JPH06178634A - Method for screening good sperm - Google Patents

Abstract

PURPOSE:To screen the good sperm by passing a sperm suspension through a PSA-affinitive glass bead column container and allow glass beads to adsorb the sperm having intact acrosome thereon. CONSTITUTION:Glass wool is spread on the bottom of a vertical container, and washed glass beans are charged in the container. A phosphoric acid buffer solution containing PSA in a concentration of 0.1-1mg/ml is filled in the container and subsequently allowed to stand at 4 deg.C for 3-21 days. A sperm suspension is passed through the obtained PSA-affinitive glass bead column container, and allowed to stand for approximately 15min at room temperature to adsorb sperms having intact acrosome. Non-adsorbed sperms are eluted with a phosphoric acid buffer solution, and subsequently a phosphoric acid buffer solution containing mannose is poured to the container at room temperature for 10min to release the adsorbed sperms for their recovery.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an excellent sperm selection method, and more particularly to an excellent sperm selection method using the normality of the sperm head as a criterion.

[0002]

BACKGROUND OF THE INVENTION AND PRIOR ART Currently, about 1 in 10 couples have infertility, and about 50% of the causes are male factor infertility. Most of the male factor infertility is unclear, and no effective treatment has been obtained yet.

Therefore, as many spermatozoa as possible are selected and collected, and artificial insemination and gifts (GIF
T) ・ Currently, the main treatment method is to achieve pregnancy by in vitro fertilization, microinsemination, etc.

[0004] For the selection of excellent sperm that has been performed so far, a swim-up method for collecting normal sperm motility and a percoll centrifugation method for selecting based on the difference in specific gravity Etc. are currently being carried out, but they are still insufficient when viewed from the therapeutic effect. One reason is that these sperm selection methods are not directly based on the normality of the sperm head, the most important part of sperm. In particular, in microinsemination, which is expected to be a powerful treatment for severe male factor infertility, sperm motility is completely unrelated, and sperm head normality is most required.

[0005]

OBJECT OF THE INVENTION It is an object of the present invention to provide a method for selecting excellent sperm directly based on sperm head normality.

According to the present invention, about half of the outer surface of the normal sperm head is not acrosome-reacted (intact).
Occupied by the acrosome (a body at the anterior end of the sperm head), pisum Sabtum agglutinin (PSA) abbreviated as PSA selectively binds only to intact acrosome and reacts with acrosome (reacted) acrosome. Is obtained by paying attention to the fact that they do not combine.

[0007]

[Configuration and Advantages of the Invention] To achieve the object above-mentioned arrangement of the invention, according to the method of the present invention, provided with glass wool in a film shape in a vertical container bottom, such as glass and plastics having a discharge opening on the lower side Spread washed glass beads on the upper side, and 0.1 to 1 mg / ml PSA
(SIGMA, L-5380 ... product name) containing phosphate buffer (GIBCO,
310-4287AG ... (trade name), and left standing at 4 ° C for 3 to 21 days to form a PSA-adsorbed film on the outer periphery of the glass beads, to prepare a PSA affinity glass bead column container. The first preparation step and the second preparation step in which the PSA solution is discharged from the PSA affinity column container and rinsed with a phosphate buffer solution, and the semen is spun down and washed to obtain a sperm suspension. Adsorption step of adhering sperm with intact acrosome by contacting with glass beads and standing at room temperature for about 15 minutes, selection step of ejecting non-adsorbed sperm with phosphate buffer, mannose (SIGMA, M-4625 (2) A phosphate buffer solution containing (trade name) is put in to release adsorbed sperm at room temperature for 10 minutes, and if necessary, pipetting is repeated a plurality of times to recover free sperm.

In the present invention, the sperm suspension is passed through a column (PSA column) packed with PSA-adhered glass beads, and sperm with a normal head are covered with an intact acrosome of sufficient area. Attached to the glass beads via the PSA. On the other hand, abnormal spermatozoa lacking acrosomes, small even if present, or reacted are unable to bind PSA and pass through the PSA-column. In this way, normal head sperm are selected. Then, mannose competes with the binding site of PSA.
The solution is added and further pipetting is performed to release and collect head normal sperm attached to the glass beads.

In the present invention, a new PSA-column was experimentally produced, and various conditions were examined so that it would be the best. Furthermore, it was confirmed that it was more effective than the swim-up method by microinsemination of hamster eggs by applying this method to severe male factor infertility.

[0010]

[Example] A PSA-affinity column was manufactured as a prototype, and a basic examination of normal head sperm selection was performed. PSA-affinity column (affinity
column) was prepared as follows. That is, as shown in FIG. 1, a 5 ml plastic syringe outer cylinder is made vertical, a well-washed glass wool is laid on the bottom in a film form, and well-washed 1 mm diameter glass beads are packed up to 1 ml line. PSA at 0, 0.1, 0.5 and 1 mg / ml (SIGMA, L-538
0) containing phosphate buffer (GIBCO, 310-4287AG) was allowed to stand at 4 ° C. for 3 to 21 days to adsorb PSA.

