Showing papers in "Reproductive Biology and Endocrinology in 2013"

Lifestyle factors and reproductive health: taking control of your fertility

Abstract: Approximately 10 to 15% of couples are impacted by infertility. Recently, the pivotal role that lifestyle factors play in the development of infertility has generated a considerable amount of interest. Lifestyle factors are the modifiable habits and ways of life that can greatly influence overall health and well-being, including fertility. Many lifestyle factors such as the age at which to start a family, nutrition, weight, exercise, psychological stress, environmental and occupational exposures, and others can have substantial effects on fertility; lifestyle factors such as cigarette smoking, illicit drug use, and alcohol and caffeine consumption can negatively influence fertility while others such as preventative care may be beneficial. The present literature review encompasses multiple lifestyle factors and places infertility in context for the couple by focusing on both males and females; it aims to identify the roles that lifestyle factors play in determining reproductive status. The growing interest and amount of research in this field have made it evident that lifestyle factors have a significant impact on fertility.

Obstetric complications in women with polycystic ovary syndrome: a systematic review and meta-analysis

Abstract: Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women of childbearing age. The risk of pregnancy and neonatal complications in women with PCOS is debatable. In order to determine the risk of pregnancy and neonatal complications, evidence regarding these risks was examined. Literature searches were performed in the electronic databases MEDLINE, EMBASE, and CENTRAL based on the established strategy and eligible tries were included according to inclusion and exclusion criteria. A systematic literature review looking at rates of gestational diabetes mellitus (GDM), pregnancy-induced hypertension (PIH), preeclampsia, premature delivery, neonatal birth weight, caesarean section and admission to a neonatal intensive care unit (NICU) was conducted in women with PCOS. Pregnancy outcomes between women with PCOS versus controls were included. Sensitivity analyses were performed to determine the reliability of the available evidence and to validate the results. The study was performed with the approval of the ethics committee of the First Affiliated Hospital of Guangxi Medical University. A total of 27studies, involving 4982 women with PCOS and 119692 controls were eligible for the meta-analysis. Women with PCOS demonstrated a significantly higher risk of developing GDM (OR3.43; 95% CI: 2.49–4.74), PIH (OR3.43; 95% CI: 2.49–4.74), preeclampsia (OR2.17; 95% CI: 1.91–2.46), preterm birth (OR1.93; 95%CI: 1.45–2.57), caesarean section (OR 1.74; 95% CI: 1.38–2.11) compared to controls. Their babies had a marginally significant lower birth weight (WMD −0.11g; 95%CI: -0.19 – -0.03), and higher risk of admission to NICU (OR 2.32; 95% CI: 1.40–3.85) compared to controls. Women with PCOS have increased risk of adverse pregnancy and neonatal complications. It is necessary to establish guidelines for supervision during pregnancy and parturition to prevent these complications.

The management of Asherman syndrome: a review of literature

Abstract: Asherman syndrome is a debatable topic in gynaecological field and there is no clear consensus about management and treatment. It is characterized by variable scarring inside the uterine cavity and it is also cause of menstrual disturbances, infertility and placental abnormalities. The advent of hysteroscopy has revolutionized its diagnosis and management and is therefore considered the most valuable tool in diagnosis and management. The aim of this review is to explore the most recent evidence related to this condition with regards to aetiology, diagnosis management and follow up strategies.

Protective effects of in vitro treatment with zinc, d-aspartate and coenzyme q10 on human sperm motility, lipid peroxidation and DNA fragmentation

Abstract: Spermatozoa are extremely vulnerable to oxidative stress caused by the unbalance between concentrations of reactive oxygen species and antioxidant scavenging systems present inside the male reproductive tract. In spite of a large number of clinical studies that claimed the beneficial effects of antioxidant oral administration on sperm physiology and fertility, only a few studies were addressed to evaluate their effects on spermatozoa in vitro. Main aims of the present study were to assess the influence of zinc, D-aspartate and coenzyme Q10, included in the dietary supplement Genadis (Merck Serono), on human sperm motility, DNA fragmentation and lipid peroxidation. Semen samples, obtained from forty-four patients (23–30 years of age) were enrolled in this study, twenty-four were normospermic and twenty patients were oligospermic. Semen samples were analysed for sperm progressive motility and kinetics through computer assisted analysis, DNA fragmentation and lipid peroxidation. Main results showed that in both normo and oligospermic samples, total and progressive sperm motility is maintained by in vitro treatment with zinc, D-aspartate and coenzyme Q10, whereas a significant decrease of these parameters occurs in parallel samples incubated in medium alone. Zinc, D-aspartate and coenzyme Q10 also prevented the decrease of sperm kinetics but such an effect was highly significant only in oligospermic samples. Moreover, they also protected spermatozoa by the increase of DNA fragmentation and lipid peroxidation. Zinc, D-aspartate and coenzyme Q10 exert a direct protective effect on human spermatozoa preventing the decrease of motility and the increase of DNA fragmentation and lipid peroxidation during in vitro culture.

Predicting live birth chances for women with multiple consecutive failing IVF cycles: a simple and accurate prediction for routine medical practice.

