Transcriptome Landscape of Human Folliculogenesis Reveals Oocyte and Granulosa Cell Interactions.

The dynamic transcriptional regulation and interactions of human germlines and surrounding somatic cells during folliculogenesis remain unknown. Using RNA sequencing (RNA-seq) analysis of human oocytes and corresponding granulosa cells (GCs) spanning five follicular stages, we revealed unique features in transcriptional machinery, transcription factor networks, and reciprocal interactions in human oocytes and GCs that displayed developmental-stage-specific expression patterns. Notably, we identified specific gene signatures of two cell types in particular developmental stage that may reflect developmental competency and ovarian reserve. Additionally, we uncovered key pathways that may concert germline-somatic interactions and drive the transition of primordial-to-primary follicle, which represents follicle activation. Thus, our work provides key insights into the crucial features of the transcriptional regulation in the stepwise folliculogenesis and offers important clues for improving follicle recruitment in vivo and restoring fully competent oocytes in vitro.

III-nitride m-plane violet narrow ridge edge-emitting laser diodes with sidewall passivation using atomic layer deposition.

A novel deep-ridge laser structure with atomic-layer deposition (ALD) sidewall passivation was proposed that enhances the optical characteristics of 8-µm ridge width III-nitride violet lasers on freestanding m-plane GaN substrates. The internal loss was determined using the variable stripe length method, where the laser structure with ALD sidewall passivation showed lower internal loss compared to the conventional shallow-ridge laser design. ALD sidewall passivation plays a critical role in device improvements; compared to the lasers without ALD sidewall passivation, the lasers with ALD sidewall passivation yield improved optoelectrical performance and longer lifetime under continuous-wave operation at high current density. This work demonstrates the importance of ALD sidewall passivation to laser performance, which enables high energy efficiency.

Bone morphogenetic protein 6 induces downregulation of pentraxin 3 expression in human granulosa lutein cells in women with polycystic ovary syndrome.

PURPOSE: To evaluate whether PTX3 is differentially expressed in the granulosa lutein cells derived from women with PCOS and whether BMP6 can regulate the expression of PTX3 in hGL cells. METHODS: The expression levels of BMP6 and PTX3 in granulosa lutein cells were evaluated by RT-qPCR. The correlation between the expression levels of BMP6 /PTX3 and oocyte quality indexes were analyzed using clinical samples. The cells were incubated with BMP6 at different concentrations and times to check the expression of PTX3 in KGN cells. TGF-ß type I inhibitors and small interfering RNA targeting ALK2/3/6,SMAD1/5/8 and SMAD4 were used to study the involvement of SMAD dependent pathways in KGN cells. RESULTS: The levels of BMP6 in hGL cells were negatively correlated with the corresponding oocyte maturation rate and high-quality embryo rate, whereas the levels of PTX3 were positively correlated with the corresponding oocyte maturation rate in PCOS. Additionally, the in vitro cell cultured results showed BMP6 significantly inhibited the expression of PTX3 in KGN cells. Furthermore, using a dual inhibition approach (kinase inhibitors and small interfering RNAs), we identified the ALK2/ALK3 type I receptors and BMPR2/ACVR2A type II receptors and the downstream SMAD1/SMAD5-SMAD4 signaling pathway were responsible for the BMP6-induced cellular activities in KGN cells. CONCLUSIONS: The suppressive effect of BMP6 on PTX3 was mediated by ALK2/ALK3 type I receptors and BMPR2/ACVR2A type II receptors in granulosa cells through the SMAD1/5-SMAD4 dependent signaling pathway in PCOS.Our findings provides new insights into the understanding of the pathogenesis of PCOS-related ovulatory disorders.

BMP2 suppresses the production of pentraxin 3 in human endometrial stromal and decidual stromal cells.

