Subscribe: Molecular Human Reproduction - recent issues
Added By: Feedage Forager Feedage Grade A rated
Language: English
cell  cells  expression  fetuin  human  mice  mouse  ndash  plusmn  results role  results  role  sperm  study design  study  vitro 
Rate this Feed
Rate this feedRate this feedRate this feedRate this feedRate this feed
Rate this feed 1 starRate this feed 2 starRate this feed 3 starRate this feed 4 starRate this feed 5 star

Comments (0)

Feed Details and Statistics Feed Statistics
Preview: Molecular Human Reproduction - recent issues

Molecular Human Reproduction - recent issues

Molecular Human Reproduction - RSS feed of recent issues (covers the latest 3 issues, including the current issue)


Progress towards human primordial germ cell specification in vitro


Primordial germ cells (PGCs) have long been considered the link between one generation and the next. PGC specification begins in the early embryo as a result of a highly orchestrated combination of transcriptional and epigenetic mechanisms. Understanding the molecular events that lead to proper PGC development will facilitate the development of new treatments for human infertility as well as species conservation. This article describes the latest, most relevant findings about the mechanisms of PGC formation, emphasizing human PGC. It also discusses our own laboratory's progress in using transdifferentiation protocols to derive human PGCs (hPGCs). Our preliminary results arose from our pursuit of a sequential hPGC induction strategy that starts with the repression of lineage-specific factors in the somatic cell, followed by the reactivation of germ cell-related genes using specific master regulators, which can indeed reactivate germ cell-specific genes in somatic cells. While it is still premature to assume that fully functional human gametes can be obtained in a dish, our results, together with those recently published by others, provide strong evidence that generating their precursors, PGCs, is within reach.

Inhibin-B secretion and FSH isoform distribution may play an integral part of follicular selection in the natural menstrual cycle


The aim of the present paper is to expand the concept on how follicular selection takes place in the follicular phase of the natural menstrual cycle. It is suggested that inhibin-B exerts a more intimate role in this process than previously understood. Inhibin-B shows a peak in the circulation around cycle day 7, simultaneous with selection of the dominant follicle, whereas levels of estradiol and inhibin-A only start to increase a few days later suggesting that inhibin-B is mainly responsible for downregulating pituitary FSH release. New data now demonstrate that the circulatory peak of inhibin-B is reflected by peak production of inhibin-B, in contrast to inhibin-A, in the selected follicle with a diameter of 10–12 mm, where concentrations are one thousand times higher than in the circulation. This high inhibin-B concentration also exerts paracrine effects, stimulating theca cell androgen production in concert with LH. New data now suggest that in the corresponding granulosa cells androgens upregulate FSH receptor (FSHR) and LH receptor (LHR) mRNA expression, which in turn stimulate CYP19a mRNA expression providing the follicles which most effectively undertake these processes with the best chance of becoming selected. Inhibin-B production is stimulated by FSH and it appears that the acidic isoforms of FSH induce inhibin-B secretion most efficiently thereby, for the first time, placing the changing FSH isoform profile during the follicular phase in a physiological context. Collectively, it appears that inhibin-B is an integral part of follicular selection in the normal menstrual cycle, exerting both endocrine and paracrine effects and facilitating continued growth of the selected follicle.

Recombinant fetuin-B protein maintains high fertilization rate in cumulus cell-free mouse oocytes


Study Question

Does fetuin-B inhibit premature zona pellucida (ZP) hardening in mouse oocytes in vitro and thus increase IVF rate?

Summary Answer

Supplementation of oocyte in vitro maturation (IVM) media with recombinant mouse fetuin-B (rmFetuB) increased fertilization rate without affecting mouse embryo development into blastocysts.

What is Known Already

Mice deficient in fetuin-B are infertile owing to premature ZP hardening. Premature ZP hardening also occurs during oocyte IVM leading to decreased fertilization rate.

Study Design, Size, Duration

We fertilized batches of 20–30 mouse metaphase II (Mll) stage oocytes from C57BL/6 mice with fresh sperm, and studied early embryo development until blastocyst hatching.

Participants/Materials, Setting, Methods

Oocytes were maintained with or without rmFetuB during IVM and IVF. Exogenous rmFetuB was added to media prior to oocyte isolation. ZP hardening was quantified by chymotrypsin digestion timing and by counting attached sperm.

Main Results and the Role of Chance

In the absence of cumulus cells, rmFetuB dose-dependently inhibited ZP hardening and increased IVF rate (P = 0.039). Fetuin-B at ≥0.03 mg/ml also inhibited physiological, fertilization-triggered ZP hardening (indicated by increased sperm binding, P = 0.0002), without increasing embryo death. Exogenous rmFetuB increased IVF rate for up to 5 hours of IVM (P = 0.02 at 1 hour, P = 0.01 at 5 hours of IVM).

Limitations, Reasons for Caution

Mll stage oocytes in this study were isolated from the ampullae of fetuin-B expressing mice. Thus, oocytes were protected against premature ZP hardening by endogenous fetuin-B. In humans and livestock, oocytes are usually isolated by follicle puncture before ovulation. In this situation, the deprivation of endogenous fetuin-B would occur earlier and the effect of exogenous fetuin-B in the IVF medium may be even more pronounced. Fertilization-triggered ZP hardening is essential for embryo development but in this study the effect of fetuin-B supplementation was only studied to blastocyst stage. Any influence of added fetuin-B on later embryo development after transplantation remains to be determined.

Wider Implications of the Findings

The astacin-type protease ovastacin triggers definitive ZP hardening by cleaving the zona pellucida protein 2. Animal sera are known to inhibit premature ZP hardening. The addition of rFetuB to the culture medium of oocytes could increase IVF rates by the inhibition of premature ZP hardening. In this regard, the results could be useful for clinical activity.

Large Scale Data


Study Funding/Competing Interest(s)

The research was supported by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University. The authors ED, JF and WJD are named inventors on a patent application of RWTH Aachen University covering the use of fetuin-B in ovary and oocyte culture.

Down-regulation of the liver-derived plasma protein fetuin-B mediates reversible female infertility


Study question Does antisense oligonucleotide (ASO)-mediated down-regulation of serum fetuin-B cause infertility like fetuin-B gene deficiency in female mice? Summary answer Pharmacological fetuin-B down-regulation by ASO therapy results in reversible infertility in female mice. What is known already Female fetuin-B deficient (Fetub–/–) mice are infertile owing to premature zona pellucida (ZP) hardening. Enzyme activity studies demonstrated that fetuin-B is a potent and highly specific inhibitor of the zona proteinase ovastacin, which cleaves ZP protein 2 (ZP2) and thus mediates definitive ZP hardening. Study design, size, duration Ten fetuin-B ASO boli (100 mg/kg) were injected s.c. over 20 days in 12 female mice, and 10 phosphate-buffered saline (PBS)-treated mice were used as control. At day 20 females were mated to evaluate fetuin-B as a potential molecular target for contraception. ASO and PBS treatment was continued for ten injections. After treatment cessation at day 50, mating was continued to investigate if infertility was reversible. Participants/materials, setting, methods We generated fetuin-B/ovastacin double deficient (Fetub–/–, Astl–/–) mice by conventional breeding to test if fertility of Fetub–/– female mice was restored when the target proteinase would likewise be deleted. At least five matings with each female genotype (Fetub–/– single deficient, Astl–/– single deficient, Fetub–/–, Astl–/– double deficient) were performed. To test the contraceptive effect of fetuin-B down-regulation, 22 female mice (6–13 weeks old) were treated with repetitive boli of 100 mg/kg fetuin-B ASO (n = 12) or PBS (n = 10) and mated continuously. Serum fetuin-B was determined by immunoblot before, during and after the ASO treatment. After 3 weeks of ASO treatment, in 6 females Fetub mRNA in liver was analyzed by PCR, and six PBS-treated females were used as control. Aspartate (AST) and alanine aminotransferase (ALT) were also measured in serum of six mice in each group. To determine the minimum permissive serum fetuin-B concentration required for successful fertilization IVF was performed in five fetuin-B ASO-treated mice. As a control, six females were injected with control oligonucleotides and six females were left untreated. Main results and the role of chance Fertility of Fetub–/– female mice was restored by additional ovastacin deficiency (Astl–/–). Unlike Fetub–/– mice, female Fetub–/–, Astl–/– mice were fertile, confirming ovastacin as a primary molecular target of fetuin-B. At day 20, after receiving 10 fetuin-B ASO boli, serum fetuin-B was down-regulated to 8 ± 6% (mean ± SD) of baseline level. Fetuin-B down-regulation was confirmed at the mRNA level. Fetuin-B ASO-treated females had 12.1 ± 3.1% of the liver Fetub mRNA level seen in PBS-treated females. In the following mating study, 11 out of 12 mated females failed to become pregnant during 50 days of ASO treatment and continuous mating from day 20 onwards. IVF of oocytes derived from ASO-treated females suggested that a serum fetuin-B level of less than 10 µg/ml was required to prevent pregnancy. Withdrawal of ASO treatment normalized serum fetuin-B and restored fertility; all female mice became pregnant and had litters within 60.3 ± 35.9 days after cessation of ASO treatment. The first litter was significantly smaller than that of control mice (4.6 ± 2.3 versus 6.7 ± 1.8 pups, n = 20, P = 0.04) but the smaller litter size was only temporary. The size of the second litter was similar to the first litter of control mice (7.6 ± 1.3 versus 6.7 ± 1.8 pups, n = 18, P = 0.25). Limitations, reasons for caution[...]