Abstract: Background: Women having experienced several consecutive failing IVF cycles constitute a critical and particular subset of patients, for which growing perception of irremediable failure, increasing costs and IVF treatment related risks necessitate appropriate decision making when starting or not a new cycle. Predicting chances of LB might constitute a useful tool for discussion between the patient and the clinician. Our essential objective was to dispose of a simple and accurate prediction model for use in routine medical practice. The currently available predictive models applicable to general populations cannot be considered as accurate enough for this purpose. Methods: Patients with at least four consecutive Failing cycles (CFCs) were selected. We constructed a predictive model of LB occurrence during the last cycle, by using a stepwise logistic regression, using all the baseline patient characteristics and intermediate stage variables during the four first cycles. Results: On as set of 151 patients, we identified five determinant predictors: the number of previous cycles with at least one gestational sac (NGS), the mean number of good-quality embryos, age, male infertility (MI) aetiology and basal FSH. Our model was characterized by a much higher discrimination as the existing models (C-statistics=0.76), and an excellent calibration. Conclusions: Couples having experienced multiple IVF failures need precise and appropriate information to decide to resume or interrupt their fertility project. Our essential objective was to dispose of a simple and accurate prediction model to allow a routine practice use. Our model is adapted to this purpose: It is very simple, combines five easily collected variables in a short calculation; it is more accurate than existing models, with a fair discrimination and a well calibrated prediction.

Proteomic analysis of human spermatozoa proteins with oxidative stress

Abstract: Background: Oxidative stress plays a key role in the etiology of male infertility. Significant alterations in the sperm proteome are associated with poor semen quality. The aim of the present study was to examine if elevated levels of reactive oxygen species cause an alteration in the proteomic profile of spermatozoa. Methods: This prospective study consisted of 52 subjects: 32 infertile men and 20 normal donors. Seminal ejaculates were classified as ROS+ or ROS- and evaluated for their proteomic profile. Samples were pooled and subjected to LC-MS/MS analysis through in-solution digestion of proteins for peptide characterization. The expression profile of proteins present in human spermatozoa was examined using proteomic and bioinformatic analysis to elucidate the regulatory pathways of oxidative stress. Results: Of the 74 proteins identified, 10 proteins with a 2-fold difference were overexpressed and 5 were underexpressed in the ROS+ group; energy metabolism and regulation, carbohydrate metabolic processes such as gluconeogenesis and glycolysis, protein modifications and oxidative stress regulation were some of the metabolic processes affected in ROS+ group. Conclusions: We have identified proteins involved in a variety of functions associated with response and management of oxidative stress. In the present study we focused on proteins that showed a high degree of differential expression and thus, have a greater impact on the fertilizing potential of the spermatozoa. While proteomic analyses identified the potential biomarkers, further studies through Western Blot are necessary to validate the biomarker status of the proteins in pathological conditions.

Proteomic analysis of seminal fluid from men exhibiting oxidative stress

Abstract: Background: Seminal plasma serves as a natural reservoir of antioxidants. It helps to remove excessive formation of reactive oxygen species (ROS) and consequently, reduce oxidative stress. Proteomic profiling of seminal plasma proteins is important to understand the molecular mechanisms underlying oxidative stress and sperm dysfunction in infertile men. Methods: This prospective study consisted of 52 subjects: 32 infertile men and 20 healthy donors. Once semen and oxidative stress parameters were assessed (ROS, antioxidant concentration and DNA damage), the subjects were categorized into ROS positive (ROS+) or ROS negative (ROS-). Seminal plasma from each group was pooled and subjected to proteomics analysis. In-solution digestion and protein identification with liquid chromatography tandem mass spectrometry (LC-MS/MS), followed by bioinformatics analyses was used to identify and characterize potential biomarker proteins. Results: A total of 14 proteins were identified in this analysis with 7 of these common and unique proteins were identified in both the ROS+ and ROS- groups through MASCOT and SEQUEST analyses, respectively. Prolactininduced protein was found to be more abundantly present in men with increased levels of ROS. Gene ontology annotations showed extracellular distribution of proteins with a major role in antioxidative activity and regulatory processes. Conclusions: We have identified proteins that help protect against oxidative stress and are uniquely present in the seminal plasma of the ROS- men. Men exhibiting high levels of ROS in their seminal ejaculate are likely to exhibit proteins that are either downregulated or oxidatively modified, and these could potentially contribute to male infertility.

Differential neutrophil gene expression in early bovine pregnancy

Abstract: In food production animals, especially cattle, the diagnosis of gestation is important because the timing of gestation directly affects the running of farms. Various methods have been used to detect gestation, but none of them are ideal because of problems with the timing of detection or the accuracy, simplicity, or cost of the method. A new method for detecting gestation, which involves assessing interferon-tau (IFNT)-stimulated gene expression in peripheral blood leukocytes (PBL), was recently proposed. PBL fractionation methods were used to examine whether the expression profiles of various PBL populations could be used as reliable diagnostic markers of bovine gestation. PBL were collected on days 0 (just before artificial insemination), 7, 14, 17, 21, and 28 of gestation. The gene expression levels of the PBL were assessed with microarray analysis and/or quantitative real-time reverse transcription (q) PCR. PBL fractions were collected by flow cytometry or density gradient cell separation using Histopaque 1083 or Ficoll-Conray solutions. The expression levels of four IFNT-stimulated genes, interferon-stimulated protein 15 kDa (ISG15), myxovirus-resistance (MX) 1 and 2, and 2′-5′-oligoadenylate synthetase (OAS1), were then analyzed in each fraction through day 28 of gestation using qPCR. Microarray analysis detected 72 and 28 genes in whole PBL that were significantly higher on days 14 and 21 of gestation, respectively, than on day 0. The upregulated genes included IFNT-stimulated genes. The expression levels of these genes increased with the progression of gestation until day 21. In flow cytometry experiments, on day 14 the expression levels of all of the genes were significantly higher in the granulocyte fraction than in the other fractions. Their expression gradually decreased through day 28 of gestation. Strong correlations were observed between the expression levels of the four genes in the granulocyte fractions obtained with flow cytometry and with density gradient separation. The expression profiles of ISG15, MX1, MX2, and OAS1 could be a useful diagnostic biomarker of bovine gestation. Assessing the expression levels of these genes in a granulocyte fraction obtained with density gradient separation is a practical way of detecting gestation in cows within three weeks of insemination.