Bone morphogenetic protein 2 (BMP2) has been shown to act as a critical regulator in the processes of embryo implantation and endometrial decidualization. The expression and production of pentraxin 3 (PTX3) is essential for successful pregnancy, and aberrant production of PTX3 is involved in the pathogenesis of several vascular complications during pregnancy. Studies have shown that several transforming growth factor ß superfamily members, including BMP2, can regulate female reproductive function by modulating the expression of PTX3 in human granulosa cells. However, to date, whether BMP2 can regulate the production of PTX3 during endometrial decidualization remains to be elucidated. In this study, we aimed to explore the effect of BMP2 on the expression and production of PTX3 and the underlying molecular mechanisms using immortalized human endometrial stromal cells (I-HESCs) and human decidual stromal cells (HDSCs). We demonstrated that treatment with exogenous BMP2 significantly suppressed PTX3 production by decreasing the mRNA level of PTX3 in both I-HESCs and HDSCs. The results also showed that BMP2 activated SMAD signaling by inducing an increase in the protein levels of phosphorylated SMAD1/5/8, and this effect could be abolished by pretreatment with the ALK2/3 inhibitor DMH-1 but not with the ALK1/4/7 inhibitor SB431542. Additionally, combined knockdown of ALK2 and ALK3 completely reversed the BMP2-induced suppressive effect on PTX3 expression, while concomitant knockdown of SMAD1 and SMAD5 or knockdown of SMAD4 completely reversed the BMP2-induced suppressive effect on PTX3 expression. Taken together, these results indicate that BMP2 suppressed PTX3 production by decreasing PTX expression, which is mediated by a canonical ALK2/3-mediated SMAD1/5-SMAD4-dependent signaling pathway. Our findings suggest that BMP2 may potentially regulate the process of endometrial decidualization by suppressing the production of PTX3 in humans.

Gonadotropin-releasing hormone analogs: Mechanisms of action and clinical applications in female reproduction.

Extra-hypothalamic GnRH and extra-pituitary GnRH receptors exist in multiple human reproductive tissues, including the ovary, endometrium and myometrium. Recently, new analogs (agonists and antagonists) and modes of GnRH have been developed for clinical application during controlled ovarian hyperstimulation for assisted reproductive technology (ART). Additionally, the analogs and upstream regulators of GnRH suppress gonadotropin secretion and regulate the functions of the reproductive axis. GnRH signaling is primarily involved in the direct control of female reproduction. The cellular mechanisms and action of the GnRH/GnRH receptor system have been clinically applied for the treatment of reproductive disorders and have widely been introduced in ART. New GnRH analogs, such as long-acting GnRH analogs and oral nonpeptide GnRH antagonists, are being continuously developed for clinical application. The identification of the upstream regulators of GnRH, such as kisspeptin and neurokinin B, provides promising potential to develop these upstream regulator-related analogs to control the hypothalamus-pituitary-ovarian axis.

The interleukin-6 trans-signaling promotes progesterone production in human granulosa-lutein cells†.

As a critical paracrine regulator of multiple reproductive functions, the cytokine interleukin-6 (IL-6) is expressed in human granulosa cells and can be detected in follicular fluid. At present, the functional role of IL-6 in the regulation of ovarian steroidogenesis is controversial. Moreover, the detailed molecular mechanisms by which IL-6 regulates the production of progesterone in human granulosa cells remain to be elucidated. In the present study, we used primary and immortalized human granulosa-lutein (hGL) cells to investigate the effects of IL-6 on progesterone synthesis and the underlying molecular mechanisms. We found that IL-6 trans-signaling by the combined addition of IL-6 and soluble IL-6 receptor (sIL-6Rα)-induced steroidogenic acute regulatory expression and progesterone production in hGL cells. Additionally, IL-6/sIL-6Rα activated the phosphorylation of Janus activated kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), and the cellular effects were abolished by AG490 (JAK2 inhibitor), C188-9 (STAT3 inhibitor), or siRNA-mediated knockdown of STAT3. IL-6 trans-signaling-induced activation of JAK2/STAT3 also upregulated the expression of suppressor of cytokine signaling 3, which, in turn, negatively regulated the JAK2/STAT3 pathway by suppressing STAT3 activation and its downstream effects. Our findings provide insight into the molecular mechanisms by which IL-6 trans-signaling modulates steroidogenesis in hGL cells.

Activin A promotes hyaluronan production and upregulates versican expression in human granulosa cells†.

