Molecular and clinical aspects of embryotoxicity induced by acetylcholinesterase inhibitors.

Acetylcholinesterase inhibitors are widely used for a variety of medical, agricultural and public health purposes. Consequently, exposure is highly possible during lifetime. However, their systematic use raises concerns for the potential impact on the fetus and newborn since these substances may affect angiogenesis, the neonatal and maternal intensive care, neuroimmune function and response, mammary growth/lactation via cholinergic/non-cholinergic central and peripheral neuroendocrine pathways. New methodologies, neuroscientific technologies and research studies are needed to harness existing knowledge along with the proper management, availability for new acetylcholinesterase inhibitors, with stable pharmacodynamics and clinical outcomes.

Expression and function of galectins in the endometrium and at the human feto-maternal interface.

Galectins are classified as lectins that share structural similarities and bind ß-galactosides via a conserved carbohydrate recognition domain. So far 16 out of 19 identified galectins were shown to be present in humans and numerous studies revealed galectins as pivotal modulators of cell death, differentiation and growth. Galectins were highlighted to interact with both the adaptive and innate immune response. In the field of reproductive medicine and placenta research different roles for galectins have been proposed. Several galectins, being abundantly present at the human feto-maternal interphase and endometrium, were hypothesized to significantly contribute to endometrial receptivity and pregnancy physiology. Hence, this review outlines selected aspects of galectin action within endometrial function and at the feto-maternal interphase. Further current knowledge on galectins in reproductive and pregnancy disorders like endometriosis, abortion or preeclampsia is summarized.

The effect of CRH and its inhibitor, antalarmin, on in vitro growth of preantral mouse follicles, early embryo development, and steroidogenesis.

In vitro growth systems of preantral follicles allow studying the effect of various endocrine, paracrine, and autocrine factors on follicular growth and oocyte maturation. CRH is a 41-amino-acid neuropeptide responsible for endocrine, autonomic, immunological, and behavioral responses of mammals to stress and has two receptors, CRH receptor type 1 (CRH-R1) and CRH-R2. Antalarmin, a CRH-R1 antagonist, has been used to elucidate the role of CRH in stress, inflammation, and reproduction. The present study describes in vitro growth of mouse preantral follicles, early embryo development, and steroidogenesis in the presence of CRH and its antagonist antalarmin. We cultured 732 follicles in control media, 1306 in CRH 10(-7) mol/liter, and 1202 in CRH 10(-7) plus antalarmin 10(-6) mol/liter. The culture medium was assayed on alternate days for 17ß-estradiol, progesterone, and ß-human chorionic gonadotropin. Total RNA was extracted from preantral follicles as well as early preimplantation embryos and was assessed by real-time RT-PCR for the expression of CRH-R1 and CRH-R2 mRNAs. Hormone analysis showed that the CRH group had lower levels of 17ß-estradiol, progesterone, and ß-human chorionic gonadotropin as the culture progressed, in comparison with the other two groups. RT-PCR demonstrated the presence of CRH-R1 and CRH-R2 in all stages of preantral follicle culture. Morula/blastocyst-stage embryos expressed only CRH-R1. In conclusion, CRH has an inhibitory effect on in vitro fertilized oocytes, resulting from cultured preantral follicles at all stages of preimplantation embryo development. Furthermore, the presence of CRH in the culture medium inhibits steroidogenesis by preantral mouse follicles cultured in vitro.

Best practices of ASRM and ESHRE: a journey through reproductive medicine.

BACKGROUND: The American Society for Reproductive Medicine (ASRM) and the European Society of Human Reproduction and Embryology (ESHRE) are the two largest societies in the world whose members comprise the major experts and professionals working in the field of reproductive medicine and embryology. These societies have never before had a joint scientific meeting. METHODS: A 3-day meeting was planned and took place in March of 2012. The goal was to present and debate key topics, as well as modes of practice in reproductive medicine and to discuss recent developments in the field. RESULTS: Presentations by members of ASRM and ESHRE were of three types: 'state of the art' lectures, 'back-to-back' presentations of two points of view and debates. CONCLUSIONS: For the first time, ASRM and ESHRE held a joint meeting where a special emphasis was given to presentations on the hottest topics in the field. Although different opinions and approaches sometimes exist on the two sides of the Atlantic, an appreciation and acceptance of these differences was evident, and there was more commonality than divergence of opinion.

Aberrant expression of corticotropin-releasing hormone in pre-eclampsia induces expression of FasL in maternal macrophages and extravillous trophoblast apoptosis.

