Harrath et al. Journal of Ovarian Research (2017) 10:77
DOI 10.1186/s13048-017-0372-x
RESEARCH
Open Access
Food restriction during pregnancy and
female offspring fertility: adverse effects of
reprogrammed reproductive lifespan
Abdel Halim Harrath1,2* , Abdulkarem Alrezaki1, Lamjed Mansour1, Saleh H. Alwasel1 and Stefano Palomba3
Abstract
Background: Food restriction during pregnancy can influence the health of the offspring during the adulthood.
The aim of the present study was to examine the effect of maternal food restriction (MFR) on the reproductive
performance in female rat offspring from the first (FR1) and second (FR2) generations.
Methods: Adult virgin Wistar female rats were given free access to tap water and were fed ad libitum on standard
rodent chow, were mated with virgin adult males, and then were randomly divided into two groups: controls (that
was fed ad libitum) and food-restricted group (FR, that was given only 50% of ad libitum food throughout gestation).
Their first (FR1) and the second (FR2) generation of offspring were fed ad libitum and sacrificed before puberty and at
adulthood. Their ovaries were removed and their histology evaluated by estimating the number of follicles (total and at
various stages of folliculogenesis), and the presence of multi-nuclei oocytes and multi-oocyte follicles.
Results: Total number of ovarian follicles was lower in FR1 females at week 4 in comparison with controls, while it was
not different in FR2 females vs. controls. The number of the primordial follicle was lower in FR1 and FR2 females vs.
controls at both week 4 and at week 8. When compared to the controls, the follicles containing multi-nuclei oocytes
were more frequent in ovaries from FR1 and FR2 females at week 4, and higher and lower respectively in ovaries form
FR1 and FR2 females at week 8.
Conclusion: MFR affects ovarian histology by inducing the development of abnormal follicles in the ovaries in first and
second generation offspring. This finding could influence the ovarian function resulting in an early pubertal onset and
an early decline in reproductive lifespan.
Keywords: Follicle, Food restriction, Ovary, Oocyte, Pregnancy
Background
Maternal nutritional status during gestation is a key determinant for the health and physiology of the offspring
at adulthood, and that influence is mainly established
during early development, i.e. well before birth [1–5].
Many adulthood diseases can be linked to the environment within which the embryo has developed, including
abnormal nutritional, environmental, and hormonal insults that may have changed the developmental trajectory
of the fetus [1, 2]. According to this hypothesis, the origins
* Correspondence: hharrath@ksu.edu.sa
1
Zoology Department, College of Science, King Saud University, Riyadh,
Saudi Arabia
2
Unit of Reproductive and Developmental Biology, Faculty of Science of
Tunis University of Tunis El Manar, Tunis, Tunisia
Full list of author information is available at the end of the article
of common diseases may be due to the environment that
the fetus directly senses via the mother [2, 5–8]. In particular, maternal food restriction (MFR) has been associated with coronary heart disease and increased arterial
blood pressure [2], reduced nephron endowment and increased renal morbidity in adulthood [9], and may affect
physical growth and neurobehavior in newborns [10].
Malnutrition during gestation has been associated with
hepatic steatosis, type 2 diabetes, and obesity during adulthood [5, 11–15]. During late gestation, MFR is associated
with metabolic signaling dysfunction in the liver, and predisposes the offspring to insulin resistance [5].
Oocyte quality is a critical determinant for the developmental trajectory of the fetus [16, 17]. Many forms of
female reproductive disruptions have been linked to the
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Harrath et al. Journal of Ovarian Research (2017) 10:77
prenatal environment, and it is likely related to early oocyte formation, which is vulnerable to numerous environmental effects [4]. In comparison with girls who were
born appropriate for the gestational age, girls born small
for the gestational age have a reduced reproductive lifespan, indicated by a decrease in ovarian size that is associated with low ovulation rates [18, 19], advanced
menarche, and early menopause [20–22]. Polycystic
ovarian syndrome (PCOS), one of the most common female endocrine disorders [23], has been suggested to
arise through a gene–environment interaction, probably
in the developmental milieu within which female gametes are formed [24]. Although maternal malnutrition is
a major factor that adversely affects fetal growth and is
associated with to lifelong consequences, relatively few
studies have investigated the effects of MFR on the reproductive outcomes of offspring [4, 25, 26]. In a large
epidemiological study women born to mothers exposed
to famine were more reproductively successful compared
to controls [27]. Moreover, malnutrition during pregnancy alters reproduction in sheep by inducing poor oocyte quality, may cause reproductive disruptions in rats
mainly by an early vaginal opening, and induce a
decrease in the primordial and antral follicle number
[28–30]. During the first trimester of pregnancy in cattle,
MFR leads to a reduced ovarian reserve in adulthood, as
observed by the increased follicle stimulating hormone
(FSH) levels [31].
