Semen:- Part 4 – Work up for Fertility (Work up of Infertile Couple)

Definition of infertility

Infertility is the inability to conceive after one year of intercourse without contraceptive devices or medications. Infertility is divided into:

1. Primary infertility is the infertile couple who had no previous successful pregnancy.
2. Secondary infertility affecting individuals who had a previous successful pregnancy but now unable to conceive.

For to evaluate infertility and fertile couple, hormones and semen workup are needed.

Male infertility factors:

1. Semen analysis:
   1. It includes count, the volume of the ejaculate, pH, and motility. This has been discussed in part 1.
2. Sperm function:
   1. The sperm should reach the ovum through directed motion, undergoes capacitation, and fuse with the oocyte membrane.
   2. Then it will incorporate into the oocyte cytoplasm.
   3. Summary of the sperm functions tests:
      

      Sperm function tests	Details of the test and interpretations
      Cervical mucus penetration	This is the observation of the cervical mucus by the sperms. Sims Hunter test can be advised.
      In vitro acrosome reaction	This is the evaluation of the acrosome to produce enzymes essential for ovum penetration.
      Hamster egg penetration	In this case, the sperms are incubated with hamster eggs. Then penetration is observed microscopically.
      Hypo-osmotic swelling	The sperms are exposed to low-sodium concentration. Then evaluated for membrane integrity and sperm viability.

3. Sperm-mucous penetration:
   1. This will tell us the ability of the sperm to travel through the vaginal mucous to reach the uterus.
   2. In vitro test to check the penetration ability:
      1. Bovine cervical mucus is taken in the capillary tube.
      2. The migration of the sperm in the tube is measured.
      3. Following factors are noticed:
         1. Migration distance.
         2. Penetration density.
         3. Duration of the progressive movement.
         4. Migration reduction.
4. Sperm motility:
   1. The presence of sperm capable of the forward, progressive movement is critical for fertility.
   2. This progressive movement will propel the sperms through cervical mucus to the uterus, fallopian tubes and reach the ovum.
   3. This should be done on well-mixed, liquefied semen within one hour of the collection.
   4. Sperm motility grading:
      

      Grade of the motility	Criteria for the motility
      Grade 4.0	There is rapid,straight-line motility.
      Grade 3.0	The speed is slower, and some lateral movement.
      Grade 2.0	There are slow forward progression and noticeable lateral movement.
      Grade 1.0	There is no forward progression.
      Grade 0	There is no movement.

5. Sperm viability testing:
   1. Decreased sperm viability will lead to infertility.
   2. Procedure to check sperm viability:
      1. Mix semen with eosin-nigrosine stain.
      2. After few minutes, make the smear.
      3. Now count the number of dead sperms per 100 sperms.
      4. Living sperms will not take the dye and will have bluish-white color.
      5. The dead sperms will stain red in color against the purple background.
   3. Normal viability requires 75% of the living sperms.

Semen viable sperm stained by the eosin-nigrosin stain

6. Seminal fructose test:
   1. Low sperm concentration is caused by the lack of support medium in the seminal fluid like fructose.
   2. If there is low or absent fructose in the seminal fluid, that will lead to low sperm concentration in the semen.
   3. The normal fructose level is 13 µmol per ejaculate.
   4. The resorcinol test judges the fructose concentration:
      1. Resorcinol reagent consists of:
         1. 50 mg resorcinol in 33 mL of concentrated HCL.
         2. Make the volume to 100 mL by adding the distle water.
      2. Procedure: Mix 1 mL of semen with 9 mL of resorcinol reagent.
      3. Boil the test tube.
      4. Result:  Observe for the orange-red color.
      5. The specimen should be tested within 2 hours of the semen collection or freeze to prevent fructolysis.
7. Antisperm antibodies:
   1. Antisperm antibodies may be present in both males and females.
   2. These antibodies may be detected in:
      1. Semen.
      2. Cervical mucosa.
      3. Serum.
   3. Antisperm antibodies are identical in the male and female infertility evaluation; these can be detected by:
      1. Agglutination.
      2. Immobilization.
      3. Radioimmunoassay (RIA).
      4. Immunofluorescent assay.
      5. Enzyme-linked immunoabsorbent assay (ELIZA).
   4. Both partners may show antisperm antibodies, but these are more common in males.
   5. Under normal conditions, the blood-testes barrier will prevent sperm exposure to the immune system.
   6. There is the exposure of the sperm to the immune system in the case of vasectomy, trauma, and infections.
   7. An antigen on the sperm will produce the antibody, which will damage the sperm.
      