Corticotropin-releasing hormone (CRH) and its receptors are expressed in human placenta. Recently, the impaired function of this system has been associated with a number of complications of pregnancy, including pre-eclampsia. The aim of the study was to test the hypothesis that CRH participates in the pathophysiology of pre-eclampsia through the induction of macrophage-mediated apoptosis of extravillous trophoblasts (EVTs). We found that the expression of CRH was increased in the EVT of the placental bed biopsy specimens from pre-eclamptic pregnancies (1.8-fold increase; P < 0.05). In addition, significantly larger numbers of apoptotic EVT were detected in pre-eclamptic placentas compared with normal ones (P < 0.05), and only in pre-eclamptic placentas, decidual macrophages were found to be Fas ligand (FasL)-positive. In vitro studies on the effect of CRH on human macrophages suggested that CRH induced the expression of the FasL protein in human macrophages and potentiated their ability to induce the apoptosis of a Fas-expressing EVT-based hybridoma cell line in co-cultures. These findings demonstrate a possible mechanism by which the aberrant expression of CRH in pre-eclampsia may activate the FasL-positive decidual macrophages, impair the physiological turnover of EVT and eventually disturb placentation.

Expression of cathepsin-D in primary breast cancer and corresponding local recurrence or metastasis: an immunohistochemical study.

BACKGROUND/AIM: The role of cathepsin-D is well established in breast cancer progression, being correlated with worse clinical outcomes. However, to our knowledge, no study has been performed investigating its expression in primary breast cancer tumors and their corresponding recurrences or metastasis. MATERIALS AND METHODS: Tissue sections from ten breast cancer cases and their corresponding local recurrences and six breast cancer cases and their corresponding metastases were immunohistochemically assessed for cathepsin-D reactivity. Cases diagnosed as either ductal carcinoma in situ (n=7), or breast carcinoma with no evidence of local recurrence or metastasis during follow-up (n=8) served as controls. RESULTS: Cathepsin-D was significantly up-regulated in all the study groups compared to controls. No difference was found between primary tumors and their corresponding recurrences or metastases. CONCLUSION: Cathepsin-D-expressing breast cancer cells seem to be involved in local recurrence or metastasis formation. Large series are needed to further verify this result with the aim of possible future molecular intervention.

Retinoid X receptor alpha (RXRα) and peroxisome proliferator-activated receptor gamma (PPARγ) expression in breast cancer: an immunohistochemical study.

BACKGROUND/AIM: The role of retinoid X receptor alpha (RXRα) and peroxisome proliferator-activated receptor gamma (PPARγ) in breast cancer has been well studied in vitro. The aim of the study was to assess the presence of these molecules in human breast cancer specimens and correlate them with major clinicopathological features. PATIENTS AND METHODS: Tissue sections from 82 breast cancer cases clustered according to histological grade, lymph node (LN) and hormone receptor (HR) status were assessed by immunohistochemistry for RXRα and PPARγ. RESULTS: RXRα was found to be strongly and moderately expressed in 11 (14.10%) and 33 (42.31%) cases, respectively. PPARγ was found to be strongly and moderately expressed in 33 (41.25%) and 25 (31.25%) cases, respectively. Only RXRα expression was inversely correlated with histological grade. Surprisingly, significantly elevated PPARγ expression was found in cases with positive LN status. Survival analysis did not yield significant results. CONCLUSION: Our data support the current thesis of RXRα being a potential target for feature molecular interventions.

Effects of phytoestrogen extracts isolated from flax on hormone production of trophoblast tumour cells Jeg 3 and BeWo.

UNLABELLED: AIM AND SETTING: To test the effects of crude extracts from flax (Linum usitatissimum) on progesterone and estradiol and ERα and ß/PR production in choriocarcinoma cell lines Jeg 3 and BeWo. Tumor trophoblast cells (Jeg 3 and BeWo) were incubated in the presence of different concentrations of the flax crude extracts. Estradiol and progesterone production was measured. Estrogen receptor α and ß as well as progesterone receptor expressions were also assessed. RESULTS: In Jeg 3 cells, progesterone production was downregulated by flax root and leaves extract, while in BeWo cells only flax root extract did manage to downregulate progesterone production. ERß expression was significantly downregulated by flax root and flax leaves extract in both cell lines; on the contrary, ERα expression was increased by flax leaves extract in BeWo cells. PR expression was downregulated by flax leaves extract in Jeg 3 and by flax root extract in BeWo cells. CONCLUSION: Flax extracts derived from leaves and especially from roots can modify progesterone and possibly estradiol production, while at the same time they seem to alter ERß expression. Further studies on animal models and adequately designed retrospective epidemiological studies are imperative to clarify this role upon progesterone.