Normalization matters: tracking the best strategy for sperm miRNA quantification


Study Question What is the most reliable normalization strategy for sperm microRNA (miRNA) quantitative Reverse Transcription Polymerase Chain Reactions (qRT-PCR) using singleplex assays? Summary Answer The use of the average expression of hsa-miR-100-5p and hsa-miR-30a-5p as sperm miRNA qRT-PCR data normalizer is suggested as an optimal strategy. What is Known Already Mean-centering methods are the most reliable normalization strategies for miRNA high-throughput expression analyses. Nevertheless, specific trustworthy reference controls must be established in singleplex sperm miRNA qRT-PCRs. Study Design, Size Duration Cycle threshold (Ct) values from previously published sperm miRNA expression profiles were normalized using four approaches: (i) Mean-Centering Restricted (MCR) method (taken as the reference strategy); (ii) expression of the small nuclear RNA RNU6B; (iii) expression of four miRNAs selected by the Concordance Correlation Restricted (CCR) algorithm: hsa-miR-100-5p, hsa-miR-146b-5p, hsa-miR-92a-3p and hsa-miR-30a-5p; (iv) the combination of two of these miRNAs that achieved the highest proximity to MCR. Participants/Materials, Setting, Methods Expression profile data from 736 sperm miRNAs were taken from previously published studies performed in fertile donors (n = 10) and infertile patients (n = 38). For each tested normalizer molecule, expression ubiquity and uniformity across the different samples and populations were assessed as indispensable requirements for being considered as valid candidates. The reliability of the different normalizing strategies was compared to MCR based on the set of differentially expressed miRNAs (DE-miRNAs) detected between populations, the corresponding predicted targets and the associated enriched biological processes. Main Results and the Role of Chance All tested normalizers were found to be ubiquitous and non-differentially expressed between populations. RNU6B was the least uniformly expressed candidate across samples. Data normalization through RNU6B led to dramatically misguided results when compared to MCR outputs, with a null prediction of target genes and enriched biological processes. Hsa-miR-146b-5p and hsa-miR-92a-3p were more uniformly expressed than RNU6B, but their results still showed scant proximity to the reference method. The highest resemblance to MCR was achieved by hsa-miR-100-5p and hsa-miR-30a-5p. Normalization against the combination of both miRNAs reached the best proximity rank regarding the detected DE-miRNAs (Area Under the Curve = 0.8). This combination also exhibited the best performance in terms of the target genes predicted (72.3% of True Positives) and their corresponding enriched biological processes (70.4% of True Positives). Large Scale Data Not applicable. Limitations, Reasons for Caution This study is focused on sperm miRNA qRT-PCR analysis. The use of the selected normalizers in other cell types or tissues would still require confirmation. Wider Implications of the Findings The search for new fertility biomarkers based on sperm miRNA expression using high-throughput assays is one of the upcoming challenges in the field of reproductive genetics. In this context, validation of the results using singleplex assays would be mandatory. The normalizer strategy suggested in this study would provide a universal option in this area, allowing for normalization of the validated data without causing meaningful variations of the results. Instead, qRT-PCR data normalization by RNU6B should be discarded in sperm-miRNA expression studies. Study Funding/Competing Interest(S) This work was supported by the 2014/SGR00524 project (Agència de Gestió d'Ajuts Universitaris i de Recerca, Generalitat de Catalunya, Spain) and UAB CF-180034 grant (Universitat Autònoma de Barcelona). Celia Corral-Vazquez is a recipient of a Personal Investigador en Formació grant UAB/PIF2015 (Universitat Aut&og[...]

Antigen unmasking enhances visualization efficacy of the oocyte activation factor, phospholipase C zeta, in mammalian sperm


Study Question Is it possible to improve clinical visualization of phospholipase C zeta (PLC) as a diagnostic marker of sperm oocyte activation capacity and male fertility? Summary Answer Poor PLC visualization efficacy using current protocols may be due to steric or conformational occlusion of native PLC, hindering antibody access, and is significantly enhanced using antigen unmasking/retrieval (AUM) protocols. What is Known Already Mammalian oocyte activation is mediated via a series of intracellular calcium (Ca2+) oscillations induced by sperm-specific PLC. PLC represents not only a potential clinical therapeutic in cases of oocyte activation deficiency but also a diagnostic marker of sperm fertility. However, there are significant concerns surrounding PLC antibody specificity and detection protocols. Study Design, Size Duration Two PLC polyclonal antibodies, with confirmed PLC specificity, were employed in mouse, porcine and human sperm. Experiments evaluated PLC visualization efficacy, and whether AUM improved this. Antibodies against two sperm-specific proteins [post-acrosomal WW-binding protein (PAWP) and acrosin] were used as controls. Participants/Materials, Setting, Methods Aldehyde- and methanol-fixed sperm were subject to immunofluorescence analysis following HCl exposure (pH = 0.1–0.5), acid Tyrode's solution exposure (pH = 2.5) or heating in 10 mM sodium citrate solution (pH = 6.0). Fluorescence intensity of at least 300 cells was recorded for each treatment, with three independent repeats. Main Results and the Role of Chance Despite high specificity for native PLC following immunoblotting using epitope-specific polyclonal PLC antibodies in mouse, porcine and human sperm, immunofluorescent visualization efficacy was poor. In contrast, sperm markers PAWP and acrosin exhibited relatively impressive results. All methods of AUM on aldehyde-fixed sperm enhanced visualization efficacy for PLC compared to visualization efficacy before AUM (P < 0.05 for all AUM interventions), but exerted no significant change upon PAWP or acrosin immunofluorescence following AUM. All methods of AUM enhanced PLC visualization efficacy in mouse and human methanol-fixed sperm compared to without AUM (P < 0.05 for all AUM interventions), while no significant change was observed in methanol-fixed porcine sperm before and after. In the absence of aldehyde-induced cross-linkages, such results suggest that poor PLC visualization efficacy may be due to steric or conformational occlusion of native PLC, hindering antibody access. Importantly, examination of sperm from individual donors revealed that AUM differentially affects observable PLC fluorescence, and the proportion of sperm exhibiting detectable PLC fluorescence in sperm from different males. Limitations, Reasons for Caution Direct correlation of fertility outcomes with the level of PLC in the sperm samples studied was not available. Such analyses would be required in future to determine whether the improved methodology for PLC visualization we propose would indeed reflect fertility status. Wider Implications of the Findings We propose that AUM alters conformational interactions to enhance PLC epitope availability and visualization efficacy, supporting prospective application of AUM to reduce misinterpretation in clinical diagnosis of PLC-linked male infertility. Our current results suggest that it is perhaps prudent that previous studies investigating links between PLC and fertility parameters are re-examined in the context of AUM, and may pave the way for future work to answer significant questions such as how PLC appears to be kept in an inactive form in the sperm. Large Scale Data Not applicable. Study Funding/Competing Interest(S) J.K. is supported by a Health Fellowship award from the National Institute for Social Care and Health Research (NISCHR). M.N. is supported by a Marie Curie Intra-Europe[...]

