Anurupa Maitra

Genetic variation in exon 17 of INSR is associated with insulin resistance and hyperandrogenemia among lean Indian women with polycystic ovary syndrome

OBJECTIVE Polycystic ovary syndrome (PCOS) is a multigenic disorder, and insulin resistance is one of its hallmark features. Polymorphisms in exon 17 of insulin receptor (INSR) gene are reported to be associated with PCOS. We investigated this association in Indian women and its putative relationship with PCOS associated traits, which has not been explored so far. METHODS In this case control study, the polymorphisms were investigated by direct sequencing in 180 women with PCOS and 144 age matched controls. Clinical, anthropometric, biochemical, and hormonal parameters were also estimated. RESULTS The silent C/T polymorphism at His1058 in exon 17 of INSR was found to be present in our study population. The polymorphic genotype (CT+TT) was significantly associated with PCOS in lean women (chi(2)=8.493, df=1, P=0.004). It showed association with higher fasting insulin levels (P=0.02), homeostasis model assessment of insulin resistance (P=0.005), free androgen index (P=0.03), and lower quantitative insulin sensitivity check index (P=0.004) in lean PCOS women. No other novel or known polymorphism was identified in exon 17 in this cohort. CONCLUSIONS The study shows significant association of C/T polymorphism at His1058 of INSR with PCOS in the lean rather than obese Indian women. Its association with indices of insulin resistance and hyperandrogenemia is also seen in the same group. The findings strengthen the concept that pathogenesis of PCOS is different in lean and obese women.

Effect of tamoxifen treatment on global and insulin-like growth factor 2-H19 locus-specific DNA methylation in rat spermatozoa and its association with embryo loss

OBJECTIVE To determine the effect of tamoxifen treatment on global and insulin-like growth factor 2-H19 imprinting control region (Igf2-H19 ICR)-specific DNA methylation in rat spermatozoa and analyze its association with postimplantation loss. DESIGN Experimental prospective study. SETTING Animal research and academic research facility. SUBJECT(S) Male and female 75-day-old Holtzman rats. INTERVENTION(S) Global and Igf2-H19 ICR-specific DNA methylation was analyzed in an epididymal sperm sample in control and tamoxifen-treated rats at a dose of 0.4 mg tamoxifen/kg/day. DNA methylation status was correlated to postimplantation loss in females mated with tamoxifen-treated males. MAIN OUTCOME MEASURE(S) Global sperm DNA methylation level, methylation status of Igf2-H19 ICR in sperm, postimplantation loss. RESULT(S) Tamoxifen treatment significantly reduced methylation at Igf2-H19 ICR in epididymal sperm. However, the global methylation level was not altered. A mating experiment confirmed a significant increase in postimplantation loss upon tamoxifen treatment and showed significant correlation with methylation at Igf2-H19 ICR. CONCLUSION(S) Reduced DNA methylation at Igf2-H19 ICR in rat spermatozoa upon tamoxifen treatment indicated a role of estrogen-associated signaling in the acquisition of paternal-specific imprints during spermatogenesis. In addition, association between DNA methylation and postimplantation loss suggests that errors in paternal imprints at Igf2-H19 ICR could affect embryo development.

CYP11A1 and CYP17 promoter polymorphisms associate with hyperandrogenemia in polycystic ovary syndrome

OBJECTIVE To analyze promoter regions of CYP11A1 and CYP17 for putative variations in a defined group of women with polycystic ovary syndrome (PCOS) and to study their association with androgen levels. DESIGN Retrospective study. SETTING A secondary referral center for infertility at National Institute for Research in Reproductive Health, Mumbai, India. PATIENT(S) One hundred women whose condition was diagnosed on the basis of the Rotterdam consensus were compared against 100 age-matched controls. INTERVENTION(S) A single sample of blood was collected after overnight fast on day 3 of the menstrual cycle. MAIN OUTCOME MEASURE(S) Plasma levels of T, androstenedione, 17alpha-hydroxyprogesterone, and DHEAS and nucleotide sequence of promoter regions of CYP11A1 and CYP17 genes. RESULT(S) Polymorphisms in promoter regions of the two key androgen-regulating genes, CYP11A1 and CYP17, were found to be significantly associated with T levels in the cohort of well-characterized PCOS cases as compared with controls. The significance was greater in the PCOS cases with both the polymorphisms. CONCLUSION(S) Our study carried out in a defined group of Indian women with PCOS suggests for the first time an individual, as well as combined, association of polymorphisms in CYP11A1 and CYP17 promoters with T levels.

Screening for FOXL2 gene mutations in women with premature ovarian failure: an Indian experience.