Associations among serum pro- and anti-inflammatory cytokines, metabolic mediators, body condition, and uterine disease in postpartum dairy cows

Abstract: Background Adipose tissue is an active endocrine organ which secretes a wide range of hormones and protein factors, collectively termed adipokines. Adipokines affect appetite and satiety, glucose and lipid metabolism, inflammation and immune functions. The objectives were to evaluate serum concentrations of adipokines (adiponectin, leptin, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6) in lactating dairy cows with postpartum uterine inflammatory conditions (metritis, clinical endometritis or subclinical endometritis) and in cows experiencing loss of body condition, and to assess the relationship of adipokines and body condition loss in the establishment of persistent uterine inflammatory conditions.

Efficacy of dehydroepiandrosterone to improve ovarian response in women with diminished ovarian reserve: a meta-analysis.

Abstract: Women with diminished ovarian reserve often respond poorly to controlled ovarian stimulation resulting in retrieval of fewer oocytes and reduced pregnancy rates. It has been proposed that pre-IVF Dehydroepiandrosterone (DHEA) adjuvant therapy may improve ovarian response and pregnancy rates in women with diminished ovarian reserve. This meta-analysis aims to investigate efficacy of DHEA as an adjuvant to improve ovarian response and IVF outcome in women with diminished ovarian reserve. Electronic databases were searched under the following terms: (DHEA) and (diminished ovarian reserve) and/or (poor response). Studies were included if they reported at least one of the following outcomes; clinical pregnancy rate, number of oocytes retrieved, miscarriage rate. We identified 22 publications determining effects of DHEA in clinical trials. Only 3 controlled studies were eligible for meta-analysis. There was no significant difference in the clinical pregnancy rate and miscarriage rates between women pre-treated with DHEA compared to those without DHEA pre-treatment (RR 1.87, 95% CI 0.96-3.64; and RR 0.59, 95% CI 0.21-1.65, respectively). The number of oocytes retrieved (WMD -1.88, 95% CI -2.08, 1.67; P < 0.001) was significantly lower in the DHEA group. In conclusion, based on the limited available evidence from a total of approximately 200 IVF cycles, there are insufficient data to support a beneficial role of DHEA as an adjuvant to controlled ovarian stimulation in IVF cycle. Well-designed, randomised controlled trials as well as more exact knowledge about DHEA mechanisms of action are needed to support use of DHEA in standard practice for poor-responders.

Functional proteomic analysis of seminal plasma proteins in men with various semen parameters

Abstract: Background: Alterations at the molecular level in spermatozoa and seminal plasma can affect male fertility. The objective of this study was to determine if analysis of differential expression of proteins in varying semen parameters can serve as potential biomarkers for male infertility. Methods: The differential expression of proteins in the seminal plasma of men based on sperm count and morphology were examined utilizing proteomic tools. Subjects were categorized based on sperm concentration and morphology into 4 groups: 1) normal sperm count and normal morphology (NN); 2) normal sperm count and abnormal morphology (NA); 3) oligozoospermia and normal morphology (ON); and 4) oligozoospermia and abnormal morphology (OA). Proteomic analysis was performed by LC-MS/MS followed by functional bioinformatics analysis. Protein distribution in the NA, ON and OA groups was compared with that of the NN group. Results: Twenty proteins were differentially expressed among the 4 groups. Among the unique proteins identified, 3 were downregulated in the NA group, 1 in the ON group and 1 in the OA group while 2 were upregulated in the ON and OA groups. The functional analysis 1) identified biological regulation as the major processes affected and 2) determined that most of the identified proteins were of extracellular origin. Conclusions: We have identified proteins that are over-or underexpressed in the seminal plasma of men with poor sperm quality. The distinct presence of some of the proteins may serve as potential biomarkers and provide insight into the mechanistic role played by these proteins in male infertility. Further studies using Western Blot analysis are required to validate these findings.

Circulating matrix metalloproteinase MMP-9 and MMP-2/TIMP-2 complex are associated with spontaneous early pregnancy failure

Abstract: Trophoblast cell (CTB) invasion into the maternal endometrium plays a crucial role during human embryo implantation and placentation. This invasion is facilitated by the activity of matrix metalloproteinases, which are regulated by tissue inhibitors of MMPs (TIMPs). This study compares the serum levels of MMP-9, MMP-2/TIMP-2 complex, TIMP-1 and TIMP-2 in 129 patients with ongoing pregnancy (n = 40) or spontaneous early pregnancy failure (n = 89). MMP-9 was markedly (p < 0.0001) elevated in missed abortions, as was MMP-2/TIMP-2 complex (p < 0.0005). However, the serum levels of TIMP-1 and TIMP-2 were markedly elevated (p < 0.0001) in ongoing pregnancies. Human placentation is mediated by fetal trophoblastic cells that invade the maternal uterine endometrium. Trophoblast invasion requires a precisely regulated secretion of specific proteolytic enzymes able to degrade the endometrial basement membrane and extracellular matrix. The elevated levels of MMP-9 and MMP-2/TIMP-2 complex may play a role in spontaneous termination of pregnancy.