At the moment, the available data regarding the relationship between the MFR and the female reproduction in the
offspring have many limitations and gaps concerning the
potential underlying mechanisms and almost all have evaluated the effects of maternal caloric restriction on the first
generation only. Based on these considerations, the aim of
the present experimental study was to investigate the
impact of MFR in rats on ovarian architecture and function
in first and second generations of female offspring.
Methods
Experimental design
First- and second-generation offspring [4, 32] of pregnant rat exposed directly (or indirectly through
germline-independent transmission) to MFR were studied. Specifically, adult virgin Wistar female rats weighing
230 ± 20 g obtained from the Animal Unit at King Saud
University were given free access to tap water and were
fed ad libitum on standard rodent chow (23% protein,
4.5% fat, 3030 kcal/kg; lab diet 5001, Brentwood, MO).
After being maintained in separate cages for four days of
adaptation, they were mated with virgin adult males, and
then were randomly divided into two groups: control
group (group C, n. 15) received ad libitum food, and
food-restricted group (group FR, n. 20) received only
50% of ad libitum food throughout gestation. The first
Page 2 of 9
generation of offspring (FR1) were fed ad libitum. After
complete weaning, FR1 and control females were sacrificed before puberty (week 4, n. 10) and at adulthood
(week 8, n. 10). The ovaries were removed and the fat
was discarded. They were weighed using a digital balance (0.0001 g) and immediately fixed in 10% neutral
buffered formalin at room temperature for classical histology. The remainder of FR1 females were allowed to
reach sexual maturity, and were treated exactly as their
mothers (FR females), i.e. 50% ad libitum food throughout gestation.
After birth, a second generation of the doubly foodrestricted group females (FR2) was obtained. The FR2
offspring females were humanely sacrificed at 4 and
8 weeks, and their ovaries were collected, weighed and
fixed exactly as before detailed for FR1 offspring
females.
Light microscopy
Ovaries were fixed in neutral buffer formalin (NBF) or
Bouin’s fluid, and were subsequently preserved in 70%
alcohol. At least three ovaries from each group have
been cut in serial sections to a thickness of 7 μm using a
Reichert-Jung microtome. Hematoxylin and eosin (H&E)
staining was used to assess ovarian architecture in the
group C, FR1, and FR2.
The effects of nutrition on folliculogenesis were evaluated by counting the number of primordial, primary,
secondary, pre-antral, and antral follicles with visible oocyte nuclei in some slides for each ovary (see below).
Specifically, ovarian follicles were classified according to
the scheme of Pedersen and Peters (1968), with modifications. Primordial follicles included oocytes surrounded
by a single layer of three to six squamous epithelial cells,
whereas primary follicles were composed of oocytes surrounded by one layer of numerous cuboidal epithelial
cells. Secondary follicles were characterized by oocytes
surrounded by more than one layer of granulosa cells
with no visible antral spaces. Antral follicles were composed of an oocyte surrounded by many layers of cuboidal granulosa cells, with many visible small antral
spaces, or one large antrum. The theca layers and cumulus oophorus may be evident.
A particular interest was given to the occurrence of
follicles containing multi-nuclei oocytes (MNOFs), key
indicator of perturbations during germ cell nest formation [33–38]. The total number of multi-oocyte follicles
(MOFs) was also counted in every section of the ovaries
from the different groups.
