Human plasma pregnancy-associated miRNAs and their temporal variation within the first trimester of pregnancy

Background During pregnancy, maternal metabolism undergoes substantial changes to support the developing fetus. Such changes are finely regulated by different mechanisms carried out by effectors such as microRNAs (miRNAs). These small non-coding RNAs regulate numerous biological functions, mostly through post-transcriptional repression of gene expression. miRNAs are also secreted in circulation by numerous organs, such as the placenta. However, the complete plasmatic microtranscriptome of pregnant women has still not been fully described, although some miRNA clusters from the chromosome 14 (C14MC) and the chromosome 19 (C19MC and miR-371-3 cluster) have been proposed as being specific to pregnancy. Our aims were thus to describe the plasma microtranscriptome during the first trimester of pregnancy, by assessing the differences with non-pregnant women, and how it varies between the 4th and the 16th week of pregnancy. Methods Plasmatic miRNAs from 436 pregnant (gestational week 4 to 16) and 15 non-pregnant women were quantified using Illumina HiSeq next-generation sequencing platform. Differentially abundant miRNAs were identified using DESeq2 package (FDR q-value ≤ 0.05) and their targeted biological pathways were assessed with DIANA-miRpath. Results A total of 2101 miRNAs were detected, of which 191 were differentially abundant (fold change < 0.05 or > 2, FDR q-value ≤ 0.05) between pregnant and non-pregnant women. Of these, 100 miRNAs were less and 91 miRNAs were more abundant in pregnant women. Additionally, the abundance of 57 miRNAs varied according to gestational age at first trimester, of which 47 were positively and 10 were negatively associated with advancing gestational age. miRNAs from the C19MC were positively associated with both pregnancy and gestational age variation during the first trimester. Biological pathway analysis revealed that these 191 (pregnancy-specific) and 57 (gestational age markers) miRNAs targeted genes involved in fatty acid metabolism, ECM-receptor interaction and TGF-beta signaling pathways. Conclusion We have identified circulating miRNAs specific to pregnancy and/or that varied with gestational age in first trimester. These miRNAs target biological pathways involved in lipid metabolism as well as placenta and embryo development, suggesting a contribution to the maternal metabolic adaptation to pregnancy and fetal growth.

Transcriptome analysis of eutopic endometrium in adenomyosis after GnRH agonist treatment

Background Adenomyosis is a chronic gynecological disease characterized by invasion of the uterine endometrium into the muscle layer. In assisted reproductive technology (ART), gonadotropin-releasing hormone agonist (GnRHa) is often used to improve pregnancy rates in patients with adenomyosis, but the underlying mechanisms are poorly understood. Methods Eutopic endometrial specimens were collected from patients with adenomyosis before and after GnRHa treatment in the midsecretory phase. RNA sequencing (RNA-Seq) of these specimens was performed for transcriptome analysis. The differentially expressed genes (DEGs) of interest were confirmed by real-time PCR and immunohistochemistry. Results A total of 132 DEGs were identified in the endometrium of patients with adenomyosis after GnRHa treatment compared with the control group. Bioinformatics analysis predicted that immune system-associated signal transduction changed significantly after GnRHa treatment. Chemokine (C-C motif) ligand 21 (CCL21) was found to be highly expressed in the eutopic endometrium after GnRHa treatment, which may be involved in the improvement of endometrial receptivity in adenomyosis. Conclusion This study suggests that molecular regulation related to immune system-associated signal transduction is an important mechanism of GnRHa treatment in adenomyosis. Immunoreactive CCL21 is thought to regulate inflammatory events and participate in endometrial receptivity in adenomyosis.

