Karyotyping
Indications for Cytogenetic Analysis
- Couples with repeated spontaneous abortions
- Men with oligozoospermia and azoospermia.
- Men with hypogonadism, breast development and lack of facial hair.
- Women with primary amenorrhoea, short stature, and streak gonads.
- Women with polycystic ovaries.
Sample: 2 ml Blood in sodium heparin vaccutainer (Green top tube), transported at room temperature.
Sperm FISH
Routine analysis of sperm chromosomes is not feasible in clinical practice. However, interphase FISH on spermatozoa offers an accurate and reliable method of analysis even in the presence of a low sperm count.
The percentage of sperm with hypo/hyperhaploidy is calculated to counsel the couple regarding chances of a normal pregnancy.
Sample : Fresh semen sample.
Outstation samples are accepted after partial processing in any andrology laboratory. The ‘swim-up’ sperm and pellet are to be separated in 2 tubes and incubated in hypotonic KCl (0.075M) for 20 minutes, following which a few drops of Carnoys fixative are added. After centrifugation, fresh fixative is added to the pellets before transportation.
Detection of Y chromosome microdeletions by PCR
Y chromosome micro-deletions like DAZ (deletion in azoospermia) and RBM (RNA binding motif gene) cause spermatogenic defects. In the Yq arm, intervals V and VI comprise an Azoospermia Factor (AZF) which contains the DAZ and RBM genes. Several genes have been identified within this region and have been proposed candidates for infertility. We now check for 20 different micro-deletions in the AZF region by multiplex PCR.
Sample: 3-5ml Blood in Potassium EDTA vaccutainer (purple top tube), transported at room temperature.
Sperm apoptosis by TUNEL method
The sperm apoptosis test is used to determine whether the male partner of an infertile couple has a high, fair, or poor fertility potential.
Apoptosis is a mode of programmed cellular death based on a genetic mechanism that induces a series of cellular, morphological and biochemical alterations, resulting in fragmentation of genomic DNA. The sensitive TUNEL method is based on the detection of single and double stranded DNA breaks occurring at early stages in apoptosis. After an enzymatic reaction, the fluorescein label incorporated at the damaged sites of DNA is visualized by fluorescence microscopy. The apoptotic sperm appear greener, while the normal non-apoptotic sperm take up more of the DAPI stain and appear blue. Partially apoptotic sperm will appear partially blue and green. This test allows the detection of apoptosis at the single cell level and may serve as an indicator for use of antioxidants prior to IVF.
Follicular fluid is a novel source of gonadal cells for detection of low-grade mosaicism in the ovaries. All women with unexplained infertility undergoing IVF could be studied to determine the chances of a successful pregnancy. If there is evidence of gonadal mosaicism, the chances of a live birth could be low. Mosaicism for the X chromosome detected rapidly by FISH on follicular fluid cells can be one more indication for preimplantation genetic diagnosis (PGD) in the same and subsequent cycles.
FISH on blood, buccal and urine cells for detection of low grade mosaicism
Mosaicism is the presence of chromosomally normal and abnormal cells in the same individual. The percentage of mosaicism is known to vary in different tissues. We can easily study cells derived from the ectoderm, endoderm and mesoderm by the FISH technique, on buccal, urine and blood cells. When >5% aneuploidy for the X chromosome is detected in the follicular fluid cells derived from the ovary, we check for aneuploidy in these other tissues as well.