The procedure for sperm selection using the PSA-affinity column is as follows. Semen is spun down and washed to give a sperm suspension. PSA-from affinity column
Drain the PSA solution and rinse with 10 ml phosphate buffer. Add 0.6 ml of sperm suspension and let it stand at room temperature for 15 minutes.
Adsorb sperm with acrosome. Phosphate buffer 15 ml
To allow non-adsorbed sperm to flow out. Mannose PS
Since it competes with the binding site of A, 16 mg / ml mannose (ma
nnose ... SIGMA, M-4625) containing 0.6 ml of phosphate buffer solution, and wait 10 minutes at room temperature to release adsorbed sperm. Pipetting
After 20 times, the free sperm fluid is collected.

[0012] Kruger's strict crit
eria) to evaluate sperm morphology and normal rate (% strict no
rmal morphology ；% SNM） ・ Normal head ratio (% normal)
Head;% NH) was examined. Data were analyzed by the χ ² test and p <0.05 was considered significant. We also examined changes in sperm concentration and motility.

The necessity of pipetting was examined as a method for releasing and recovering normal head sperm adsorbed on PSA-adhered glass beads. Also, mannose (ma
nnose) to investigate the effect of 0, 1.6, 16 and 160
The sperm were cultured for 2 hours in a phosphate buffer containing mg / ml mannose, and the decrease in motility was compared.

In order to confirm the effect of the present invention, a hamster perivitelline intestinal sperm injection experiment was carried out in severe male factor infertility.

Sperm was selected by PSA-affinity column or swim-up method in 3 cases of severe male infertility in which no fertilized egg was obtained by in vitro fertilization more than 3 times, and sperm was stimulated by calcium ionophore treatment. Then, microinsemination was performed in the perivitelline cavity of hamster eggs, and the fertilization rates were compared. First, sperm is spun down and washed to prepare a sperm suspension. This was divided into two, and one was a PSA-affinity column (0.5 mg /
Sperm selection was performed as described above by using ml PSA).

On the other hand, a phosphate buffer solution was layered on the sperm suspension and incubated at 37 ° C. for 45 minutes, the sperm were allowed to swim up, and the upper layer was collected and sperm selection was performed. In both groups, the obtained sperm was treated with 5 μM calcium ionophore A 2318.
7 (Calbiochem, 100105) was treated at 37 ° C for 10 minutes. Immediately spin down for 5 minutes, remove the ionophore solution, and add 30 mg / ml bovine serum albumin.
in) added HTF culture solution was added to neutralize the ionophore. 5
After culturing for 2 hours at 37 ° C. in 95% CO ₂ 95% air, sperm was injected into the perivitelline cavity of hamster eggs by a micromanipulator. After 5 hours, the cells were fixed and stained, and the fertilization was confirmed by observing the swollen sperm head or two or more pronuclei and the sperm tail, and the fertilization rates by the two sperm selection methods were compared.

The results of the basic examination of the selection of normal head sperm by the PSA-affinity column are as follows. Fig. 2 shows% SNM and% NH before and after semen / PSA-column. PSA-column significantly increased% SNM and% NH, 0.5 mg / ml PSA
The maximum was 1.7 times and 1.6 times in the treated column.

FIGS. 3A and 3B show the sperm morphology before and after the 0.5 mg / ml PSA column. As for sperm concentration and motility, semen is 96
× 10 ⁶ / ml · 75%, 0, 0.1, 0.5 and 1.0 mg / ml P
After SA-column, 5 × 10 ⁶ / ml ・ 30%, 26 ×
10 ⁶ / ml ・ 45%, 28 × 10 ⁶ / ml ・ 50%, 25 × 10 ⁶ / ml ・
It was 40%.

The effect of pipetting on the recovery of sperm was that when pipetting + mannose treatment, four times as much sperm was recovered as compared to mannose treatment alone.

The effect of mannose on sperm motility is
The motility before culturing (normal semen / immediately after swim-up) was 90%, but the motility was 70% after culturing in phosphate buffer containing 0, 1.6, 16 and 160 mg / ml mannose for 2 hours.
%, 75%, 70% and 70%, no effect was observed.

The results of the hamster perivitelline intestinal sperm injection test in severe male factor infertility are as follows. PSA-affinity column or swim was used for 3 cases of severe male factor infertility in which no fertilized eggs were obtained by in vitro fertilization more than 3 times.
After selecting sperm by the -up method and stimulating the sperm by calcium ionophore treatment, microfertilization was performed in the perivitelline cavity of hamster eggs, and fertility rates were compared. The results are summarized in Table 1. 0.5 mg / ml, although no statistically significant difference was obtained
The fertilization rate (29%) when sperm selection was performed using the PSA-column is
It was higher than the fertilization rate (10%) when sorted by swim-up.
This is shown in the table below.