Endometrial injury concurrent with hysteroscopy increases the expression of Leukaemia inhibitory factor: a preliminary study

Objective It is not known by which mechanism endometrial injury increases pregnancy rates. Leukaemia inhibitory factor (LIF) is a cytokine involved in wound healing and implantation. The aim of this study was to determine the change in endometrial LIF mRNA expression before and after mechanical injury during hysteroscopy. Methods Forty patients with a history of two or more unsuccessful implantations who decided to undergo hysteroscopy in the proliferative phase were divided into two equal groups: one with endometrial injury (scratching group) and the other with noninjury (control group). Endometrial sampling was conducted before injury on the patients in the scratching group, and then injury was performed with monopolar needle forceps. Only diagnostic hysteroscopy was performed on the patients in the control group. Endometrial tissues were collected using a Pipelle catheter between Days 20 and 23 of the mid-luteal phase of the next cycles in both the scratching and control groups. Endometrial LIF mRNA expression was evaluated with the use of reverse-transcription polymerase chain reactions. Results Relative changes in mRNA expression levels of the LIF gene in endometrial samples taken before and after injury were calculated using the 2-ΔΔCt method, and the fold changes obtained were compared between and within the groups. Compared with preinjury values, an 11.1-fold increase was found in postinjury LIF mRNA expression in patients with monopolar forceps injury (p < 0.001). There was a 3.9-fold significant increase in postinjury LIF mRNA levels compared with those in the control group (p < 0.02). Conclusions The fertility-promoting effect of hysteroscopy-guided mechanical endometrial injury may be mediated by LIF mRNA.

Polycystic ovary syndrome as a plausible evolutionary outcome of metabolic adaptation

As a common endocrinopathy of reproductive-aged women, polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, oligo-anovulation and polycystic ovarian morphology. It is linked with insulin resistance through preferential abdominal fat accumulation that is worsened by obesity. Over the past two millennia, menstrual irregularity, male-type habitus and sub-infertility have been described in women and confirm that these clinical features of PCOS were common in antiquity. Recent findings in normal-weight hyperandrogenic PCOS women show that exaggerated lipid accumulation by subcutaneous (SC) abdominal stem cells during development to adipocytes in vitro occurs in combination with reduced insulin sensitivity and preferential accumulation of highly-lipolytic intra-abdominal fat in vivo. This PCOS phenotype may be an evolutionary metabolic adaptation to balance energy storage with glucose availability and fatty acid oxidation for optimal energy use during reproduction. This review integrates fundamental endocrine-metabolic changes in healthy, normal-weight PCOS women with similar PCOS-like traits present in animal models in which tissue differentiation is completed during fetal life as in humans to support the evolutionary concept that PCOS has common ancestral and developmental origins.

EndMT: New findings on the origin of myofibroblasts in endometrial fibrosis of intrauterine adhesions

Background Intrauterine adhesion (IUA) is one of the leading causes of infertility and the main clinical challenge is the high recurrence rate. The key to solving this dilemma lies in elucidating the mechanisms of endometrial fibrosis. The aim of our team is to study the mechanism underlying intrauterine adhesion fibrosis and the origin of fibroblasts in the repair of endometrial fibrosis. Methods Our experimental study involving an animal model of intrauterine adhesion and detection of fibrosis-related molecules. The levels of molecular factors related to the endothelial-to-mesenchymal transition (EndMT) were examined in a rat model of intrauterine adhesion using immunofluorescence, immunohistochemistry, qPCR and Western blot analyses. Main outcome measures are levels of the endothelial marker CD31 and the mesenchymal markers alpha-smooth muscle actin (α-SMA) and vimentin. Results Immunofluorescence co-localization of CD31 and a-SMA showed that 14 days after moulding, double positive cells for CD31 and a-SMA could be clearly observed in the endometrium. Decreased CD31 levels and increased α-SMA and vimentin levels indicate that EndMT is involved in intrauterine adhesion fibrosis. Conclusions Endothelial cells promote the emergence of fibroblasts via the EndMT during the endometrial fibrosis of intrauterine adhesions.

Oocyte cryopreservation review: outcomes of medical oocyte cryopreservation and planned oocyte cryopreservation