To estimate the number of slides to be read for
each ovary we used sample size calculations using the
2
following formula [39]: n≅z21‐α=2 hs 2 where: s = sample
standard deviation from an initial number (n0) of
Harrath et al. Journal of Ovarian Research (2017) 10:77
replications (n0 = 11), Z1 − α/2 = the value retrieved
from the normal standard distribution, corresponding
to the 1-α/2 probability (we choose α = 0.05) and h:
the half width of the confidence interval.
Page 3 of 9
week 4 (P = 0.0014 and P < 0.0001, respectively), and at
week 8 (P = 0.0002 and P = 0.0044, respectively) (Fig. 2b).
Primary follicles
For data analysis of follicle number, we used the
GraphPad prism version 5. Statistical comparisons
were made using a two-tailed t-test. All values are
presented as the mean ± standard deviation (SD). Significance was set at P value less than 0.05.
The number of primary follicles was significantly (P =
0.034 and 0.003, respectively) higher in FR1 and FR2 vs.
controls at week 4. That statistical (P = 0.0072) differences were sustained at week 8 only in FR1 females vs.
controls (Fig. 2c). A significant (P = 0.0014) difference in
the number of primary follicles was observed in FR2 vs.
FR1 females at week 8.
Results
Secondary follicles
Ovary weight
The number of secondary follicles was significantly (P =
0.0002) lower in the ovaries of FR1 females compared to
controls only at week 4 (Fig. 2d). Both at week 4 and 8,
the number of secondary follicles was significantly (P =
0.0010 and P = 0.0002, respectively) higher in FR2 than
in FR1 females.
Statistical analysis
No difference in mean ovary weight was detected between FR1 and FR2 females vs. controls in 4-week-old.
Conversely, in 8-week-old animals a significant (P =
0.0003) difference between intervention and control
group in ovarian weight for FR2 females only (Fig. 1). A
significant (P = 0.0011) difference was also observed in
ovary weight in FR2 vs. FR1 females at week 8 (Fig. 1).
Number of follicles
Total follicles
Total number of ovarian follicles was significantly (P =
0.0006) lower in FR1 females at week 4 in comparison
with controls, while it was not different in FR2 females
when compared to the controls (Fig. 2a). At week 8, the
total number of follicles in ovaries from both FR1 and FR2
females resulted significantly (P = 0.0485 and P = 0.0013,
respectively) lower than in controls. The total number of
follicles was significantly (P = 0.0020 and P = 0.0074, respectively) higher and lower in FR2 vs. FR1 females at
week 4 and 8, respectively.
Primordial follicles
The amount of the primordial follicle was significantly
lower in FR1 and FR2 females than in controls at both
Antral follicles
When compared to controls, the number of antral follicles was significantly lower (P = 0.0023) in the ovaries of
FR1 females at week 4, and significantly higher (P =
0.0084) in the ovaries of FR2 females at week 8 (Fig. 2e).
A significant (P = 0.0008 and P < 0.0001, respectively)
difference in the number of antral follicles was detected
between FR2 and FR1 females at week 4 and 8.
Ovarian histopathology
MOFs
MOFs population was found in all the studied groups and
at all follicular stages, from the primordial to the large antral stage; these MOFs contained two or more oocytes
(Fig. 3a-f). The architecture of the ovaries from FR1 and
FR2 females was mainly characterized by more growing
follicles when compared to controls. In most of the cases,
these were adjacent to each other, suggesting that a fusion
has occurred (Fig. 3a). Furthermore, we even reported
joining follicles, characterized by the displacement of the
oocyte from one follicle into another (Fig. 3b).
When compared to the controls, the MOFs were significantly (P = 0.0044 and P = 0.006, respectively) more
frequent in ovaries from FR1 and FR2 females at week 4
(Fig. 2f ). Nevertheless, while this number was significantly higher (P = 0.0013) in FR1 females compared to
control at week 8, it was significantly (P = 0.0075) lower
in ovaries from FR2 females.
