Deepak Modi

Decidual Control of Trophoblast Invasion

At the time of implantation, the trophoblast cells of the embryo adhere and then invade into the maternal endometrium and eventually establish placentation. The endometrium at the same time undergoes decidualization, which is essential for successful pregnancy. While the NK cells of the decidua have been implicated to play a key role in trophoblast invasion, few evidence are now available to demonstrate a pro‐invasive property of decidual stromal cells. Secretions from decidualized endometrial stromal cells promote invasion of primary trophoblasts and model cell lines by activating proteases and altering expression of adhesion‐related molecules. The decidual secretions contain high amounts of pro‐invasive factors that include IL‐1β, IL‐5, IL‐6, IL‐7, IL‐8, IL‐9, IL‐13, IL‐15, Eotaxin CCL11, IP‐10 and RANTES, and anti‐invasive factors IL‐10, IL‐12 and VEGF. It appears that these decidual factors promote invasion by regulating the protease pathways and integrin expression utilizing the STAT pathways in the trophoblast cells. At the same time the decidua also seem to secrete some anti‐invasive factors that are antagonist to the matrix metalloproteinases and/or are activators of tissue inhibitors of metalloproteinases. This might be essential to neutralize the effects of the invasion‐promoting factors and restrain overinvasion. It is tempting to propose that during the course of pregnancy, the decidua must balance the production of these pro and anti‐invasive molecules and such harmonizing production would allow a timely and regulated invasion.

Decidualized endometrial stromal cell derived factors promote trophoblast invasion

OBJECTIVE To evaluate the effects of decidua-derived factors on trophoblast invasion. DESIGN Experimental study. SETTINGS Research institute. PATIENT(S) In vitro decidualized human endometrial cells, trophoblast cell lines JEG-3, and ACH-3P. INTERVENTION(S) The effect of decidual conditioned medium (DCM) on the invasion of trophoblast cells lines via JEG-3 and ACH-3P was investigated using a Matrigel invasion assay. The changes in expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and integrins in response to DCM in the trophoblast cells was also evaluated. MAIN OUTCOME MEASURE(S) Response of the trophoblast cells to the conditioned medium from decidual cells in terms of their invasive capability, and expression on invasion related molecules was measured. RESULT(S) DCM increased the invasion of both the cell lines by approximately 1.8-2.2-fold, compared with control condition medium. The increase in invasion was associated with elevated levels of MMP2, MMP3, and MMP9 mRNA and increased activity of MMP2 and MMP9 in DCM-treated ACH-3P, but not JEG-3 cells. DCM treatment led to a reduction in TIMP1 and TIMP3 and increased TIMP2 mRNA in JEG-3, cells but not ACH-3P cells. Compared with CCM-treated controls, DCM treatment led to a significant increase in the mRNA expression of integrin α5 and α6, but not integrin αV subunit in both cell lines. CONCLUSION(S) Decidua-derived factors increase the invasiveness of trophoblast cell lines and alter the expression of integrins, MMPs, and TIMPs.

Follicle stimulating hormone receptor gene variants in women with primary and secondary amenorrhea

PurposeThis retrospective study was designed to analyze the FSHR gene variants in subjects with primary and secondary amenorrhea with hypergonadotropic hypogonadism.Materials and methodsEighty six women with primary or secondary amenorrhea and 100 normally cycling proven fertile women of Indian origin were retrospectively studied. These subjects were systematically screened for entire FSHR gene.ResultsThe frequency distribution of polymorphism at −29 position of FSHR gene is altered in women with primary and secondary amenorrhea as compared to controls. AA genotype at −29 position of FSHR gene seems to be associated with increased serum FSH levels in the study subjects. We have identified a novel homozygous mutation C1723T (Ala575Val) in one woman with primary amenorrhea.ConclusionsOur findings suggest that increased serum FSH levels in subjects with primary amenorrhea correlated to FSHR genotype at position −29. We identified a novel homozygous mutation C1723T (Ala575Val) in a woman with primary amenorrhea.