Vitamin A prevents round spermatid nuclear damage and promotes the production of motile sperm during in vitro maturation of vitrified pre-pubertal mouse testicular tissue


STUDY QUESTION Does vitamin A (retinol, Rol) prevent round spermatid nuclear damage and increase the production of motile sperm during in vitro maturation of vitrified pre-pubertal mouse testicular tissue? SUMMARY ANSWER The supplementation of an in vitro culture of ~0.75 mm3 testicular explants from pre-pubertal mice with Rol enhances spermatogenesis progression during the first spermatogenic wave. WHAT IS KNOWN ALREADY The production of functional spermatozoa in vitro has only been achieved in the mouse model and remains a rare event. Establishing an efficient culture medium for vitrified pre-pubertal testicular tissue is now a crucial step to improve the spermatic yield obtained in vitro. The role of Rol in promoting the differentiation of spermatogonia and their entry into meiosis is well established; however, it has been postulated that Rol is also required to support their full development into elongated spermatids. STUDY DESIGN, SIZE, DURATION A total of 60 testes from 6.5 days post-partum (dpp) mice were vitrified/warmed, cut into fragments and cultured for 30 days: 20 testes were used for light microscopy and histological analyses, 20 testes for DNA fragmentation assessment in round spermatids and 20 testes for induced sperm motility assessment. Overall, 16 testes of 6.5 dpp were used as in vitro fresh tissue controls and 12 testes of 36.5 dpp mice as in vivo controls. Testes were vitrified with the optimal solid surface vitrification procedure and cultured with an in vitro organ culture system until Day 30 (D30). Histological analysis, cell death, degenerating round spermatids, DNA fragmentation in round spermatids and induced sperm motility were assessed. Testosterone levels were measured in media throughout the culture by radioimmunoassay. MAIN RESULTS AND THE ROLE OF CHANCE At D30, better tissue development together with higher differentiation of spermatogonial stem cells, and higher global cell division ability were observed for vitrified/warmed testicular fragments of ~0.75 mm3 with a culture medium supplemented with Rol compared to controls. During in vitro culture of vitrified pre-pubertal testicular tissue, Rol enhanced and maintained the entry of spermatogonia into meiosis and promoted a higher spermatic yield. Furthermore, decreased round spermatid nuclear alterations and DNA damage combined with induced sperm motility comparable to in vivo highlight the crucial role of Rol in the progression of spermatogenesis during the first wave. LIMITATIONS, REASONS FOR CAUTION Despite our promising results, the culture media will have to be further improved and adapted within the context of a human application. WIDER IMPLICATIONS OF THE FINDINGS The results have potential implications for the handling of human pre-pubertal testicular tissues cryopreserved for fertility preservation. However, because some alterations in round spermatids persist after in vitro culture with Rol, the procedure needs to be optimized before human application, bearing in mind that the murine and human spermatogenic processes differ in many respects. LARGE SCALE DATA None. STUDY FUNDING AND COMPETING INTEREST(S) This study was supported by a Ph.D. grant from the Normandy University and a financial support from ‘la Ligue nationale contre le cancer’ (both awarded to L.D.), funding from Rouen University Hospital, Institute for Research and Innovation in Biomedicine (IRIB) and Agence de la Biomédecine. The authors declare that there is no conflict of interest. [...]

Uterosome-like vesicles prompt human sperm fertilizing capability


STUDY QUESTION Does the rapid transit through the uterine environment modulate the sperm physiological state? SUMMARY ANSWER The uterosome-like vesicles (ULVs) secreted by endometrial epithelial cells (EECs) in vitro are able to fuse with human spermatozoa, prompting their fertilizing capacity. WHAT IS KNOWN ALREADY Early studies suggest that sperm capacitation begins in the uterus and ends in the oviduct, and that a synergistic effect of both female organs may accelerate this process. Although it has been reported that co-incubation of human spermatozoa with endometrial cell-conditioned medium (CM) stimulates sperm capacitation, the mechanism mediating this communication is unknown. STUDY DESIGN, SIZE, DURATION Human ULVs secreted by EECs were characterized and their effect on human sperm physiology was analysed. Spermatozoa were incubated with EEC-derived CM or ULV, after which sperm capacitation was evaluated at different time points. In addition, the interaction of spermatozoa with ULV was analysed. PARTICIPANTS/MATERIALS, SETTING, METHODS ULVs were isolated by ultracentrifugation and identified using electron microscopy and Western blotting to assess the presence of specific protein markers. Following seminal plasma removal, human spermatozoa were incubated CM or ULV, after which sperm capacitation was evaluated as the ability of the sperm to undergo the induced acrosome reaction and the level of protein tyrosine phosphorylation (PY) determined by Western blot and immunocytochemistry. The interaction of spermatozoa with labelled ULV was analysed by fluorescence microscopy. In all cases, at least three biological replicates from different sperm donors were performed for each set of experiments. Significant differences between mean values were determined by one-way ANOVA followed by Tukey's post hoc test. Differences between treatments were considered statistically significant at P ≤ 0.05. MAIN RESULTS AND THE ROLE OF CHANCE The level of capacitated spermatozoa and those recruited by chemotaxis increased 3- to 4-fold when spermatozoa were incubated in the presence of CM for 4 h. Even a 15 min incubation of spermatozoa with CM was also enough to increase the level of capacitated cells 3- to 4-fold (P < 0.05). Furthermore, a short co-incubation of spermatozoa with ULV stimulates sperm capacitation, as determined by the increase in the level of induced acrosome reaction and the induction of PY. In addition, after the co-incubation of spermatozoa with fluorescent labelled ULV, the sperm cells acquired the fluorescent staining which indicates that ULV might be transferred to the sperm surface by a fusion mechanism. LIMITATIONS, REASONS FOR CAUTION This is an in vitro study performed with human biological material, spermatozoa and endometrial derived cells; the latter being a cell line originally isolated from a uterine adenocarcinoma. WIDER IMPLICATIONS OF THE FINDINGS The capability of spermatozoa to briefly interact with ULVs supports the hypothesis that any step of sperm transport may have physiological consequences, despite the interaction lasting for only a limited period of time. This way of communication of spermatozoa with cell products of uterine origin opens new frontiers of investigation (e.g. the signalling molecules involved), shedding light on the sperm processes that prepare the male gamete for fertilization, which might have implications for human infertility treatment. LARGE SCALE DATA N/A. STUDY FUNDING AND COMPETING INTEREST(S) The project was financially supported by SECyT-UNC. The authors declare no conflict of interest. [...]

A grafted ovarian fragment rescues host fertility after chemotherapy



Can host fertility be rescued by grafting of a fragment of a healthy ovary soon after chemotherapy?


We found that grafting a green fluorescent protein (GFP)-positive fragment from a healthy isogenic ovary to the left ovary of a chemo-treated host rescued function and fertility of the grafted host ovary, and resulted in the production of host-derived offspring as late as the sixth litter after chemotherapy (CTx) treatment, whereas none of the ungrafted controls produced a second litter.


In women and girls undergoing chemotherapy, infertility and premature ovarian failure are frequent outcomes. There are accumulating reports of improved endocrine function after autotransplantation of an ovarian fragment, raising the possibility that the transplant is beneficial to the endogenous ovary.


We first established a CTx treatment regimen that resulted in the permanent loss of fertility in 100% of female mice of the FVB inbred strain. We grafted an isogenic ovary fragment from a healthy female homozygous for a GFP transgene to the left ovary of 100 CTx-treated hosts, and compared fertility to 39 ungrafted controls in 6 months of continuous matings, using GFP to distinguish offspring derived from the graft, and those derived from the host.