Premature ovarian failure (POF) occurs in 1% of the general population and affects approximately 10% of non-ovulating women, resulting in infertility and sex steroid deficiency. The forkhead domain transcription factor (FOXL2) gene is one of the candidate genes associated with POF. This case-control study was designed for mutational analysis of the coding region of the FOXL2 gene in 80 cases of POF patients, 50 controls and 17 family members of 11 index cases using restriction fragment length polymorphism, single-stranded conformational polymorphism, heteroduplex analysis and direct DNA sequencing. A 738C-->T transition and a 773C-->G transversion were detected in two of the 80 patients and a family member of one index case, but in none of the 50 controls screened. No other alterations in the coding region of FOXL2 gene were detected. These data suggest that FOXL2 gene mutations are a rare occurrence in isolated POF cases and may not be involved in the pathogenesis of POF.

Clinical and laboratory evaluation of idiopathic male infertility in a secondary referral center in India

The genetic basis of infertility has received increasing recognition in recent years, particularly with the advent of assisted reproductive technology. It is now becoming obvious that genetic etiology for infertility is an important cause of disrupted spermatogenesis. Y‐chromosome microdeletions and abnormal karyotype are the two major causes of altered spermatogenesis. To achieve biological fatherhood, intracytoplasmic sperm injection (ICSI) is performed in cases of severe infertility with or without genetic abnormalities. There is a concern that these genetic abnormalities can be transmitted to the male progeny, who may subsequently have a more severe phenotype of infertility. A total of 200 men were recruited for clinical examinations, spermiograms, hormonal profiles, and cytogenetic and Yq microdeletion profiles. Testicular biopsy was also performed whenever possible and histologically evaluated. Genetic abnormalities were seen in 7.1% of cases, of which 4.1% had chromosomal aberrations, namely Klinefelters mosaic (47XXY) and Robertsonian translocation, and 3.0% had Yq microdeletions, which is very low as compared to other populations. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) were significantly increased in men with nonobstructive azoospermia (NOA) as compared to severe oligoasthenozoospermia (P<0.0001), whereas testosterone levels were significantly decreased in men with microdeletions as compared to men with no microdeletions (P<0.0083). Low levels of androgen in men with microdeletions indicate a need to follow‐up for early andropause. Patients with microdeletions had more severe testicular histology as compared to subjects without deletions. Our studies showed a significant decrease (P<0.002) in the serum inhibin B values in men with NOA, whereas FSH was seen to be significantly higher as compared to men with severe oligoasthenozoospermia (SOAS), indicating that both the Sertoli cells as well the germ cells were significantly compromised in cases of NOA and partially affected in SOAS. Overall inhibin B in combination with serum FSH would thus be a better marker than serum FSH alone for impaired spermatogenesis. In view of the genetic and hormonal abnormalities in the group of infertile men with idiopathic severe oligozoospermia and NOA cases, who are potential candidates for ICSI, genetic testing for Y‐chromosome microdeletions, karyotype, and biochemical parameters is advocated. J. Clin. Lab. Anal. 22:29–38, 2008.

Study of NAT2 Gene Polymorphisms in an Indian Population

AbstractMethods: NAT2 genotyping of 201 subjects was carried out by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing. Acetylation phenotypes were predicted from NAT2 genotypes. The association of au]|NAT2 genotypes with plasma isoniazid concentrations was determined by measuring the plasma levels in tuberculosis patients at different time points using reverse-phase high-performance liquid chromatography (HPLC). Results: The predominant alleles found in this study population were NAT2*5B and NAT2*6A, while NAT2*5B/*6A and NAT2*6A/*6A were the most frequent genotypes; the frequency varied widely from other reported studies in the Indian population. The distribution of slow, intermediate, and fast acetylators was 55%, 32%, and 13%, respectively. We observed relatively higher plasma concentrations of isoniazid in our patients than those reported in other similar studies, and they correlated well with the au]|NAT2 genotypes. Conclusion: The results suggested high variation in the frequencies of au]|NAT2 alleles and genotypes within Indian populations, which influence plasma isoniazid concentrations. Further studies of the relationship between NAT2 genotypes and adverse drug events are required to make genotyping a helpful tool for optimizing the isoniazid therapeutic response and minimizing adverse drug reactions, particularly in countries with a high burden of tuberculosis.

Exposure of adult rats to estradiol valerate induces ovarian cyst with early senescence of follicles.