MNOFs (Heterokaryon)
Fig. 1 Effect of MFR on ovarian weight results in a significant reduction
in 8-week-old FR2 females when compared to controls (P = 0.0003), and
to FR1 females (P = 0.0011); no effect is observed in 4-week-old females
A high frequency of MNOFs was detected in the ovaries
of FR1 and FR2 females compared to controls (Fig. 4ab). A detailed analysis of these MNOFs provided clues
on how they were generated. Specifically, we detected
Harrath et al. Journal of Ovarian Research (2017) 10:77
Page 4 of 9
Fig. 2 Effect of MFR on the number of follicles per section of ovarian tissue. a Total number of follicles: MFR significantly affects the total number
of follicles in ovaries from FR1 females at 4 weeks (P = 0.0006) and 8 weeks (P = 0.0485), and from FR2 females at 8 weeks (P = 0.0013). The total
number of follicles from FR2 at week 4 is higher vs. FR females (P = 0.0020), whereas it is lower in FR2 females at week 8 compared to FR females
(P = 0.0074). b Primordial follicles: significant effect of MFR on the number of primordial follicles in FR1 and FR2 females at week 4 when compared to
control (P = 0.0014 and P < 0.0001, respectively) and in FR2 females at week 8 (P = 0.0002). The total number of primary follicles from FR2 at both week
4 and 8 is significantly lower when compared to FR1 females (P = 0.0020 and P = 0.003, respectively). c Primary follicles are significantly higher in
number in ovaries of 4-week-old females in both FR1 and FR2 vs. controls (P = 0.034 and P = 0.003). At week 8, FR1 females have significantly higher
number of primary follicles vs. control (P = 0.0072), whereas FR2 females have significantly lower number of primary follicles vs. FR1 (P = 0.0014).
d Secondary follicles: FR1 females at week 4 have significantly lower number of secondary follicles vs. control (P = 0.0002) and vs. FR2 (P = 0.001),
whereas at week 8 FR2 have significantly higher number of secondary follicles vs. FR1 females (P = 0.0002). e Antral follicles: MFR has the same effect as
in secondary follicles, except for a significant increase in FR2 females vs. control at week 8 (P = 0.008). f MOFs: The number of MOFs is significantly
higher in ovaries from FR and FR2 females vs. control at week 4 (P = 0.0044 and P = 0.006, respectively); it is also significantly higher in FR1 females at
week 8 (P = 0.0013), and again significantly lower in ovaries from FR2 females vs. control (P = 0.0075). FR2 females at week 8 have significantly lower
number of MOFs when compared to FR1 females (P < 0.0001). All results are given as mean ± SD; P < 0.05, *FR1 vs. controls (C) and FR2
vs. controls, * with error bars: FR1 vs. FR2
many MOFs in which oocytes were frequently observed
very close to each other (Fig. 4d), suggesting they had
fused to form a heterokaryon. In some cases, oocytes
within the same follicle were apparently undergoing
such a joining process (Fig. 4e).
Discussion
The fertile reproductive lifespan of female mammals is
mainly linked to the initial ovarian reserve of primordial
follicles that reaches its maximum level around the time
of birth, and is gradually depleted during reproductive
life [40–42]. At prepubertal age (week 4), we found that
the number of primordial follicles was significantly decreased in FR1 and FR2 rats, suggesting that MFR affects
the ovarian reserve of primordial follicles during early
fetal life. The significant decrease in the number of
primordial follicles at week 4 was associated with an increase in the number of primary follicles among the
Harrath et al. Journal of Ovarian Research (2017) 10:77
Fig. 3 (See legend on next page.)
Page 5 of 9
Harrath et al. Journal of Ovarian Research (2017) 10:77
Page 6 of 9
(See figure on previous page.)
Fig. 3 Hematoxylin and eosin staining of paraffinized ovarian sections from FR1 and FR2 females showing the generation of MOFs. a Ovaries
from FR1 and FR2 females are mainly characterized by many growing follicles that are adjacent to each other, indicating their fusion. b-d Follicles
in the process of merging (arrowheads); we can see in (d) the displacement of the oocyte from one follicle into the second one. e Primordial
follicle with two oocytes. f primordial follicle with three oocytes. g Primary follicle with two oocytes, (h) Primary follicle with three oocytes. i
Secondary follicle with two oocytes. (J) Antral follicle with two oocytes; (k) Antral follicle with three oocytes; (l) Antral follicle merging with an
early secondary follicle (arrowhead), the large arrow is showing the oocyte position of the antral follicle. Scale bar = 200 μm
different studied groups suggesting that MFR might
cause an early menarche by inducing early folliculogenesis. This hypothesis seems consistent with previous
studies [26, 43].
