biochemical miscarriage % 16.7 (7/42) 17.9 (7/39) 0.88 Clinical miscarriage % 17.1 (6/35) 15.6 (5/32) 0.87 Study question: The aim of this work was to evaluate if there was any difference when the assisted hatching (AH) was carried out at the level of the inner cell mass (ICM) or the trophoectoderm (TRP) Summary answer: The results appear to point towards a favourable trend in applying assisted hatching techniques at the level of the ICM and may explain the role played by the ICM in the embryo hatching process What is known already: In order for implantation to take place, the blastocyst must be hatched from the zona pellucida (ZP). In fact, different ART techniques try to simulate this process in vitro, a process which is known as ‘Assisted Hatching’ (AH). Despite the reported results, the effectiveness of AH dose not seem entirely clear Study design, size, duration: Prospectively, 148 treatments from frozen-thawed day-ﬁve blastocysts were randomly selected in the period since 2010 to undergo AH treatment in the area corresponding to the ICM or TRP Participants/materials, setting, methods: Blastocysts were vitriﬁed-devitriﬁed using Kuwayama method and the laser-zona-thining technique was carried out randomly in the area corresponding to the ICM or TRP after their re–expansion. Clinical outcomes were analysed computationally and t-student signiﬁcance was deﬁned when p , 0.05 Main results and the role of chance: In the clinical outcomes no statistic differences were observed when either MCI or TRP assisted hatching was performed . However a small trend in the pregnancy rate was shown (table 1)Table 1 Limitations, reason for caution: This study could be limited by the number or treatments and our vitriﬁcation system so further prospective and randomised studies must be necessary in order to provide new evidence Wider implications of the findings: Although no statistically signiﬁcant differences were observed, the pregnancy rate results appear to point towards a favourable trend in applying assisted hatching techniques at the level of the inner cell mass. These results would be in agreement with the results obtained by other groups that also defend the role played by the ICM in the embryo hatching process, but further studies will be necessary in order to provide proof. Study funding/competing interest(s): This study was supported by IVI Murcia SL. Murcia. Spain Trial registration number: No trial registration number P-204 Does gender affect pre-implantation embryonic developmental rates?- looking back at the baby pictures S. Duffy1, A. Campbell2, S. Montgomery1, C.F.L. Hickman3, and S. Fishel2 CARE Fertility, Embryology, Manchester, United Kingdom, 2CARE Fertility, Embryology, Nottingham, United Kingdom, 3Mouwasat CARE Fertility, Embryology, Damamm, Saudi Arabia 1 Study question: The study objective was to establish whether time-lapse derived morphokinetic variables differ between male and female embryos. Summary answer: Only 1 out of 11 morphokinetic variables studied showed a signiﬁcant difference according to embryo gender. A trend for male embryos to develop at a faster rate up to 8 cells, take less time to achieve full compaction, but to blastulate and expand at a slower rate was observed. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 P-202 AH ICM ........................................................................................ i200 P-205 Bovine embryo development in vitro: a sensitive assay to evaluate plga peg nanoparticle toxicity I. Fiorentino1, R. Gualtieri1, V. Barbato 1, S. Braun1, V. Mollo2, P. Netti2, and R. Talevi1 1 University of Naples Federico II, structural and functional biology, Naples, Italy, 2 University of Naples Federico II, Materials and Production Engineering, Naples, Italy Study question: The heterogeneity of nanoparticles (NPs) developed for biomedical use emphasizes the need of sensitive toxicity tests. Data on somatic cells were controversial and only a few studies were done on extremely sensitive processes such as embryo development. Summary answer: Biodegradable 65nm rhodaminated poly(d,l-lactic-co-glycolic acid)-block-polyethylene glycol (PLGA-PEG) NPs exert a cytotoxic effect on embryo development, directly affecting 8 cell embryo (day 3) and blastocyst (day 8) rates, in a dose-dependent manner. What is known already: Most studies were performed on somatic cells. NPs, depending on their chemical and physical features, may promote sperm apoptosis and ROS production in a dose and size-dependent manner. NPs may also impair folliculogenesis in mouse. Toxicity assays on zebraﬁsh indicate that NPs may damage embryo development, but no data are available in mammals. Study design, size, duration: The study was performed on 594 oocytes. Cleavage and 8 cell embryo rates were scored at day 3, blastocyst rates at day 8. Moreover, NP internalization, blastocyst mean cell numbers (MCN) and percentage of nuclei with fragmented DNA were assessed by confocal and transmission electron microscopy (TEM). Participants/materials, setting, methods: Fertilized oocytes treated with NPs (10 or 50 mg/mL) or vehicle for 8 days, were assessed for cleavage and 8 cell embryos (day +3) and blastocyst rates (day +8). Blastocyst MCN was analyzed through labelling with Hoechst 33342; DNA fragmentation was investigated through TUNEL assay and NPs internalization by TEM. Main results and the role of chance: NPs treatment did not affect cleavage competence at day 3 after insemination. NPs at 50ug/ml reduced 8 cell embryo and blastocyst rates (treated vs control: 8 cell, 40 vs 60%, P , 0,05; blastocyst, 34 vs 46,6%, P , 0,05) but MCN and DNA fragmentation were not inﬂuenced (treated vs control: MCN, 134+ 40 vs 132 + 65; DNA fragmentation, 7,06%+ 3,44 vs 7,09% + 4,14). The confocal z-sectioning showed that PLGA/PEG-NPs were efﬁciently internalized by embryos and at higher magniﬁcation their localization was cytoplasmic and particulated. In agreement with confocal data, TEM analysis showed NPs localized in cytoplasmic vacuoles and vesicles. Limitations, reason for caution: NPs behavior toward biological systems and their toxic effects are not fully understood. Contrasting ﬁnding could depend on the chemical-physical nature of NPs, the variety of cell types and experimental conditions used in different studies. Wider implications of the findings: In conclusion, PLGA-PEG-NPs exert a cytotoxic effect on embryo development in a dose-dependent manner. In vitro embryo development in animal models may represent a sensitive and predictive assay of NP toxicity before their application in biomedicine. Study funding/competing interest(s): Department of Biology, Department of Materials and Production Engineering Trial registration number: none P-206 The relationship of morphokinetic events and euploidy in human cleavage-stage embryos A. Bayram1, N. Findikli2, M. Serdarogullari1, O. Sahin1, U. Ulug3, S.B. Tosun3, and M. Bahceci4 1 Bahceci Umut IVF Center, Embryology, Istanbul, Turkey, 2Bahceci Fulya IVF Center, Embryology, Istanbul, Turkey, 3Bahceci Umut IVF Center, Clinical Department, Istanbul, Turkey, 4Bahceci Fulya IVF Center, Clinical Department, Istanbul, Turkey Study question: This study questions whether there is a link between early morphokinetic parameters and aneuploidy/euploidy status of human embryos in preimplantation genetic screening (PGS) cycles. Summary answer: Our results show that