Showing papers in "Reproductive Biology and Endocrinology in 2009"

Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics.

Abstract: The assessment of oocyte quality in human in vitro fertilization (IVF) is getting increasing attention from embryologists. Oocyte selection and the identification of the best oocytes, in fact, would help to limit embryo overproduction and to improve the results of oocyte cryostorage programs. Follicular fluid (FF) is easily available during oocyte pick-up and theorically represents an optimal source on non-invasive biochemical predictors of oocyte quality. Unfortunately, however, the studies aiming to find a good molecular predictor of oocyte quality in FF were not able to identify substances that could be used as reliable markers of oocyte competence to fertilization, embryo development and pregnancy. In the last years, a well definite trend toward passing from the research of single molecular markers to more complex techniques that study all metabolites of FF has been observed. The metabolomic approach is a powerful tool to study biochemical predictors of oocyte quality in FF, but its application in this area is still at the beginning. This review provides an overview of the current knowledge about the biochemical predictors of oocyte quality in FF, describing both the results coming from studies on single biochemical markers and those deriving from the most recent studies of metabolomics

Potential markers of preeclampsia – a review

Abstract: Preeclampsia is a leading cause of maternal and fetal/neonatal mortality and morbidity worldwide. The early identification of patients with an increased risk for preeclampsia is therefore one of the most important goals in obstetrics. The availability of highly sensitive and specific physiologic and biochemical markers would allow not only the detection of patients at risk but also permit a close surveillance, an exact diagnosis, timely intervention (e.g. lung maturation), as well as simplified recruitment for future studies looking at therapeutic medications and additional prospective markers. Today, several markers may offer the potential to be used, most likely in a combinatory analysis, as predictors or diagnostic tools. We present here the current knowledge on the biology of preeclampsia and review several biochemical markers which may be used to monitor preeclampsia in a future, that, we hope, is not to distant from today.

New discoveries on the biology and detection of human chorionic gonadotropin.

Abstract: Human chorionic gonadotropin (hCG) is a glycoprotein hormone comprising 2 subunits, alpha and beta joined non covalently. While similar in structure to luteinizing hormone (LH), hCG exists in multiple hormonal and non-endocrine agents, rather than as a single molecule like LH and the other glycoprotein hormones. These are regular hCG, hyperglycosylated hCG and the free beta-subunit of hyperglycosylated hCG. For 88 years regular hCG has been known as a promoter of corpus luteal progesterone production, even though this function only explains 3 weeks of a full gestations production of regular hCG. Research in recent years has explained the full gestational production by demonstration of critical functions in trophoblast differentiation and in fetal nutrition through myometrial spiral artery angiogenesis. While regular hCG is made by fused villous syncytiotrophoblast cells, extravillous invasive cytotrophoblast cells make the variant hyperglycosylated hCG. This variant is an autocrine factor, acting on extravillous invasive cytotrophoblast cells to initiate and control invasion as occurs at implantation of pregnancy and the establishment of hemochorial placentation, and malignancy as occurs in invasive hydatidiform mole and choriocarcinoma. Hyperglycosylated hCG inhibits apoptosis in extravillous invasive cytotrophoblast cells promoting cell invasion, growth and malignancy. Other non-trophoblastic malignancies retro-differentiate and produce a hyperglycosylated free beta-subunit of hCG (hCG free beta). This has been shown to be an autocrine factor antagonizing apoptosis furthering cancer cell growth and malignancy. New applications have been demonstrated for total hCG measurements and detection of the 3 hCG variants in pregnancy detection, monitoring pregnancy outcome, determining risk for Down syndrome fetus, predicting preeclampsia, detecting pituitary hCG, detecting and managing gestational trophoblastic diseases, diagnosing quiescent gestational trophoblastic disease, diagnosing placental site trophoblastic tumor, managing testicular germ cell malignancies, and monitoring other human malignancies. There are very few molecules with such wide and varying functions as regular hCG and its variants, and very few tests with such a wide spectrum of clinical applications as total hCG.

Altered microRNA expression in patients with non-obstructive azoospermia.