      Formation of antisperm antibody Ab

   8. The damaged sperm produce antibodies in the female.
   9. In the male sperm, antibodies are suspected when there is clumping of the sperms seen in routine semen analysis.
   10. The presence of antisperm antibodies in the female can be seen:
       1. Mix the sperms with the cervical mucosa or the serum.
       2. Observe the agglutination.
   11. Other tests for the antisperm antibodies are:
       1. Mixed agglutination reaction.
          1. This is a screening procedure to detect the IgG antibodies.
          2. Semen containing motile sperm s is incubated with IgG antihuman globulin (AHG).
          3. Latex suspension or treated RBCs coated with IgG.
          4. The bivalent AHG will bind the sperm and another side to latex coated particles (or IgG coated  RBCs).
          5. This reaction will give rise to microscopically agglutination or clumping of the sperms.
          6. Result: <10% of the motile sperms attached to the latex particles are considered normal.
       2. Immunobed test.
          1. This is a more specific test, and it can detect IgG, IgM, and IgA antibodies.
          2. This will also show which area of the sperm, head. Neck or tail is affected.
             1. Antibodies against the head will interfere with the penetration of the sperms to the ovum or cervical mucosa.
             2. Procedure: Sperms are mixed with polyacrylamide beads coated with either anti-IgG, anti-IgM, or anti-IgA.
                1. Examine under the microscope, which will show the beads attached to the sperm at a particular area.
                2. Result:  The presence of the beads <20% of the sperms is normal.
   12. Simes-Hunter test:
       1. This is the post-coital test to rule out any factors present in the cervical mucosa.
       2. Procedure: This is usually done in the mid-cycle ovulatory phase when there is maximum cervical mucus.
          1. After 2 to 3 hours of the post-coital period (after the intercourse).
          2. Expose the cervix, and take mucus from the endocervix.
          3. Examined the aspirated material under the microscope.
          4. Result: Report the total number of the sperms/HPF and the percentage of motile sperms.
   13. Evaluation of the level of the hormones:
       1. Serum testosterone level:
          1. This is important when the secondary sex characters’ development is deficient.
          2. For evaluating the Leydig cell function, inject  I/M 5000 IU of CG (Chorionic gonadotropins).
          3. Measure the serum testosterone level between  48 hours and 96 hours after the injection.
          4. Male with hypogonadism will have a decreased testosterone response to the injection.
       2. FSH level to be measured in patients with a count of 5 to 10 million/mL semen.
          1. Elevated FSH level = Sertoli cell dysfunction.
             1. Male may show azoospermia.
             2. Primary germinal cell failure.
             3. Klinefelter syndrome.
          2. Elevated FSH + LH + low testosterone + oligospermia in males = Primary testicular failure or andropause (decreased sexual satisfaction, reduced body hairs).
   14. In the abnormal semen can advise these test:
       1. Decreased count of sperm:
          1. There may be a lack of seminal fluid nutrients for the sperm-like fructose.
          2. Advise fructose level and test.
       2. Decreased motility with normal count:
          1. There is a viability issue.
          2. Advise Eosin-nigrosin test.
       3. Normal count with continued infertility:
          1. Check for the female antisperm antibodies.
          2. Advise immunobead test.
          3. Check for the sperm agglutination with female serum.
       4. Decreased motility with clumping:
          1. There may be male antisperm antibodies.
          2. Mix sperms with male serum for agglutination.
          3. Advise Immunobead test.
          4. Advise mixed agglutination reaction.

Summary of male infertility factors:

Female infertility factors

In the female, mostly the infertility factors are contributed to ovary and hormones almost 30% of the cases. While the pelvic factors including fallopian tubes, cervical. and uterine diseases account for almost 50% of the cases. Immunological factors are involved in roughly 5% of infertile ladies.

Female infertility causes

1. Ovarian dysfunction can develop regardless of the female has normal menses, making it difficult to diagnose ovarian dysfunction.
2. Product of conception (PCO) results in androgen excess is the most common cause of anovulation.
3. Ovulatory dysfunction is caused by liver and thyroid diseases.

Evaluation of female infertility:

1. It includes the detailed history of the patient and physical examination.
2. If you find the obvious cause, then treat that.
3. In the case of abnormal menstrual history, further evaluation of the hormones is done.
4. Post-coital test:
   1. The post-coital test is the quick assessment of multiple factors affecting fertility.
   2. Procedure: Collect the post-coital cervical mucus in the middle of the cycle.
      1. Place it on the glass slide with two coverslips.
      2. Mucus with adequate estrogen stimulation is clear and thin.
      3. This mucus forms a 6 cm or greater thread in length when the coverslips are separated from the slide.
      4. Now examine under the microscope, air-dried mucus, which has an adequate estrogen effect, will form a fern-like pattern.
      5. Before mucus gets dried, >20 motile sperms are seen under microscope HPF.
5. Progesterone Measurement: 
   1. Measurement of the progesterone level is the primary assay used to evaluate ovulation.
   2. After the ovulation, the blood progesterone level rises, and the peak is within 5 to 9 days ( day 21 to day 23).
   3. In case there is no ovulation, then there is no formation of the corpus luteum and no rise in the progesterone hormone.
   4. Progesterone level of >10 ng/ml suggests normal ovulation.
   5. While progesterone level of <10 ng/mL indicates an anovulatory cycle.
      

      Female infertility, progesterone, and LH surge to guide for ovulation

6. luteinizing hormone (LH) surge:
   1. LH appears in the urine just after the physiologic LH surge; this is 24 to 36 hours before the ovulation.
   2. LH surge confirms the ovulation and serves as the guide used to time for intercourse.
   3. There are kits available to check the LH surge to find the time of ovulation.
7. Hormonal changes:
   1. FSH (Follicular stimulating hormone):
      1. In the case of primary ovarian failure (Hypergonadotropic hypogonadism), there repeatedly elevated basal FSH level (>30 IU/L).
      2. Or a single elevated level of >40 IU/L.
      3. Patients with hypergonadotropic hypogonadism also have low estrogen levels (HYpoestrogenic). These patients have estradiol <20 IU/L.
   2. Hypogonadotropic hypogonadism case show:
      1. Estradiol = <40 pg/mL  (110 pmol/L).
      2. LH = <10 IU/L (LH level is decreased).
      3. FSH = <10 IU/L (FSH level is decreased).
   3. Prolactine level: Hyperprolactinemia causes hypergonadotropic hypogonadal infertility in these patients.
      1. Prolactin levels may be high in patients taking drugs like antidepressants, cimetidine, and methyldopa.
      2. Prolactin level will be raised in products of conception (PCOS).
   4. TSH (Thyroid-stimulating hormone) level should be measured to rule out hypothyroidism.
   5. Pituitary gland:
      1. Radiographic imaging is needed to rule out pituitary adenoma or empty sella syndrome.

Summary of the female infertility factors: