JP2974525B2 - Selection and evaluation method of sperm head normality - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for selecting excellent sperm, and more particularly to a method for selecting and evaluating the normality of sperm heads based on the normality of sperm heads.

[0002]

BACKGROUND OF THE INVENTION AND PRIOR ART Patent No. 1651369 registered on March 30, 1992 (right holder: Pharmacia AB, inventor: Bailman Pell et al., Japanese Patent Publication No. 3-17492) Discloses a "method and means for selecting motile sperm", and a patent has been granted to an invention made particularly with a focus on sperm motility.

[0003] The present invention relates to a method for evaluating sperm quality based on the normality of the head rather than the motility of sperm.

[0004] The screening and evaluation of excellent spermatozoa performed so far include a swim-up method for collecting normal sperm motility and a percoll centrifugation for screening based on the difference in specific gravity. Laws are currently being implemented, but are still inadequate in view of the therapeutic effect. One reason is that these sperm sorting 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 not related at all, and sperm head normality is most required.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for screening and evaluating sperm head normality based directly on sperm head normality.

According to the present invention, the normal sperm head does not undergo an acrosome reaction on about half of its outer surface (intact).
When occupied by acrosomes (acrosomes: the frontal bodies of the sperm head), the pismum sativum aglytinin (agglutinin) abbreviated PSA selectively binds only to intact acrosomes and reacts with acrosomes (reacted). Are obtained by noting 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 Then, washed glass beads are spread on the upper side, and 0.1-1 mg / ml PSA
(SIGMA, L-5380… trade name) containing phosphate buffer (GIBCO,
310-4287AG… filled with trade name) and allowed to stand at 4 ° C for 3 to 21 days to form a PSA-adsorbed film on the outer periphery of the glass beads to produce a PSA affinity glass bead column container. A first preparation step, a second preparation step of discharging the PSA solution from the PSA affinity column container and rinsing with a phosphate buffer, and centrifuging and washing the semen to form a sperm suspension; Contacting the beads with glass beads and allowing them to stand at room temperature for about 15 minutes to adsorb the spermatozoa having intact acrosomes, selecting the non-adsorbed spermatozoa with a phosphate buffer, and separating the mannose (SIGMA, M-4625 ... a phosphate buffer solution containing (trade name) is added, the adsorbed spermatozoa are released at room temperature for 10 minutes, and if necessary, pipetting is performed a plurality of times to recover the free spermatozoa.

In the present invention, a sperm suspension is passed through a column (PSA column) packed with glass beads to which PSA has been attached, and the sperm having a normal head is covered with a sufficient area of intact acrosome. Therefore, it adheres to glass beads via PSA. On the other hand, abnormal spermatozoa that lack acrosomes, are small, if any, or reactant cannot bind to PSA and pass through the PSA-column. Thus, normal head sperm are selected. Next, a mannose that competes with the binding site of PSA
The solution is added and pipetting is further performed to release and recover the normal head sperm attached to the glass beads.

In the present invention, a new PSA-column was experimentally manufactured, and various conditions were examined so as to be the best. Furthermore, we applied this method to severe male factor infertility, performed microinsemination on hamster eggs, and confirmed that it was more effective than the swim-up method.

[0010]

【Example】

A prototype PSA-affinity column was prepared, and a basic study on the selection of normal head sperm was performed. The PSA-affinity column was prepared as follows. That is, as shown in FIG. 1, a 5 ml plastic syringe barrel is set upright, a well-washed glass wool is spread on the bottom in a film form, and a well-washed 1 mm diameter glass
Pack the beads to a 1 ml line. 0, 0.1, 0.5 and 1mg
/ Ml of phosphate buffer (GIBC) containing PSA (SIGMA, L-5380)
O, 310-4287AG) and leave at 4 ° C for 3-21 days
PSA was adsorbed.

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

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

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

In order to confirm the effect of the present invention, a sperm injection experiment was performed in the hamster's perivitelline in severe male factor infertility.

[0015] Spermatozoa were selected by PSA-affinity column or swim-up method from 3 cases of infertility of severe males who could not obtain fertilized eggs at all in three or more in vitro fertilizations and stimulated by calcium ionophore treatment. Thereafter, the hamster eggs were microinseminated into the perivitelline space, and the fertilization rates were compared. First, the semen is spun down and washed to prepare a sperm suspension. This was divided into two, one of which was a PSA-affinity column (0.5 mg /
ml PSA) to perform sperm sorting as described above.