At week 8, the significant decrease in ovarian weight
related to the total number of follicles in both first and
second generation offspring after MFR is mainly caused
by the significant decrease in primordial follicles in the
ovarian reserve compared to controls. This could suggest an early menopause in FR2, which is less likely in
FR1 animals. In fact, previous studies reported that
MFR is associated with early menarche and menopause [26, 43–45]. To this regard, it is possible to
hypothesize that MFR provided first and second generation offspring with a phenotype that is better
suited for the lack of food. That new phenotype is
consistent with the trade-off theory, i.e. an increase in
fertility and a decrease in reproductive lifespan may
lead to an increase in the chances of an organism to
reproduce successfully [46]. In fact, fetal growth restriction can be considered as a part of the life history strategy for FR1 and FR2 females that were in
utero when their mothers underwent food restriction.
Since prenatal undernutrition leads to reduced longevity in mice [47], these females may anticipate a
shorter life because of a higher risk of extrinsic mortality. It is possible to hypothesize that they may have
to adjust their reproductive aptitude by changing the
intensity and duration of their lifespan, the timing of
the stages of folliculogenesis, as well as the age at
which they should reach reproductive maturity. Due
to the lack of food sensed through nutritional or
endocrine signaling during fetal life [48], and to ensure reproductive success before death, these females
have probably programmed their reproductive lifespan
to be very intensive but relatively limited in time,
which is consistent with population regulation in the
theory of life history [49]. Thus, when they reach
Fig. 4 Hematoxylin and eosin staining of paraffinized ovarian sections from FR1 and FR2 females showing the generation of MNOFs. a Primordial
follicle with two nuclei- oocyte. b Secondary follicle containing one oocyte with two proportional nuclei (c) Secondary follicle containing one
merged oocyte with four disproportional nuclei. d Secondary follicle with two oocytes that are very close to each other, suggesting that they will
probably fuse soon to form a heterokaryon. e Secondary follicle containing two semi-fused oocytes (arrowhead). f The same secondary follicle in
(e) but at another level of section. Scale bar = 200 μm
Harrath et al. Journal of Ovarian Research (2017) 10:77
prepubertal age, they may upregulate the expression
of genes involved in steroidogenesis, which in turn induces folliculogenesis for a greater number of primordial follicles while concomitantly explaining the
significant higher number of in primary follicles in 4week-old FR1 and FR2 females vs. control. The relatively large number of induced follicles undergoing
folliculogenesis at one time makes follicles adjacent to
each other and, consequently, highly increases the
probability that they will merge to form MOFs [34].
This may have decreased the number of growing oocytes (secondary and antral follicles), and may explain
the lower total number of follicles, and the higher
number of MOFs in 4-week-old FR1 females (vs. controls). This strategy is also associated with a faster decline in ovarian function with aging, that is clearly
supported by the significant decrease in the number
of primordial follicles in the ovarian reserve, and also
by the significant decrease in the total number of
follicles in both FR1 and FR2 females at an early age
(8-week), that corresponds in normal females to a
high reproductive performance.
Of note, a higher number of MOFs in ovaries from 4week-old first and second generation females was observed. Many follicles at these stages may have fused to
form MOFs, which explains their significant higher
number in FR1 females at four weeks, while this is less
likely in FR2 females. While the mechanism of MOFs
formation during nest breakdown has been described
[33, 50], this is the second study that clearly confirms a
new mechanism for the generation of MOFs through the
fusion of follicles in the mammalian ovary, and their incidence increased sharply at prepubertal age [34]. Inversely, we found that the number of secondary and
antral follicles was significantly lower in FR1 and FR2 at
prepubertal age when compared to controls. Previous
findings have reported that most cases of MOFs represent a fusion between secondary follicles, or a fusion between one secondary and one large antral follicle [34].
Furthermore, the number of secondary/antral follicles
observed in 8-week-old females was higher than in controls when the number of MOFs was lower. This result
is also consistent with the finding of Perez-Sanz and coworkers that showed that the number of MOFs declines
significantly in female mice when they become sexually
mature [33].
