Saikat Kumar Jana

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.

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.

Studies on the phenolic profiling, anti-oxidant and cytotoxic activity of Indian honey: in vitro evaluation

Commercial honey types were screened for phenolic profile and anti-oxidant capacity. Phenolic profiling was done using high performance liquid chromatography, which was further corroborated with electro spray ionisation-mass spectroscopy. Dihydroxy benzoic acid, caffeic acid, ferulic acid and cinnamic acid were the major phenolic constituents found in the honey samples. The anti-oxidant content and free-radical scavenging effect of honey were established using various assays. Total anti-oxidant potential and free-radical scavenging ability varied among the honey varieties and exhibited significant correlation with their phenolic content. Further, honey samples with richly abundant phenolic content were found to limit oxidant-induced cell death more effectively. Cytotoxic studies of a selected sample on a breast cancer cell displayed growth inhibition, depending on the concentration used. Cell cycle analysis indicated increasing accumulation of cells at the sub-G1 phase. These results summarise the phenolic profile and anti-oxidant and cytotoxic potential of Indian honey samples for the first time.

1H NMR Based Targeted Metabolite Profiling for Understanding the Complex Relationship Connecting Oxidative Stress with Endometriosis

Accumulating evidence indicates the active role of oxidative stress in the development of endometriosis; however, the mechanism of reactive oxygen species generation is poorly understood. Metabonomics/metabolomics is a scientific discipline that can be used to study changes in metabolite ensembles associated with disease pathophysiology. The present study focuses on the use of proton nuclear magnetic resonance spectroscopy based targeted metabolite profiling approach to explore dysregulation in metabolites expression in women with endometriosis. Further, association of oxidative stress with the metabolite ensembles, if any, is investigated. Using multivariate statistics, partial least square discriminant analysis model was generated which could classify endometriosis patients with sensitivity and specificity of 92.83% and 100%, respectively, and with a classification rate of 96.4%. In conjunction with increased glucose metabolism, citrate and succinate were found to be elevated in endometriosis patients. Higher levels of reactive oxygen species, lipid peroxidation, and advanced oxidation protein products and lower levels of total antioxidant capacity, superoxide dismutase, catalase, and glutathione were also observed. Increased glucose metabolism and defects in the mitochondrial respiratory system are suggested to be the possible sources of excessive reactive oxygen species generation in endometriosis.

Cut-off Value of Reactive Oxygen Species for Predicting Semen Quality and Fertilization Outcome

Though the role of reactive oxygen species (ROS) in male infertility is widely investigated worldwide, there is a lack of adequate information on the cut-off value of ROS beyond which ART outcome may be adversely affected. The objective of the present study is to establish an upper level of ROS in semen samples which can be considered as a potential marker of good semen quality. Semen ejaculates were randomly collected from 338 male partners of infertile couples. The upper critical limit (UCL) of ROS was calculated from the control chart of normozoospermic patients and found to be 0.75×106 counted photons per minute (cpm)/10 million cells. This was further validated by estimating ROS in 17 proven fertile men and 38 patients undergoing intra cytoplasmic sperm injection (ICSI). As expected, all abnormal semen samples exhibited higher ROS as compared to nomozoospermic and proven fertile samples. All semen samples from proven fertile volunteers were found to be <0.075×106 cpm/10 million cells. On the basis of the calculated UCL, ICSI patients were divided into two groups: Group I (UCL). The semen parameters, fertilization rate and pregnancy outcome were found to be significantly affected in Group II. Significant difference in live birth-rates was observed between Group I (47.6%) and Group II (17.6%) while no live-birth was recorded for ROS level >0.1×106 cpm/10 million cells. It is concluded that the upper cut-off value of normal semen samples that correlates with good semen quality is, therefore, in the order of 0.075–0.1×106 cpm/10 million cells. In addition to the WHO [32] semen analysis, this range is expected to assist andrologists and clinicians in predicting semen quality and fertilization outcome in patients with male factor infertility undergoing ICSI.

Preparation of albumin based nanoparticles for delivery of fisetin and evaluation of its cytotoxic activity

Fisetin is a well known flavonoid that shows several properties such as antioxidant, antiviral and anticancer activities. Its use in the pharmaceutical field is limited due to its poor aqueous solubility which results in poor bioavailability and poor permeability. The aim of our present study is to prepare fisetin loaded human serum albumin nanoparticles to improve its bioavailability. The nanoparticles were prepared by a desolvation method and characterized by spectroscopic and microscopic techniques. The particles were smooth and spherical in nature with an average size of 220 ± 8 nm. The encapsulation efficiency was found to be 84%. The in vitro release profile showed a biphasic pattern and the release rate increases with increase in ionic strength of solution. We have also confirmed the antioxidant activity of the prepared nanoparticles by a DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. Further its anticancer activity was evaluated using MCF-7 breast cancer cell lines. Our findings suggest that fisetin loaded HSA nanoparticles could be used to transfer fisetin to target areas under specific conditions and thus may find use as a delivery vehicle for the flavonoid.

