FERTILITY TESTING

Frequencies of Defects among infertile couple Ovulation Defects Semen Abnormality 30% 22% Tubal defects other disorders Endometriosis 17% 12% 5%

Most Commonly used diagnostic steps for infertility Men: 1. Semen analysis incld sperm antibody 2. Ultrasonographic scan of the pathway 3. Sperm function test 4. Testosterone level and other hormones Women: 1. Endometrial biopsy 2. Serum progesterone estimation 3. Laparoscopy 4. Hysterosalpingogram 5. Post coital test

I. SEMEN Analysis - failure to conceive after 6mos. of unprotected intercourse - sexual abstinence of 3 days but not longer than 5 days - longer abstinence = inc. semen vol. but reduced sperm motility collect 2 nd specimen in another 2 hours - collect by masturbation - evacuate bladder prior to ejaculation - use pre-weighed sterile plastic container with screw top - deliver specimen w/in an hour of collection

- if suspecting retrograde ejaculation get post ejaculate urine - collect and analyse 2 specimens at 2 to 3 wks interval (+) marked difference, collect additional specimens

Initial Macroscopic exam done after liquefaction - liquefy in <20min at room temp.

failure to liquefy = inadequate prostatic secretion add proteolytic enzymes (bromelin, plasmin or chymotrypsin) - thoroughly mix specimen before subsequent exam and viscosity recorded.

Macroscopic • Volume – 2 to 5 ml • Color – white or gray-white yellowish hue = pyospermia rust color = small bleedings in the seminal vesicle 3. pH – 7.2 to 7.8

8.0 or above = acute infection in the prostate, SV or epididymis 7.0 or lower = contamination with urine = obstruction in the ED = mainly prostatic fluid

Initial Microscopic Exam 1. Estimate of Sperm concentration Depth of 20um 4uL of sample Hemocytometer Total sperm ct = concentration x volume (20 to 50 mil/mL) (2 to 5 mL)

2. Motility = 50% or above = done on warm stage Grade 4 = rapid movement in a straight line with little yaw and lateral movement Grade 3 = slower Grade 2 = slower (normal range) Grade 1 = no forward progression -motility of <50% = use viability stain of eosin with nigrosin (red dye accumulation in the head of non-motile sperm) - samples with no visible sperm = centrifuge entire sample = repeat analysis in another 4 to 6 months

3. Agglutination - motile sperm stick to each other in a variety of orientation - suggestive of immunologic cause of infertility - record description of the type agglutination Other Cells: 1. Round Cells immature germ cells (single or double highly condensed nucleus with larger area of surrounding cytoplasm) 2. Bacteria 3. Epithelial cells PMN (smaller and inc. N/C ratio) (+) peroxidase staining

-Negative sperm with low semen volume fructose test examine post ejaculate urine Teratozoospermic index –average number of defects/spermatozoa - acrosomal cap should be >1/3 of head surface - cytoplasmic droplet of < half of the head size - tail should be >45um long

II. Immunologic Assays - antibody binding to head and tail is considered specific for immunologic infertility.

- usually of the

IgA

or IgG and rarely IgM

Latex bead

- MAR test and immunobead assay

Human IgG + =

Increased risk of antibody in men: (a) vasectomy (b) repeated infections (c) obstruction of the ducts (d) cryptochidism (e) varicocele (f) testicular biopsy (g) trauma (h) torsion (I) cancer (j) genetic predisposition in women: (a) intense mucosal inflammation of the genital tract

Microbiologic Assays - E.coli can cause sperm agglutination and immobilization - >1000 cfu/ml C/S

Biochemical Assays Prostate gland – Zinc, Citric Acid, pH, & acid phosphatase activity Seminal Vesicle – fructose Ejaculatory duct obstruction, Agenesis of Vas defferens and SV - low volume, low pH, no coagulation and no characteristic semen odor Epididymis – neutral alpha glucosidase

Fertilizing Function or Sperm Function Test 1. Sperm Penetration Assay (SPA) - tests sperm fertilizing capacity -capacitation -acrosome rxn -Speroolemma fusion -sperm incorporation in the ooplasm -decondensatin of sperm chromatin

2. Hemizona Assay - uses unfertilized oocytes - strict criteria morphology and IVF rates

3. Acrosin Assay - trypsin-like serine proteinase

4. Hypo-osmotic swelling test (HOS) - membrane integrity of sperm membrane - correlates with SPA

5. Cervical Mucus Penetration Assay - at midcycle - inc . estrogen - inc. water - inc. Ca +2 - inc. pH (7 – 8.5)

Therapeutic Procedures 1. Sperm Wash - IVF, GIFT, intrauterine insemination, retrograde ejaculation, (+) sperm antibody - separation within an hour of ejaculation 2. Semen Cryopreservation - sperm banking - before beginning cancer tx, before surgical sterilization, life threatening situation, low sperm count