On the other hand, a phosphate buffer was layered on the sperm suspension and cultured for 45 minutes at 37 ° C., the sperm was allowed to swim up, the upper layer was recovered, and sperm was selected. In both groups, the obtained spermatozoa were treated with 5 μM calcium ionophore A2318.
7 (Calbiochem, 100105) for 10 minutes at 37 ° C. Immediately centrifuge for 5 minutes, remove the ionophore solution, and add 30 mg / ml bovine serum albumin.
in) Ionophore was neutralized by adding an added HTF culture solution. 5
After culturing at 37 ° C. for 2 hours under 95% air with 95% CO ₂ , spermatozoa were injected into the perivitelline 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 sorting methods were compared.

The results of a basic study on the selection of normal head sperm using a 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 with 0.5 mg / ml PSA
The maximum was 1.7 times and 1.6 times respectively in the processing column.

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

The effect of pipetting on the recovery of sperm was four times that of the pipetting and mannose treatment as compared to the mannose treatment alone.

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

The results of the intestinal ovarian sperm injection test for 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 in three or more in vitro fertilizations.
Sperm was selected by the -up method, sperm was stimulated by calcium ionophore treatment, and then microinseminated into the perivitelline of hamster eggs, and the fertilization rates were compared. Table 1 summarizes the results. Although no statistically significant difference was obtained, 0.5 mg / ml
Fertilization rate (29%) when sorting sperm by PSA-column
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. The normal sperm head has about half of its outer surface occupied by intact acrosomes. PSA selectively binds only to intact acrosomes, but not to acrosome-reacted acrosomes. Therefore, when a sperm suspension is passed through a column filled with glass beads to which PSA is attached (PSA-column Fig. 1), the sperm having a normal head is covered with an intact acrosome with a sufficient area. Therefore, it adheres to glass beads via PSA. On the other hand, abnormal spermatozoa that lack acrosomes, are small if any, or have undergone an acrosome reaction cannot bind to PSA and pass through the PSA-column. Thus, normal head sperm are selected. Mannose is PSA
The normal head sperm attached to the glass beads is released by adding a mannose solution and further pipetting to recover. Thus, normal head spermatozoa are selectively collected.

The sperm sorting method using a PSA-affinity column can selectively recover normal head spermatozoa and can be applied to cases with a motility of 0%, and is expected to be useful as a sperm sorting evaluation method. Also other methods with different sorting mechanisms, such as sw
It is thought that the selection accuracy can be further improved by combining with im-up method or percoll centrifugation method.

[Brief description of the drawings]

FIG. 1. Principle diagram of PSA-affinity column, PSA selectively binds to acrosomes, so normal sperm is adsorbed to glass beads, and abnormal spermatozoa with missing or too small acrosomes pass through the column.

FIG. 2: Semen / PSA-% strict normal morphology and sperm head normal rate (% normal head) before and after the column. 3 homology experiments. # *: P <0.
01vs semen (χ ² test)

FIG. 3: 0.5 mg / ml PSA-Sperm morphology before (A) and after (B) the column. In A, sperm with abnormalities are mixed in the sperm head, acrosome, midpiece and tail, while in B, normal sperm are selected. References 1. World Health Organization: WHO Laboratory Manual for the Examination of Human
Semen and Semen-Cervical 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 poormorphology 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.

Claims (1)

(57) [Claims] 1. A glass wool film is provided at the bottom of a vertical container made of glass, plastic or the like having a discharge port on the lower side, and washed glass beads are spread over the upper side thereof.
~ 1mg / ml of Pisum satitom agglutinin
vum agglutinin (abbreviated PSA)
At a temperature of 3 ° C. for 3 to 21 days to form a membrane in which PSA is adsorbed on the outer periphery of the glass beads to prepare a PSA affinity glass bead column container. A second preparation step of draining the liquid and rinsing with a phosphate buffer; centrifuging and washing normal semen to form a sperm suspension; bringing the suspension into contact with the PSA glass beads; Adsorbing spermatozoa having intact acrosomes, separating the non-adsorbed spermatozoa with a phosphate buffer, and adding a phosphate buffer containing mannose to release the adsorbed spermatozoa at room temperature for 10 minutes. A method for selecting and evaluating normality of a sperm head, comprising a collecting step of collecting free sperm by performing pipetting a plurality of times as necessary.