Pregnancy-related low back pain.

Pregnancy related low back pain is a common complaint among pregnant women. It can potentially have a negative impact on their quality of life. The aim of this article is to present a current review of the literature concerning this issue.By using PubMed database and low back pain, pelvic girdle pain, pregnancy as keywords, abstracts and original articles in English investigating the diagnosis treatment of back pain during pregnancy were searched and analyzedLow back pain could present as either a pelvic girdle pain between the posterior iliac crest and the gluteal fold or as a lumbar pain over and around the lumbar spine. The source of the pain should be diagnosed and differentiated early.The appropriate treatment aims to reduce the discomfort and the impact on the pregnant womans quality of life. This article reveals the most common risk factors, as well as treatment methods, which may help to alleviate the pain. Some suggestions for additional research are also discussed.

Corticotropin-releasing hormone, stress and human reproduction: an update.

The stress system has suppressive effects on female and male reproductive function. Corticotrophin-releasing hormone (CRH), the principal regulator of stress, has been identified in the female and male reproductive system. Reproductive CRH participates in various reproductive functions that have an inflammatory component, where it serves as an autocrine and paracrine modulator. These include ovarian and endometrial CRH, which may participate in the regulation of steroidogenesis and the inflammatory processes of the ovary (ovulation and luteolysis) and the endometrium (decidualization and blastocyst implantation) and placental CRH, which is secreted mostly during the latter half of pregnancy and is responsible for the onset of labor. It has been suggested that there is a "CRH placental clock" which determines the length of gestation and the timing of parturition and delivery. The potential use of CRH-antagonists is presently under intense investigation. CRH-R1 antagonists have been used in animal studies to elucidate the role of CRH in blastocyst implantation and invasion, early fetal immunotolerance and premature labor. The present review article focuses on the potential roles of CRH on the physiology and pathophysiology of reproduction and highlights its participation in crucial steps of pregnancy, such as implantation, fetal immune tolerance, parturition and fetal programming of the hypothalamic-pituitary-adrenal (HPA) axis.

Glycoprotein and carbohydrate binding protein expression in the placenta in early pregnancy loss.

Glycoproteins expressed at the fetal-maternal interface have been shown to exert immunomodulating effects. Glycodelin, hCG and transferrin have been used in in vitro experiments as ligands to block E-selectin-mediated cell adhesion. We found that glycodelin is a strong inhibitor of the E-selectin-mediated cell adhesion with a 10(3)-fold increase in potency compared to the monovalent tetrasaccharide sialyl Lewis X. HCG with distinct carbohydrate expression is also an effective selectin antagonist, whereas the potency of transferrin is low. This could indicate a possible role of glycodelin, hCG and transferrin in preventing leukocyte adhesion to the fetal trophoblast. In decidual tissue of abortion patients, glycodelin expression was significantly reduced compared to normal gestation. These results were confirmed by in situ hybridization. Moreover, glycodelin expression in endometrial cells in vitro could be stimulated by addition of hCG. Because hCG is down-regulated in women with abortion, we speculate that hCG could be one of the factors regulating glycodelin expression. Galectins are structurally related proteins with the ability to bind beta-galactosides through a conserved carbohydrate recognition domain. Galectin-1 (gal-1) expression in the syncytiotrophoblast is down-regulated in early pregnancy loss. Gal-1 recognizes the Thomsen-Friedenreich disaccharide (Galbeta1-3GalNAc-) on the syncytiotrophoblast and extravillous trophoblast. Gal-1 also inhibited trophoblast cell proliferation but did not induce apoptosis in BeWo cells. Ligation of Gal-1 on trophoblast cells may have regulatory effects on trophoblast cell differentiation. Decreased expression of Gal-1 may partly explain disturbed trophoblast differentiation during early placentation leading to early pregnancy loss.

CRH-like peptides in human reproduction.

CRH and Urocortins 1, 2 and 3 comprise the, so far identified, members of the CRH family of peptides in humans. Their actions are mediated through two distinct receptors, CRHR1 and CRHR2, encoded by different genes. CRH-like peptides and their receptors have been identified in reproductive tissues, such as the ovary, uterus as well as fetal and placental membranes. The participation of the "CRH family" of peptides and receptors in the physiology of these organs is currently under intense investigation. During the estrus cycle, endometrial CRH acts as a fine tuner of stromal cells decidualization. CRH is produced by embryonic trophoblast and maternal decidual cells and plays important roles in implantation. CRH also participates in the control of trophoblast invasion. Furthermore, placental CRH and Urocortin are involved in the mechanisms controlling maintenance of pregnancy and the onset of labor. The level of participation of urocortins 2 and 3 in these phenomena is currently under investigation. This review will focus on existing data on the expression and regulation of the CRH family of peptides and their receptors in the female reproductive system, as well as in their potential biologic role(s) in human reproductive functions.