Follicle-stimulating hormone receptor polymorphism (Thr307Ala) is associated with variable ovarian response and ovarian hyperstimulation syndrome in Indian women

OBJECTIVE To evaluate the association of FSH receptor polymorphism and ovarian response. DESIGN Retrospective study. SETTING Academic research institute and private IVF clinic. PATIENT(S) Fifty women were recruited in an assisted reproductive technology program (ART) and 100 proven fertile women of Indian origin. INTERVENTION(S) Polymerase chain reaction, restriction fragment-length polymorphism for detecting polymorphisms at T(307)A and N(680)S. MAIN OUTCOME MEASURE(S) FSH receptor polymorphisms, serum FSH, and estradiol levels, amount of FSH administered, occurrence of ovarian hyperstimulation syndrome (OHSS). RESULT(S) Prevalence of polymorphism at 307 position was 24%, 53%, and 23% in controls and 24%, 62%, and 14% in ART subjects for TT, TA, and AA, respectively, whereas at position 680, it was 31%, 56%, and 13% in controls and 42%, 46%, and 12% in ART subjects for NN, NS, and SS, respectively. The amount of FSH required for ovulation induction was low in AA compared with TT and TA subjects; the estradiol levels before and on the day of hCG administration were significantly higher. Eighty-five percent of the subjects with AA genotype developed OHSS. CONCLUSION(S) In Indian women, the subjects with AA genotype require low amounts of FSH for ovarian stimulation and have an increased risk of developing OHSS.

Stage-specific Localization and Expression of c-kit in the Adult Human Testis

The c-kit receptor (KIT) and its ligand, stem cell factor (SCF), represent one of the key regulators of testicular formation, development, and function and have been extensively studied in various animal models. The present study was undertaken to characterize the pattern of localization and expression of c-kit in normal adult human testis. Immunohistochemical analysis showed that KIT is expressed in the cytoplasm of spermatogonia, acrosomal granules of spermatids, and Leydig cells. Interestingly, a rather heterogenous pattern of expression of the protein along the basement membrane was observed. Intense protein localization in spermatogonia was detected in stages I–III, whereas low expression was observed in stages IV–VI of the seminiferous epithelium, indicating that the expression of the molecule was stage specific. In situ hybridization studies revealed that the transcripts of the gene were also localized in a similar non-uniform pattern. To the best of our knowledge, such a stage-specific expression of KIT has not been reported previously in the human testis. The results of the present study may expand current knowledge about the c-kit/SCF system in human spermatogenesis.

Progesterone utilizes the PI3K-AKT pathway in human spermatozoa to regulate motility and hyperactivation but not acrosome reaction.

Progesterone is a physiologic regulator of sperm hyperactivation and acrosome reaction and it does so by activating a range of kinases present in the spermatozoa. In the present study, the involvement of the AKT- phosphatidylinositol 3-kinase (PI3K) signaling pathway in mediating progesterone response in human spermatozoa was investigated. In capacitated spermatozoa, progesterone transiently and concentration dependently lead to phosphorylation of AKT at both Thr 308 and Ser 473 in the tail region. This phosphorylation was inhibited by the PI3K inhibitor wortmannin, suggesting that progesterone leads to activation of PI3K-AKT pathway. The activation of AKT in response to progesterone is calcium dependent and the CatSper channel inhibitor mibefradil significantly reduced progesterone mediated AKT phosphorylation. Preincubation of spermatozoa with wortmannin inhibited the progesterone mediated increase in tyrosine phosphorylation and also attenuated the increase in number of motile, progressively motile and hyperactive spermatozoa but not the number of acrosome reacted spermatozoa. These observations imply that progesterone via CatSper activates the PI3K-AKT pathway required for motility and hyperactivation but not for acrosome reaction.

Differential concentration and time dependent effects of progesterone on kinase activity, hyperactivation and acrosome reaction in human spermatozoa

Progesterone has been identified to be one of the physiological regulators of sperm hyperactivation and acrosome reaction. However, the high sensitivity of human spermatozoa to progesterone implies that many may undergo premature hyperactivation and acrosome reaction thereby compromising their ability to fertilize. We hypothesized that if a spermatozoon has to preclude the occurrence of these events prematurely, there should be differential dose- and time-dependent effects on motility and acrosome reaction. We observed that low concentrations of progesterone (10 and 100 nm) induce sperm motility and activate tyrosine kinase; higher concentrations (1-10 μm) are required to induce extracellular signal regulated kinases 1/2 (Erk1/2), p90 ribosomal S6 kinase (p90RSK), p38 mitogen-activated protein kinase (p38MAPK), c-Jun N-terminal kinase (JNK1) and AKT phosphorylation, hyperactivation and acrosome reaction. The induction of acrosome reaction and tyrosine phosphorylation in response to higher concentration of progesterone is not absolutely dependent on activation of T-type voltage-gated Ca(2+) channel or CatSper as Mibefradil did not completely abrogate progesterone-mediated effects. These results imply that although the spermatozoa are sensitive to low concentrations of progesterone, they only activate motility and tyrosine kinase activation; higher concentrations are required to induce hyperactivation and acrosome reaction probably by activating multiple kinase pathways including the MAPK and AKT.