Abstract: MicroRNAs (miRNAs), a class of small non-coding RNA molecules, are indicated to play essential roles in spermatogenesis However, little is known about the expression patterns or function of miRNAs in human testes involved in infertility In this study, the miRNA expression profiles of testes of patients with non-obstructive azoospermia (NOA) and normal controls were performed by using microarray technologies Altered microRNA expression in NOA patients was found, with 154 differentially down-regulated and 19 up-regulated miRNAs These findings have been confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) assays on select miRNAs, including miR-302a, miR-491-3p, miR-520d-3p and miR-383 Several down-regulated miRNA clusters in patients with NOA were identified, such as the oncogenic potential of the mir-17-92 cluster and mir-371,2,3 cluster This is the first report that the expression of miRNAs is altered in testicular tissues of patients with NOA, suggesting a role of miRNAs in regulating spermatogenesis in human males

Pathophysiology of cell phone radiation: oxidative stress and carcinogenesis with focus on male reproductive system

Abstract: Hazardous health effects stemming from exposure to radiofrequency electromagnetic waves (RF-EMW) emitted from cell phones have been reported in the literature. However, the cellular target of RF-EMW is still controversial. This review identifies the plasma membrane as a target of RF-EMW. In addition, the effects of RF-EMW on plasma membrane structures (i.e. NADH oxidase, phosphatidylserine, ornithine decarboxylase) and voltage-gated calcium channels are discussed. We explore the disturbance in reactive oxygen species (ROS) metabolism caused by RF-EMW and delineate NADH oxidase mediated ROS formation as playing a central role in oxidative stress (OS) due to cell phone radiation (with a focus on the male reproductive system). This review also addresses: 1) the controversial effects of RF-EMW on mammalian cells and sperm DNA as well as its effect on apoptosis, 2) epidemiological, in vivo animal and in vitro studies on the effect of RF-EMW on male reproductive system, and 3) finally, exposure assessment and dosimetry by computational biomodeling.

Removal of spermatozoa with externalized phosphatidylserine from sperm preparation in human assisted medical procreation: effects on viability, motility and mitochondrial membrane potential

Abstract: Externalization of phosphatidylserine (EPS) occurs in apoptotic-like spermatozoa and could be used to remove them from sperm preparations to enhance sperm quality for assisted medical procreation. We first characterized EPS in sperms from infertile patients in terms of frequency of EPS spermatozoa as well as localization of phosphatidylserine (PS) on spermatozoa. Subsequently, we determined the impact of depleting EPS spermatozoa on sperm quality. EPS were visualized by fluorescently-labeled annexin V binding assay. Double staining with annexin V and Hoechst differentiates apoptotic from necrotic spermatozoa. We used magnetic-activated cell sorting using annexin V-conjugated microbeads (MACS-ANMB) technique to remove EPS spermatozoa from sperm prepared by density gradient centrifugation (DGC). The impact of this technique on sperm quality was evaluated by measuring progressive motility, viability, and the integrity of the mitochondrial membrane potential (MMP) by Rhodamine 123. Mean percentages of EPS spermatozoa were 14% in DGC sperm. Four subpopulations of spermatozoa were identified: 70% alive, 3% early apoptotic, 16% necrotic and 11% late apoptotic or necrotic. PS were localized on head and/or midpiece or on the whole spermatozoa. MACS efficiently eliminates EPS spermatozoa. MACS combined with DGC allows a mean reduction of 70% in EPS and of 60% in MMP-disrupted spermatozoa with a mean increase of 50% in sperm survival at 24 h. Human ejaculates contain EPS spermatozoa which can mostly be eliminated by DGC plus MACS resulting in improved sperm long term viability, motility and MMP integrity. EPS may be used as an indicator of sperm quality and removal of EPS spermatozoa may enhance fertility potential in assisted medical procreation.

Expression of genes associated with immunity in the endometrium of cattle with disparate postpartum uterine disease and fertility

Abstract: Contamination of the uterine lumen with bacteria is ubiquitous in cattle after parturition. Some animals develop endometritis and have reduced fertility but others have no uterine disease and readily conceive. The present study tested the hypothesis that postpartum cattle that develop persistent endometritis and infertility are unable to limit the inflammatory response to uterine bacterial infection. Endometrial biopsies were collected several times during the postpartum period from animals that were subsequently infertile with persistent endometritis (n = 4) or had no clinical disease and conceived to first insemination (n = 4). Quantitative PCR was used to determine the expression of candidate genes in the endometrial biopsies, including the Toll-like receptor (TLR 1 to 10) family of innate immune receptors, inflammatory mediators and their cognate receptors. Selected proteins were examined by immunohistochemistry. The expression of genes encoding pro-inflammatory mediators such as interleukins (IL1A, IL1B and IL6), and nitric oxide synthase 2 (NOS2) were higher during the first week post partum than subsequently. During the first week post partum, there was higher gene expression in infertile than fertile animals of TLR4, the receptor for bacterial lipopolysaccharide, and the pro-inflammatory cytokines IL1A and IL1B, and their receptor IL1R2. The expression of genes encoding other Toll-like receptors, transforming growth factor beta receptor 1 (TGFBR1) or prostaglandin E2 receptors (PTGER2 and PTGER4) did not differ significantly between the animal groups. Gene expression did not differ significantly between infertile and fertile animals after the first week postpartum. However, there were higher ratios of IL1A or IL1B mRNA to the anti-inflammatory cytokine IL10, during the first week post partum in the infertile than fertile animals, and the protein products of these genes were mainly localised to the epithelium of the endometrium. Cattle may maintain fertility by limiting the inflammatory response to postpartum bacterial infection in the endometrium during the first week after parturition.