RUNX2, GPX3 and PTX3 gene expression profiling in cumulus cells are reflective oocyte/embryo competence and potentially reliable predictors of embryo developmental competence in PCOS patients

Abstract: Background Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women. The developmental competence of oocytes and embryos in PCOS patients is reduced to a certain extent (comparing to non-PCOS patients, the high quality embryo rate was decreased by 16% from the data of our centre) during the in vitro fertilization (IVF) process. Cross-talk between the oocyte and cumulus cells is critical for oocyte maturation and embryo competence. In this study, we have evaluated the transcription of specific genes in cumulus cells harvested from pre-ovulatory follicles of PCOS patients before IVF, according to individual oocyte nuclear maturity and developmental competence. Seven genes (RUNX2, PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, and ITGB5) were targeted from our previous cDNA microarray data which isolated genes related to oocyte nuclear maturation in PCOS patients. Two additional genes which had been found to be associated with oocyte maturation or embryo quality in non-PCOS patients (GPX3 and PTX3) were also studied.

Anti-Mullerian-hormone levels during pregnancy and postpartum

Abstract: The number of unintentionally childless couples is increasing as more couples seek to conceive for the first time in the third or fourth decade of the woman’s life. Determination of ovarian reserve is an essential component of infertility assessment. The Anti-Mullerian-Hormone (AMH) seems to be the most reliable predictor of ovarian reserve. In this study we analyzed AMH in a cohort of pregnant women without fertility impairment to determine age-dependent decline and possible AMH fluctuations during pregnancy and postpartum. A total of 554 healthy women aged 16 to 47 years without history of infertility or previous surgery on the ovaries were enrolled in the study between 1995 and 2012. In 450 women, a single measurement of AMH was taken during pregnancy, allowing for cross sectional analysis of trimester- and age-related differences in AMH levels. For another 15 women longitudinal data on AMH levels for all trimesters was recorded. In addition, for 69 women AMH was measured at the time just before and after delivery, and for another 20 AMH was measured just before delivery and once on each of the first four days after delivery. We used AMH-Gen-II ELISA (Beckman Coulter, Immunotech, Webster, USA) for the assessment of AMH levels. Non-parametric statistical tests were used to compare AMH levels between age groups, trimesters and postpartum. Comparison between the trimesters revealed a significant difference in AMH values at each trimester (first trimester: 1.69 ng/ml (IQR 0.71–3.10), second trimester: 0.8 ng/ml (IQR 0.48–1.41), third trimester: 0.5 ng/ml (IQR 0.18–1.00)). AMH significantly dropped during the course of pregnancy and immediately after delivery, whereas an increase was observed over the first four days postpartum. Women, greater than or equal to 35 years, showed significant lower AMH levels than those <35 years across all trimesters. AMH levels decrease during pregnancy. The decline in AMH levels during pregnancy indicates ovarian suppression. AMH levels recover quickly after delivery. AMH levels assessed in pregnant women are not an accurate indicator of ovarian reserve, since AMH levels during pregnancy seem not to be independent of gestational age.

Protective effects of nuclear factor erythroid 2-related factor 2 on whole body heat stress-induced oxidative damage in the mouse testis

Abstract: Background: Whole body heat stress had detrimental effect on male reproductive function. It's known that the nuclear factor erythroid 2-related factor 2 (Nrf2) activates expression of cytoprotective genes to enable cell adaptation to protect against oxidative stress. However, it’s still unclear about the exactly effects of Nrf2 on the testis. Here, we investigate the protective effect of Nrf2 on whole body heat stress-induced oxidative damage in mouse testis. Methods: Male mice were exposed to the elevated ambient temperature (42°C) daily for 2 h. During the period of twelve consecutive days, mice were sacrificed on days 1, 2, 4, 8 and 12 immediately following heat exposure. Testes weight, enzymatic antioxidant activities and concentrations of malondialdehyde (MDA) and glutathione (GSH) in the testes were determined and immunohistochemical detection of Nrf2 protein and mRNA expression of Nrf2regulated genes were analyzed to assess the status of Nrf2-antioxidant system. Results: Heat-exposed mice presented significant increases in rectal, scrotal surface and body surface temperature. The concentrations of cortisol and testosterone in serum fluctuated with the number of exposed days. There were significant decrease in testes weight and relative testes weight on day 12 compared with those on other days, but significant increases in catalase (CAT) activity on day 1 and GSH level on day 4 compared with control group. The activities of total superoxide dismutase (T-SOD) and copper-zinc SOD (CuZn-SOD) increased significantly on days 8 and 12. Moreover, prominent nuclear accumulation of Nrf2 protein was observed in Leydig cells on day 2, accompanying with up-regulated mRNA levels of Nrf2-regulated genes such as Nrf2, heme oxygenase 1 (HO-1), γ-Glutamylcysteine synthetase (GCLC) and NAD (P) H: quinone oxidoreductase 1 (NQO1)) in heat-treated groups. Conclusions: These results suggest that Nrf2 displayed nuclear accumulation and protective activity in the process of heat treated-induced oxidative stress in mouse testes, indicating that Nrf2 might be a potential target for new drugs designed to protect germ cell and Leydig cell from oxidative stress.