Poor ovarian response to gonadotrophin stimulation is associated with FSH receptor polymorphism

Similarities in the phenotype observed in women with FSH receptor mutation and in FSH receptor knockout mice have clearly established a critical role of this protein in normal gonadal function. Two common single nucleotide polymorphisms in the exonic region of the FSH receptor gene have been shown to be associated with altered ovarian response in subjects undergoing gonadotrophin treatment. Recent in-vitro studies have shown that the A allele at the -29 position in the 5 untranslated region of the FSH receptor gene is associated with impaired transcriptional activity. Differential expression of the FSH receptor and its function may be one of the factors responsible for altered ovarian response. These observations prompted a study of the association between FSH receptor genotype at the -29 position and ovarian response in women undergoing gonadotrophin treatment. Analysis of the data revealed that the subjects with AA genotype at the -29 position required the highest amount of exogenous FSH for ovulation induction, and oestradiol concentrations before the day of human chorionic gonadotrophin administration were significantly lower (P = 0.015) compared with the GA genotype. The number of pre-ovulatory follicles and retrieved oocytes were lowest in the subjects with AA genotype. These results indicate that the AA genotype at position -29 may be associated with the poor ovarian response.

Regulation of homeobox A10 expression in the primate endometrium by progesterone and embryonic stimuli

Homeobox A10 (HOXA10), a member of abdominal B subclass of homeobox genes, is responsible for uterine homeosis during development. Intriguingly, in the adult murine uterus, HOXA10 has been demonstrated to play important roles in receptivity, embryo implantation, and decidualization. However, the roles of HOXA10 in the primate endometrium are not known. To gain insights into the roles of HOXA10 in the primate endometrium, its expression was studied in the endometria of bonnet monkey (Macaca radiata) in the receptive phase and also in the endometria of monkeys treated with antiprogestin onapristone (ZK98.299) or in conception cycle where the presence of preimplantation stage blastocyst was verified. In addition, the mRNA expression of HOXA11 and insulin-like growth factor-binding protein 1 (IGFBP1) was evaluated by real-time PCR in these animals.The results revealed that HOXA10 in the luteal phase primate endometrium is differentially expressed in the functionalis and the basalis zones, which is modulated in vivo by progesterone and also by the signals from the incoming embryo suggesting the involvement of HOXA10 in the process of establishment of pregnancy in primates. In addition, the results also demonstrated that the expression of IGFBP1 but not HOXA11 is coregulated with HOXA10 in the endometria of these animals. The pattern of changes in the expression of HOXA10 in response to the two stimuli suggests that endometrial receptivity and implantation not only requires a synchrony of maternal and embryonic signaling on endometrial cells in the primates but there also exists a controlled differential response among the cells of various uterine compartments.

Regulation of decidualization, interleukin-11 and interleukin-15 by homeobox A 10 in endometrial stromal cells

Cytokine production by the endometrial stromal and decidual cells is essential for successful differentiation of the endometrial stromal cells and uterine leukocytes to sustain pregnancy. Interleukin-11 and -15 (IL-11 and IL-15) secreted by the stromal and decidual cells are two key modulators of the process of decidualization and natural killer cell (NK) activity in the uterus and are essential for pregnancy. However, limited information exists on the maternal factors that regulate the production of these cytokines by the stromal cells. In this study, we investigated the role of homeobox A10 (HOXA10) in the regulation of expression of genes encoding the decidualization markers insulin-like growth factor binding protein-1 (IGFBP1), prolactin and the cytokines IL-11 and IL-15 by endometrial stromal and decidual cells in vitro. The results demonstrated that the expression of IGFBP1, Prolactin (PRL), HOXA10, IL11, and IL15 are co-regulated during steroid hormone-mediated decidualization of human endometrial stromal cells in vitro. In the predecidual cells, downregulation of HOXA10 by siRNA suppresses IGFBP1 and IL15, but increases IL11 expression. In the decidualized cells, knocking down HOXA10 inhibits IGFBP1 and PRL expression but elevates the expression of IL11 and IL15. In addition, our data also demonstrate that transient inhibition of HOXA10 expression in the predecidual cells does not influence its ability to subsequently decidualize or affect cytokine expression, suggesting that steroid hormone-mediated decidualization and cytokine production in vitro does not require HOXA10 preconditioning.