Baidyanath Chakravarty

Mutational screening of the coding region of growth differentiation factor 9 gene in Indian women with ovarian failure

Objective:To establish the risk associated with mutations in the coding region of GDF9 gene in Indian women with ovarian failure. Design:This case-control study was designed for mutational analysis of the GDF9 coding region in a cohort of women with premature ovarian failure (n = 127), primary amenorrhea (n = 58), and secondary amenorrhea (n = 10) compared with controls (n = 220). Results:This case-control study revealed eight mutations in the GDF9 gene, including five novel mutations: c.1-8C>T, c.199A>C (p.Lys67Glu), c. 205C>T, c.646G>A (p.Val216Mat), and c.1353C>T, and three documented mutations: c.398-39C>G, c.447C>T, and c.546G>A. Missense mutation c.199A>C was present in 4 of 127 premature ovarian failure (POF) cases and 1 of 10 secondary amenorrhea cases. The c.646G>A mutation was present in two POF cases. Both missense mutations were absent in controls. Genotype distribution of c.447C>T was significantly different in POF cases than controls (χ2 = 5.93, P = 0.05). We chose two frequent single-nucleotide polymorphisms (c.398-39C>G, c.447C>T) for haplotyping and found that the C-T haplotype was significantly higher in patients (P = 0.03), whereas the C-C haplotype was representative of the control group. Conclusions:We report two rare missense mutations, c.199A>C and c.646G>A, which are associated with ovarian failure. The presence of the c.447>T mutation might indicate a higher risk for POF. Haplotype C-T was significantly associated with ovarian failure, whereas the C-C haplotype was representative of the control group.

Upper control limit of reactive oxygen species in follicular fluid beyond which viable embryo formation is not favorable.

Though the role of reactive oxygen species (ROS) in female infertility has been a subject of rigorous research worldwide, there is inadequate information on the cut-off value of ROS in the oocyte microenvironment beyond which ART outcome may be adversely affected. An upper ROS level in follicular fluid (FF) samples of women undergoing IVF beyond which good quality embryo formation is unlikely, is established. ROS, lipid peroxidation and total antioxidant capacity were estimated. The upper cut-off ROS level beyond which viable embryo formation is not favorable was found to be approximately 107 cps/400 microl FF. This level, determined in women with tubal factor infertility, was further validated in women with endometriosis and PCOS and correlated with fertilization and pregnancy rate and embryo quality. Summarizing, a threshold level in FF has been established for the first time beyond which ROS may be considered toxic for viable embryo formation and pregnancy outcome.

Effect of Follicular Fluid Oxidative Stress on Meiotic Spindle Formation in Infertile Women with Polycystic Ovarian Syndrome

Background: The effect of follicular fluid (FF) oxidative stress (OS) on meiotic spindle (MS) formation in oocytes and subsequent outcome in women with polycystic ovarian syndrome (PCOS) are evaluated in this study. Methods: 326 oocytes from 35 PCOS women (group A) and 208 oocytes from 32 women with tubal infertility (group B) were visualized for MS using PolScope. FF was analyzed for OS markers including reactive oxygen species (ROS), lipid peroxidation and total antioxidant capacity (TAC). Group A was further classified into groups A1 and A2, and group B into groups B1 and B2 depending upon the presence or absence of MS, respectively. Results: MS formation was absent in a significantly higher number of oocytes in group A compared to group B (p ≤ 0.05). OS markers were significantly higher in group A compared to group B (p ≤ 0.05). Fertilization rate, number of good quality embryos and clinical pregnancy rates were higher in group B compared to group A, though not statistically significant. FF ROS was significantly higher and TAC significantly lower in groups A2 and B2 compared to groups A1 and B1 (p < 0.001). Conclusion: The presence of MS and oocyte maturation in PCOS women showed a positive correlation with low levels of OS.

Markers of oxidative stress in follicular fluid of women with endometriosis and tubal infertility undergoing IVF.

