Jaslok Hospital

Characterization of chromosome anomalies, Y microdeletions and Cystic Fibrosis mutations in male infertility.
A significant proportion (60%) of male infertility is due to an underlying genetic cause. The use of intracytoplasmic sperm injection (ICSI) raises a possibility of transgenerational transmission of genetic defects to the offspring where the father has a genetic abnormality such as Y chromosome microdeletions. For proper management of these couples, counseling after clinical investigation, semen analysis, a karyotype and a DNA analysis to rule out Y chromosome microdeletions were carried out.

At least three distinct non-overlapping regions on the Y chromosome, named as AZFa, AZFb and AZFc for azoospermia factors a, b and c are critical for germ-cell differentiation. A fourth locus AZFd has been suggested afterwards which lies between loci AZFb and AZFc. Y chromosome microdeletion detection was carried out on 100 males with a history of either azoospermia, severe oligozoospermia, oligozoospermia or oligoasthenoteratozoospermia (OAT). Multiplex PCR was set up using the Promega kit Version 1.1 for 18 mutations in the AZF (azoospermia factor) a, b, c & d regions. The PCR products were analyzed on 2.5% agarose gel by electrophoresis. Y chromosome microdeletions were observed in 12 out of 100 patients (12%).

We are now using the modified version of Y chromosome microdeletion detection kit as Version 2.0 from Promega which includes 20 mutations.
Cytogenetic and Molecular Analysis of the Fragile X Syndrome.
Fragile-X syndrome is a genetic condition mainly leading to mental retardation in males. It is caused by a fragile site at the tip of the long arm of the X chromosome, which is demonstrable on karyotyping using special tissue culture media. However, cytogenetic methods are not very reliable for Fragile X detection, compared to molecular techniques. Molecular studies have shown the presence of an increasing number of CGG trinucleotide repeats in every generation. A carrier mother may have 60-200 repeats and an affected son will have >200 repeats in the FMR-1 gene. Southern blotting with radioactive detection and PCR are used for molecular diagnosis. There is hardly any data on Fragile X patients from western India. The aim of this study is to try and develop a non-radioactive molecular method for detection of Fragile X, using Silver staining detection for PCR analysis and chemiluminescence detection for Southern hybridization. Children from special schools will be studied to identify families at risk of transmitting this condition and offer prenatal diagnosis to prevent the birth of other affected children.
FISH on Follicular Fluid to detect Gonadal Sex Chromosome Mosaicism in women undergoing infertility treatment
Gonadal chromosome mosaicism is difficult to diagnose as a gonadal biopsy is required. This cannot be used as a routine procedure. In infertile females undergoing in vitro fertilization (IVF) follicular fluid is obtained during oocyte pick-up. After collecting the oocytes for IVF, the surrounding follicular fluid is generally discarded. We studied the cumulus cells present in follicular fluid by the fluorescence in situ hybridization (FISH) technique and found that this was a novel way of detecting aneuploidy in the ovarian tissue. We could detect Turner syndrome mosaicism in this tissue in a cytogenetically proven case of Turner syndrome mosaicism undergoing IVF. The percentage of chromosomal mosaicism is known to vary in different tissues. We could demonstrate that follicular fluid cells are an easily available alternate source of cells to detect chromosome mosaicism by FISH, in patients undergoing IVF. The study is ongoing to collect more data on follicular fluid cells and compare it with karyotyping from blood and FISH on buccal smears.
In Vitro Maturation of Germinal Vesicle Oocytes
In an IVF cycle, about 8.5% of retrieved oocytes are immature, at the germinal vesicle stage. Complete maturation of oocytes is essential for the developmental competence of embryos. Human oocyte maturation is considered as the re-initiation and completion of the first meiotic division from the germinal vesicle stage (prophase I) to metaphase II and the accompanying cytoplasmic maturation, for fertilization and early embryonic development. Immature human oocytes obtained from patients undergoing oocyte retrieval can be matured and fertilized in vitro. In vitro maturation is slowly becoming established as an alternative treatment option to stimulated IVF for women requiring assisted conception. Germinal vesicle stage cumulus-oocyte complexes collected from patients undergoing oocyte retrieval are being cultured in maturation medium with supplements and those that reach the M2 stage undergo intra-cytoplasmic sperm injection (ICSI). Clevage-stage embryos are obtained. The overall maturation, fertilization and cleavage rates after ICSI of 150 germinal vesicle stage cumulus-oocyte complexes will be determined.
Fluorescent in situ hybridization (FISH) in slowly cleaving and arrested embryos.
Reproductive wastage is a natural phenomenon to eliminate defective embryos. This has been confirmed by cytogenetic studies on spontaneous abortions and recently, on arrested embryos. The Fluorescence in situ Hybridization (FISH) technique enables rapid detection of common aneuploidies. A contributory cause of IVF failure is a high number of zygotes with missing or extra chromosomes. Individual blastomeres from slowly cleaving and arrested embryos which are of no use in IVF are biopsied using a non-contact diode laser beam, treated with hypotonic solution and fixed on slides with an effort to remove the cytoplasm and expose the intact nucleus. The nucleii are located under phase contrast and photographed. FISH for common aneuploidies is performed on these single cells. The signals are analysed to detect aneuploid, mosaic and chaotic embryo