Epigenetic regulation in mammalian preimplantation embryo development

Abstract: Preimplantation embryo development involves four stages: fertilization, cell cleavage, morula and blastocyst formation. During these stages, maternal and zygotic epigenetic factors play crucial roles. The gene expression profile is changed dramatically, chromatin is modified and core histone elements undergo significant changes. Each preimplantation embryo stage has its own characteristic epigenetic profile, consistent with the acquisition of the capacity to support development. Moreover, histone modifications such as methylation and acetylation as well as other epigenetic events can act as regulatory switches of gene transcription. Because the epigenetic profile is largely related to differentiation, epigenetic dysfunction can give rise to developmental abnormalities. Thus, epigenetic profiling of the embryo is of pivotal importance clinically. Given the importance of these aspects, this review will mainly focus on the epigenetic profile during preimplantation embryo development, as well as interactions between epigenetic and genetic regulation in these early developmental stages.

Biological versus chronological ovarian age: implications for assisted reproductive technology.

Abstract: Women have been able to delay childbearing since effective contraception became available in the 1960s. However, fertility decreases with increasing maternal age. A slow but steady decrease in fertility is observed in women aged between 30 and 35 years, which is followed by an accelerated decline among women aged over 35 years. A combination of delayed childbearing and reduced fecundity with increasing age has resulted in an increased number and proportion of women of greater than or equal to 35 years of age seeking assisted reproductive technology (ART) treatment. Literature searches supplemented with the authors' knowledge. Despite major advances in medical technology, there is currently no ART treatment strategy that can fully compensate for the natural decline in fertility with increasing female age. Although chronological age is the most important predictor of ovarian response to follicle-stimulating hormone, the rate of reproductive ageing and ovarian sensitivity to gonadotrophins varies considerably among individuals. Both environmental and genetic factors contribute to depletion of the ovarian oocyte pool and reduction in oocyte quality. Thus, biological and chronological ovarian age are not always equivalent. Furthermore, biological age is more important than chronological age in predicting the outcome of ART. As older patients present increasingly for ART treatment, it will become more important to critically assess prognosis, counsel appropriately and optimize treatment strategies. Several genetic markers and biomarkers (such as anti-Mullerian hormone and the antral follicle count) are emerging that can identify women with accelerated biological ovarian ageing. Potential strategies for improving ovarian response include the use of luteinizing hormone (LH) and growth hormone (GH). When endogenous LH levels are heavily suppressed by gonadotrophin-releasing hormone analogues, LH supplementation may help to optimize treatment outcomes for women with biologically older ovaries. Exogenous GH may improve oocyte development and counteract the age-related decline of oocyte quality. The effects of GH may be mediated by insulin-like growth factor-I, which works synergistically with follicle-stimulating hormone on granulosa and theca cells. Patients with biologically older ovaries may benefit from a tailored approach based on individual patient characteristics. Among the most promising adjuvant therapies for improving ART outcomes in women of advanced reproductive age are the administration of exogenous LH or GH.

Miscarriage rates after dehydroepiandrosterone (DHEA) supplementation in women with diminished ovarian reserve: a case control study

Abstract: Background Dehydroepinadrosterone (DHEA) supplementation improves pregnancy chances in women with diminished ovarian reserve (DOR), by possibly reducing aneuploidy. Since a large majority of spontaneous miscarriages are associated with aneuploidy, one can speculate that DHEA supplementation may also reduce miscarriage rates.