Immunofluoresece and western blot analysis of 39 treated ovaries during and 15 days after CTx treatment revealed elevated apoptosis, rapid loss of granulosa cells and an increased recruitment of growing follicles. Using immunofluorescence and confocal imaging, we tracked the outcome of the grafted tissue over 4 months and its effect on the adjacent and contralateral ovary of the host.


Fifty-three percent of grafted females produced a second litter whereas none of the ungrafted females produced a second litter. The likelihood that this could occur by chance is very low (P < 0.0001).


These results are shown only in mice, and whether or how they might apply to chemotherapy patients subjected to different CTx regimens is not yet clear.


Our experiments prove that rescue of a chemo-treated ovary is possible, and establish a system to investigate the mechanism of rescue and to identify the factors responsible with the long-term goal of developing therapies for preservation of ovarian endocrine function and fertility in women undergoing chemotherapy.


No large datasets were produced.


Duke University Medical Center Chancellor's Discovery Grant to BC; ESJ was supported by an NRSA 5F31CA165545; SK was supported by NIH RO1 GM08033; RWT was supported by the Duke University School of Medicine Ovarian Cancer Research Fellowship; XBM was supported by CONICYT. The authors have no conflicts of interest to declare.

Sphingosine-1-phosphate restores endothelial barrier integrity in ovarian hyperstimulation syndrome


StUDY QUESTION Are follicular fluid (FF) sphingosine-1-phosphate (S1P) levels in patients at risk of developing ovarian hyperstimulation syndrome (OHSS) altered and in part responsible for the high vascular permeability observed in these patients. STUDY ANSWER FF S1P levels are lower in FF from patients at risk of OHSS and treatment with S1P may reduce vascular permeability in these patients. WHAT IS KNOWN ALREADY Although advances have been made in the diagnosis, and management of OHSS and in basic knowledge of its development, complete prevention has proven difficult. StUDY DESIGN, SIZE, DURATION A total of 40 FF aspirates were collected from patients undergoing ART. The women (aged 25–39 years old) were classified into a control group (n = 20) or a group at risk of OHSS (n = 20). The EA.hy926 endothelial cell line was used to assess the efffects of FF from patients at risk of OHSS with or without the addition of S1P. An animal model that develops OHSS in immature Sprague-Dawley rats were also used. PaRTICIPANTS/MATERIALS, SETTING, METHODS Migration assays, confocal microscopy analysis of actin filaments, immunoblotting and quail chorioallantoic membrane (CAM) assays of in-vivo angiogenesis were performed and statistical comparisons between groups were made. MAIN RESULTS AND THE ROLE OF CHANCE The S1P concentration was significantly lower in FF from patients at risk of OHSS (P = 0.03). The addition of S1P to this FF decreased cell migration (P < 0.05) and prevented VE-cadherin phosphorylation in endothelial cells (P < 0.05). S1P in the FF from patients at risk of OHSS increased the levels of VE-cadherin (P < 0.05), N-cadherin (P < 0.05) and β-catenin (P < 0.05), and partially reversed actin redistribution in endothelial cells. The addition of S1P in FF from patients at risk of OHSS also decreased the levels of vascular endothelial growth factor (VEGF121; P < 0.01) and S1P lyase (SPL; P < 0.05) and increased the levels of S1PR1 (P < 0.05) in endothelial cells. In CAMs incubated with FF from patients at risk of OHSS with S1P, the number of vessel branch points decreased while the periendothelial cell coverage increased. Additionally, in a rat OHSS model, we demonstrated that vascular permeability and VEGF121 and its receptor KDR expression were increased in the OHSS group compared to the control group and that S1P administration decreased these parameters. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION The results of this study were generated from an in-vitro system. This model reflects the microvasculature in vivo. Even though the ideal model would be the use of human endothelial cells from the ovary, it is obviously not possible to carry out this kind of approach in ovaries of patients from ART. More studies will be necessary to delineate the effects of S1P in the pathogenesis of OHSS. Hence, clinical studies are needed in order to choose the most appropriate method of prevention and management. WIDER IMPLICATIONS OF THE FINDINGS The use of bioactive sphingolipid metabolites may contribute to finding better and safer therapeutic strategies for the treatment of OHSS and other human diseases that display aberrant vascular leakage. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants ANPCyT (PICT 2012-897), CONICET (PIP 5471), Roemmers and Baron Foundation, Argentina. The authors declare no conflict of interest. [...]

Improved cryotolerance and developmental potential of in vitro and in vivo matured mouse oocytes by supplementing with a glutathione donor prior to vitrification


STUDY QUESTION Can supplementation of media with a glutathione (GSH) donor, glutathione ethyl ester (GEE), prior to vitrification protect the mouse oocyte from oxidative damage and critical changes in redox homeostasis, and thereby improve cryotolerance? SUMMARY ANSWER GEE supplementation supported redox regulation, rapid recovery of spindle and chromosome alignment after vitrification/warming and improved preimplantation development of mouse metaphase II (MII) oocytes. WHAT IS KNOWN ALREADY Cryopreservation may affect mitochondrial functionality, induce oxidative stress, and thereby affect spindle integrity, chromosome segregation and the quality of mammalian oocytes. GEE is a membrane permeable GSH donor that promoted fertilization and early embryonic development of macaque and bovine oocytes after IVM. STUDY DESIGN, SIZE, DURATION Two experimental groups consisted of (i) denuded mouse germinal vesicle (GV) oocytes that were matured in vitro in the presence or absence of 1 mM GEE (IVM group 1) and (ii) in vivo ovulated (IVO) MII oocytes that were isolated from the ampullae and exposed to 1 mM GEE for 1 h prior to vitrification (IVO group 2). Recovery of oocytes from both groups was followed after CryoTop vitrification/warming for up to 2 h and parthenogenetic activation. PARTICIPANTS/MATERIALS, SETTING, METHODS Reactive oxygen species (ROS), spindle morphology and chromosome alignment were analyzed by confocal laser scanning microscopy (CLSM) and polarization microscopy in control and GEE-supplemented MII oocytes. The relative overall intra-oocyte GSH content was assessed by analysis of monochlorobimane (MBC)-GSH adduct fluorescence in IVM MII oocytes. The GSH-dependent intra-mitochondrial redox potential (Em GSH) of IVM MII oocytes was determined after microinjection with specific mRNA at the GV stage to express a redox-sensitive probe within mitochondria (mito-Grx1-roGFP2). The absolute negative redox capacity (in millivolts) was determined by analysis of fluorescence of the oxidized versus the reduced form of sensor by CLSM and quantification according to Nernst equation. Proteome analysis was performed by quantitative 2D saturation gel electrophoresis (2D DIGE). Since microinjection and expression of redox sensor mRNA required removal of cumulus cells, and IVM of denuded mouse oocytes in group 1 induces zona hardening, the development to blastocysts was not assessed after IVF but instead after parthenogenetic activation of vitrified/warmed MII oocytes from both experimental groups. MAIN RESULTS AND ROLE OF CHANCE IVM of denuded mouse oocytes in the presence of 1 mM GEE significantly increased intra-oocyte GSH content. ROS was not increased by CryoTop vitrification but was significantly lower in the IVM GEE group compared to IVM without GEE before vitrification and after recovery from vitrification/warming (P < 0.001). Vitrification alone significantly increased the GSH-dependent intra-mitochondrial redox capacity after warming (Em GSH, P < 0.001) in IVM oocytes, presumably by diffusion/uptake of cytoplasmic GSH into mitochondria. The presence of 1 mM GEE during IVM increased the redox capacity before vitrification and there was no further increase after vitrification/warming. None of the reproducibly detected 1492 spots of 2D DIGE separated proteins were significantly altered by vitrification or GEE supplementation. However, IVM of denuded oocytes significantly affected spindle integrity and chromosome alignment right after warming from vitrification (0 h) in group 1 and spindle integrity in group 2 (P < 0.05). GEE improved recovery in IVM group as numbers of oocytes with unaligned chromosomes and aberrant spindles was not significantly increased compared to unvitrified c[...]