embryos that are eligible for embryo biopsy in PGS cycles reveal similar cleavage proﬁles irrespective of the euploidy status. The possible use of time lapse technology in PGS cycles in order to pinpoint euploidy status of embryos may therefore require larger sample size and a different study design that includes post compaction follow-up and analysis until blastocyst stage. What is known already: Recent studies indicate that embryos carrying chromosomal abnormalities can show a variety of cleavage disturbances during preimplantation development. However, current literature correlating the developmental competence and euploidy status of human embryos are mostly performed on static embryo culture hence data regarding early cleavage division characteristics of such embryos are very limited. Study design, size, duration: Embryos obtained from 43 PGS cycles in which chromosomes 13, 15, 16, 17, 18, 21, 22, X and Y were analyzed by ﬂuorescent in situ hybridization have been monitored in a special tri-gas incubator with built-in time-lapse monitoring system. The morphokinetic characteristics of each embryo were traced until day 3 of embryo development. All embryo biopsy procedures were performed on day 3. The data consisted of embryos and PGS results of patients that were admitted in our clinics between March 2011-December 2012. Participants/materials, setting, methods: Morphokinetic parameters of 254 biopsied day 3 embryos were retrospectively analyzed and grouped according to the chromosomal status after PGS as euploid (normal) or aneuploid (including monosomies, trisomies and complex abnormalities). Grouped data were than compared for each time points (T2 through T8) as well as the duration between these cleavages such as S2, S3 and cc2. Data has been analyzed with Student’s t test and a P-value less than 0.05 has been considered statistically signiﬁcant. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 What is known already: No studies published to-date using time lapse cite a relationship between embryo morphokinetics and offspring gender. Alfarawati et al (2011) discussed a possible connection between blastocyst morphology and embryo gender when considering a correlation between embryo morphology and chromosomal status. Results of their study demonstrated that male embryos achieved full blastulation at a faster rate than female embryos and a signiﬁcantly increased number of male embryos were of a higher grade than the female cohort. Study design, size, duration: This observational study included the retrospective analysis of the morphokinetic parameters of 29 ICSI embryos following embryo transfer and live birth. These embryos had undergone time-lapse imaging in the EmbryoscopeTM (Unisense Fertilitech, Denmark) for between 3 and 5 days before being selected for embryo transfer. Participants/materials, setting, methods: Patient data was obtained from 58 fresh ICSI cycles that had utilized EmbryoScopeTM culture in a private IVF clinic setting and had achieved a live birth. Only embryo transfers that resulted in 100% implantation were included in the data analysis to ensure complete accuracy of embryo gender. Main results and the role of chance: The time-points in hours post insemination (hpi) of the ﬁrst cleavage (t2) up to the 9+ cell stage (t9+) and the time to reach morula (tM), full blastocyst (tB) and expanded blastocyst (tEB) were recorded for embryos of known gender. The mean time-points for male and female embryos respectively were; (t2) 23.01 vs 24.2, (t3) 33.54 vs 35.14, (t4) 35.41 vs 36.77, (t5) 45.44 vs 47.61, (t6) 47.34 vs 49.94, (t7) 50.72 vs 54.02, (t8) 53.47 vs 55.07, (t9+) 62.17 vs 59.71, (tM) 73.96 vs 77.42, (tB) 102.08 vs 101.37, (tEB) 110.65 vs 107.71. A two sample t-test was performed to statistically compare the mean time-points of known gender embryos using a p value ,0.05. Statistical signiﬁcance was observed only at t7( p ¼ 0.037). Limitations, reason for caution: Data set size is a limiting factor, making reliable statistical analyses difﬁcult. Morphokinetic analyses of known gender embryos is ongoing as more live births occur, which, in addition to the continual availability of time lapse in this clinic setting, will allow analysis of a larger data set to be presented. Wider implications of the findings: The concept of differing morphokinetic values according to embryo gender is of scientiﬁc interest. The results of our preliminary analysis, disagree with recent publications, but this may alter as the data set increases. The morphokinetic differences between the known gender embryos, although too subtle to be observed in standard incubation, may be observed in embryos cultured in other time lapse systems. Study funding/competing interest(s): N/A Trial registration number: N/A Abstracts i201 Abstracts Euploid Monosomy Trisomy Complex Abnormality ............................................................................................................................................................................................... T2 27,03 + 3,58 (45) 27,41 + 3,42 (56) 26,82 + 4,069 (46) T3 37,21 + 5,085 (45) 37,22 + 5,07 (56) 37,41 + 4,767 (46) 28,044 + 4,038 (89) 35,73 + 5,59 (89) T4 39,99 + 4,50 (45) 40,23 + 6,468 (56) 39,77 + 5,479 (46) 39,22 + 5,95 (89) T5 50,8 + 7,44 (44) 49,24 + 7,28 (54) 49,07 + 6,78 (43) 46,251 + 8,138 (86) T6 53,6 + 6,058 (43) 51,48 + 7,40 (53) 52,16 + 6,393 (42) 50,128 + 7,467 (81) T7 55,48 + 5,45 (40) 54,54 + 6,30 (46) 54,92 + 5,427 (38) 53,343 + 7,296 (74) T8 56,75 + 5,89 (35) 56,11 + 6,31 (39) 56,94 + 5,531 (35) 55,149 + 6,685 (63) 10,17 + 4,024 (45) 9,59 + 4,98 (56) 2,78 + 3,764 (45) 3,001 + 4,37 (56) 2,356 + 3,717 (46) 3,537 + 5,096 (88) S3 (t8-t5) 6,93 + 5,81 (35) 8,423 + 6,28 (39) 8,448 + 6,28 (35) 9,628 + 6,502 (63) Main results and the role of chance: Morphokinetic parameters of 45 euploid and 209 aneuploid embryos carrying single or complex chromosomal abnormalities shows similar cleavage time characteristics on each time points and parameters analyzed as shown in the Table. This may indicate that although they have different chromosome constitutions, embryos that are selected for embryo biopsy with a certain selection criteria are indifferent in their cell division kinetics until day 3 embryo development. p . 0.05 for all the parameters compared among groups. Limitations, reason for caution: Our study includes only embryos that are suitable for biopsy hence the possible correlation for embryos with developmental abnormalities could not be assessed in this setting. Data including extended embryo culture parameters as well as reanalysis of PGS results on day 5 could also contribute more to our study. Wider implications of the findings: Our results can indicate that morphokinetic parameters on early developmental stages fail to indicate any distinct chromosomal abnormality pattern hence cannot be used as an additional indicator for aneuploidy in good quality cleavage stage embryos. Study funding/competing interest(s): This study received no funding and there are no conﬂicts of interests to be declared. Trial registration number: This study was not an RCT and therefore there is no registration number. 