Letrozole and Curcumin Loaded-PLGA Nanoparticles: A Therapeutic Strategy for Endometriosis

Clinical treatment of endometriosis, a common gynaecological disorder, still remains a challenge. Hormonal suppression, pain relief medication and surgical intervention are the conventional treatment modalities; however, frequent recurrence of the disease renders the results unsatisfactory. Given the fact that oxidative stress, angiogenesis, excessive matrix degradation, and aromatase activity are associated with endometriosis, we were motivated to use the combinatorial effect of two drugs, Curcumin (Cur) and Letrozole (Let) encapsulated in PLGA and test their efficacy in mice induced with the disease. The nanoparticles (NPs) were synthesized using solvent evaporation method and characterization indicated the spherical particles to be monodispersed, polymorphic, small in size with high encapsulation efficiency, without having a tendency for significant aggregation or adhesion. Following standardization, 40mg/kg body weight of NPs was administered in endometriotic mice. Oxidative stress parameters, angiogenic markers, matrix degrading molecules, estrogen levels and endometriotic lesions, were assessed and compared before and after administration. Let-Cur NPs treatment in vivo, in addition to decreasing these parameters significantly, was also successful in reducing endometrial glands and micro-vessels density in the peritoneum to a considerable extent, thereby indicating significant regression of the disease. This is a proof of concept study. Pre-clinical non-human primate studies for testing the safety and efficacy of these NPs at larger doses in endometriosis is suggested.

Alteration in Endometrial Remodeling: A Cause for Implantation Failure in Endometriosis?

The process of implantation is an interactive cascade of events between the embryo and the endometrium. It is a dynamic process consisting of three distinct phases. They are (i) apposition of embryo (ii) attachment with the epithelial lining of the endometrium and (iii) invasion into the endometrial stroma gaining access to the maternal circulatory system. Embryo implantation failure may occur due to embryonic defect or unsupportive endometrium. Advances in Assisted Reproductive Technology (ART) have made it possible to obtain good quality embryos; however, successful implantation remains the bottleneck for a successful pregnancy. The endometrium remodels before attaining a state of receptivity. Endometrium remains receptive during a limited period, when it is favourable for blastocyst attachment and implantation. In women, there is clinical evidence of a brief period of optimal uterine receptivity which allows for blastocyst implantation. This period, called the implantation window, is related to changes in the endometrial epithelial morphology. Inappropriate morphological development leads to unreceptive endometrium that causes defective endometrial /embryonic cross talk. This is generally agreed to be one of the main reasons for implantation failure. Endometriosis, characterized by benign growth of endometrial tissue outside the uterus, affects approximately 20%–48% of women during their reproductive years. The occurrence of aberrant hormonal, immunological, genetic and pathophysiological events associated with endometriosis is attributed to the heterogeneous etiology of the disease. The symptoms of endometriosis do not depend on the severity or stage of the disease. Women with even mildest degree of endometriosis can have a 3 – 4 fold reduction in their annual birth rate compared to normal non-endometriotic women.

A low cost short-term sperm preservation procedure for double IUI in controlled ovarian hyperstimulation cycles

Unavailability of the male partner during double Intrauterine Insemination (dIUI) often poses a problem when there is a requirement for semen collection on two consecutive days. Ejaculatory disorder may be another limiting factor. Cryopreservation, though a popular option, is known to adversely affect sperm parameters. A low-cost short-term sperm preservation technique is proposed. Material and Methods: 180 out of 570 couples undergoing IUI were randomly selected to evaluate the efficacy of the new preservation technique (Group A). One part of the semen sample was processed for immediate insemination (pre ovulatory day 1 IUI); the other part was preserved under liquid paraffin for 18 hours for post ovulatory-day 2 IUI. 386 couples undergoing routine dIUI where fresh semen ejaculates were collected on two consecutive days were considered as controls (Group B). Semen parameters, reactive oxygen species (ROS) and acrosomal integrity (AI) were assessed on day 1 and day 2 in both the groups. Primary outcome measure included pregnancy rate per IUI cycle. Results: Count, motility, ROS levels, DNA damage and AI did not shown any significantly difference on day 2 following preservation under liquid paraffin as compared to day 1 in Group A. No significant differences were observed on comparing count, motility and AI in Group B. However, DNA damage and ROS levels were significantly affected in day 2 ejaculates of the control group. Clinical pregnancy rate per cycle was comparable in Group A (10.9%), Group B (11.7%). Conclusions: This new preservation method for dIUI appears to be a promising alternative to the conventional cryopreservation technique.