The BSA-induced Ca(2+) influx during sperm capacitation is CATSPER channel-dependent

Abstract: Serum albumin is a key component in mammalian sperm capacitation, a functional maturation process by which sperm become competent to fertilize oocytes. Capacitation is accompanied by several cellular and molecular changes including an increased tyrosine phosphorylation of sperm proteins and a development of hyperactivated sperm motility. Both of these processes require extracellular calcium, but how calcium enters sperm during capacitation is not well understood. BSA-induced changes in intracellular calcium concentration were studied using Fluo-4 and Fura-2 calcium imaging with wild-type and Catsper1 knockout mouse sperm. We found that the fast phase of the BSA-induced rises in intracellular calcium concentration was absent in the Catsper1 knockout sperm and could be restored by an EGFP-CATSPER1 fusion protein. The calcium concentration increases were independent of G-proteins and phospholipase C but could be partially inhibited when intracellular pH was clamped. The changes started in the principal piece and propagated toward the sperm head. We conclude that the initial phase of the increases in intracellular calcium concentration induced by BSA requires the CATSPER channel, but not the voltage-gated calcium channel. Our findings identify the molecular conduit responsible for the calcium entry required for the sperm motility changes that occur during capacitation.

Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes

Abstract: Maintaining the integrity of sperm DNA is vital to reproduction and male fertility. Sperm contain a number of molecules and pathways for the repair of base excision, base mismatches and DNA strand breaks. The presence of Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues has recently been shown in male germ cells, specifically during stage VII of spermatogenesis. High PARP expression has been reported in mature spermatozoa and in proven fertile men. Whenever there are strand breaks in sperm DNA due to oxidative stress, chromatin remodeling or cell death, PARP is activated. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Therefore, cleaved PARP (cPARP) may be considered a marker of apoptosis. The presence of higher levels of cPARP in sperm of infertile men adds a new proof for the correlation between apoptosis and male infertility. This review describes the possible biological significance of PARP in mammalian cells with the focus on male reproduction. The review elaborates on the role played by PARP during spermatogenesis, sperm maturation in ejaculated spermatozoa and the potential role of PARP as new marker of sperm damage. PARP could provide new strategies to preserve fertility in cancer patients subjected to genotoxic stresses and may be a key to better male reproductive health.

The endogenous production of hydrogen sulphide in intrauterine tissues

Abstract: Hydrogen sulphide is a gas signalling molecule which is produced endogenously from L-cysteine via the enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). The possible role of hydrogen sulphide in reproduction has not yet been fully investigated. It has been previously demonstrated that hydrogen sulphide relaxes uterine smooth muscle in vitro. The aim of the present study was to investigate the endogenous production of hydrogen sulphide in rat and human intrauterine tissues in vitro. The production of hydrogen sulphide in rat and human intrauterine tissues was measured in vitro using a standard technique. The expression of CBS and CSE was also investigated in rat and human intrauterine tissues via Western blotting. Furthermore, the effects of nitric oxide (NO) and low oxygen conditions on the production rates of hydrogen sulphide were investigated. The order of hydrogen sulphide production rates (mean +/- SD, n = 4) for rat tissues were: liver (777 +/- 163 nM/min/g) > uterus (168 +/- 100 nM/min/g) > fetal membranes (22.3 +/- 15.0 nM/min/g) > placenta (11.1 +/- 4.7 nM/min/g), compared to human placenta (200 +/- 102 nM/min/g). NO significantly increased hydrogen sulphide production in rat fetal membranes (P < 0.05). Under low oxygen conditions the production of hydrogen sulphide was significantly elevated in human placenta, rat liver, uterus and fetal membranes (P < 0.05). Western blotting (n = 4) detected the expression of CBS and CSE in all rat intrauterine tissues, and in human placenta, myometrium, amnion and chorion. Rat and human intrauterine tissues produce hydrogen sulphide in vitro possibly via CBS and CSE enzymes. NO increased the production of hydrogen sulphide in rat fetal membranes. The augmentation of hydrogen sulphide production in human intrauterine tissues in a low oxygen environment could have a role in pathophysiology of pregnancy.

Human male gamete endocrinology: 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates different aspects of human sperm biology and metabolism

Abstract: Background A wider biological role of 1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D3, in tissues not primarily related to mineral metabolism was suggested. Recently, we evidenced the ultrastructural localization the 1,25(OH)2D3 receptor in the human sperm. However, the 1,25(OH)2D3 action in human male reproduction has not yet been clarified.