Background The utilization of oocyte cryopreservation (OC) has become popularized with increasing numbers of reproductive-aged patients desiring to maintain fertility for future family building. OC was initially used for fertility preservation in postmenarchal patients prior to gonadotoxic therapies; however, it is now available to patients to circumvent age-related infertility and other diagnoses associated with early loss of ovarian reserve. The primary aim of this paper is to provide a narrative review of the most recent and robust data on the utilization and outcomes of OC in both patient populations. Summary OC results in similar oocyte yield in patients facing gonadotoxic therapies and patients undergoing planned OC. Available data are insufficient to predict the live birth rates or the number of oocytes needed to result in live birth. However, oocyte yield and live birth rates are best among patients < 37.5 years old or with anti-mullerian hormone levels > 1.995 ng/dL, at the time of oocyte retrieval. There is a high ‘no use’ rate (58.9%) in patients using planned OC with 62.5% returning to use frozen oocytes with a spouse. The utilization rate in medical OC patients is < 10%. There is currently no data on the effects of BMI, smoking, or ethnicity on planned OC outcomes. Conclusion It is too early to draw any final conclusions on outcomes of OC in medical OC and planned OC; however, preliminary data supports that utilization of OC in both groups result in preservation of fertility and subsequent live births in patients who return to use their cryopreserved eggs. Higher oocyte yield, with fewer ovarian stimulation cycles, and higher live birth rates are seen in patients who seek OC at younger ages, reinforcing the importance of age on fertility preservation. More studies are needed in medical OC and planned OC to help guide counseling and decision-making in patients seeking these services.

Preeclampsia at delivery is associated with lower serum vitamin D and higher antiangiogenic factors: a case control study

Background Preeclampsia is characterized by decreased trophoblastic angiogenesis leading to abnormal invasion of spiral arteries, shallow implantation and resulting in compromised placentation with poor uteroplacental perfusion. Vitamin D plays an important role in pregnancy influencing implantation, angiogenesis and placental development. The objective of this study was to determine whether there is an association between serum vitamin D levels, and anti-angiogenic factors at the time of delivery and the occurrence of preeclampsia. Methods This nested case control study analyzed frozen serum samples at the time of delivery and related clinical data from women with singleton liveborn pregnancies who had participated in studies of the NICHD Stillbirth Collaborative Research Network. Women with a recorded finding of preeclampsia and who had received magnesium sulfate treatment prior to delivery were considered index cases (N = 56). Women without a finding of preeclampsia were controls (N = 341). Results Women with preeclampsia had 14.5% lower serum vitamin D levels than women in the control group (16.5 ng/ml vs. 19 ng/ml, p = 0.014) with 64.5% higher sFlt-1 levels (11,600 pg/ml vs. 7050 pg/ml, p < 0.001) and greater than 2 times higher endoglin levels (18.6 ng/ml vs. 8.7 ng/ml, < 0.001). After controlling for gestational age at delivery and maternal BMI, vitamin D levels were 0.88 times lower (P = 0.051), while endoglin levels were 2.5 times higher and sFlt-1 levels were 2.1 times higher than in control pregnancies (P < 0.001). Conclusions Women with preeclampsia at time of delivery have higher maternal antiangiogenetic factors and may have lower maternal serum vitamin D levels. These findings may lead to a better understanding of the underlying etiology of preeclampsia as well as possible modifiable treatment options which could include assuring adequate levels of maternal serum vitamin D prior to pregnancy.

Polycystic ovary syndrome phenotype does not have impact on oocyte morphology

Purpose The primary objective of the present study of women participating in an ICSI program was to determine whether the morphologic quality of oocytes was related to the polycystic ovary syndrome (PCOS) phenotype. Methods We performed a retrospective cohort study in the IVF unit at the Lille University Medical Center (Lille, France) between 2006 and 2015. Oocyte morphology (fragmented first polar body, abnormal zona pellucida, large perivitelline space, material in perivitelline space, abnormal shape of oocyte, granular cytoplasm and intracytoplasmic vacuoles) was evaluated in PCOS women and according to different subgroup (depending on the presence or absence of the cardinal features polycystic ovarian morphology (PCOM), hyperandrogenism (HA), and oligo-anovulation (OA)). Results A total of 1496 metaphase II oocytes (n = 602 for phenotype A combining PCOM + HA + OA, n = 462 oocytes for phenotype C: PCOM + HA, and n = 432 for phenotype D: PCOM + OA) were assessed. The phenotypes A, C and D did not differ significantly with regard to the proportion of normal oocytes (adjusted percentages (95%CI): 35.2% (31.5 to 39.1%), 25.8% (21.9 to 29.9%) and 34.0% (29.7 to 38.6%), respectively: adjusted p = 0.13). Likewise, there were no significant intergroup differences in oocyte morphology. The ICSI outcome was not significantly associated with the PCOS phenotype. Conclusion The present study is the first to show that the PCOS phenotype (notably the presence vs. absence of OA and/or HA) is not significantly associated with the morphological quality of oocytes.