Age-associated telomere shortening in mouse oocytes

Abstract: Background Oocytes may undergo two types of aging. The first is induced by exposure to an aged ovarian microenvironment before being ovulated, known as ‘reproductive or maternal aging’, and the second by either a prolonged stay in the oviduct before fertilization or in vitro aging prior to insemination, known as ‘postovulatory aging’. However, the molecular mechanisms underlying these aging processes remain to be elucidated. As telomere shortening in cultured somatic cells triggers replicative senescence, telomere shortening in oocytes during reproductive and postovulatory aging may predict developmental competence. This study aimed to ascertain the mechanisms underlying altered telomere biology in mouse oocytes during reproductive and postovulatory aging.

MicroRNAs expression profiling of eutopic proliferative endometrium in women with ovarian endometriosis

Abstract: Background: The eutopic endometrium of women with endometriosis, compared with disease-free individuals, contains certain molecular alterations, including the differential expression of microRNA (miRNA). The aim of the study was to compare the expression of the most relevant miRNAs in the eutopic endometrium of women with and without ovarian endometriosis. Methods: A total of 46 regularly menstruating patients, 21 patients with ovarian endometriosis and 25 controls, underwent surgery in the proliferative phase of the cycle. The eutopic endometrium was collected through aspirating biopsy prior to laparoscopy. Only patients with advanced (stage III and IV) histopathologically confirmed ovarian endometriosis were included. TaqMan MicroRNA Array Cards were applied to examine the expression of 667 human miRNAs in 10 patients with endometriosis and 10 controls. Custom-made, low-density real-time PCR arrays were used to confirm the expression of 15 selected molecules in 21 endometriosis patients and 25 diseasefree individuals. Results: Of 667 miRNAs, 2 were highly likely to be upregulated and 13 were downregulated in the eutopic endometrium of patients with endometriosis compared with the controls. Validation using real-time PCR showed that hsa-miR-483-5p (p = 0.012) and hsa-miR-629* (p = 0.02) are significantly downregulated in patients with endometriosis. Conclusions: Changes in the expression of select miRNAs might lead to or be a consequence of an early defect in the physiological activity of the proliferative endometrium, ultimately resulting in the overgrowth of this tissue outside the uterus.

A common polymorphic allele of the LH beta-subunit gene is associated with higher exogenous FSH consumption during controlled ovarian stimulation for assisted reproductive technology

Abstract: V-betaLH is a common genetic variant of LH caused by two polymorphic base changes in the beta subunit gene, altering the amino acid sequence (Trp8Arg and Ile15Thr). In a previous-preliminary trial performed in women undergoing IVF, it was demonstrated that carriers of v-betaLH show sub-optimal ovarian response to a standard long GnRH-agonist down -regulation protocol when stimulated with pure recombinant FSH (r-hFSH). The aim of this study was to confirm the hypothesis that women with v-betaLH display hypo-sensitivity to exogenous FSH in a larger IVF population and to explore the frequency of this variant in a Danish female population. In the present study, the effect of v-betaLH was retrospectively investigated in a larger series of women undergoing controlled ovarian stimulation (COS) and, for the first time, in a Danish IVF population. A total of 220 normogonadotrophic women following a long GnRH-agonist down-regulation protocol received an individualized dose of r-hFSH (100 IU and 375 IU s.c. daily) according to antral follicle count, baseline FSH, body mass index and age. The LH genotype was assessed in all patients by immunofluorometric assay. V-betaLH was present in 11% of patients, whereas the allelic frequency was 12%. The study population was divided into two groups according to their LH genotype. Group A consisted of 196 wt/wt women. Group B included 24 individuals with v-betaLH (21 heterozygous and 3 homozygous). No statistically significant differences in the mean number of oocytes retrieved, fertilization rate and pregnancy rate per cycle were observed between groups. However, Group B received a significantly higher cumulative-dose of r-hFSH than Group A (2435.86 +/− 932.8 IU versus 1959.8 +/− 736.45 p = 0.048). When one-way ANOVA in a within design was applied, the LH genotype had a statistically significant effect (p < 0.01) on the cumulative dose of r-hFSH, showing a progressive increase from wt/wt (1959.8 +/− 736.45 IU) to v-betaLH hetero- (2267.5 +/− 824.3) and homozygotic women (3558.3 +/− 970.9). These results confirm that carriers exhibit hypo-sensitivity to exogenous FSH during COS, documenting that the frequency of v-betaLH in Denmark is similar to a number of European countries.

Optimal usage of the GnRH antagonists: a review of the literature.

Abstract: Gonadotropin-releasing hormone (GnRH) antagonists, which became commercially available from 1999, have been used for the prevention of premature luteinizing hormone (LH) surges in controlled ovarian stimulation for in vitro fertilization or intracytoplasmic sperm injection. This review focuses on the recent literature on the use of GnRH antagonists and provides guidelines for optimal use in light of increasing evidence showing that GnRH antagonists are safe and effective, allowing flexibility of treatment in a wide range of patient populations. This includes patients undergoing first-line controlled ovarian stimulation, poor responders, and women diagnosed with polycystic ovary syndrome. The GnRH antagonist offers a viable alternative to the long agonists, providing a shorter duration of treatment with fewer injections and with no adverse effects on assisted reproductive technology outcome. This results in a significantly lower amount of gonadotropins required, which is likely to lead to improved patient compliance.