Differences in cumulus cell gene expression indicate the benefit of a pre-maturation step to improve in-vitro bovine embryo production


STUDY QUESTION Does the gene expression profile of cumulus cells (CC) accompanying oocytes with different degrees of chromatin compaction within the germinal vesicle (GV) reflect the oocyte's quality and response in culture during in-vitro embryo production (IVP). SUMMARY ANSWER The transcriptomic profile of the CC is related to oocyte competence, setting the stage for the development of customized pre-maturation strategies to improve IVP. WHAT IS KNOWN ALREADY Oocytes complete the acquisition of their competence during antral follicle development. During this period, the chromatin configuration within the GV changes dynamically and is indicative of oocyte's developmental potential. The interactions between somatic and germ cells modulate chromatin morphology and function and are critical for acquisition of oocyte competence. STUDY DESIGN, SIZE, DURATION Bovine cumulus–oocyte complexes (COC) were isolated from 0.5 to 6 mm antral follicles. Surrounding CC were separated from the oocyte and classified as GV0, GV1, GV2 and GV3 according to the degree of the oocyte's chromatin compaction. PARTICIPANTS/MATERIALS, SETTING, METHOD RNA extracted from CC of each group was amplified and hybridized on a bovine embryo-specific 44 K Agilent slide. The CC_GV1, CC_GV2 and CC_GV3 classes were each hybridized against the CC_GV0 class, representing an early oocyte differentiation stage with poor development competence. The data were normalized and fold changes of the differentially expressed genes were determined. Microarray data were validated using quantitative RT-PCR on selected targets. Microarray data were further analyzed through: (i) between-group analysis (BGA), which classifies the samples according to their transcriptomic profiles; (ii) cluster analysis according to the expression profile of each gene; and (iii) Ingenuity Pathway Analysis (IPA) to study gene regulation patterns and predicted functions. Furthermore, CC of each GV group were cultured and apoptotic cells were assessed after 3 h by caspase analysis. Finally, based on the analysis of CC transcriptomic profiles and the relationship between morphological features of the COC and the oocyte chromatin configuration, a customized, stage-dependent oocyte pre-maturation (pre-IVM) system was used to improve oocyte developmental potential before IVM. For this, the blastocyst rate and quality were assessed after in-vitro maturation and fertilization of pre-matured oocytes. MAIN RESULTS AND THE ROLE OF CHANCE Overall, quantitative RT-PCR results of a subset of five selected genes were consistent with the microarray data. Clustering analysis generated 16 clusters representing the main profiles of transcription modulation. Of the 5571 significantly differentially expressed probes, the majority (25.49%) best fitted with cluster #6 (downregulation between CC_GV0 and CC_GV1 and stable low levels in successive groups). IPA identified the most relevant functions associated with each cluster. Genes included in cluster #1 were mostly related to biological processes such as ‘cell cycle’ and ‘cell death and survival’, whereas genes included in cluster #5 were mostly related to ‘gene expression’. Interestingly, ‘lipid metabolism’ was the most significant function identified in clusters #6, #9 and #12. IPA of gene lists obtained from each contrast (i.e., CC_GV0 vs. CC_GV1; CC_GV0 vs. CC_GV2; CC_GV0 vs. CC_GV3) revealed that the main affected function in each contrast was ‘cell death and survival’. Importantly, apoptosis was predicted to be inhibited in CC_GV1 and CC_GV2, but activated in CC_GV3. Caspase analysis indicated that a low percentage of CC_GV0 was prone to undergo apoptosis but apopto[...]

Identification of genes differentially expressed in menstrual breakdown and repair


STUDY QUESTION Does the changing molecular profile of the endometrium during menstruation correlate with the histological profile of menstruation. SUMMARY ANSWER We identified several genes not previously associated with menstruation; on Day 2 of menstruation (early-menstruation), processes related to inflammation are predominantly up-regulated and on Day 4 (late-menstruation), the endometrium is predominantly repairing and regenerating. WHAT IS KNOWN ALREADY Menstruation is induced by progesterone withdrawal at the end of the menstrual cycle and involves endometrial tissue breakdown, regeneration and repair. Perturbations in the regulation of menstruation may result in menstrual disorders including abnormal uterine bleeding. STUDY DESIGN, SIZE DURATION Endometrial samples were collected by Pipelle biopsy on Days 2 (n = 9), 3 (n = 9) or 4 (n = 6) of menstruation. PARTICIPANTS/MATERIALS, SETTING, METHODS RNA was extracted from endometrial biopsies and analysed by genome wide expression Illumina Sentrix Human HT12 arrays. Data were analysed using ‘Remove Unwanted Variation-inverse (RUV-inv)’. Ingenuity pathway analysis (IPA) and the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 were used to identify canonical pathways, upstream regulators and functional gene clusters enriched between Days 2, 3 and 4 of menstruation. Selected individual genes were validated by quantitative PCR. MAIN RESULTS AND THE ROLE OF CHANCE Overall, 1753 genes were differentially expressed in one or more comparisons. Significant canonical pathways, gene clusters and upstream regulators enriched during menstrual bleeding included those associated with immune cell trafficking, inflammation, cell cycle regulation, extracellular remodelling and the complement and coagulation cascade. We provide the first evidence for a role for glutathione-mediated detoxification (glutathione-S-transferase mu 1 and 2; GSTM1 and GSTM2) during menstruation. The largest number of differentially expressed genes was between Days 2 and 4 of menstruation (n = 1176). We identified several genes not previously associated with menstruation including lipopolysaccharide binding protein, serpin peptidase inhibitor, clade B (ovalbumin), member 3 (SERPINB3) and -4 (SERPINB4), interleukin-17C (IL17C), V-set domain containing T-cell activation inhibitor 1 (VTCN1), proliferating cell nuclear antigen factor (KIAA0101/PAF), trefoil factor 3 (TFF3), laminin alpha 2 (LAMA2) and serine peptidase inhibitor, Kazal type 1 (SPINK1). Genes related to inflammatory processes were up-regulated on Day 2 (early-menstruation), and those associated with endometrial repair and regeneration were up-regulated on Day 4 (late-menstruation). LIMITATIONS, REASONS FOR CAUTION Participants presented with a variety of endometrial pathologies related to bleeding status and other menstrual characteristics. These variations may also have influenced the menstrual process. WIDER IMPLICATIONS OF THE FINDINGS The temporal molecular profile of menstruation presented in this study identifies a number of genes not previously associated with the menstrual process. Our findings provide valuable insight into the menstrual process and may present novel targets for therapeutic intervention in cases of endometrial dysfunction. LARGE SCALE DATA All microarray data have been deposited in the public data repository Gene Expression Omnibus (GSE86003). STUDY FUNDING AND COMPETING INTEREST(S) Funding for this work was provided by a National Health and Medical Research Council of Australia (NHMRC) Project Grant APP1008553 to M.H., P.R. and J.G. M.H. is supported by an NHMRC Practitioner Fe[...]

Cystatin-related epididymal spermatogenic subgroup members are part of an amyloid matrix and associated with extracellular vesicles in the mouse epididymal lumen