10,593 + 3,46 (46) 7,928 + 5,218 (88) Study design, size, duration: Three experiments were designed between 2010 and 2012. In the ﬁrst experiment 86 treatments from our oocyte donation program were analyzed. Furthermore, 28 metaphase II matured oocytes remainig from donor treatments were studied morphometrically. In the third experiment oocyte hardening was assessed in 47 fresh vs 37 vitriﬁed oocytes Participants/materials, setting, methods: The metaphase II matured oocytes were vitriﬁed-devitriﬁed using the Kuwayama method and the clinical outcomes treatments were studied depending on oocyte origin.The morphometric measurements were performed using an image capture software and to study the ZP hardening the time of digestion mediated by an acidiﬁc tyrodes solution was evaluated Main results and the role of chance: No clinical otucomes differences were observed when different oocytes were used (Table 1). Focusing on the morphometry, no differences were found (Table 2). when oocyte hardening was studied no differences were observed in fresh vs vitriﬁed oocytes (23.1 sec vs 19.8 sec; p ¼ 0.18 ). Statistical differences were analysed computationally and t-student signiﬁcance was deﬁned when P , 0.05.Table 1 Table 2 Vitrified oocytes Fresh oocytes p ......................................................................................... P-207 Vitrification effect on the oocyte morphometry and the hardening of zona pellucida Age 40.4 39.8 Fertilization % 80.5 77.9 0.34 A. Sanchez Leon1, D. Gumbao 1, J. Marcos 1, M. Mollá1, B. Amorocho1, M. Nicolás2, L. Fernández2, and J. Landeras2 1 IVI Murcia, IVF Laboratory, Murcia, Spain, 2IVI Murcia, Gynecologist, Murcia, Spain Clinical pregnancy % 77.8 66.0 0.27 Implantation % 56.5 55.8 0.94 Biochemical % 16 5.4 0.21 Study question: The aim of this study was to focus on the possible morphological and developmental alterations in our oocyte vitriﬁcation system, paying particular attention to the oocyte zona pellucida (ZP) and its morphological peculiarities, whilst studying different oocyte morphometric measurements and the possible hardening effect associated with the cryopreservation process Summary answer: We may conclude that in our oocyte vitriﬁcation system no negative effect was evidenced on the embryo clinical outcomes when either fresh or vitriﬁed oocytes were used and neither morphometric nor ZP hardening changes after vitriﬁcation were caused What is known already: The ability to cryopreserve human oocytes confers signiﬁcant advantages in IVF-ICSI cycles, not only for the improvement of clinical results but also when considering ethical and legal aspects. Since the ﬁrst human vitriﬁcation studies different alterations have been reported and it is clear that all oocytes and embryos could suffer considerable morphological and functional damage during the cryopreservation process that may affect in vitro embryo development and IVF results. Clinical miscarriage % 19 11.4 0.24 Vitrified oocytes Fresh oocytes p ......................................................................................... ZP diameter (mm) Oocyte diameter (mm) 19.4 163 19.1 162 0.91 0.74 Ooleme diameter (mm) 113 112 0.89 Polar body size (mm2) 332.2 355.1 0.57 Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 cc2 (t3-t2) S2 (t4-t3) i202 Abstracts Limitations, reason for caution: These studies could be limited to our vitriﬁcation system and because of the results obtained further prospective and randomised studies must be necessary in order to provide new evidence Wider implications of the findings: The results show that, in our oocyte vitriﬁcation system, no negative effect was seen in embryo development when either fresh or vitriﬁed oocytes were used. So we may conclude that the vitriﬁcation method works well adn also no morphometric changes after vitriﬁcation were found. On the other hand, the hardening associated process was discarted in order to know if any hatching assisted method would be necessary to use Study funding/competing interest(s): This study was supported by IVI Murcia SL. Murcia. Spain Trial registration number: No trial registration number The influence of sequential scoring in determining the best embryo classification for transfer when not using time lapse technology M.C.A. Cardoso1, A.P.S. Aguiar1, C. Sartorio2, A. Evangelista2, P. Gallo-Sá2, and M.C. Erthal-Martins2 1 Vida Centro de Fertilidade da Rede D’Or, IVF Laboratory, Rio de Janeiro, Brazil, 2Vida Centro de Fertilidade da Rede D’Or, Clinic, Rio de Janeiro, Brazil Study question: What are the beneﬁts of removing the embryos out of the incubator on day 2 (D2) to fulﬁll the classiﬁcation scores for the before transfer? Summary answer: The ASEBIR morphological classiﬁcation assesses embryo evolution from D2 to day 3 (D3): similar embryos can be graded differently on D3 depending on classiﬁcation they had on D2. On the other hand, taking the embryos out of the incubator increases stress on them, probably decreasing their chance in implanting. What is known already: Embryo morphology classiﬁcation is still an important tool of selection for transfer, no matter if the transfer is on day 2, day 3 or blastocyst. The ASEBIR embryo morphological classiﬁcation takes in account the embryo evolution from day 2 to day 3. This is the method for embryo classiﬁcation which best correlated with the pregnancy outcomes in our clinic and it was the chosen method, since 2010. Study design, size, duration: Cases were randomly selected to be either classiﬁed or not on D2. PGD cases, D2 transfers, oocyte donation and when all the embryos were vitriﬁed (no fresh transfer) were not included. A total of 112 IVF cycles were analyzed. The study was conducted from April to December of 2012. Participants/materials, setting, methods: The study groups were divided in two: Group 1 ¼ D2 + D3 scoring and Group 2 ¼ D3 only scoring. Clinical pregnancy and implantation rates were compared between groups. In Group 1, a blind reclassiﬁcation was made as if the D2 assessment had not been done. Main results and the role of chance: Group 1 had 59 cycles and Group 2, 53 cycles. The groups were similar among patients age (34,3 and 35 years). The pregnancy rate and implantation rate were 22.0% and 15.5% for Group 1, and 34.0% and 24.5% for Group 2 (p ¼ 0.15 and p ¼ 0.09 respectively). Group 1 had 116 embryos transferred in which 40.5% were grade A, 26.7% grade B, 19.0% grade C and 13.8% grade D. Group 2 had 101 embryos transferred in which in which 69.6% were grade A, 17.7% grade B, 7.8% grade C and 4.9% grade D. In Group 1, if the classiﬁcation had been done only in D3, the score would be different (higher) in 11.2% of embryos transferred. Limitations, reason for caution: When assessments were made only on Day 3, it was presumed that the embryos had the best score on Day 2, which falsely increases the scores of some embryos. Wider implications of the findings: There was no signiﬁcant difference between Groups 1 and 2 in pregnancy and implantation rates, but the validity of considering the embryo development proﬁle until the day of transfer, while not having a timelapse incubator, is still questionable. Subtracting one day of observation diminishes exposure of the embryos. The loss in accuracy on embryo grading affects only a few percentage of them, not impairing their implantation potential. Study funding/competing interest(s): This study was conducted in a private clinic without third party sponsorship. Trial registration number: None P-209 embryos Factors affecting the transcriptome of human preimplantation Study question: What is the relative effect of common environmental and