The role and molecular mechanism of D-aspartic acid in the release and synthesis of LH and testosterone in humans and rats

Abstract: D-aspartic acid is an amino acid present in neuroendocrine tissues of invertebrates and vertebrates, including rats and humans Here we investigated the effect of this amino acid on the release of LH and testosterone in the serum of humans and rats Furthermore, we investigated the role of D-aspartate in the synthesis of LH and testosterone in the pituitary and testes of rats, and the molecular mechanisms by which this amino acid triggers its action For humans: A group of 23 men were given a daily dose of D-aspartate (DADAVIT®) for 12 days, whereas another group of 20 men were given a placebo For rats: A group of 10 rats drank a solution of either 20 mM D-aspartate or a placebo for 12 days Then LH and testosterone accumulation was determined in the serum and D-aspartate accumulation in tissues The effects of D-aspartate on the synthesis of LH and testosterone were gauged on isolated rat pituitary and Leydig cells Tissues were incubated with D-aspartate, and then the concentration (synthesis) of LH and cGMP in the pituitary and of testosterone and cAMP in the Leydig cells was determined In humans and rats, sodium D-aspartate induces an enhancement of LH and testosterone release In the rat pituitary, sodium D-aspartate increases the release and synthesis of LH through the involvement of cGMP as a second messenger, whereas in rat testis Leydig cells, it increases the synthesis and release of testosterone and cAMP is implicated as second messenger In the pituitary and in testes D-Asp is synthesized by a D-aspartate racemase which convert L-Asp into D-Asp The pituitary and testes possesses a high capacity to trapping circulating D-Asp from hexogen or endogen sources D-aspartic acid is a physiological amino acid occurring principally in the pituitary gland and testes and has a role in the regulation of the release and synthesis of LH and testosterone in humans and rats

Vasodilator factors in the systemic and local adaptations to pregnancy

Abstract: We postulate that an orchestrated network composed of various vasodilatory systems participates in the systemic and local hemodynamic adaptations in pregnancy. The temporal patterns of increase in the circulating and urinary levels of five vasodilator factors/systems, prostacyclin, nitric oxide, kallikrein, angiotensin-(1–7) and VEGF, in normal pregnant women and animals, as well as the changes observed in preeclamptic pregnancies support their functional role in maintaining normotension by opposing the vasoconstrictor systems. In addition, the expression of these vasodilators in the different trophoblastic subtypes in various species supports their role in the transformation of the uterine arteries. Moreover, their expression in the fetal endothelium and in the syncytiotrophoblast in humans, rats and guinea-pigs, favour their participation in maintaining the uteroplacental circulation. The findings that sustain the functional associations of the various vasodilators, and their participation by endocrine, paracrine and autocrine regulation of the systemic and local vasoactive changes of pregnancy are abundant and compelling. However, further elucidation of the role of the various players is hampered by methodological problems. Among these difficulties is the complexity of the interactions between the different factors, the likelihood that experimental alterations induced in one system may be compensated by the other players of the network, and the possibility that data obtained by manipulating single factors in vitro or in animal studies may be difficult to translate to the human. In addition, the impossibility of sampling the uteroplacental interface along normal pregnancy precludes obtaining longitudinal profiles of the various players. Nevertheless, the possibility of improving maternal blood pressure regulation, trophoblast invasion and uteroplacental flow by enhancing vasodilation (e.g. L-arginine, NO donors, VEGF transfection) deserves unravelling the intricate association of vasoactive factors and the systemic and local adaptations to pregnancy.

Women with endometriosis improved their peripheral antioxidant markers after the application of a high antioxidant diet

Abstract: Oxidative stress has been identified in the peritoneal fluid and peripheral blood of women with endometriosis. However, there is little information on the antioxidant intake for this group of women. The objectives of this work were 1) to compare the antioxidant intake among women with and without endometriosis and 2) to design and apply a high antioxidant diet to evaluate its capacity to reduce oxidative stress markers and improve antioxidant markers in the peripheral blood of women with endometriosis. Women with (WEN, n = 83) and without endometriosis (WWE, n = 80) were interviewed using a Food Frequency Questionnaire to compare their antioxidant intake (of vitamins and minerals). Then, the WEN participated in the application of a control (n = 35) and high antioxidant diet (n = 37) for four months. The high antioxidant diet (HAD) guaranteed the intake of 150% of the suggested daily intake of vitamin A (1050 μg retinol equivalents), 660% of the recommended daily intake (RDI) of vitamin C (500 mg) and 133% of the RDI of vitamin E (20 mg). Oxidative stress and antioxidant markers (vitamins and antioxidant enzymatic activity) were determined in plasma every month. Comparison of antioxidant intake between WWE and WEN showed a lower intake of vitamins A, C, E, zinc, and copper by WEN (p < 0.05, Mann Whitney Rank test). The selenium intake was not statistically different between groups. During the study, the comparison of the 24-hour recalls between groups showed a higher intake of the three vitamins in the HAD group. An increase in the vitamin concentrations (serum retinol, alpha-tocopherol, leukocyte and plasma ascorbate) and antioxidant enzyme activity (superoxide dismutase and glutathione peroxidase) as well as a decrease in oxidative stress markers (malondialdehyde and lipid hydroperoxides) were observed in the HAD group after two months of intervention. These phenomena were not observed in the control group. WEN had a lower intake of antioxidants in comparison to WWE. Peripheral oxidative stress markers diminished, and antioxidant markers were enhanced, in WEN after the application of the HAD.

