Diagnosis of immunologically caused infertility

During a long married life without the use of contraceptives, and in the presence of micro-in mucous membranes of the genital can occur gradually spot immunization antigens of spermatozoa. The physical examination of the spouses, consisting in a barren marriage, can not determine the cause of infertility in 10-15 % cases. It is in this group of infertile spouses when applying special methods of investigation secret cervical canal, semen plasma and serum sperm antibodies are often detected (ASAT), Uses qualitative and quantitative tests of immunological research.

Postcoital test (FCT)

Postcoital test (FCT) It refers to qualitative immunoassay. A negative result indicates the absence of sperm in the cervical canal secret, but you can not identify the cause of their absence.

Method. By the number of motile sperm in the cervical canal secret, obtained after coitus, establish the extent of violations of their penetrating power in infertility or immunity to them mucus of the cervical canal.

FCT is necessary to conduct the following conditions: The study should be conducted in the period of ovulation, which revealed colpocytologic or other method, after prior abstinence spouses within 2-3 days.

The methodology of the. After the usual coitus for a woman 30 minutes should lie on his back, bending your knees. Douching and cleaning the prohibited, need only sanitary napkin, which is removed during the capture for the study of mucus of the cervical canal. After 2-4 hours, permissible and after 6-10 hours (time, elapsed before the study, indicated in the analysis) after intercourse cervix exposed using a mirror Cuzco and endocervical mucus aspirated or tuberculin syringe (without needle), a pipette with a rubber bulb, or polyethylene catheter.

When washing the instrument should not be used disinfectants, as they affect sperm motility. One drop of the obtained mucus of the cervical canal is transferred onto a warmed glass slide, coated coverslip, Territory lubricated with liquid paraffin to leak. Formulation immediately mikroskopiruyut (eyepiece 7X, 40X objective), determining the number and nature of sperm motility. The drug can be detected with active sperm progressive motility, oscillating motion (the phenomenon of swing) and fixed.

Accepted provide five types of conclusions FCT.

• Great (the appropriate rate): in five fields of view up to 15-20 with active sperm progressive mobility and more. Sperm with the phenomenon of oscillation is less than 25 %.
• Satisfactory: in five fields of view to 10 active sperm progressive mobility and less 25 % sperm with the phenomenon of swing.
• Bad: less 5 spermatozoa in five fields of view.
• Negative: absence of sperm.
• Doubtful: in five fields of view more 5 active-motile sperm and more 25 % the phenomenon of swing.

When the fifth type of FCT often observed immune incompatibility of spouses, therefore necessary to determine the ACAT. To identify ACAT secret endocervical, plasma, semen or blood serum and display them (agglutination or immobilized) It requires the appointment of quantitative immunoassays.

A negative PBC studies begin with a definition of penetrating (penetration) the ability of the sperm via penetrative test (Fri.) and susceptibility to sperm secretion of the cervical canal using a contact test of sperm and cervical mucus (TKSTSS).

A penetration test (TP)

The thin capillary length 10 see during ovulation typed mucus of the cervical canal. One open end of the capillary closes clay, and placed in a second vial with the test fresh ejaculate (no more 3 hours after ejaculation). With clay capillary vertically attached to tube. The sample is incubated in a thermostat at a temperature 37 ° C. 1 no. On the quality of the ejaculate is judged by the depth of penetration of sperm in the mucus of the cervical canal. There are three types of GHG.

• Positive - The depth of penetration (the greatest distance in centimeters, traveled at least single sperm) above 5 cm; the number of sperm in the point of maximum penetration more 5 in sight, and the number of motile spermatozoa in the upper third of the capillary is greater than 50 %.
• Doubtful - A depth of penetration up to 5 See less sperm count 5, their mobility in the upper third of the capillary reaches 50%.
• Negative - The depth of penetration of sperm reaches 1 cm.

The ability of sperm to penetration It depends on the phase of the menstrual cycle. She begins to emerge from the 9th day of the menstrual cycle,, gradually activating, It reaches its maximum expression in the period of ovulation, held at this level for two days, and then gradually decreases.

Test contact semen and cervical mucus (TKSTSS)

On the slide is put a small drop of mucus from the cervical canal, taken from women during ovulation, and a number of the same size drop of ejaculate. Both drops are covered with a cover glass, which is slightly pressed down for, to drop connected. The drug is placed in a moist chamber (Petri dish with moistened cotton swab) and incubated in a thermostat at a temperature 37 ° C. 1 no.

Under these conditions, normal sperm penetrate the secret of the normal cervical canal, as can be seen under a microscope at a magnification 200X. To account for the reaction near the boundary between two media counts the number of penetrating the secret of the cervical canal sperm in two adjacent fields of view.

With excellent TKSTSS in two fields of view reveals more 25 actively motile sperm in the phenomenon of swing less 25 %. If satisfactory TKSTSS in two fields of view revealed more 15 actively motile sperm, in poor TKSTSS - less 15. A negative absent penetration TKSTSS, if questionable - a phenomenon of oscillation exceeds 25 %.