The presence of MNOFs is exceptional and represents a challenge for future studies. Two different
mechanisms could explain the origin of this
phenomenon: the nest breakdown–follicle assembly,
and the fusion of more than one oocyte within the
same multi-oocyte follicle. Based on our findings, the
second mechanism appears the most probable since it
is suggested that MOFs are most likely generated by
Page 7 of 9
the assembly of follicles [34] rather than being produced early on during the formation of the ovary.
The presence of more than one oocyte in direct contact within the same follicle highly increases the possibility of their fusion, and leads to the formation of
MNOFs. In fact, any cell brought into contact with
another cell and given the right conditions (such as
sufficient amounts of fusogen proteins, simultaneously
present on each of the two cell surfaces) will fuse
with the second cell, even when the latter is foreign
[51–53]. This fusion can be beneficial mainly during
embryonic development, and for cell-based therapies,
and represents a well-known process during
reproduction when gametes (spermatozoa and oocytes) unite during fertilization to form the zygote. It
has also been described in muscle cells, macrophages
and nerve cells [54–58]. When cell fusion is blocked
during embryonic development, defects in organogenesis, embryonic lethality, and postembryonic defects
can increase [53, 59] suggesting that fused cells are
hybrid cells or chimera that function efficiently during
embryonic development, driving correct organ formation [54]. In Caenorhabditis elegans, particularly in
the proliferative zone of germ cells, two or more
crescent shaped nuclei have been observed [60].
MNOFs have also been described during the initial
stages of oogenesis in some amphibian species. For
example, in Ascaphus truei, oogenesis involves eight
nuclei [61], whereas frog oocytes with two nuclei have
been described in Leiopelma hochstetteri [62]. The
most evident example of the necessity for multinucleated cells is the syncytiotrophoblast, which is
formed when embryonic cytotrophoblast cells fuse
with the maternal endometrial epithelium [63, 64].
These cells represent the most important cell type in
the placenta, and there is a strong correlation between a successful pregnancy and healthy syncytiotrophoblasts, likely due to their multi-nuclear state [64].
Conclusions
Current data suggest that MFR influences ovarian histopathology and, in turn, the reproductive health of first
and second generation female offspring during fetal development, and they have been probably programmed to
have an early menarche by inducing early folliculogenesis, and an early decline in ovarian function thereby decreasing the reproductive lifespan, and leading to an
early menopause.
Abbreviations
FR1: Female rat offspring from the first generation; FR2: Female rat offspring
from the second generation; FSH: Follicle stimulating hormone;
H&E: Hematoxylin and eosin; MFR: Maternal food restriction; MNOFs: Follicles
containing multi-nuclei oocytes; MOFs: Multi-oocyte follicles; NBF: Neutral
buffer formalin; SD: Standard deviation
Harrath et al. Journal of Ovarian Research (2017) 10:77
Acknowledgements
We would like to thank Dr. Deborah Sloboda from the Departments of
Biochemistry and Biomedical Sciences, Obstetrics and Gynecology, and
Pediatrics, McMaster University, Hamilton, Canada, for the constructive
criticism on an earlier draft of the manuscript.
Funding
The authors would like to extend their sincere appreciation to the Deanship of
Scientific Research at King Saud University for funding this research RG-164.
Availability of data and materials
There are no shared the data and material for this manuscript.
Authors’ contributions
AHH and SA designed the study. AHH, AA and LM performed the experiments
and interpreted the data of the work. AHH, SA and AA wrote the first draft. SP
strongly revised the manuscript for intellectual content, and improved the first
draft. All authors approved the final version of the manuscript.
Ethics approval and consent to participate
The study was approved by the Research Ethics Committee at King Saud
University.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
Zoology Department, College of Science, King Saud University, Riyadh,
Saudi Arabia. 2Unit of Reproductive and Developmental Biology, Faculty of
Science of Tunis University of Tunis El Manar, Tunis, Tunisia. 3Unit of
Gynecology and Obstetrics, Grande Ospedale Metropolitano “Bianchi –
Melacrino – Morelli”, Reggio Calabria, Italy.
1
Received: 11 September 2017 Accepted: 15 December 2017
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