Impact of female age and male infertility on ovarian reserve markers to predict outcome of assisted reproduction technology cycles.

Abstract: This study was designed to assess the capability of ovarian reserve markers, including baseline FSH levels, baseline anti-Mullerian hormone (AMH) levels, and antral follicle count (AFC), as predictors of live births during IVF cycles, especially for infertile couples with advanced maternal age and/or male factors. A prospective cohort of 336 first IVF/ICSI cycles undergoing a long protocol with GnRH agonist was investigated. Patients with endocrine disorders or unilateral ovaries were excluded. Among the ovarian reserve tests, AMH and age had a greater area under the receiving operating characteristic curve than FSH in predicting live births. Furthermore, AMH and age were the sole predictive factors of live births for women greater than or equal to 35 years of age; while AMH was the major determinant of live births for infertile couples with absence of male factors by multivariate logistic regression analysis. However, all the studied ovarain reserve tests were not preditive of live births for women < 35 years of age or infertile couples with male factors. The serum AMH levels were prognostic for pregnancy outcome for infertile couples with advanced female age or absence of male factors. The predictive capability of ovarian reserve tests is clearly influenced by the etiology of infertility.

Specific subsets of immune cells in human decidua differ between normal pregnancy and preeclampsia--a prospective observational study.

Abstract: Background Changes in the balance of decidual leucocyte populations may lead to an unfavourable uterine microenvironment which may be associated with the development of preeclampsia (PE). In this study, we therefore investigated the leucocyte subpopulations in decidual tissues of 33 women with preeclampsia and 66 control patients.

A retrospective study on IVF outcome in euthyroid patients with anti-thyroid antibodies: effects of levothyroxine, acetyl-salicylic acid and prednisolone adjuvant treatments

Abstract: Background Anti-thyroid antibodies (ATA), even if not associated with thyroid dysfunction, are suspected to cause poorer outcome of in vitro fertilization (IVF).

Expression of nodal signalling components in cycling human endometrium and in endometrial cancer.

Abstract: The human endometrium is unique in its capacity to remodel constantly throughout adult reproductive life. Although the processes of tissue damage and breakdown in the endometrium have been well studied, little is known of how endometrial regeneration is achieved after menstruation. Nodal, a member of the transforming growth factor-beta superfamily, regulates the processes of pattern formation and differentiation that occur during early embryo development. In this study, the expression of Nodal, Cripto (co-receptor) and Lefty A (antagonist) was examined by RT-PCR and immunohistochemistry across the menstrual cycle and in endometrial carcinomas. Nodal and Cripto were found to be expressed at high levels in both stromal and epithelial cells during the proliferative phase of the menstrual cycle. Although immunoreactivity for both proteins in surface and glandular epithelium was maintained at relatively steady-state levels across the cycle, their expression was significantly decreased within the stromal compartment by the mid-secretory phase. Lefty expression, as has previously been reported, was primarily restricted to glandular epithelium and surrounding stroma during the late secretory and menstrual phases. In line with recent studies that have shown that Nodal pathway activity is upregulated in many human cancers, we found that Nodal and Cripto immunoreactivity increased dramatically in the transition from histologic Grade 1 to histologic Grades 2 and 3 endometrial carcinomas. Strikingly, Lefty expression was low or absent in all cancer tissues. The expression of Nodal in normal and malignant endometrial cells that lack Lefty strongly supports an important role for this embryonic morphogen in the tissue remodelling events that occur across the menstrual cycle and in tumourogenesis.

