A significant proportion 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 genetic abnormalities 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 are 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 later. This lies between loci AZFb and AZFc. Y chromosome microdeletion detection carried out on 245 males with a history of either azoospermia, severe oligozoospermia, oligozoospermia or oligoasthenoteratozoospermia (OAT) using the Promega Kit showed microdeletions in 20% men with azoospermia and 5% men in the OAT group.
Sperm aneuploidy detection by FISH in 48 cases showed 3-20% aneuploidy in 15% men.
Structural and numerical chromosome abnormalities were detected in 3% of over 500 men karyotyped.
Fragile-X syndrome is a genetic condition 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 was 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. More than 300 children were studied to identify families at risk of transmitting this condition and offer prenatal diagnosis to prevent the birth of other affected children.
It was observed that the Fragile X carrier females show premature ovarian failure. Hence women with premature ovarian failure should be tested for Fragile X carrier status before planning a pregnancy.
Gonadal chromosome mosaicism is difficult to diagnose as a gonadal biopsy is required. This cannot be performed routinely. In infertile females undergoing in vitro fertilization (IVF), follicular fluid is obtained during oocyte retrieval. 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 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 can be used as an alternative treatment option to a stimulated IVF cycle in a select group of women requiring Assisted Conception. Germinal vesicle stage cumulus-oocyte complexes collected from patients undergoing oocyte retrieval are cultured in maturation medium with supplements and those that reach the MII 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 was determined.
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 were of no use in IVF were 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 were located under phase contrast and photographed. FISH for common aneuploidies was performed on these single cells. The signals were analysed to detect aneuploid, mosaic and chaotic embryos.
This study explored the biology of the cumulus cells at the molecular level so as to establish the role of cytokines and growth factors secreted by the cumulus cells in embryonic development and also in providing a mechanism to improve embryo-uterine adhesion.
We have screened the cumulus cells for the production of 6 main factors, namely VEGF, LIF, IL-6, TGF-b, IGF-1 and integrins since they have been identified as the main factors to impact embryo development as well as implantation.
Chromosome polymorphisms or variants are known to occur. They are demonstrated as changes in size of particular areas of certain chromosomes, and are seen mainly in the following regions:
- Centromeric heterochromatin of chromosomes 1, 9, 16 and distal heterochromatin of Y. There is either increase or decrease in the length of the heterochromatic region. Pericentric inversions in these chromosomes are also considered as variants.
- Polymorphisms include increase in the length of short arms and stalks or size of satellites of the acrocentric chromosomes (13, 14, 15, 21 and 22)..
There is limited published data worldwide on the frequency of polymorphic chromosome variants in the normal fertile population, as most surveys have been carried out on newborns. Karyotyping of normal fertile and infertile individuals is being carried out in our laboratory to help add to our knowledge regarding associated causes of infertility, without bias of laboratory variation and geographic distribution. Our Research work and research paper on polymorphisms has been sited by more then 100 scientists world wide.
Currently, our team is concentrating on oocyte cryopreservation, a procedure which can help women with some types of cancer to freeze their genetic material before oophorectomy, before radiation of the ovaries and prior to chemotherapy. We have frozen oocytes for more then 200 women.