biological factors on the transcriptome changes during human preimplantation development? Summary answer: Developmental stage and maternal age have a larger effect on the global gene expression proﬁle of human preimplantation embryos than the culture medium or oxygen concentration used in in-vitro culture. What is known already: Studies on mouse and bovine embryos have shown that different conditions in the in-vitro culture of embryos can lead to changes in transcriptome proﬁles. For humans this is not yet known. An effect of developmental stage on the transcriptome proﬁle of embryos has been demonstrated, but studies on the effect of maternal age or various culture conditions are lacking. Study design, size, duration: Human preimplantation embryos were randomised to two culture-media (G5-medium or HTF-medium) and to two oxygen concentrations (5% or 20%), with stratiﬁcation for maternal age. Next to these variables, developmental stage after culture was also taken into account in the transcriptome analysis. Participants/materials, setting, methods: After thawing, donated human embryos that were cryopreserved on day four, were cultured for two days under the randomized conditions (N¼ 89). Only embryos developing to morula or blastocyst stage (N ¼ 39) were assessed for genome-wide gene expression using microarrays and a regression model was built to select the contributing factors. Main results and the role of chance: Based on the number of differentially expressed genes (DEGs), developmental stage (3,519 DEGs) and maternal age (1,258 DEGs) had a larger effect on the global gene expression proﬁle of human preimplantation embryos than either tested culture medium (596 DEGs) or oxygen concentration (492 DEGs) used during in-vitro culture. Interactions between the factors were found, indicating that culture conditions might have a different effect depending on the developmental stage or the maternal age of the embryos. Affected pathways included metabolism, cell cycle processes and oxidative phosphorylation. Limitations, reason for caution: Culture of embryos for only two days might have limited the effect on global gene expression by the investigated culture conditions. Earlier stages of development (day 0 until day 4) were not analyzed and might respond differently to the experimental conditions. Wider implications of the findings: Our results show that when studying gene expression in single human preimplantation embryos under various experimental conditions, one should take into account the confounding effect of biological variables, such as developmental stage and maternal age. This makes these experiments different from gene expression experiments where these variables can be tightly controlled, for example when using cell lines. Study funding/competing interest(s): This study received no external funding and there were no competing interests. Trial registration number: None P-210 Can completion of compaction predict implantation outcome? E. Power, S. Montgomery, S. Duffy, K. Jordan, A. Campbell, and S. Fishel CARE Fertility, Manchester embryology, Manchester, United Kingdom Study question: If cellular material is excluded from the embryo at the stage of compaction (deﬁned as incomplete compaction), does this affect the ability of the embryo to implant successfully? Summary answer: Signiﬁcantly more embryos where compaction was incomplete failed to implant, compared to embryos with complete compaction (p ¼ 0.033). What is known already: There is evidence with conventional microscopy showing snap shots of embryo development, that incomplete compaction may be related to the ability to form a blastocyst (Ivec et. al.). Time lapse technology Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 P-208 E. Mantikou1, M.J. Jonker2, M. de Jong2, K.M. Wong1, A.P.A. van Montfoort3, T.M. Breit2, S. Repping1, and S. Mastenbroek1 1 Center for Reproductive Medicine, Academic Medical Center University of Amsterdam, Amsterdam, The Netherlands, 2Swammerdam Institute for Life Sciences Faculty of Science (FNWI) University of Amsterdam, MicroArray Department and Integrative Bioinformatics Unit (MAD-IBU), Amsterdam, The Netherlands, 3Maastricht University Medical Center, Obstetrics and Gynaecology, Maastricht, The Netherlands i203 Abstracts P-211 Vitrification/warming before embryo biopsy can positively contribute to clinical outcome in Preimplantation Genetic Screening cases N. Findikli1, T. Aksoy1, M. Gultomruk1, A. Aktan2, C. Goktas1, U. Ulug3, and M. Bahceci2 1 Bahceci Fulya IVF Centre, Embryology Laboratory, Istanbul, Turkey, 2Bahceci Fulya IVF Centre, Clinical Department, Istanbul, Turkey, 3Bahceci Umut IVF Centre, Clinical Department, Istanbul, Turkey Study question: Does vitriﬁcation/warming before embryo biopsy have any effect or additional role in cases where Preimplantation Genetic Screening (PGS) is performed? Summary answer: The use of vitriﬁed/warmed cleavage stage embryos before biopsy can positively contribute to cycle outcome compared to cases in which only embryos obtained in fresh cycles are biopsied. What is known already: Numerous studies report that cryopreserving embryos following biopsy for PGS may affect the clinical outcome. However, the data about the efﬁciency of vitriﬁcation/warming of cleavage stage embryos before biopsy for PGS and subsequent embryo transfer are scarce and contains limited data. Study design, size, duration: This retrospective cohort study includes 222 patients undergoing a total of 231 PGS cycles performed for numerical and structural chromosomal abnormalities between August 2010 – December 2012 in Fulya and Umut Bahceci Assisted Reproductive Technology Centers. Participants/materials, setting, methods: PGS cycles that were included in this study were grouped into three according to the nature of biopsied embryos: Group 1 includes 110 PGS cycles in which 792 fresh embryos were biopsied, group 2 consists of 38 PGS cycles in which a total of 350 fresh and vitriﬁed/warmed embryos were biopsied and group 3 includes 83 PGS cycles that utilized only vitriﬁed/ warmed embryos (803 embryos biopsied). Upon availability of chromosomally competent embryos, cycles in group 1 and group 2 proceeded with fresh embryo transfers, embryos in 3 were transferred in frozen embryo transfer setting with proper endometrial preparation. Main results and the role of chance: A total of 1945 embryos (fresh and vitriﬁed/ warmed) were biopsied and an informative PGS result were obtained in 1903/1945 (97.8%). Depending on the availability of chromosomally normal embryos, 71 cycles in group 1 (65%), 25 cycles in group 2 (66%) and 65 cycles in group 3 (79%) reached embryo transfer stage. Average number of embryos transferred in each group was 1.36, 1.52 and 1.46 respectively. Likewise, clinical pregnancy rates in group 1, 2 and 3 were 38%, 48%, 53.8% and implantation rates were found to be 29%, 34% and 41%, respectively. In group 2 and 3, a total of 1060 embryos were warmed in which 952 were utilized for embryo biopsy on day 3 of embryo development (90.8%). Limitations, reason for caution: This study includes cases in which ﬂuorescent in situ hybridisation (FISH)-based PGS were performed for 5, 7 or 9 chromosomes, respectively. Clinical outcome can be improved if array comparative genomic hybridization (aCGH) or similar technologies can be utilized in the same approach. Wider implications of the findings: This study may be generalized in PGS cases in which the number of embryos in a fresh cycle is very limited and there is a high probability of not ﬁnding a chromosomally normal embryo after analysis. In such cases, embryos of consecutive IVF cycles can be vitriﬁed and pooled. Also, PGS in combination with frozen embryo transfer programmes can produce better clinical outcome with more physiological endometrial environment. Study funding/competing interest(s): This study received no funding and there are no conﬂicts of interests to be declared. Trial registration number: This study was not an RCT and therefore there is no registration number. P-212 Impact of HIV infection in women submitted to ICSI cycles R. Petracco, L. Okada, R. Azambuja, F. Badalotti, J. Michelon, V. Reig, D. Kvitko, A. Tagliani-Ribeiro, M. Badalotti, and A. Petracco Fertilitat, Centro de Medicina Reprodutiva, Porto Alegre, Brazil Study question: Analyze the human immunodeﬁciency virus (HIV) infection in women submitted to assisted reproduction technology on the oocyte quality, embryo quality and overall pregnancy rate. Summary answer: Our data showed that HIV patients have no difference when submitted to intracytoplasmic sperm injection (ICSI) cycles regarding to stimulation response, laboratory and pregnancy outcome. What is known already: There are 34.2 million people infected by HIV around the world. Half of this population is women and three quarters are in their reproductive years. Considering best quality of life and higher survival rates using anti retroviral therapies, the patients are considering pregnancy planning using assisted reproductive technologies. Cumulative evidence suggests that ART is safe and effective for avoiding horizontal and vertical transmission in HIV serodiscordant couples. The literature is controversial about the HIV patient results, but some of the publications relates worse IVF/ICSI outcome in this population. Study design, size, duration: Retrospective cohort study with 11 HIV serodiscordant couples, whose women were infected, submitted to ICSI between 2002 and 2012. Participants/materials, setting, methods: The HIV patients were matched to two different control patients according to infertility cause, age, semen quality and ovarian stimulation protocol. In the HIV patients group, mean age was 38.7 years old and 22 cycles were performed, in the control groups was 39 years old and were performed 44 cycles. The results were compared using t test (P , 0.05). Main results and the role of chance: The number of oocytes retrieved, mature (MII) oocytes, oocytes fertilized, number of embryos transferred and embryo quality were compared. In the serodiscordant group, there were 18 cycles with embryo transfer. Out of those, there were 9 pregnancies (50%), from those 1 biochemical and 8 clinical. Out of the clinical pregnancy there were 2 miscarriages, 2 ongoing pregnancy and 4 newborn. In the control group there were 41 cycles with embryo transfer. Out of those, there were 18 pregnancies (43%), from these 4 biochemical and 14 clinical. Out of the clinical pregnancies 3 miscarriage, 2 are ongoing and 9 newborn. There were no statistical differences between the groups. Limitations, reason for caution: The study was conducted with a small number of patients. Wider implications of the findings: Our data showed that HIV patients presented similar results to the control group regarding to stimulation, laboratory and pregnancy outcome, however the number of embryos transferred had a trend to signiﬁcance in the control group (p ¼ 0,054). Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 has allowed us to study this in greater detail and retrospectively compare this to implantation outcome. Study design, size, duration: The use of time lapse technology in the IVF laboratory has facilitated the ability to study embryo development as a dynamic process and to retrospectively assess how compaction may affect embryo development and ultimately implantation. Using the EmbryoScopeTM (Unisense Fertilitech, Denmark) it has been possible to compare the process and degree of compaction, whether cellular material is excluded, in those embryos that are known to have implanted to those that are known to have not. Participants/materials, setting, methods: Embryos cultured in the EmbryoScope (ES) with a minimum of 5% fragmentation (by volume) at the cleavage stage (up to 8 cell) that had developed to full blastocyst stage were included. This group was further restricted to embryos of known implantation outcome (KID) where a single embryo was replaced and implanted, or 2 embryos were replaced and both implanted (KID +) and those where no implantation occurred (KID -). A database of the embryo development videos was set up in the ES viewer and all were viewed and annotated according to whether or not any cellular material was excluded at compaction. The end point of compaction was taken as the start of blastulation. The data was then exported to excel and compared to implantation outcome. Main results and the role of chance: In the 75 embryos studied, there was a signiﬁcant difference (p ¼ 0.033) in the number of embryos that completely compacted and went on to implant (48% 29/61), compared to the number with incomplete compaction that went on to implant (14% 2/14). Limitations, reason for caution: The embryo numbers are restricted by the fact that the embryos from many double embryo transfers where a single implantation occurred have been excluded. Data from the KID embryos will continue to be collected. Wider implications of the findings: This may be a useful clinical tool, in conjunction with time lapse technology, in de-selecting embryos with a reduced potential to implant. Study funding/competing interest(s): N/A Trial registration number: N/A i204 Study funding/competing interest(s): There were no competing interests in this study Trial registration number: Not available P-213 Synchronicity of cleavage cycles predicts blastocyst formation and quality C. Pirkevi, M. Cetinkaya, H. Yelke, Y. Kumtepe, Z. Atayurt, and S. Kahraman Istanbul Memorial Hospital, Assisted Reproductive Technologies and Reproductive Genetics Center, Istanbul, Turkey P-214 The relationship between homocysteine, embryo quality and pregnancy in embryo culture medium in assisted reproductive techniques B. Aydin and I. Cepni Cerrahpasa Tip Fakültesi (Medical Faculty), Obstetric and Gynecology, Fatih Istanbul, Turkey Study question: Can homocysteine in embryo culture medium affect the embryo quality? Summary answer: Homocysteine in embryo culture medium affect the embryo quality. What is known already: Homocysteine is an oxidant aminoacid product that affect the reproductive process. Study design, size, duration: Our study is acase control study. The study has been completed in 1 year.40 cases are included.23 cases are non pregnant and 16 cases are pregnant. Participants/materials, setting, methods: 40 cases which admitted to our clinic with fertility desire were included into study. In each case; the level of homocysteine in culture medium was measured by spectrophotometry. Also the correlation between Hcy levels in culture medium and embryo quality were investigated. Main results and the role of chance: Age, the duration of infertility, infertility type, VKI, FSH levels in 3 th day of the menstruation, serum AMH levels were not statistically signiﬁcant between pregnant and non-pregnant group in which Hcy levels were detected in embryo culture medium. And also total dosage of gonadotropin, the number of total oocyte and oocyte which ICSI was performed, the daytime in which the stimulation performed were not signiﬁcant between two groups. . Positive correlation was detected between Hcy in embryo culture medium and embryo grade (r:0,376, p , 0.034). 