Intra- and inter-observer analysis in the morphological assessment of early-stage embryos

Abstract: Background The aim of this study was to determine the intra- and inter-observer variability in the evaluation of embryo quality. Multilevel images of embryos on day 1, day 2 and day 3, were analysed using different morphological parameters.

Racial disparity in pathophysiologic pathways of preterm birth based on genetic variants

Abstract: Objective To study pathophysiologic pathways in spontaneous preterm birth and possibly the racial disparity associating with maternal and fetal genetic variations, using bioinformatics tools.

Distribution and hormonal regulation of membrane progesterone receptors β and γ in ciliated epithelial cells of mouse and human fallopian tubes

Abstract: Background: The controlled beating of cilia of the fallopian tube plays an important role in facilitating the meeting of gametes and subsequently transporting the fertilized egg to its implantation site. Rapid effects of progesterone on ciliary beat frequency have been reported in the fallopian tubes of cows, but the identity of the receptors mediating this non-genomic action of progesterone is not known. We recently identified a member of the non-genomic membrane progesterone receptor family, mPR gamma, as a candidate for mediating these actions of progesterone. Here, we investigated the possible presence of a related receptor, mPR beta, in the fallopian tubes of mice and women as well as the possible hormonal regulation of mPR beta and gamma. Methods: Western blot and immunohistochemistry with specific antibodies were used to characterize the expression and cellular localization of the mPRs in mouse and human tissues. Taqman (Quantitative Polymerase Chain Reaction) assays were used to quantify mRNA levels in the fallopian tubes of two different mouse models after injections with different hormones and specific antagonists. Results: In the fallopian tubes of both mouse and human, the expression of mPR beta and mPR gamma proteins was exclusively found in the ciliated cells. Whereas mPR beta was found on the cilia, mPR gamma was localized at the base of the same ciliated cells, as previously reported. In gonadotropin-primed mice, both mPRs genes were down-regulated after an injection with progesterone. Treatment with estradiol rapidly down-regulated the level of mPR beta mRNA and protein in immature mice. The mPR gamma protein was down-regulated around the time of ovulation in cycling women, similar to the regulation observed in mice stimulated to ovulate via gonadotropin injections. Conclusion: Our findings show the presence and hormonal regulation of two distinct mPRs associated with the cilia of the fallopian tubes in both mice and women. It is hypothesized that these receptors are involved in the control of ciliary movement and, thus, gamete transport in the fallopian tubes of mammals.

Antioxidant enzymes and lipid peroxidation in endometrium of patients with polyps, myoma, hyperplasia and adenocarcinoma

Abstract: Background Oxidative stress and impaired antioxidant system have been proposed as a potential factors involved in the pathophysiology of diverse disease states, including carcinogenesis. In this study, we explored the lipid peroxidation levels and antioxidant enzyme activities in women diagnosed with different forms of gynecological diseases in order to evaluate the antioxidant status in endometrium of such patients.

Reduced levels of intracellular calcium releasing in spermatozoa from asthenozoospermic patients.

Abstract: Asthenozoospermia is one of the most common findings present in infertile males characterized by reduced or absent sperm motility, but its aetiology remains unknown in most cases. In addition, calcium is one of the most important ions regulating sperm motility. In this study we have investigated the progesterone-evoked intracellular calcium signal in ejaculated spermatozoa from men with normospermia or asthenozoospermia. Human ejaculates were obtained from healthy volunteers and asthenospermic men by masturbation after 4–5 days of abstinence. For determination of cytosolic free calcium concentration, spermatozoa were loaded with the fluorescent ratiometric calcium indicator Fura-2. Treatment of spermatozoa from normospermic men with 20 micromolar progesterone plus 1 micromolar thapsigargin in a calcium free medium induced a typical transient increase in cytosolic free calcium concentration due to calcium release from internal stores. Similar results were obtained when spermatozoa were stimulated with progesterone alone. Subsequent addition of calcium to the external medium evoked a sustained elevation in cytosolic free calcium concentration indicative of capacitative calcium entry. However, when progesterone plus thapsigargin were administered to spermatozoa from patients with asthenozoospermia, calcium signal and subsequent calcium entry was much smaller compared to normospermic patients. As expected, pretreatment of normospermic spermatozoa with both the anti-progesterone receptor c262 antibody and with progesterone receptor antagonist RU-38486 decreased the calcium release induced by progesterone. Treatment of spermatozoa with cytochalasin D or jasplakinolide decreased the calcium entry evoked by depletion of internal calcium stores in normospermic patients, whereas these treatments proved to be ineffective at modifying the calcium entry in patients with asthenozoospermia. Our results suggest that spermatozoa from asthenozoospermic patients present a reduced responsiveness to progesterone.