Study Question Do the CRES (cystatin-related epididymal spermatogenic) subgroup members, including CRES2, CRES3 and cystatin E2, contribute to the formation of a nonpathological, functional amyloid matrix in the mouse epididymal lumen? Summary Answer CRES2, CRES3 and cystatin E2 self-assemble with different aggregation properties into amyloids in vitro, are part of a common amyloid matrix in the mouse epididymal lumen and are present in extracellular vesicles. What is Known Already Although previously thought only to be pathological, accumulating evidence has established that amyloids, which are highly ordered protein aggregates, can also carry out functional roles in the absence of pathology. We previously demonstrated that nonpathological amyloids are present in the epididymis; specifically, that the reproductive cystatin CRES forms amyloid and is present in the mouse epididymal lumen in a film-like amyloid matrix that is intimately associated with spermatozoa. Because the related proteins CRES2, CRES3 and cystatin E2 are also expressed in the epididymis, the present studies were carried out to determine if these proteins are also amyloidogenic in vitro and in vivo and thus may coordinately function with CRES as an amyloid structure. Study Design, Samples/Materials, Methods The epididymides from CD1 and Cst8 (CRES)129SvEv/B6 gene knockout (KO) and wild-type mice and antibodies that specifically recognize each CRES subgroup member were used for immunohistochemical and biochemical analyzes of CRES subgroup proteins. Methods classically used to identify amyloid, including the conformation-dependent dyes thioflavin S (ThS) and thioflavin T (ThT), conformation-dependent antibodies, protein aggregation disease ligand (which binds any amyloid independent of sequence) and negative stain electron microscopy (EM) were carried out to examine the amyloidogenic properties of CRES subgroup members. Immunofluorescence analysis and confocal microscopy were used for colocalization studies. Main Results and the Role of Chance Immunoblot and immunofluorescence analyzes showed that CRES2, CRES3 and cystatin E2 were primarily found in the initial segment and intermediate zone of the epididymis and were profoundly downregulated in epididymides from CRES KO mice, suggesting integrated functions. Except for CRES3, which was only detected in a particulate form, proteins were present in the epididymal lumen in both soluble and particulate forms including in a film-like matrix and in extracellular vesicles. The use of amyloid-specific reagents determined that all CRES subgroup members were present as amyloids and colocalized to a common amyloid matrix present in the epididymal lumen. Negative stain EM, dot blot analysis and ThT plate assays showed that recombinant CRES2, CRES3 and cystatin E2 formed amyloid in vitro, albeit with different aggregation properties. Together, our studies demonstrate that a unique amyloid matrix composed of the CRES family of reproductive-specific cystatins and cystatin C is a normal component of the mouse epididymal lumen and may play a functional role in sperm maturation by coordinating interactions between the luminal fluid and spermatozoa. Limitations, Reasons for Caution The structures examined in our studies were isolated from luminal fluid obtained by puncture of the epididymis and therefore we cannot rule out some contamination by epithelial cells. Although our studies show CRES family members are associated with extracellular vesicles, we have yet to determine if proteins are present on the surface or are within the vesicles. We also have not established if narrow/apical cells are the source of the CRES family extracellular ve[...]

Irinotecan metabolite SN38 results in germ cell loss in the testis but not in the ovary of prepubertal mice


STUDY QUESTION Does the Irinotecan metabolite 7-ethyl-10-hydroxycamptothecan (SN38) damage the gonads of male and female prepubertal mice? SUMMARY ANSWER The Irinotecan metabolite SN38 reduces germ cell numbers within the seminiferous tubules of mouse testes at concentrations that are relevant to cancer patients, while in contrast it has little if any effect on the female germ cell population. WHAT IS KNOWN ALREADY Little is known about the role of the chemotherapeutic agent Irinotecan on female fertility, with only one article to date reporting menopausal symptoms in perimenopausal women treated with Irinotecan, while no data are available either on adult male fertility or on the impact of Irinotecan on the subsequent fertility of prepubertal cancer patients, female or male. STUDY DESIGN SIZE, DURATION Male and female gonads were obtained from postnatal day 5 C57BL/6 mice and exposed in vitro to a range of concentrations of the Irinotecan metabolite SN38: 0.002, 0.01, 0.05, 0.1 or 1 µg ml–1 for the testis and 0.1, 1, 2.5 or 5 µg ml–1 for the ovary, with treated gonads compared to control gonads not exposed to SN38. SN38 was dissolved in 0.5% dimethyl sulfoxide, with controls exposed to the same concentration of diluent. The number of testis fragments used for each analysis ranged between 3 and 9 per treatment group, while the number of ovaries used for each analysis ranged between 4 and 12 per treatment group. PARTICIPANTS/MATERIALS, SETTING, METHODS Neonatal mouse gonads were developed in vitro, with tissue analysed at the end of the 4–6 day culture period, following immunofluorescence or hematoxylin and eosin staining. Statistical analyses were performed using one-way ANOVA followed by Bonferroni post-hoc test for normally distributed data and Kruskal-Wallis test followed by Dunns post-test for non-parametric data. MAIN RESULTS AND THE ROLE OF CHANCE Abnormal testis morphology was observed when tissues were exposed to SN38, with a smaller seminiferous tubule diameter at the highest concentration of SN38 (1 µg ml–1, p < 0.001 versus control) and increased number of Sertoli cell-only tubules at the two highest concentrations of SN38 (0.1 µg ml–1, p < 0.001; 1 µg ml–1, p < 0.0001, both versus control). Within seminiferous tubules, a dose response decrease was observed in both germ cell number (mouse vasa homologue (MVH)-positive cells) and in proliferating cell number (bromodeoxyuridine (BrdU)-positive cells), with significance reached at the two highest concentrations of SN38 (0.1 µg ml–1, p < 0.01 for both; 1 µg ml–1, p < 0.001-MVH, p < 0.01-BrdU; all versus control). No change was seen in protein expression of the apoptotic marker cleaved caspase 3. Double immunofluorescence showed that occasional proliferating germ cells were present in treated testes, even after exposure to the highest drug concentration. When prepubertal ovaries were treated with SN38, no effect was seen on germ cell number, apoptosis or cell proliferation, even after exposure to the highest drug concentrations. LIMITATIONS REASONS FOR CAUTION As with any study using in vitro experiments with an experimental animal model, caution is required when extrapolating the present findings to humans. Differences between human and mouse spermatogonial development also need to be considered when assessing the effect of chemotherapeutic exposure. However, the prepubertal testes and ovaries used in the present studies contain germ cell populations that are representative of those found i[...]

Human fallopian tube epithelium co-culture with murine ovarian follicles reveals crosstalk in the reproductive cycle


Study question Do interactions between human fallopian tube epithelium and murine follicles occur during an artificial reproductive cycle in a co-culture system in vitro? Summary answer In a co-culture system, human fallopian tissues responded to the menstrual cycle mimetic by changes in morphology and levels of secreted factors, and increasing murine corpus luteum progesterone secretion. What is known already The entire fallopian tube epithelium, including ciliated and secretory cells, can be regulated in the reproductive cycle. Currently, there are no in vitro culture models that can monitor fallopian tissues in real time in response to factors produced by the ovary. In addition, there are no reports on the impact of fallopian tissue on ovarian function during the menstrual cycle. Study design, samples/materials, methods Human fallopian tissue (n = 24) was obtained by routine hysterectomies from women (aged 26–50 years, mean age = 43.6) who had not undergone exogenous hormonal treatment for at least 3 months prior to surgery. CD1 female mice were used for ovarian follicle isolation. The human fallopian epithelium layers were either co-cultured with five murine multilayer secondary follicles (150–180 μm follicles, encapsulated in one alginate gel bead) for 15 days or received stepwise steroid hormone additions for 13 days. The fallopian tissue morphology and cilia beating rate, as measured by an Andor Spinning Disk Confocal, were investigated. Oviduct-specific glycoprotein 1 (OVGP1), human insulin-like growth factor 1 (hIGF1), vascular endothelial growth factor A (VEGF-A) and interleukin 8 (IL8) as biological functional markers were measured either by ELISA or western blot to indicate dynamic changes in the fallopian epithelium during the reproductive cycle generated by mouse follicles or by stepwise steroid hormone induction. Three or four patients in each experiment were recruited for replicates. Data were presented as mean ± SD and further analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. Main results and the role of chance The cultured fallopian tube epithelium responded to exogenous steroid hormone stimulation, as demonstrated by enhanced cilia beating rate (~25% increase, P = 0.04) and an increase in OVGP1 secretion (P = 0.02) in response to 1 nM estradiol (E2) treatment when compared with 0.1 nM E2. Conversely, 10 nM progesterone plus 1 nM E2 suppressed cilia beating rate by ~30% (P = 0.008), while OVGP1 secretion was suppressed by 0.1 nM E2 plus 50 nM progesterone (P = 0.002 versus 1 nM E2 alone). Human fallopian tube epithelium was co-cultured with murine secondary follicles to mimic the human menstrual cycle. OVGP1 and VEGF-A secretion from fallopian tissue was similar with stepwise hormone treatment and when cultured with murine follicles. However, the secretion patterns of hIGF1 and IL8 differed in the luteal phase when comparing steroid treatment with follicle co-culture. In co-culture, hIGF1 secretion was suppressed in the luteal versus follicular phase (P = 0.005) but stepwise hormone treatment had no effect on hIGF1. In co-culture, IL8 secretion was also suppressed on luteal phase day 15 (P = 0.013) versus follicular phase day 7, but IL8 secretion increased continuously under high E2/progesterone treatment (P = 0.003 for D13 versus D3). In the co-culture system, the corpus luteum continuously produced progesterone in the presence of fallopian tube tissue until Day 18 while, without fallopian tissue, progesterone started to dr[...]