[0022]

[Table 1]

The principle of the PSA-affinity column will be inferred with reference to FIG. About half of the outer surface of the normal sperm head is occupied by intact acrosome. PSA selectively binds to intact acrosomes only, but not to acrosome-reacted acrosomes. Then, when the sperm suspension was passed through a column packed with glass beads to which PSA was attached (PSA-column Figure 1), sperm with normal heads were covered with intact acrosomes of sufficient area. Attached to the glass beads via the PSA. On the other hand, abnormal spermatozoa that lack acrosome, are small or have acrosome reaction cannot bind to PSA and pass through the PSA-column. In this way, normal head sperm are selected. Mannose is PSA
The head normal spermatozoa attached to the glass beads are recovered by adding mannose solution and releasing by pipetting. Thus, head normal sperm are selectively collected.

The sperm selection method using the PSA-affinity column selectively recovers normal sperm from the head and can be applied to cases with a motility rate of 0%. For artificial insemination, GIFT, in vitro fertilization, and microinsemination. It is expected to be useful as a sperm selection method. Other methods with different sorting mechanisms, such as swim-up method and pe
We think that it is possible to further improve the sorting accuracy by combining it with rcoll centrifugation.

[Brief description of drawings]

[Figure 1] Principle of PSA-affinity column, PSA selectively binds to acrosome, so that normal sperm are adsorbed on glass beads, and abnormal sperm lacking or too small acrosome passes through the column.

[Fig. 2] Normal rate of sperm morphology (% strict normal morphology) and normal rate of sperm head (% normal head) before and after semen / PSA-column. Three homologous experiments. # ・ *: P <0.
01vs semen (χ ² test)

FIG. 3: Sperm morphology before (A) and after (B) 0.5 mg / ml PSA-column. In A, sperm with abnormalities in sperm head, acrosome, midpiece and tail are mixed, but in B, normal sperm are selected. References 1. World Health Organization: WHO Laboratory Man
ual for the Examination of Human Semen and Semen-C
ervical Mucus Interaction. Cambridge University Press. Cambridge. 1987. 2. Kruger TF. Menkveld R. Stander FSH. Lombard CJ. Van der Merwe JP. Van Zyl JA and Smith
K: Sperm morpho-logic features as a prognostic fa
ctor in vitro fertilization. Fertil Steril. 46: 1118-1123, 1986. 3. Trowbridge. IS: Isolation and chemical cha
racterization of amitogenic lectin from pisum sati
vum. J. Biol. Chem., 249: 6004. 1974. 4. Cross. NL, Morales, P., Overstreet, JW a
nd Hanson, FW: Twosimple methods for detecting
acrosome-reacted human sperm. GameteRes., 15: 213,
1986. 5. Liu DY and Baker HWG: The proportion of human
sperm with poor morphology but normal intact acroso
mes detected with pisum sativumagglutinin correlat
es with fertilization in vitro. Fertil Steril. 50: 288-293. 1988. 6. Jinno. M., Kobayashi. T., Sugimura. K., Nozaw
a. S., Katayama. E., Iida. E .: IVF / Sperm Morpho
logy. Mol. Androl., 2: 161-168. 1990.

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Claims (1)

[Claims] 1. A glass-wool film is provided at the bottom of a vertical container of glass, plastic, etc. having a discharge port on the lower side, and washed glass beads are laid on the upper side of the film, and
~ 1mg / ml of Pisum sativ agglutinin
vum agglutinin Abbreviation PSA; SIGMA, L-5380 (trade name)
Filled with phosphate buffer solution (GIBCO, 310-4287AG ... trade name) containing and containing it on the outer circumference of the glass beads by leaving it at 4 ° C for 3 to 21 days.
The first preparatory step of forming a PSA-adsorbed membrane to prepare a PSA affinity glass / bead column container, and the second preparatory step of discharging the PSA solution from the PSA affinity column container and rinsing with a phosphate buffer solution. , An adsorption step in which normal semen is spun down and washed to make a sperm suspension, and this suspension is brought into contact with the PSA glass beads and allowed to stand at room temperature for about 15 minutes to adsorb sperm having intact acrosomes. And a selection step of discharging non-adsorbed sperm with a phosphate buffer solution, and adding a phosphate buffer solution containing mannose (SIGMA, M-4625 ... trade name) to release the adsorbed sperm at room temperature for 10 minutes, and if necessary, multiple times An excellent sperm selection method comprising a collecting step of collecting free sperm by performing petting.