The new Rapid-i carrier is an effective system for human embryo vitrification at both the blastocyst and cleavage stage

Abstract: The Rapid-i is a new FDA cleared closed carrier for embryo vitrification. The cooling rate of - 1220°C/min is far lower than that reported with open vitrification systems such as the cryoloop (−15,000°C/min). Little published data is currently available on this device. This study presents our initial clinical data, as well as live birth outcomes, with the Rapid-i. The efficacy of this device for the cryopreservation of cleavage, as well as blastocyst stage human embryos is also analyzed. We further compare outcomes to those achieved with the cryoloop, an “open” vitrification system routinely used in our laboratory. Human embryos were vitrified at either the 8–10 cell stage or else the blastocyst stage. The vitrification protocol was: 7.5% DMSO/7.5% ethylene glycol (EG) (2–3 min) followed by incubation in 15% DMSO /15% EG (45 sec) before loading on the vitrification carrier. Cryoprotectant was removed during warming by sequential washes in 0.25 M and 0.125 M sucrose in culture medium. Clinical outcome data for frozen cycles between January 2011 and August 2012 were stratified according to carrier and cell stage. The student t-test and chi square test were used to compare results. P value of < 0.05 was considered significant. A total of 486 vitrified-warmed embryos were assessed and 92% of them were transferred. The clinical pregnancy rate (CPR) and implantation rate (IR) with Rapid-i vitrified blastocysts were 59% and 49%, versus 47% and 37%, respectively for cleavage stage embryos. This was not statistically different from results with the cryoloop vitrified blastocysts (CPR 46%, IR 38%) nor the cleavage stage vitrified embryos (CPR 49%, IR 35%). To date, there have been 31 deliveries of 34 healthy infants from Rapid-i vitrified embryos, with another 12 pregnancies still on-going. The Rapid-i offers an excellent alternative to existing open vitrification devices for embryo cryopreservation at the 8–10 cell stage as well as the blastocyst stage. Use of this type of “closed” sealed system that prevents direct contact between the embryos and liquid nitrogen reduces the potential risk of sample cross-contamination or infection. These preliminary data and live birth outcomes have paved the way toward transitioning to a closed vitrification system in our own IVF program.

The influence of circulating anti-Müllerian hormone on ovarian responsiveness to ovulation induction with gonadotrophins in women with polycystic ovarian syndrome: a pilot study

Abstract: Background: Women with polycystic ovarian syndrome (PCOS) are known to have elevated circulating AntiMullerian hormone (AMH), which has been found to desensitize ovarian follicles to follicle stimulating hormone (FSH). The purpose of this study was to investigate the impact of high circulating AMH on ovarian responsiveness to ovulation induction with gonadotrophins in PCOS women. Methods: This prospective observational pilot study was conducted in two collaborating Fertility Centres in the UK and Egypt. The study included 20 consecutive anovulatory women with PCOS who underwent 34 cycles of human menopausal gonadotrophin (hMG) ovarian stimulation using chronic low-dose step up protocol. Blood samples were collected for the measurement of serum AMH concentrations in the early follicular (day 2-3) phase in all cycles of hMG treatment. The serum levels of AMH were compared between cycles with good vs. poor response. The good response rates and the total dose and duration of hMG treatment were compared between cycles with high vs. low serum AMH concentrations. Results: Cycles with poor response (no or delayed ovulation requiring >20 days of hMG treatment) had significantly (p = .007) higher median{range} serum AMH concentration (6.5{3.2-13.4}ng/ml) compared to that (4.0 {2.2-10.2}ng/ml) of cycles with good response (ovulation within 20 days of hMG treatment). ROC curve showed AMH to be a useful predictor of poor response to hMG stimulation (AUC, 0.772; P = 0.007). Using a cut-off level of 4.7 ng/ml, AMH had a sensitivity of 100% and specificity of 58% in predicting poor response. The good response rate was significantly (p = 4.7 ng/ml (100% vs. 35%, respectively). All cycles with markedly raised serum AMH levels (> 10.2 ng/ml) were associated with poor response. Cycles with high AMH (> = 4.7 ng/ml) required significantly (p < .001) greater amounts (median {range}, 1087{450-1650}IU) and longer duration (20 {12-30}days) of hMG stimulation than cycles with lower AMH (525 {225-900}IU and 8{6-14}days). Conclusions: PCOS women with markedly raised circulating AMH seem to be resistant to hMG ovulation induction and may require a higher starting dose.

Changes in the transcriptome of morula-stage bovine embryos caused by heat shock: relationship to developmental acquisition of thermotolerance

Abstract: Background: While initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3’ tag digital gene expression (3’DGE) and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h) that could be associated with thermotolerance. Results: Using 3’DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5), decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (P<0.05) and tended to increase expression of HSP90AA1 (P<0.07) but had no effect on expression of SOD1 or CAT. Conclusions: Changes in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene networks involved in embryonic development. It is likely that the increased resistance of morula-stage embryos to heat shock as compared to embryos at earlier stages of development is due in part to developmental acquisition of mechanisms to prevent accumulation of denatured proteins and free radical damage.

L-Arginine enhances cell proliferation and reduces apoptosis in human endometrial RL95-2 cells.