The expression of receptivity markers in the fallopian tube epithelium.

Pinopodes represent the morphological and integrins, the biomolecular markers of endometrial receptivity. We studied using scanning electron microscopy, the expression of pinopodes on tubal samples and their corresponding endometria, from 21 women of reproductive age (7 from proliferative phase, 7 from day LH +5 and 7 from day LH +7). In addition, we examined the immunohistochemical staining of integrins alpha v beta 3, alpha v beta 5 and their ligands, fibronectin (FN) and osteopontin (OPN) in the same tubal epithelium samples. Pinopodes were detected on the tubal epithelium exclusively during day LH +7, coincident with their formation in the endometrium and synchronous to alpha v beta 3 sharp increase in the oviduct epithelium, suggesting a regulation similar to the endometrium. In contrast, alpha v beta 5, FN and OPN remained unchanged during the cycle. These results show for the first time the formation of pinopodes in the tubal epithelium at the time of endometrial receptivity and correlate it with the upregulation of the intact dimmer alpha v beta 3 in the tubes.

Skewed X-chromosome inactivation in Greek women with idiopathic recurrent miscarriage.

OBJECTIVES: To investigate if skewed X-chromosome inactivation (XCI) is associated with unexplained recurrent miscarriage (RM) in Greek women. METHODS: This was a prospective case-control study. A methylation-sensitive assay was used to investigate the X-inactivation pattern of women with unexplained RM and controls. RESULTS: Fifty-six of the 74 patients (75.7%) and 55 of 80 controls (68.8%) were informative. Among the informative cases, 6/56 (10.7%) women showed extreme XCI (>90%) and among the informative controls, 2/55 (3.6%) showed extreme XCI. CONCLUSIONS: In the present study, women with unexplained RM showed a statistically nonsignificant increase in skewed XCI prevalence (10.7%) compared with control women (3.6%; p = 0.271).

Abortion is associated with increased expression of FasL in decidual leukocytes and apoptosis of extravillous trophoblasts: a role for CRH and urocortin.

Human reproduction is remarkably inefficient, with more than half of spontaneous conceptions failing to complete the first trimester. However, little is known on the molecular events that take place at the implantation site during abortion. Here, we examined the hypothesis that the expression of the proapoptotic Fas/FasL system at the implantation site is impaired in abortions. We found that, in contrast to normal pregnancy, abortive deciduas contain leukocytes that are positive for FasL and extravillous trophoblasts (EVTs), which show increased expression of Fas and increased rates of apoptosis. In addition, the neuropeptides, corticotropin-releasing hormone and urocortin, were elevated in placental material obtained from abortions. In vitro, these peptides induced the expression of FasL in decidual lymphocytes (DL) obtained from elective termination of pregnancy placentas and thus potentiated the cells' ability to induce Fas-mediated apoptosis in an EVT-based hybridoma cell line. Finally, DL from abortion sites effectively induced apoptosis of EVT without prior treatment. It is possible that these events may impede successful early placentation and thus contribute to the pathophysiology of human abortion.

Intratumoral CRH modulates immuno-escape of ovarian cancer cells through FasL regulation.

Although corticotropin-releasing hormone (CRH) and Fas ligand (FasL) have been documented in ovarian carcinoma, a clear association with tumour progression and immuno-escape has not been established. FasL plays an important role in promoting tumour cells' ability to counterattack immune cells. Here, we examined immunohistochemically the expression of CRH, CRHR1, CRHR2 and FasL in 47 human ovarian cancer cases. The ovarian cancer cell lines OvCa3 and A2780 were further used to test the hypothesis that CRH might contribute to the immune privilege of ovarian tumours, by modulating FasL expression on the cancer cells. We found that CRH, CRHR1, CRHR2 and FasL were expressed in 68.1, 70.2, 63.8 and 63.8% of the cases respectively. Positivity for CRH or FasL expression was associated with higher tumour stage. Finally, CRH increased the expression of FasL in OvCa3 and A2780 cells through CRHR1 thereby potentiated their ability to induce apoptosis of activated peripheral blood lymphocytes. Corticotropin-releasing hormone produced by human ovarian cancer might favour survival and progression of the tumour by promoting its immune privilege. These findings support the hypothesis that CRHR1 antagonists could potentially be used against ovarian cancer.

The role of corticotropin-releasing hormone in blastocyst implantation and early fetal immunotolerance.