TNF{alpha}-induced IKK{beta} complex activation influences epithelial, but not stromal cell survival in endometriosis


Study question Can the activity of the IB kinase (IKKβ) complex in endometriotic cells contribute to endometriotic lesion survival? Summary answer There is a constitutive activity of the IKKβ catalytic complex in peritoneal and deeply infiltrating lesions that can influence epithelial, but not stromal cell viability. What is known already Endometriotic lesions exist in an inflammatory microenvironment with higher local concentrations of cytokines, such as tumour necrosis factor α (TNFα). TNFα stimulates the activation of the IKKβ complex, an important nodal point in multiple signalling pathways that influence gene transcription, proliferation and apoptosis. However, few data on the regulation of IKKβ in endometriotic tissue are currently available. Study design, size, duration A retrospective analysis of endometriotic tissue from peritoneal, ovarian and deeply infiltrating lesions from 37 women. Participants/materials, setting, methods Basal and activated (phosphorylated) IKKβ concentrations were analysed by western blotting and immunohistochemistry. The relationship between the expression and activation of these proteins and peritoneal fluid (TNFα) concentrations, measured via ELISA, was examined. A subsequent in vitro analysis of TNFα treatment on the activation of IKKβ and the effect on epithelial and stromal cell viability by its inhibition with PS1145 was also performed. Main results and role of chance Levels of the phosphorylated IKKβ complex in endometriotic lesions had a significant positive correlation with peritoneal fluid TNFα concentrations. Phosphorylated IKKβ complex was more prevalent in peritoneal and deeply infiltrating endometriosis lesions compared with ovarian lesions. IKKβ was present in both epithelial and stromal cells in all lesions but active IKKβ was limited to epithelial cells. TNFα stimulated an increased expression of phosphorylated IKKβ and the inhibition of this kinase with PS1145 significantly influenced ectopic epithelial cells viability but not eutopic epithelial cells, or endometrial stromal cells. Limitations, reasons for caution In vitro analysis on epithelial cells was performed with immortalized cell lines and not primary cell cultures and only low sample numbers were available for the study. Wider implications of the findings The regulation of aberrant signalling pathways represents a promising yet relatively unexplored area of endometriosis progression. The IKKβ complex is activated by inflammation and is critical nodal point of numerous downstream kinase-signalling pathways, including NFB (nuclear factor B), mTOR (mammalian target of rapamycin) and BAD (Bcl2-antagonist of cell death). This study shows a significant relationship between peritoneal fluid TNFα and IKKβ activation in epithelial cells that will have significant consequences for the continued survival of these cells at ectopic locations through the regulation of downstream pathways. Large scale data None. Study funding/competing interest(s) The study was funded by the Swiss National Science Foundation (Grant Number 320030_140774). The authors have no conflict of interest to declare. [...]

Preimplantation maternal stress impairs embryo development by inducing oviductal apoptosis with activation of the Fas system


STUDY QUESTION What are the mechanisms by which the preimplantation restraint stress (PIRS) impairs embryo development and pregnancy outcome? SUMMARY ANSWER PIRS impairs embryo development by triggering apoptosis in mouse oviducts and embryos,and this involves activation of the Fas system. WHAT IS KNOWN ALREADY Although it is known that the early stages of pregnancy are more vulnerable than later stages to prenatalstress, studies on the effect of preimplantation stress on embryo developmentare limited. Furthermore, the mechanisms by which psychological stress impairs embryo development are largely unknown. These issues are worth exploring using the mouse PIRS models because restraint of mice is an efficient experimental procedure developed for studies of psychogenic stress. STUDY DESIGN, SIZE AND DURATION Mice of Kunming strain, the generalized lymphoproliferative disorder (gld) mice with a germline mutation F273L in FasL in a C57BL/6J genomic background and the wild-type C57BL/6J mice were used. Female and male mice were used 8–10 weeks and 10–12 weeks after birth, respectively. Female mice showing vaginal plugs were paired by weight and randomly assigned to restraint treatments or as controls. For restraint treatment, an individual mouse was put in a micro-cage with food and water available. Control mice remained in their cages with food and water during the time treated females were stressed. PARTICIPANTS/MATERIALS, SETTING, METHODS Female mice were exposed to PIRS for 48 h starting from 16:00 on the day of vaginal plug detection. At the end of PIRS, levels of glucorticoids (GC), corticotropin-releasing hormone (CRH)and redox potential were measured in serum, while levels of GC, GC receptor (GR), CRH, CRH receptor (CRHR), Fas and Fas ligand (FasL) protein, mRNAs for brain derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1), oxidative stress (OS) and apoptosis were examined in oviducts. Preimplantation development and levels of GR, Fas, redox potential and apoptosis were observed in embryos recovered at different times after the initiation of PIRS. The gld mice were used to confirm a role for the Fas system in triggering apoptosis of embryos and oviducts. MAIN RESULTS AND THE ROLE OF CHANCE Compared to those in control mice, while the number of blastocysts/mouse (5.0 ± 0.7 versus 11.1 ± 0.5), cell number/blastocyst (49.1 ± 1.3 versus 61.5 ± 0.9), percentages of term pregnancy (37.5% versus 90.9%) and litter size (3.7 ± 0.1versus 9.6 ± 0.6) decreased, blood CRH (560 ± 23 versus 455 ± 37 pg/ml), cortisol (27.3 ± 3.4 versus 5 ± 0.5 ng/ml) and OS index (OSI: 2.8 versus 1.7) increased significantly (all P < 0.05) following PIRS. In the oviduct, while levels of CRH (1175 ± 85 versus 881 ± 33 pg/100 mg), cortisol (28.9 ± 1.7 versus14 ± 4 ng/g), CRHR (2.3 ± 0.3 versus 1.0 ± 0.0), FasL (1.31 ± 0.06 versus 1.08 ± 0.05 ng/g), Fas (1.42 ± 0.13 versus 1.0 ± 0.0) and apoptotic cells (19.1 ± 0.5% versus 8.4 ± 0.4%) increased, levels of GR proteins (0.67 ± 0.14 versus 1.0 ± 0.0) and Igf-1 (0.6 ± 0.09 versus 1.0 ± 0.0) and Bdnf (0.73 ± 0.03 versus 1[...]

DNA methylation mediated up-regulation of TERRA non-coding RNA is coincident with elongated telomeres in the human placenta


Study Question What factors regulate elongated telomere length in the human placenta? Summary Answer Hypomethylation of TERRA promoters in the human placenta is associated with high TERRA expression, however, no clear mechanistic link between these phenomena and elongated telomere length in the human placenta was found. What is Known Already Human placenta tissue and trophoblasts show longer telomere lengths compared to gestational age-matched somatic cells. However, telomerase (hTERT) expression and activity in the placenta is low, suggesting a role for an alternative lengthening of telomeres (ALT). While ALT is observed in 10–15% of human cancers and in some mouse stem cells, ALT has never been reported in non-cancerous human tissues. Study Design, Samples/Materials, Methods Human term placental tissue and matched cord blood mononuclear cells (CBMCs) were collected as part of the Peri/Postnatal Epigenetic Twins study (PETS). In addition, first trimester placental villi, purified cytotrophoblasts, choriocarcinoma cell lines and a panel of ALT-positive cancer cell lines were tested. Telomere length was determined using the Terminal Restriction Fragment (TRF) assay and a relative quantitative PCR method. DNA methylation levels at several CpG rich subtelomeric TERRA promoters were determined using bisulfite conversion and the SEQUENOM EpiTYPER platform. Expression of TERRA and hTERT was determined using quantitative RT-PCR. ALT was assessed using the C-circle assay (CCA). Main Results and the Role of Chance The human placenta tissue and purified first trimester trophoblasts showed low subtelomeric (TERRA) DNA methylation compared to matched CBMCs and other somatic cells. Interestingly placental TERRA methylation was lower than ALT-cancer cell lines, previously reported to be hypomethylated at these loci. Low TERRA methylation was associated with higher expression of TERRA RNA in placenta compared to matched CBMCs. Detectable levels of C-circles were observed in first trimester placental villi, but not term placenta, suggesting that the ALT mechanism may be active in specific placental cells in early gestation. C-circle analysis of purified first trimester trophoblasts and ALT-associated PML bodies (APB) staining of first trimester villi cross-sections failed to identify this specific cell type population. Limitations, Reasons for Caution While first trimester villi showed detectable levels of C-circles, these levels were very low compared with those observed in ALT-positive tumours and cell lines. This is consistent with a small sub-population of ALT-positive cells but this requires further investigation. Finally, no mechanistic link was established between TERRA DNA methylation, the presence of C-circles and longer telomere length. Wider Implications of the Findings Given the previously described role of TERRA ncRNA as a negative regulator of telomerase, the finding of elevated TERRA and long telomeres is counterintutive. ALT as a mechanism for telomere length maintenance has only been reported in certain human cancers, and recently in mouse embryonic stem cells and embryos. As with many aspects of cancer, it appears that ALT activity in tumours may be the inappropriate activation of a pathway found in very specific cell types in human development. Our data are the first supportive evidence for ALT in a non-cancerous human tissue, a result that requires further investigation and replication. The level of TERRA methylation in the human placenta is significantly lower than found in ALT cancer cell line[...]

A role for the endocannabinoid system in premature luteal regression and progesterone withdrawal in lipopolysaccharide-induced early pregnancy loss model


STUDY QUESTION What is the role of the endocannabinoid system (eCS) in the alterations of the endocrine system in a murine model of lipopolysaccharide (LPS)-induced miscarriage? SUMMARY ANSWER In 7-days pregnant wild type, but not cannabinoid receptor type 1 knockout (CB1-KO) mice, LPS increased COX-2 expression and prostaglandin F2α (PGF2α) production in the uterus leading to lower expression of prolactin receptor in the ovary and a marked regression of corpora lutea (CL), suggesting that the eCS mediates the deleterious effects of LPS on reproductive events. WHAT IS KNOWN ALREADY Appropriate systemic progesterone levels are critical for a successful pregnancy outcome. Precocious loss of luteal progesterone (P4) secretion leads to miscarriage in rodents. We have previously shown that LPS administration to pregnant mice induces embryonic resorption accompanied by a dramatic decrease in systemic progesterone levels in a murine model of inflammatory miscarriage, with the eCS mediating these LPS-induced deleterious effects. STUDY DESIGN SAMPLES/MATERIALS, METHODS CD1 wild-type (WT) and CB1-KO mice were randomly allocated to Vehicle (saline; i.p.) or LPS (0.5 μg/g body weight; i.p.) treated groups: (WT-Vehicle; WT-LPS; CB1-KO-Vehicle and CB1-KO-LPS). A single injection was given on day 7 of pregnancy and tissues (blood, ovary, uterus) were collected 6, 12, 24 and 48 h later. P4 and PGF2α plasma levels were determined by radioimmunoassay. Cyclooxygenase-2 (COX-2) mRNA (RT-PCR) and protein (Western blot) content in uterus was assayed. COX-2 and prolactin receptor (PrlR) mRNA levels in the ovary were assayed by RT-PCR. Tissue morphology of the CL was assessed by haematoxylin–eosin staining. MAIN RESULTS AND THE ROLE OF CHANCE Treatment of 7-day pregnant WT mice with LPS induced a P4 withdrawal (p < 0.05), increased in uterine COX-2 mRNA and protein expression (p < 0.05) as well as an increase in uterine PGF2α production (p < 0.05). These changes were absent in LPS-treated 7-day pregnant CB1-KO mice. In ovarian tissues, LPS treatment to 7-day pregnant WT mice induced a downregulation of PrlR mRNA expression (p < 0.05) together with an increase in COX-2 mRNA expression (p < 0.05) and PGF2α content (p < 0.05). These effects were absent in the CB1-KO mice. Collectively, our results suggest a role for the eCS mediating LPS-induced deleterious effects on reproductive tissues. LIMITATIONS, REASONS FOR CAUTION An important caveat of this study is the endocrine differences between mice and humans during pregnancy (e.g. P4 is produced by the CL throughout pregnancy in mice, whereas this is not the case in humans), which limits the extrapolation of the results presented here. WIDER IMPLICATIONS OF THE FINDINGS Our findings provide new insights in the role of the endocannabinoid system in the physiopathology of reproduction as well as the role of this endogenous system as a mediator of LPS deleterious effects on reproductive tissues. LARGE SCALE DATA None. STUDY FUNDING AND COMPETING INTEREST(S) Dr Ana María Franchi was funded by Agencia Nacional para la Promoción Científica y Tecnológica (PICT 2010/0813 and PICT 2013/0097) and by Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2012/0061). The authors have no competing interests. [...]

mtDNA diversity in human populations highlights the merit of haplotype matching in gene therapies


STUDY QUESTION Does mitochondrial DNA (mtDNA) diversity in modern human populations potentially pose a challenge, via mtDNA segregation, to mitochondrial replacement therapies? SUMMARY ANSWER The magnitude of mtDNA diversity in modern human populations is as high as in mammalian model systems where strong mtDNA segregation is observed; consideration of haplotype pairs and/or haplotype matching can help avoid these potentially deleterious effects. WHAT IS KNOWN ALREADY In mammalian models, substantial proliferative differences are observed between different mtDNA haplotypes in cellular admixtures, with larger proliferative differences arising from more diverse haplotype pairings. If maternal mtDNA is ‘carried over’ in human gene therapies, these proliferative differences could lead to its amplification in the resulting offspring, potentially leading to manifestation of the disease that the therapy was designed to avoid—but existing studies have not investigated whether mtDNA diversity in modern human populations is sufficient to permit significant amplification. STUDY DESIGN, SIZE, DURATION This theoretical study used over 7500 human mtDNA sequences from The National Center for Biotechnology Information (NCBI), a range of international and British mtDNA surveys, and 2011 census data. PARTICIPANTS/MATERIALS, SETTING, METHODS A stochastic simulation approach was used to model random haplotype pairings from within different regions. In total, 1000 simulated pairings were analysed using the basic local alignment search tool (BLAST) for each region. Previous data from mouse models were used to estimate proliferative differences. MAIN RESULTS AND THE ROLE OF CHANCE Even within the same haplogroup, differences of around 20–80 single-nucleotide polymorphisms (SNPs) are common between mtDNAs admixed in random pairings. These values are sufficient to lead to substantial segregation in mouse models over an organismal lifetime, even given low starting heteroplasmy, inducing increases from 5% to 35% over 1 year. Substantial population mixing in modern UK cities increases the expected genetic differences. Hence, the likely genetic differences between humans randomly sampled from a population may well allow substantial amplification of a disease-carrying mtDNA haplotype over the timescale of a human lifetime. We report ranges and mean differences for all statistics to quantify uncertainty in our results. LIMITATIONS/REASONS FOR CAUTION The mapping from mouse and other mammalian models to the human system is challenging, as timescales and mechanisms may differ. Reporting biases in NCBI mtDNA data, if present, may affect the statistics we compute. We discuss the robustness of our findings in the light of these concerns. WIDER IMPLICATIONS OF THE FINDINGS Matching the mtDNA haplotypes of the mother and third-party donor in mitochondrial replacement therapies is supported as a means of ameliorating the potentially deleterious results of human mtDNA diversity. We present a chart of expected SNP differences between mtDNA haplogroups, allowing the selection of optimal partners for therapies. LARGE SCALE DATA N/A STUDY FUNDING/COMPETING INTERESTS The authors report no external funding sources or conflicts of interest. [...]