Oxidative stress and trace elements in the oocytes environment is explored in endometriosis and impact on in vitro fertilization (IVF) outcome assessed. Follicular fluid was aspirated at the time of oocyte retrieval from endometriosis (n=200) and tubal infertility (n=140) and the analytes measured using spectroscopy and HPLC. Increased concentration of reactive oxygen species (ROS), nitric oxide (NO), lipid peroxidation (LPO), iron, lead, cadmium and reduced levels of total antioxidant capacity (TAC), superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR), vitamins A, C, E, copper, zinc and selenium was observed compared to tubal infertility. Increased ROS and NO in endometriosis and tubal infertility associated with poor oocytes and embryo quality. Increased levels of ROS, NO, LPO, cadmium and lead were observed in women who did not become pregnant compared to women who did. Intrafollicular zinc levels were higher in women with endometriosis who subsequently became pregnant following IVF.

Oxidative status in granulosa cells of infertile women undergoing IVF

Studies on elevated reactive oxygen species (ROS) levels in granulosa cells (GC) and its subsequent effect on fertilization are limited. Oxidative stress (OS) mediated alterations in GC of infertile women undergoing in vitro fertilization (IVF) and embryo transfer (ET) was investigated. GC were obtained from 28 women with endometriosis (Group A), 26 women with polycystic ovary syndrome (PCOS) (Group B), and 32 women with tubal factor infertility (Group C). GC characteristics including cell count, viability, morphology and number of oocytes retrieved, and oocyte quality were assessed. OS parameters such as ROS, mitochondrial membrane potential (MMP), and DNA fragmentation were also studied and IVF outcome parameters assessed. An ∼20 fold increase in GC ROS generation was observed in Group B as compared to Group C. Though not as high as Group B, Group A also showed significantly high ROS levels compared with Group C. More than 100-fold decrease in MMP in Group B compared with Group C was observed. A similar trend was observed in Group A, where MMP decreased 7 fold. Significant apoptosis was evident in Groups A and B supported by depolarization of MMP and significant increase in DNA damage. IVF outcome parameters including fertilization rate, good quality embryo formation rate, and pregnancy outcome were adversely affected in Group B. It is hypothesized that ∼20 fold increase in ROS generation in GC of PCOS women plays an adverse role in affecting the IVF success rate. It was of note that the IVF outcome parameters of women with endometriosis were not affected.

PROINFLAMMATORY CYTOKINES INDUCED ALTERED EXPRESSION OF CYCLOOXYGENASE-2 GENE RESULTS IN UNRECEPTIVE ENDOMETRIUM IN WOMEN WITH IDIOPATHIC RECURRENT SPONTANEOUS MISCARRIAGE

OBJECTIVE To investigate the expression pattern of proinflammatory, anti-inflammatory, and angiogenic cytokines and their effect on various mediators of endometrial receptivity in women with idiopathic recurrent spontaneous miscarriage (IRSM). DESIGN A prospective study. SETTING Tertiary care hospital and reproductive health research unit. PATIENT(S) Thirty-six women with IRSM (<35 years) and 30 fertile women as controls matched by age and body mass index undergoing sterilization. INTERVENTION(S) Endometrial biopsies in all women corresponding to the window of implantation. MAIN OUTCOME MEASURE(S) Assessment of endometrial expression of proinflammatory, anti-inflammatory, and angiogenic cytokines, mediators of matrix turnover and angiogenesis, markers of receptivity. RESULT(S) A statistical significantly higher level of proinflammatory cytokines, mediators of matrix turnover and angiogenesis, and a reduced expression of anti-inflammatory and angiogenic cytokines were observed in women with IRSM. Additionally, the markers of endometrial receptivity were poorly expressed in women with IRSM. CONCLUSION(S) Aberrant expression of proinflammatory, anti-inflammatory, and angiogenic cytokines during implantation window in women with IRSM is one of the key factors that adversely affect endometrial development, as evidenced by the inadequate expression of various endometrial receptivity markers.

Subclinical hypothyroidism: a determinant of polycystic ovary syndrome.

The present study was an endeavor to explore whether and how hypothyroidism plays a role in the etiology of polycystic ovarian syndrome (PCOS). A composite picture of the hormone profile was assessed in different groups of subjects (control women and hypothyroid women with or without PCOS). Comparative analysis of the results suggests that hypothyroidism is invariably followed by a lowering of sex hormone binding globulin and an increment in the free testosterone level, but further metabolism of testosterone (T) may or may not be directed towards an overproduction of estriol (E3). The factors that dictate the route of T metabolism, and the way by which E3 acts to rescue the ovaries from the development of PCOS under the hypothyroid state are discussed.