Hyperandrogenism and menstrual imbalance are the best predictors of metformin response in PCOS patients

Background Moving from the correlation between insulin-resistance and PCOS, metformin has been administered in some PCOS women improving ovulatory and metabolic functions and decreasing androgen levels. Inconsistency and unpredictability of response to metformin limit its extensive use. Aim of this study was to identify reliable predictors of response to metformin therapy for weight loss and reduction in plasma androgen levels using ANNs (artificial neural networks). Methods One hundred eight consecutive women with PCOS (ESHRE/ASRM 2003 Rotterdam criteria) treated with metformin 1500 mg/day, at inclusion and every 6 months underwent to a complete clinical, endocrine/metabolic assessment and ultrasonographic evaluation. Therapy outcomes were BMI reduction (≥1 kg/m2) in overweight/obese and free-androgen-index (FAI) decrease (≥1%) in hyperandrogenemic women. Semantic connectivity maps (SCMs) were obtained through Auto-CM, a fourth generation ANN, to compare patients’ baseline clinical features to the treatment outcomes. Multivariate logistic regression analysis was used to assess the major predictor in drop-out patients and the associated risk. Results At 6 months 54 out of 103 (52,4%) obese patients showed BMI reduction and 45 out of 89 (50,6%) hyperandrogenemic women showed FAI decrease. The further response rates at 12 months were 30,6 and 47%, respectively. SCMs showed a clear polarization for both the outcomes with elevated accuracy. Treatment responsiveness resulted strictly related to oligo-amenorrhea and hyperandrogenemia at baseline. In addition, lower serum testosterone levels at baseline were found to be the major predictor of treatment discontinuation. Conclusions In women with PCOS, menstrual pattern imbalance and ovarian androgens excess are the best predictors of metformin response. They may pave the way for a rethinking of the criteria for evaluating hyperandrogenism in order to better define the large population included in the diagnosis of PCOS. Baseline plasma testosterone level can serve as a sensitive marker to predict treatment compliance.

Proteomic analysis of extracellular vesicles secreted by primary human epithelial endometrial cells reveals key proteins related to embryo implantation

Background Successful implantation is dependent on coordination between maternal endometrium and embryo, and the role of EVs in the required cross-talk cell-to-cell has been recently established. In this regard, it has been reported that EVs secreted by the maternal endometrium can be internalized by human trophoblastic cells transferring their contents and enhancing their adhesive and invasive capacity. This is the first study to comprehensively evaluate three EV isolation methods on human endometrial epithelial cells in culture and to describe the proteomic content of EVs secreted by pHEECs from fertile women. Methods Ishikawa cells and pHEECs were in vitro cultured and hormonally treated; subsequently, conditioned medium was collected and EVs isolated. Ishikawa cells were used for the comparison of EVs isolation methods ultracentrifugation, ExoQuick-TC and Norgen Cell Culture Media Exosome Purification Kit (n = 3 replicates/isolation method). pHEECs were isolated from endometrial biopsies (n = 8/replicate; 3 replicates) collected from healthy oocyte donors with confirmed fertility, and protein content of EVs isolated by the most efficient methodology was analysed using liquid chromatography–tandem mass spectrometry. EV concentration and size were analyzed by nanoparticle tracking analysis, EV morphology visualized by transmission electron microscopy and protein marker expression was determined by Western blotting. Results Ultracentrifugation was the most efficient methodology for EV isolation from medium of endometrial epithelial cells. EVs secreted by pHEECs and isolated by ultracentrifugation were heterogeneous in size and expressed EV protein markers HSP70, TSG101, CD9, and CD81. Proteomic analysis identified 218 proteins contained in these EVs enriched in biological processes involved in embryo implantation, including cell adhesion, differentiation, communication, migration, extracellular matrix organization, vasculature development, and reproductive processes. From these proteins, 82 were selected based on their functional relevance in implantation success as possible implantation biomarkers. Conclusions EV protein cargos are implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs. Identified proteins may define new biomarkers of endometrial receptivity and implantation success.