Abstract: L-arginine is considered to be one of the most versatile amino acids due to the fact that it serves as a precursor for many important molecules in cellular physiology. When supplemented in the diet, L-arginine can increase the number of implantation sites in mice and rats, suggesting an effect at the level of the endometrium. To this end, this study determined the effect that L-arginine has on apoptosis and cell proliferation in human endometrial RL95-2 cells. L-arginine at physiological (200 micromol/L) and supra-physiological (800 micromol/L) concentrations increased cell proliferation at days 2 and 4 post-treatment with a dose-dependent effect being observed on day 2. Additionally, inhibition of nitric oxide (NO) synthase and arginase, which are responsible for the conversion of L-arginine to NO and polyamines, respectively, reduced the proliferative effect of L-arginine. L-arginine also decreased the proportion of cells with TUNEL positive nuclei and increased the ratio of cells with healthy mitochondria compared to cells with a disrupted mitochondrial membrane potential, indicating that L-arginine prevents mitochondrial mediated apoptosis in endometrial RL95-2 cells. Furthermore, exposure to L-arginine did not affect total BAD protein expression; however, L-arginine increased the abundance of phosphorylated BAD protein. In summary, L-arginine added to the culture media at physiological (200 micromol/L) and supraphysiological concentrations (800 micromol/L) enhanced endometrial RL95-2 cell proliferation through mechanisms mediated by NO and polyamine biosynthesis. In addition, L-arginine reduced endometrial RL95-2 mitochondrial mediated apoptosis through increased phosphorylation of BAD protein.

Efficient induction of spawning of northern leopard frogs (Lithobates pipiens) during and outside the natural breeding season.

Abstract: Background: Amphibian declines are now recognized globally. It is also well known that many anurans do not reproduce easily in captivity, especially when held over long periods, or if they require hibernation before breeding. A simple method to induce spawning and subsequent development of large numbers of healthy tadpoles is therefore required to meet research and conservation goals. Methods: The method is based on simultaneous injection of both female and male leopard frogs, Lithobates pipiens (formerly called Rana pipiens) with a cocktail of a gonadotropin-releasing hormone agonist (GnRH-A) and a dopamine antagonist. We call this the AMPHIPLEX method, which is derived from the combination of the words amphibian and amplexus. Following injection, the animals are thereby induced, and perform amplexus and natural fertilization under captive conditions. Results: We tested combinations of a GnRH agonist with 2 different dopamine antagonists in L. pipiens in the breeding season. The combination of des-Gly 10 , D-Ala 6 , Pro-NHEt 9 -GnRH (0.4 micrograms/g body weight; GnRH-A) with metoclopramide hydrochloride (10 micrograms/g body weight; MET) or domperidone (DOM) were equally effective, producing 89% and 88% successful spawning, respectively. This yielded more than 44,000 eggs for the 16/18 females that ovulated in the GnRH-A+MET group, and more than 39,000 eggs for the 15/17 females that ovulated in the GnRH-A+DOM group. We further tested the GnRH-A+MET in frogs collected in the wild in late autumn and hibernated for a short period under laboratory conditions, and report a low spawning success (43%). However, GnRH-A priming 24 hours prior to injections of the GnRH-A+MET cocktail in animals hibernated for 5–6 weeks produced out-of-season spawning (89%) and fertilization (85%) comparable to those we observed for inseason spawning. Assessment of age and weight at metamorphosis indicated that L. pipiens tadpoles resulting from out-of-season spawning grew normally and metamorphosed successfully. Conclusion: We provide evidence for successful captive breeding of the leopard frog, L. pipiens. This simple protocol can be used to obtain large numbers of eggs in a predictable, timed manner.

Differential expression of beta-catenin and dickkopf-1 in the third trimester placentas from normal and preeclamptic pregnancies: a comparative study

Abstract: Background Beta-catenin is a key nuclear effector of Wnt signaling which could be antagonized by dickkopf-1(DKK1). Beta-catenin and DKK1 are involved in a variety of biological processes; however, their expression in the placenta with severe preeclampsia (PE) has not been elucidated. This study was aimed to detect the localization and compare the expression of beta-catenin and DKK1 in normal and preeclamptic placenta.

Normal and cancer stem cells of the human female reproductive system

Abstract: The female reproductive system (FRS) has a great capacity for regeneration. The existence of somatic stem cells (SSC) that are likely to reside in distinct tissue compartments of the FRS is anticipated. Normal SSC are capable of regenerating themselves, produce a progeny of cells that differentiate and maintain tissue architecture and functional characteristics, and respond to homeostatic controls. Among those SSC of the FRS that have been identified are: a) undifferentiated cells capable of differentiating into thecal cells and synthesizing hormones upon transplantation, b) ovarian surface epithelium stem cells, mitotically responsive to ovulation, c) uterine endometrial and myometrial cells, as clonogenic epithelial and stromal cells, and d) epithelial and mesenchymal cells with self-renewal capacity and multipotential from cervical tissues. Importantly, these cells are believed to significantly contribute to the development of different pathologies and tumors of the FRS. It is now widely accepted that cancer stem cells (CSC) are at the origin of many tumors. They are capable of regenerating themselves, produce a progeny that will differentiate aberrantly and do not respond adequately to homeostatic controls. Several cell surface antigens such as CD44, CD117, CD133 and MYD88 have been used to isolate ovarian cancer stem cells. Clonogenic epithelial and stromal endometrial and myometrial cells have been found in normal and cancer tissues, as side population, label-retaining cells, and CD146/PDGF-R beta-positive cells with stem-like features. In summary, here we describe a number of studies supporting the existence of somatic stem cells in the normal tissues and cancer stem cells in tumors of the human female reproductive system.

Profiles of cytokines secreted by isolated human endometrial cells under the influence of chorionic gonadotropin during the window of embryo implantation

Abstract: Background Several studies have indicated that human pre-implantation embryo-derived chorionic gonadotropin (hCG) may influence the implantation process by its action on human endometrial epithelial and stromal cells. Despite reports indicating that hCG acts on these cells to affect the production of several cytokines and growth factors (e.g., MIF, IGF-I, VEGF, LIF, IL-11, GMCSF, CXL10 and FGF2), our understanding of the integral influence of hCG on paracrine interactions between endometrial stromal and epithelial cells during implantation is very limited.

