FERTILITY TESTING
Frequencies of Defects among infertile couple
Ovulation Defects
-
30%
Semen Abnormality -
22%
Tubal defects
-
17%
other disorders
-
12%
Endometriosis
-
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 2nd 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
PMN
(single or double highly condensed nucleus
with larger area of surrounding cytoplasm)
(smaller and inc. N/C ratio)
(+) peroxidase staining
2. Bacteria
3. Epithelial cells
-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
- MAR test and immunobead assay
Latex bead
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