24 of 40 patients were not pregnant and 16 of 40 were pregnant. Also signiﬁcant correlation was found between being not able to conceive and Hcy levels in culture medium (r:0,5131, p , 0.0004). Limitations, reason for caution: Our case numbers are so low. We can redouble the case numbers and also the validity of the study. Wider implications of the findings: In other studies, homocysteine in semen and follicular ﬂuid has been also found as an oxidant product that affect inversly the reproductive parameters. Study funding/competing interest(s): Homocysteine is an important marker for embryo quality. Trial registration number: There is no registration number. P-215 Comparison of gender-specific human embryo development characteristics by time lapse technology M. Serdarogullari1, N. Findikli2, A. Bayram1, C. Goktas2, O. Sahin1, U. Ulug3, and M. Bahceci4 1 Bahceci Umut IVF Centre, Embryology Laboratory, Istanbul, Turkey, 2Bahceci Fulya IVF Centre, Embryology Laboratory, Istanbul, Turkey, 3Bahceci Umut IVF Centre, Clinical Department, Istanbul, Turkey, 4Bahceci Fulya IVF Centre, Clinical Department, Istanbul, Turkey Study question: This study asks whether there exist gender-speciﬁc embryo development kinetics or parameters between human male and female embryos that can be observed and distinguished by time lapse technology. Summary answer: Our results indicate that the cavitation time of a female embryo was slightly earlier compared to the male embryos. However, other morphokinetic parameters used, such as early cleavage time points, duration between each cleavages, early signs of compaction, cavitation and blastocoel expansion do not show any variation. What is known already: Numerous studies in laboratory animals and mammals indicate that there might be differences in embryo growth dynamics between male and female embryos. However, current data and literature that had analyzed gender-speciﬁc preimplantation growth parameters in humans so far are scarce and the results are inconclusive or conﬂicting. Study design, size, duration: Embryos were cultured in special tri-gas incubators with built-in time-lapse monitoring system in 116 cycles. In these cycles, gender of the off-springs was also assessed through ultrasonography performed at 14-16 weeks of gestation. Study included data from cycles performed between March 2011-December 2012 in Bahceci Assisted Reproductive Technology Centers. Participants/materials, setting, methods: Embryos of 18 cycles were excluded from analysis since they were twin pregnancies of different genders. Remaining 114 embryos were analysed for parameters including cleavage time points and duration in each cleavage from two cells - until hatching blastocyst stages (t2 to tHB), time interval between cleavages (cc2, S2 andS3). Main results and the role of chance: Morphokinetic parameters 62 female and 52 male embryos from a total of 98 cycles processed for data analysis. Table shows the time kinetics of each observed parameter and the number of embryos scored. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: Can we predict high quality blastocyst formation potential from day3 embryos using morphokinetics? Summary answer: The ﬁve formulas cited below were used to identify cleavage synchronicity: (F1): (t8-t2)/((t3-t2) + (t5-t4)); (F2): (t8-t5)/(t8-t4); (F3): (t4-t3)/ (t4-t2); (F4): (t4-t2)/(t8-t4); (F5): (t3-t2), additionally two criteria (irregular divisions and evenness of blastomeres at 2 and 4-cell stages) were applied to build an additive embryo scoring model predicting high quality blastocyst formation. What is known already: The blastocyst formation was found to correlate with cc2 (time between division to 2 cells and division to 3 cells), s2 (time between division to 3 cells and subsequent division to 4 cells) and with the duration of the ﬁrst cytokinesis (Wong, 2010). Cruz et al. have added t4 and t5 and Dal Canto et al. used different subtractions giving the duration of cell cycles (t3-t2; t4-t3; t4-t2; t8-t4; t8-t5) to predict blastocyst quality. Study design, size, duration: This retrospective cohort study was conducted between October 2011 and October 2012 in a private ART Center and approved by the institutional review board. The study included 1199 embryo having a (t8) from 190 infertile patients transferred on day5 (Female age: 31.7 + 4.4; Previous ART cycles: 1.6 + 1.8; BMI: 24.4 + 6.2; MII: 8.2 + 3.4). Participants/materials, setting, methods: Embryos were cultured in a single step culture medium, which was refreshed on day3. Incubation was performed under oil at 378C, 5%O2 and 6%CO2. Each time of cleavage was recorded. Blastocysts were scored before transfer according to Gardner’s classiﬁcation (115-120h post ICSI). Main results and the role of chance: Embryos should mainly stay in even cell stages, which should be balanced when compared to each other, ideally reﬂecting cleavage synchronicity. Each formula was applied and embryos were classiﬁed in 5%groups and good/ top quality blastocyst rate (BR) was computed for the 20intervals. In order to follow mathematically the natural trends obtained in BR, the average of ≥3consecutive 5%groups was subtracted from the average of the 2following groups, and divided to the initial BR until the theoretical threshold set at 0.15 was reached. Each class had a mean BR, which was subtracted from the initial BR, giving a positive or negative score. Obtained scores were added for the ﬁnal prediction of the blastocyst formation potential. The logistic regression model built gave an AUC ¼ 0.841 (95%CI 0.819-0.861). Limitations, reason for caution: The model described may depend on culture conditions applied in this particular IVF laboratory (low O2, single step medium, mainly antagonist ovarian stimulation protocol). The cohort studied involved only infertile patients (female, male or combined) and is in this respect a heterogeneous population. Wider implications of the findings: Time points deﬁning precise embryo cleavage events may not be generalized to infertile patients with different etiologies. However, using ratios based on selected cleavage cycles deﬁning synchronicity of embryos, allows in this retrospective cohort study an individualized analysis giving high predictivity of blastocyst formation and quality. The proposed model has to be further tested in a randomized prospective trial to evaluate its ef?cacy, and may in the future improve embryo selection in IVF treatments. Study funding/competing interest(s): Authors have nothing to disclose. Trial registration number: Not applicable Abstracts i205 Abstracts Female Final outcome of embryo transfers on day 3, 4 and 5 using own and donated oocytes D. Rodrı́guez-Arnedo1, J. Ten1, J. Guerrero1, I. Ochando1, M. Pérez 2, and R. Bernabeu3 1 Instituto Bernabeu, Embryology Dept., Alicante, Spain, 2Instituto Bernabeu, Embryology Dept., Cartagena, Spain, 3Instituto Bernabeu, Reproductive Medicine Dept., Alicante, Spain Study question: Is transfer on day 4 a reasonable alternative in ART (assisted reproduction treatments) and are there differences between own or donated oocytes in term of clinical pregnancy and implantation rates? Summary answer: Transfer on day 4 is a reasonable alternative to day 5 in treatments using own and donated oocytes considering that at this time of development the embryo has full genomic activation. What is known already: The day 4 human embryo appears as a mass of cells composed of 16-32 blastomeres; the morula. The blastomere adherence process is mediated by changes in distribution of E-cadherin proteins from the cytoplasm to the cell membrane. This process has been linked to activation of the embryonic genome (Desai et al., 2000). Transfer on day 4 occurs postgenome activation and allows us to select the highest developmental potential embryo from a cohort. Study design, size, duration: A cross-sectional study was performed in a retrospective analysis of 2,685 IVF/ICSI treatments with own oocytes (670) and donated oocytes (2015) between January 2008 and December 2012. Embryo transfers were performed on day 3, 4 or 5. Participants/materials, setting, methods: A multivariant analysis was performed including confusing factors like maternal age, number of oocytes collected, embryo quality on day 3, number of transferred embryos and their quality. A multiple lineal regression for quantitative dependent variables and binary logistic regression for categorical dependent variables were applied. We compared clinical pregnancy rates (CPR) and implantation rates (IR) in 3 groups: day 3, 4 and 5 transfers. Main results and the role of chance: In case of own oocytes, there were no statistically signiﬁcant differences between CPR and transfers on day 3, 4 and 5 (43.3%; 49.4%; 50.5%, respectively). In terms of IR, we found a signiﬁcant difference between day 3 and day 5 (28.00 vs 38.86, respectively, p ¼ 0.004). With donated eggs, there were statistically signiﬁcant differences between day 3 vs. day 4 and 5 with respect to CPR (44.1%, 51.0%, 56.4% respectively), p ¼ 0.044(day 3 with day 4), p ¼ 0.001 (day 3 with day 5), IR (28.71%, 37.26%, 42.57% respectively), p ¼ 0.002 (day 3 with day 4), p ¼ 0.001 (day 3 with day 5) and number of useful embryos (transferred plus frozen) (3.26, 3.68, 3.82, respectively), p¼ 0.003(day 3 with day 4), p ¼ 0.001 (day 3 with day 5). Limitations, reason for caution: According to our results, day 4 is a reasonable alternative for transfer in treatments of own and donated oocytes, but this study is retrospective and has some limitations. A prospective randomized study should be done to conﬁrm our ﬁndings. Wider implications of the findings: These results conﬁrm the contributions of other authors on the subject, considering the transfer on day 4 as an alternative to transfer on day 5 without reducing success rates. In this case we have analyzed a total of 2,685 cycles, coming from day 3 transfers (901), day 4 (430) and day 5 (1354); probably the greatest number of cases analyzed. P T2 25,5 +2,7(62) 25,6 + 3,51(52) 0.86 T3 36,3 + 4,1 (62) 36,4 + 3,93 (52) 0.89 T4 37,5 + 3,5 (62) 37,96 + 3,52 (51) 0.48 T5 48,3 + 6,5 (61) 48,83 + 6,14 (48) 0.66 T6 51,1 + 4,8 (61) 51,21 + 4,95 (48) 0.90 T7 53,2 + 5,2 (61) 53,1 + 5,5(47) 0.92 T8 55,9 + 5,6 (61) 56,98 + 8,2 (46) 0.42 T9 65,07 + 7,6 (54) 66,91 + 8,22 (45) 0.25 70,4 + 8,2 (48) 71,14 + 9,84 (41) 0.69 Tcomp P-216 Male ......................................................................................... Tm Tcav 81,25 + 8,6 (46) 81,4 + 12,4 (41) 0.94 90,9+ 8,1 (42) 94,4 + 9,65 (37) 0.08 Teb 103,1 +6,7 (40) 104,7+ 6,1 (33) 0.29 Thb 112,2 + 6,05( 14) 112,2 + 2,06 (4) 1.0 cc2 (t3-t2) 10,8 + 2,2 (62) 10,72 + 3,08 (52) 0.87 S2 (t4-t3) 1,2 +2,1 (62) 1,58 + 2,61 (51) 0.67 S3 (t8-t5) 7,5 +5,9 (61) 8,35 + 7,63 (46) 0.51 Study funding/competing interest(s): Conﬂicts of interest and source of funding not declared. Trial registration number: There is not trial registration number P-217 Pregnancy rates comparison between fresh versus frozen blastocyst transfers L. Okada, R. Petracco, R. Azambuja, F. Badalotti, J. Michelon, V. Reig, A. Tagliani-Ribeiro, D. Kvitko, M. Badalotti, and A. Petracco Fertilitat-Centro de Medicina Reprodutiva, Center for Medicine Reproductive, Porto Alegre, Brazil Study question: To compare the pregnancy rates of transferred blastocysts derived from fresh or frozen cycles. Summary answer: Our data suggest that frozen blastocyst transfer does not reduce the chances of pregnancy What is known already: The embryo cryopreservation has become a fundamental technique in assisted reproduction to store indeﬁnitely and viably the surplus embryos. The culture of embryos to the blastocyst stage has shown to be a more efﬁcient way of selection and with better chances of implantation. Study design, size, duration: Retrospective cohort study with only cycles transferring blastocysts embryos (fresh ¼ 116 or frozen ¼ 58), during the year 2012. Participants/materials, setting, methods: The results of blastocyst transfer cycles were evaluated. The mean age of patients in the fresh cycle and FET (frozen embryo transfer) were 33.8 and 34.8, respectively. The technique of cryopreservation was vitriﬁcation (Kuwayama et al. 1998). Statistical analysis was performed using Fisher’s exact test (p ,0.05). Main results and the role of chance: The number of embryos transferred in both groups (fresh and FET) were 238 and 109, respectively, with an average of 2.1 and 1.9 per patient. The rates of pregnancies and clinical pregnancies were 45.7% and 40.5% for fresh embryos and 50.0% and 43.1% transferring frozen/thawed blastocysts. No statistically signiﬁcant difference (p. 0.05) was observed in the results obtained. Limitations, reason for caution: The study was conducted with a small number of patients in a short period of time. Wider implications of the findings: The results suggest that frozen blastocyst transfer does not reduce the chances of pregnancy, according to published data. Study funding/competing interest(s): There were no competing interests in this study Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Limitations, reason for caution: The number of embryos and cycles included in the study. To use of time lapse technology in predicting the gender-speciﬁc development of the human embryos may require further research and larger sample size with the current parameters and technical setting. Wider implications of the findings: Results of this study may indicate that although there is a trend of early cavitation in females, there are no distinct genderspeciﬁc differences in embryo development kinetics in human preimplantation embryos. Therefore, either novel dynamic embryo culture and selection or conventional embryo culture and scoring systems are not expected to change sex-ratio of the resulting pregnancies irrespective of the day of embryo transfer. Study funding/competing interest(s): This study received no funding and there are no conﬂicts of interests to be declared. Trial registration number: This study was not an RCT and therefore there is no registration number. i206 Trial registration number: Not available P-218 Transfer of frozen/thawed embryos: pregnancy comparison between blastocysts and cleavage stage embryos V. Reig, D. Kvitko, A. Tagliani-Ribeiro, L. Okada, R. Azambuja, R. Petracco, J. Michelon, F. Badalotti, A. Petracco, and M. Badalotti Fertilitat, Centro de Med. Reprodutiva, Porto