During blastocyst implantation, the maternal endometrial response to the invading semi-allograft has characteristics of an acute, aseptic inflammatory response. However, once implanted, the embryo suppresses this response and prevents rejection. Simultaneously, the mother's immune system prevents a graft VS. host reaction deriving from the fetal immune system. We have shown that embryonic trophoblast and maternal decidua cells, i.e., cells located in the interface between the fetal placenta and the maternal endometrium, produce corticotropin-releasing hormone (CRH) and express Fas ligand. CRH may play a crucial role in the implantation and the anti-rejection process that protects the fetus from the maternal immune system, primarily by killing activated T cells through the Fas-FasL interaction. In experimental animals, type 1 CRH receptor (CRH-R1) blockade by antalarmin, a specific type 1 CRH receptor antagonist, decreased implantation sites by approximately 70%. CRH is also involved in controlled trophoblast invasion, by downregulating the synthesis of the carcinoembryonic antigen-related cell adhesion molecule 1 by extravillous trophoblast cells. IN VITRO findings showed that CRH-R1 blockade by antalarmin increased trophoblast invasion by approximately 60%. Defective uterine CRH/CRH-R1 system during early pregnancy may be implicated in the pathophysiology of recurrent miscarriage, placenta accreta, and preeclampsia.

Maternal serum corticotropin-releasing hormone and ACTH levels as predictive markers of premature labor.

OBJECTIVE: To investigate whether corticotropin-releasing hormone (CRH) and corticotropin (ACTH) plasma concentrations in women diagnosed with preterm labor are of potential clinical value in the assessment of the risk of preterm birth. METHOD: Plasma samples of 79 women diagnosed with preterm labor were used in this study. Samples were divided into three groups based on the week of gestation (24th-28th, 29th-32nd, 33rd-37th). CRH and ACTH values were determined by ELISA. RESULT: Mean maternal peripheral plasma values of CRH and ACTH were significantly higher (p<0.001) in women who were initially diagnosed with preterm labor and finally delivered a preterm birth, compared to women with the same diagnosis but with term birth. CONCLUSION: CRH and ACTH serum levels in women diagnosed with preterm labor could be used as predictors for the timing of parturition.

Expression of urocortin in the extravillous human trophoblast at the implantation site.

Urocortin (UCN) is a 40 amino acid peptide which is closely related to corticotropin-releasing hormone and binds with high affinity to both CRH type 1 and type 2 receptors. UCN is expressed in human reproductive tissues including endometrium, ovary, and placenta. This study was designed to investigate the cellular localization of UCN at the implantation site of the human blastocyst, as well as the regulation of the UCN promoter by two major intracellular signaling pathways, the cAMP/PKA and diacylglycerol/PKC pathways, in cells of placental origin. For this reason, immunohistochemistry was performed on tissue sections from paraffin-embedded human first trimester placentas and freshly isolated human invasive extravillous trophoblast cells (EVT) were analyzed for UCN expression using RT-PCR and immunofluorescence. Finally, UCN promoter activity was analyzed in the JEG3 human choriocarcinoma cell line. Immunohistochemistry revealed expression of UCN in the cytotrophoblast, the EVT and decidual cells. Both UCN mRNA and peptide were detectable in freshly isolated EVT. Finally, a human UCN promoter luciferase reporter construct transfected into JEG3 cells was significantly inducible by phorbol ester plus ionomycin, but not by phorbol ester alone or by forskolin. Collectively, the present study reports the expression of UCN in EVT and the activation of the UCN gene promoter by the diacylglycerol/PKC pathway. The functional significance of urocortin for the physiology of EVT requires further investigation.

CRF receptor antagonists: utility in research and clinical practice.

CRF, CRF-related peptides and CRF receptors constitute a complex physiological system which has a key role in facilitating the adaptation of the organism to the stressful stimuli of the environment. The behavioral, endocrine, autonomic and immune branches of stress response are considered to be under the coordinating effects of CRF and its related peptides. The effects of these peptides are mediated through two distinct receptors, types 1 and 2 CRF receptors (CRF(1) and CRF(2)). The two receptors are encoded by separate genes and belong to the G-coupled receptor superfamily. The wide influence of the CRF system on physiological processes in both brain and periphery, suggests the implication of the respective peptides in the pathophysiology of numerous disorders which involve dysregulated stress responses. The potential use of CRF antagonists in such disorders is currently under intense investigation. Furthermore, such compounds have been invaluable in elucidating the physiology of the CRF system. This review will focus on existing data on the structural and pharmacological characteristics as well as the experimental and potential clinical uses of non-peptide, small molecule CRF antagonists.