Environmental and therapeutic estrogens are known to play an important role in modulating the reproductive life and pubertal maturation in males as well as in females. Animal studies have shown that exogenously administered estrogen induces follicular cysts. However, the probable mechanisms underlying this abnormal ovarian development and its impact on steroidogenesis have been ill defined. The present study was therefore carried out to understand the ontogeny of ovarian pathology owing to adult estrogenisation. Regularly cycling female Holtzman rats were sacrificed at one week, two weeks, three weeks and four weeks after a subcutaneous administration of 2 mg of estradiol valerate (E(2)V). The effect of this supra-physiological estrogen on serum endocrine profiles, development of follicular cysts, follicular apoptosis and expression of markers of folliculogenesis viz., estrogen receptor (ER)-beta, inhibin A and progesterone receptor (PR) were studied. Results indicate a temporal augmentation of steroidogenesis, which was associated with induction of follicular cyst with theca cell hyperplasia and induction of apoptosis in the primary and secondary follicles of the ovaries. Immuno-histochemical localization showed an increase in inhibin A with a reduction in ER-beta and PR indicating early maturation, poor follicle growth and granulosa cell differentiation. Results indicate that exposure to exogenous estrogen in adulthood can have deleterious effects on the ovarian physiology and endocrinology which may ultimately lead to cystogenesis, loss of follicle pool and early senescence.

CGG repeat sizing in the FMR1 gene in Indian women with premature ovarian failure

The CGG repeat stretch in the FMR1 gene is polymorphic, ranging from 5 to 50 repeats in the normal population. Expansion of the repeats to the premutation range (50-200) has been associated with premature ovarian failure (POF). This case-control study was conducted to enumerate CGG repeats in the FMR1 gene in 80 Indian women with non-familial, non-syndromic POF, and 70 controls from the same ethnicity. A possible association between CGG repeats and endocrine profile of these cases was investigated. All patients and controls had CGG repeats in the normal polymorphic range. Serum FSH concentrations were significantly raised in both POF cases and controls having CGG repeats in the 31-40 repeats range (P < 0.0001). POF cases and controls had FSH concentrations of 133.7 versus 84.2 mIU/ml and 16.0 versus 6.2 mIU/ml for >30 repeats versus <30 repeats respectively. Inhibin B concentrations were not associated with CGG repeats. The results of this study indicate that FMR1 premutations are rare in sporadic cases of POF with no family history of fragile X syndrome. However, although in the normal polymorphic range, expansion of the CGG repeat tract to beyond 30 repeats was associated with serum FSH concentrations in both POF cases and controls.

Longer CAG repeat length in the androgen receptor gene is associated with premature ovarian failure

BACKGROUND Premature ovarian failure (POF) is a disorder characterized by lack of ovulation and elevated serum gonadotrophin levels before the age of 40 years. The cause of POF in most cases is unknown. As mice lacking the Androgen receptor (Ar) gene reportedly have a POF-like phenotype, we hypothesize that, variations in the AR gene maybe one of the causative factors for POF in humans. Thus the objective of the study is to evaluate the number of CAG repeats in exon 1 of the AR gene in non-familial, non-syndromic cases of POF. METHODS A clinic-based case-control study. Seventy-eight patients with non-familial, non-syndromic POF, and 90 controls were recruited to investigate the CAG repeat numbers in exon 1 of the AR gene by PCR and Gene Scan analysis. RESULTS The mean CAG repeat length in exon 1 of the AR gene of women with POF was 23.6 +/- 3.8, which was significantly higher than controls (20.08 +/- 3.45) (P < 0.001). The biallelic mean CAG repeat ranged from 11 to 32 in the control women, compared to 16 to 30 in the POF patients. The 22 CAG repeat allele followed by the 24 CAG repeat allele was found to be at highest frequency (15.38 and 12.8%) in POF cases, although the 19 CAG repeat allele was observed at highest frequency (12.2%) in controls. CONCLUSIONS The observation suggests that the CAG repeat length is increased in women with POF as compared with controls, and may be pathogenic for POF, at least in a subset of Indian women.

Obesity and polycystic ovary syndrome: association with androgens, leptin and its genotypes

Obesity and hyperandrogenaemia are key features of polycystic ovary syndrome (PCOS). The aim of this study was to investigate whether leptin and androgens are associated with obesity in PCOS subjects and identify whether there exist any genetic alterations in leptin gene in women with PCOS. The results reveal that leptin levels are elevated in women with PCOS and associate with BMI. However, irrespective of the obesity status leptin levels are higher in PCOS cases indicating that increased BMI/obesity may not be the only factor contributing to elevated levels of leptin. With regard to testosterone and androstenedione, the levels were increased in obese individuals irrespective of PCOS status. No correlation between leptin and androstenedione or testosterone was observed in controls and PCOS subjects. The single-nucleotide polymorphism G19A detected in the untranslated exon 1 of leptin gene was not associated with PCOS and does not contribute to elevated levels of leptin. The results overall suggest that androgen and leptin levels are increased in PCOS and obesity. It demonstrates that obesity is a confounding factor for hyperandrogenaemia irrespective of their PCOS status. The study rules out role of obesity status and leptin genotype in increase in leptin levels observed in PCOS cases.