Alegre, Brazil Endometriosis, endometrium, implantation and fallopian tube P-219 Down-regulation of dipeptidyl peptidase 4 under hypoxia could enhance endometrial stromal cell migration in endometriosis C.W. Tan1, Y.H. Lee1, M. Choolani2, H.H. Tan3, L. Grifﬁth 4, and J. Chan5 1 Singapore-MIT Alliance for Research and Technology (SMART), BioSystems and Micromechanics, Singapore, Singapore Rep. of, 2National University of Singapore (NUS), Yong Loo Lin School of Medicine, Singapore, Singapore Rep. of, 3KK Women & Children Hospital, Department of Reproductive Medicine, Singapore, Singapore Rep. of, 4Massachusetts Institute of Technology (MIT), Department of Biological & Mechanical Engineering, Boston, U.S.A, 5Duke NUS Graduate Medical School, Cancer & Stem Cell Biology Program, Singapore, Singapore Rep. of Study question: To determine whether hypoxic microenvironment might be a factor in inﬂuencing cell migration of endometrial stromal cells in endometriosis Summary answer: Endometrial stromal cells (ESCs) derived from endometriotic patients could migrate and invade more aggressively than control ESCs under hypoxia and this phenomenon may act through a downregulation of dipeptidyl peptidase 4 (DPPIV/CD26)- involved pathway and enhanced expression of angiogenesis- related genes. What is known already: Previous reports showed that hypoxia-induciblefactor-1(HIF-1) was signiﬁcantly higher in endometriotic women. Besides, increased expression of focal adhesion kinase (FAK) in endometriotic tissue also alluded to altered cell motility in endometriosis, yet there is little known about the relationship between hypoxia and cellular migration in the pathogenesis of such lesions. Study design, size, duration: ESCs were isolated from endometrial curettage samples obtained from women with endometriosis (Stage IV AFS, n ¼ 3) or other benign gynaecological disease (Stage 0 AFS, n ¼ 3) undergoing laparoscopic surgery. Participants/materials, setting, methods: Cells were exposed to hypoxia (2% O2) and assayed for motility with Oris Pro Cell migration/ invasion assay. Total RNA was extracted for PCR Array Human Cell Motility (SA Biosciences). DPPIV/CD26 expression was determined by ﬂow cytometry whereas conditioned medium was applied to Human Angiogenesis Antibody Array (R&D Systems). Main results and the role of chance: Endometriotic ESCs could migrate and invade through collagen gel (p , 0.005) more under hypoxic condition as compared to ESCs derived from healthy patients. PCR array revealed down-regulation of migration inhibitors (Fibroblast activation protein (FAP), DPPIV/CD26) in patient ESCs under hypoxia and was conﬁrmed via ﬂow cytometry (normoxia: 30.62% vs hypoxia: 12.37%; p ¼ 0.004) Protein array studies showed that angiogenesisrelated genes such as TIMP-1, angiogenin and IGFBP-3 was aberrantly upregulated in endometriotic cells when being exposed to hypoxic environment. This could imply that ESCs from endometriosis patients may be enhanced their cell motility under hypoxic microenvironment, leading to up- regulation of migration/ angiogenesisrelated genes while keeping the migratory inhibitors at low concentration. Limitations, reason for caution: One limitation is the ability to test downstream gene expression of DPPIV/CD26 pathway. Since DPPIV/CD26 is proven to modulate Stromal Cell Derived Factor-1 (SDF-1 / CXCL12) and its receptor, CXCR4, inhibiting DPPIV/CD26 may be possible to test whether DPPIV/ CD26 down- regulation of in endometriosis is SDF- 1/ CXCL12-CXCR4 axis dependent. Wider implications of the findings: This is the ﬁrst study to show a statistically signiﬁcant difference in DPPIV/CD26 expression associated with this disease. DPPIV/CD26, a membrane-bound extracellular peptidase, is found to be able to cleave CXCL12 and thereby immobilize hematopoietic stem and progenitor cell (HSCs/HPCs) populations. Overexpressing DPPIV/CD26 to modulate SDF-1 / CXCL12 and their subsequent chemotactic activity may represent new targets for novel therapies in women with endometriosis. Study funding/competing interest(s): The authors declare no competing ﬁnancial interests. Trial registration number: N.A. P-220 Inhibition of annexin A2 by prostaglandin E2 results in reduced phagocytic ability of peritoneal macrophages and contributes to the development of endometriosis P.C. Chuang1, M.H. Wu2, Y.J. Lin3, and S.J. Tsai4 1 Chang-Gung Memorial Hospital Kaohsiung, Department of Medical Research, Kaohsiung City, Taiwan R.O.C, 2National Cheng Kung University Medical College, Department of Obstetrics and Gynecology, Tainan City, Taiwan R.O.C, 3 Chung Hwa University of Medical Technology, Department of Nursing, Tainan City, Taiwan R.O.C, 4National Cheng Kung University Medical College, Department of Physiology, Tainan City, Taiwan R.O.C Study question: Is annexin A2 involved in the reduced phagocytic ability of macrophages in endometriosis? Summary answer: Data from women with endometriosis and a murine model of the disease show that expression of annexin A2 in peritoneal macrophages is inhibited by prostaglandin E2 (PGE2) and this impairs the phagocytic ability of macrophages. What is known already: Endometriosis is a chronic inﬂammatory disease that recruits many immune cells, especially macrophages, to the peritoneal cavity. The phagocytic ability of peritoneal macrophages isolated from women with endometriosis is reduced. Downloaded from https://academic.oup.com/humrep/article/28/suppl_1/i149/660892 by guest on 22 January 2023 Study question: To compare the pregnancy rate in patients submitted to FET (Frozen Embryos Transfer) of cleavage stage embryos (Group 1), or at the blastocyst stage (Group 2). Summary answer: Our data showed that the transfer of frozen blastocysts has a better pregnancy rate than cleavage embryos. What is known already: The ﬁrst birth of a baby arising from a cryopreserved embryo occurred in 1983. Since then, improvements in embryo cryopreservation have occurred. However since early 90′ s a vitriﬁcation technique has also been developed to cryopreserve embryos in any stage of development. Study design, size, duration: Retrospective cohort study during 2012, with a total of 132 cycles. Participants/materials, setting, methods: In the group 1 the embryos had been frozen on the 2nd, 3rd or 4th day after fertilization, while in the group 2 the embryos were frozen on 5th or 6th day at blastocyst stage. All embryos were cryopreserved by vitriﬁcation followed Kuwayama et al, 1998 protocol. The pregnancy rates were compared through the chi-square test (p , 0.05). Main results and the role of chance: The Group 1 consisted of 74 patients, which pregnancy and clinical pregnancy rate were 27.0% (20/74) and 21.6% (16/74), respectively. The Group 2 had 58 patients, and pregnancy and clinical pregnancy rate were 50.0% (29/58) and 43.1% (25/58) respectively. There was statistical difference between the groups (P¼ 0.0107). The mean age were 34.8 and years old for groups 1 and 2 respectively. Limitations, reason for caution: The study was conducted during a short period of time. Wider implications of the findings: This result suggests that the transfer of embryos at the blastocyst stage would be the best choice for IVF clinics. Study funding/competing interest(s): There were no competing interests in this study. Trial registration number: Not Available Abstracts

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