Recurrent Pregnancy Loss in Polycystic Ovary Syndrome: Role of Hyperhomocysteinemia and Insulin Resistance

Recurrent pregnancy loss (RPL) in polycystic ovary syndrome (PCOS), which occurs in ∼50% of total pregnancies is a frequent obstetric complication. Among the several hypotheses, insulin resistance (IR), obesity and hyperhomocysteinemia (HHcy) play significant role/s in RPL. This study was conducted to assess the link between elevated levels of homocysteine and IR in PCOS-associated women with RPL in Kolkata, India. A retrospective study was conducted of one hundred and twenty six PCOS women (<30 years) who experienced two or more spontaneous abortions during the first trimester presenting to Institute of Reproductive Medicine (IRM) in Kolkata during the period of March 2008 through February 2011. One hundred and seventeen non-PCOS subjects with matching age range were randomly chosen as controls. Incidence of HHcy and IR was 70.63% (n = 89) and 56.34% (n = 71), respectively, in RPL-affected PCOS population which was significantly higher (p<0.04; p<0.0001) when compared to the non-PCOS set (HHcy: 57.26%; IR: 6.83%). Rates of miscarriage were significantly higher (p<0.008; p<0.03) in hyperhomocysteinemia-induced miscarriage when compared to the normohomocysteinemic segment (PCOS: 70.63% vs.29.36% & non-PCOS: 57.26% vs. 42.73%) along with the insulin resistant (p<0.04; p<0.0001) population (PCOS: 70.63% vs. 56.34% & non-PCOS: 57.26% vs. 6.83%) in both groups. A probabilistic causal model evaluated HHcy as the strongest plausible factor for diagnosis of RPL. A probability percentage of 43.32% in the cases of HHcy- mediated RPL suggests its increased tendency when compared to IR mediated miscarriage (37.29%), further supported by ROC-AUC (HHcy: 0.778vs. IR: 0.601) values. Greater susceptibility towards HHcy may increase the incidence for miscarriage in women in India and highlights the need to combat the condition in RPL control programs in the subcontinent.

Role of Latent Genital Tuberculosis in Repeated IVF Failure in the Indian Clinical Setting

Genital tuberculosis is reported to be a major pelvic factor causing infertility in Indian women and often exists without any apparent signs and symptoms. The role of latent tuberculosis in repeated IVF failure in unexplained infertility is examined. 81 women with unexplained infertility having repeated IVF failure tested for Mycobacterium tuberculosis using PCR, ZN staining and BACTEC-460 culture were selected. Fresh IVF-ET or frozen embryo transfer (FET) was attempted on patients successfully treated with anti-tubercular drugs (ATD). ATD-treated fresh cycles (group A1) and frozen cycles (group B1) were compared to previously failed fresh cycles (group A2) and FET attempts (group B2), respectively. Main outcome measures were gonadotropin required, terminal E2, number of oocytes retrieved, fertilization rate, embryo quality, endometrial thickness and sub-endometrial blood flow (Vmax). Gonadotropin required in group A1 was significantly less as compared to group A2. Number of oocytes retrieved and grade I embryos, endometrial thickness and Vmax were significantly higher in group A1. Endometrial thickness and Vmax were significantly increased in group B1 as compared to B2. The study indicates that latent tuberculosis should be considered in young Indian patients presenting with unexplained infertility with apparently normal pelvic and non-endometrial tubal factors and repeated IVF failure.

Comparison of oral dydrogestrone with progesterone gel and micronized progesterone for luteal support in 1,373 women undergoing in vitro fertilization: a randomized clinical study.

OBJECTIVE To compare the efficacy of oral dydrogesterone with that of micronized vaginal P gel and micronized P capsule for luteal supplementation. DESIGN Prospective, randomized clinical study. SETTING Institute of Reproductive Medicine, Kolkata, India. PATIENT(S) A total of 1,373 infertile women undergoing IVF participated. INTERVENTION(S) Micronized P gel, P capsule, and oral dydrogesterone were administered for luteal support and compared. MAIN OUTCOME MEASURE(S) Demographic profile and pregnancy and miscarriage rates. RESULT(S) The overall pregnancy rate and miscarriage rate were comparable among the three groups. CONCLUSION(S) Oral dydrogesterone seems to be a promising drug for luteal support in woman undergoing IVF.