A novel variant of ER-alpha, ER-alpha36 mediates testosterone-stimulated ERK and Akt activation in endometrial cancer Hec1A cells

Abstract: Endometrial cancer is one of the most common gynecologic malignancies and its incidence has recently increased. Experimental and epidemiological data support that testosterone plays an important role in the pathogenesis of endometrial cancer, but the underlying mechanism has not been fully understood. Recently, we identified and cloned a variant of estrogen receptor (ER) alpha, ER-alpha36. The aim of the present study was to investigate the role of ER-alpha36 in testosterone carcinogenesis. The cellular localization of ER-alpha36 was determined by immunofluorescence. Hec1A endometrial cancer cells (Hec1A/V) and Hec1A cells with siRNA knockdown of ER-alpha36 (Hec1A/RNAi) were treated with testosterone, ERK and Akt phosphorylation was assessed by Western blot analysis. Furthermore, the kinase inhibitors U0126 and LY294002 and the aromatase inhibitor letrozole were used to elucidate the pathway underlying testosterone-induced activities. Immunofluorescence shows that ER-alpha36 was localized on the plasma membrane of the both ER-alpha- and androgen receptor-negative endometrial cancer Hec1A cells. Testosterone induced ERK and Akt phosphorylation, which could be abrogated by ER-alpha 36 shRNA knockdown or the kinase inhibitors, U0126 and LY294002, and the aromatase inhibitor letrozole. Testosterone induces ERK and Akt phosphorylation via the membrane-initiated signaling pathways mediated by ER-alpha36, suggesting a possible involvement of ER-alpha 36 in testosterone carcinogenesis.

Maternal celiac disease autoantibodies bind directly to syncytiotrophoblast and inhibit placental tissue transglutaminase activity.

Abstract: Background Celiac disease (CD) occurs in as many as 1 in 80 pregnant women and is associated with poor pregnancy outcome, but it is not known if this is an effect on maternal nutrient absorption or, alternatively, if the placenta is an autoimmune target. The major autoantigen, tissue transglutaminase (tTG), has previously been shown to be present in the maternal-facing syncytiotrophoblast plasma membrane of the placenta.

Characterization of a novel telomerase-immortalized human endometrial stromal cell line, St-T1b

Abstract: Background Coordinated differentiation of the endometrial compartments in the second half of the menstrual cycle is a prerequisite for the establishment of pregnancy. Endometrial stromal cells (ESC) decidualize under the influence of ovarian progesterone to accommodate implantation of the blastocyst and support establishment of the placenta. Studies into the mechanisms of decidualization are often hampered by the lack of primary ESC. Here we describe a novel immortalized human ESC line.

The impact of cocaine and heroin on the placental transfer of methadone

Abstract: Methadone is the therapeutic agent of choice for the treatment of opiate addiction in pregnancy. The co-consumption (heroin, cocaine) which may influence the effects of methadone is frequent. Therefore, the impact of cocaine and heroin on the placental transfer of methadone and the placental tissue was investigated under in vitro conditions. Placentae (n = 24) were ex-vivo perfused with medium (m) (control, n = 6), m plus methadone (n = 6), m plus methadone and cocaine (n = 6) or m plus methadone and heroin (n = 6). Placental functionality parameters like antipyrine permeability, glucose consumption, lactate production, hormone production (hCG and leptin), microparticles release and the expression of P-glycoprotein were analysed. Methadone accumulated in placental tissue. Methadone alone decreased the transfer of antipyrine from 0.60 +/- 0.07 to 0.50 +/- 0.06 (fetal/maternal ratio, mean +/- SD, P < 0.01), whereas the combination with cocaine or heroin increased it (0.56 +/- 0.08 to 0.68 +/- 0.13, P = 0.03 and 0.58 +/- 0.21 to 0.71 +/- 0.24; P = 0.18). Microparticles (MPs) released from syncytiotrophoblast into maternal circuit increased by 30% after cocaine or heroin (P < 0.05) and the expression of P-glycoprotein in the tissue increased by ≥ 49% after any drug (P < 0.05). All other measured parameters did not show any significant effect when methadone was combined with cocaine or heroine. The combination of cocaine or heroin with methadone increase antipyrine permeability. Changes of MPs resemble findings seen in oxidative stress of syncytiotrophoblast.