Castration-induced testosterone deficiency increases fasting glucose associated with hepatic and extra-hepatic insulin resistance in adult male rats.

Abstract: Testosterone deficiency is associated with insulin resistance. However, how testosterone deficiency affects insulin actions remains unclear. The aim of this study was to investigate the influence of castration-induced testosterone deficiency on the metabolic kinetics of glucose and to evaluate the hepatic and extra-hepatic insulin sensitivity, in advanced-age male Sprague–Dawley (SD) rats. Ten-week-old male SD rats were randomly divided into three groups: (1) a control group (n = 10) in which the rats underwent sham castration (2) a castrated group (TD group for testosterone deficiency, n = 10) in which the rats underwent bilateral orchidectomy surgery and (3) a castrated group given testosterone propionate via intraperitoneal injection (25 mg/kg/day) to supplement androgen (TD + TP group, n = 10). At ten weeks after castration in the noted groups, all rats were subjected to an oral glucose tolerance test (OGTT), a pyruvate tolerance test (PTT) and an insulin tolerance test (ITT). Twenty weeks following that treatment, all rats underwent a hyperinsulinemic-euglycemic clamp procedure in conjunction with isotope--labeled glucose and glycerol tracer infusions. The rate of appearance (Ra) of glucose, glycerol and gluconeogenesis (GNG), hepatic glucose production and the rate of glucose disappearance (Rd) were assessed. Glucose uptake was determined by measuring the 2-deoxy-D-14C-glucose in the gastrocnemius muscles. Ten weeks after castration in the TD group, the fasting blood glucose and insulin levels were significantly increased (p < 0.01), the glucose-- induced insulin secretion was impaired and ITT revealed a temporarily increased whole body insulin sensitivity compared with the control group; 30 weeks after castration, the Ra of glucose, Ra of glycerol, as well as the HGP and GNG were also increased (p < 0.01), while the exogenous glucose infusion rate and uptake glucose in the muscle markedly decreased (p < 0.01). Castration-induced testosterone deficiency primarily increases fasting blood glucose levels. The clamp experiments revealed a clear insulin resistance both at the hepatic and extra-hepatic levels.

Neonatal outcomes after the transfer of vitrified blastocysts: closed versus open vitrification system

Abstract: Background Increasing evidence indicates that closed vitrification has been successfully used in the cryopreservation of human oocytes and embryos. Little information is available regarding the neonatal outcome of closed blastocysts vitrification. The aim of this study was to evaluate the effectiveness and safety of blastocyst vitrification using a high-security closed vitrification system compared with an open vitrification system.

Combination of a GnRH agonist with an antagonist prevents flare-up effects and protects primordial ovarian follicles in the rat ovary from cisplatin-induced toxicity: a controlled experimental animal study

Abstract: With the continuous improvement of surgery and chemotherapeutic treatments, many tumour patients increasingly achieve long-term survival and can even be completely cured. However, platinum-containing drugs, which are widely used to treat a variety of types of cancer, cause menstrual disorders and ovarian failure, which in turn lead to infertility. Thus far, gonadotropin releasing hormone (GnRH) agonist (GnRHa) and antagonist (GnRHant) are reported to act as protective agents of the ovary in chemotherapy through the inhibition of the female gonadal axis. Nevertheless, they both have disadvantages that limit their use. GnRHa causes a flare-up effect during the first week after administration, and no long-acting GnRHant agent is available. GnRHa combined with GnRHant may prevent the flare-up effect of GnRHa and rapidly inhibit the female gonadal axis. Several clinical studies with small sample sizes have reported controversial conclusions. In this strictly controlled animal study, we investigated the advantages of combination treatment with GnRHa and GnRHant. Rats aged 12 weeks were divided into six groups: Control, cisplatin (CDDP), GnRHa, GnRHant, Combination (sht, short-term) and Combination (lng, long-term) of GnRHa and GnRHant. The last four groups received Triptorelin (1 mg/kg·d, for 14 days), Cetrorelix (0.5 mg/kg·d, for 10 days), a combination of Triptorelin (1 mg/kg·d, for 10 days) and Cetrorelix (0.5 mg/kg·d, for 10 days) in the long-term group and for 3 days in the short-term group. The Control and CDDP groups received saline (1 ml/kg·d, for 10 day). Then, all groups apart from the Control group received cisplatin (1 mg/kg·d, for 10 days), and the Control group received another 10 days of saline as described above. Blood samples were collected to detect the serum levels of E2, LH and FSH. Observation of oestrous cyclicity was also performed after drug administration. Finally, bilateral ovaries were collected for histological study and follicle counting. We observed a flare-up effect in rats treated with GnRHa, but not in any of the combination groups. The percentage of normal cyclicity increased from 0% in the CDDP group to 25.0%, 33.3%, 66.7% and 41.7%, in the GnRHa, GnRHant, combination (lng) and combination (sht) groups, respectively. Pretreatment with GnRHa, GnRHant and combination (lng) significantly protected the primordial follicles from destruction by preserving 57.6%, 63.4%, 87.1% and 60.4% of the follicles, respectively. The combination of a GnRH agonist with antagonist completely prevented the flare-up effect and enhanced the protective effect of the ovary from cisplatin-induced gonadotoxicity in rats.