In this test, as in PCT, the phenomenon of swing, exceeding 25 %, likely, It indicates the presence of ACAT and requires the appointment of quantitative immunoassays.

Using semen and cervical secretions channel infertile and fertile men in various amounts to using TKSTSS identify infertile husband.

• Example 1. Taken a secret channel of the cervix in women infertile couples and control ejaculate healthy men. Excellent TKSTSS indicates a weak penetration ability of sperm of her husband, and the susceptibility of normal secretion of the cervical canal.
• Example 2. Taken full secret of the cervical canal healthy woman and her husband ejaculate of infertile couples. Bad TKSTSS indicates a weak penetration of sperm.
• Example 3. Taken secret endocervical women infertile couples and control ejaculate healthy men. Bad TKSTSS indicates inferiority secret cervical canal, which obviously do not penetrate normal spermatozoa.

Quantitative immunological tests

Mikroagglyutinatsionny test Friberg

Mikroagglyutinatsionny test Friberg (TF) sufficiently sensitive and is available for clinical diagnostic laboratories. Using TF can determine the presence of ACAT and agglutination titer in various materials (plasma ejaculate, extracts of mucus of the cervical canal and in the serum of both spouses).

Reagents:

1. Phosphate buffered saline (FSB) 0,01 М pH 7,2—7,4.

2. Petrolatum.

Equipment: microscope, tuberculin syringe, mikroagglyutinatsionnye pipetter 1 ml, micropipette capacity 0,1 ml.

Method. The study serum inactivated by incubation in an incubator at 56 ° C. 30 m. Ejaculate, obtained by masturbation, immediately diluted so that FSB, the concentration of sperm was 40 million. in 1 ml.

Because of the material (plasma ejaculate, extract the secret of the cervical canal or serum) is preparing a series of dilutions 1:4 to 1:128 using as a liquid for dilution FSB. In wells with paraffin oil mikroagglyutinatsionnyh planchettes tuberculin syringe with a needle is introduced (under petrolatum) Small drops of each dilution of the test material. After each sample is washed four times the syringe. In each dilution is added to the 5 times smaller drop of ejaculate, and by 4 h at room temperature detected agglutinates. Last breeding, whereby under a microscope at 100X magnification detected 3 agglutinate under review and more, agglutination titer corresponds ACAT.

Spermoimmobilizuyuschy test Izodzhima (SIT).

Titer immobilizing ACAT serum, plasma or semen extract secret cervical canal is set by the number of motile sperm in the presence of antigens (sperm).

Components.

1. Ejaculate, which is produced and prepared similarly, for TF using FSB; motility exceed 70 %.

2. Komplementsoderzhaschaya serum of guinea pigs.

3. Immune human serum (IChS), giving in the presence of complement through 1 h incubation immobilization 90 % sperm.

4. Normal human serum (NChS), inactivated by heating at a temperature 56 ° C. 30 m.

5. The tested inactivated serum, plasma and semen extract secret cervical canal.

Method. To evaluate complement activity immobilizing the 1st tube is introduced 0,5 ml of isotonic sodium chloride solution, to determine the changes in sperm motility without ASAT the 2nd test tube - 0,5 ml NHS, control immobilization third tube - 0,5 ml of ICHS and to determine the ACAT immobilizing the 4th tube - 0,5 ml of serum, or plasma of the ejaculate, extract or secretion endocervical.

Then, each tube is introduced by 0,1 ml complement. The last thing to all tubes is added 0,05 ml sperm suspension in a concentration 40 million. in 1 ml. Tripod tubes were placed in an incubator at a temperature 37 ° C 1 no. The tubes are removed from the oven and carefully shaken to reflect settled agglutinated sperm.

To evaluate the results under high magnification microscope (400X) calculated the percentage of motile sperm in a drop of the test tube, deposited on a glass slide and covered with a cover. In the same way set the percentage of motile sperm in the droplets of the test tubes with the NHS and the ICHS. The result of the reaction into account by the formula

SIT = K / A

where K - the number of motile sperm in the control with the ICHS and the NHS;

About - the number of motile sperm in the test tube (serum test, or plasma эяkulяta, or the secret of the cervical canal).

With average SIT<2 the test is negative (number of motile sperm in control and in the test tubes of approximately the same), if the index CTI>2 - The test is positive, t. it is. in the material (serum, plasma or semen extract secret cervical canal) are immobilized ACAT.

• Example 1. Ground control to ICHS - 10 % motile sperm. The second control with NHS - 80% motile sperm. The two controls together - 90 % motile sperm. In the test tube - 80 % motile sperm.

SIT = R / D = 90/80 = 1.1

The test is negative, immobilized ACAT absent.

• Example 2. In the test tube - 40 % motile sperm, However, in the control, in Example 1.

KN = 90/40 = 2.2

The test positive. There is immobilized antibody (ASAT), through which sperm motility significantly reduced.