Hyaluronan is a structural component of the expanded cumulus matrix, and hyaluronan synthase 2 is the major enzyme for the synthesis of hyaluronan in humans. Versican cross-links the hyaluronan-rich matrix to cumulus cells and is critical for successful ovulation. Activin A is a critical intrafollicular regulator of ovarian function. Although activin A has been shown to promote cumulus matrix expansion in mice, the functional role of activin A in the regulation of cumulus expansion in the human ovary remains to be elucidated. Using primary and immortalized human granulosa-lutein cells as study models, we provide the first data showing that activin A increased the production of hyaluronan by upregulating the expression of hyaluronan synthase 2 in these cells. Additionally, activin A also promoted the expression of the hyaluronan-binding protein versican. Moreover, using inhibitor- and small interfering RNA-mediated inhibition approaches, we found that these stimulatory effects of activin A are most likely mediated through the type I receptor activin receptor-like kinase (ALK4)-mediated Sma- and Mad-related protein (SMAD2)/SMAD3-SMAD4 signaling pathway. Notably, the chromatin immunoprecipitation analyses demonstrated that SMAD4 could bind to human hyaluronan synthase 2 and VERSICAN promoters. The results obtained from this in vitro study suggest that locally produced activin A plays a functional role in the regulation of hyaluronan production and stabilization in human granulosa-lutein cells.

Connective tissue growth factor mediates bone morphogenetic protein 2-induced increase in hyaluronan production in luteinized human granulosa cells.

BACKGROUND: Hyaluronan is the main component of the cumulus-oocyte complex (COC) matrix, and it maintains the basic structure of the COC during ovulation. As a member of the transforming growth factor ß (TGF-ß) superfamily, bone morphogenetic protein 2 (BMP2) has been identified as a critical regulator of mammalian folliculogenesis and ovulation. However, whether BMP2 can regulate the production of hyaluronan in human granulosa cells has never been elucidated. METHODS: In the present study, we investigated the effect of BMP2 on the production of hyaluronan and the underlying molecular mechanism using both immortalized (SVOG) and primary human granulosa-lutein (hGL) cells. The expression of three hyaluronan synthases (including HAS1, HAS2 and HAS3) were examined following cell incubation with BMP2 at different concentrations. The concentrations of the hyaluronan cell culture medium were determined by enzyme-linked immunosorbent assay (ELISA). The TGF-ß type I receptor inhibitors (dorsomorphin and DMH-1) and small interfering RNAs targeting ALK2, ALK3, ALK6 and SMAD4 were used to investigate the involvement of TGF-ß type I receptor and SMAD-dependent pathway. RESULTS: Our results showed that BMP2 treatment significantly increased the production of hyaluronan by upregulating the expression of hyaluronan synthase 2 (HAS2). In addition, BMP2 upregulates the expression of connective tissue growth factor (CTGF), which subsequently mediates the BMP2-induced increases in HAS2 expression and hyaluronan production because overexpression of CTGF enhances, whereas knockdown of CTGF reverses, these effects. Notably, using kinase inhibitor- and siRNA-mediated knockdown approaches, we demonstrated that the inductive effect of BMP2 on the upregulation of CTGF is mediated by the ALK2/ALK3-mediated SMAD-dependent signaling pathway. CONCLUSIONS: Our findings provide new insight into the molecular mechanism by which BMP2 promotes the production of hyaluronan in human granulosa cells.

Bone morphogenetic protein 2 upregulates SERPINE2 expression through noncanonical SMAD2/3 and p38 MAPK signaling pathways in human granulosa-lutein cells.

Serine protease inhibitor-E2 (SERPINE2) is highly expressed in the granulosa cells of growing follicles and the dynamic changes in SERPINE2 expression are correlated with follicular development and ovulation in several mammals, including mice, cattle, sheep, and humans. Bone morphogenetic proteins (BMPs) and their functional receptors are extensively expressed in the ovary and play critical roles in the regulation of ovarian folliculogenesis and luteal function. To date, whether BMPs regulate the expression of SERPINE2 during human follicular development remains to be elucidated. The aim of this study was to investigate the effects of BMPs on the regulation of SERPINE2 expression (a major regulator of plasminogen activators [PA]) and the underlying mechanisms using primary and immortalized human granulosa-lutein (hGL) cells. Our results demonstrated that these BMPs (BMP2, BMP4, BMP6, BMP7, and BMP15) induced differential upregulation of SERPINE2 expression. In this regard, BMP2 is the major modulator that has the best cellular activity, which further decreased the production of urokinase PA and tissue PA in hGL cells. In addition to canonical SMAD1/5/8 signaling, BMP2 also activates noncanonical SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) signaling. Using two inhibition approaches (kinase receptor inhibitors and siRNA-mediated knockdown), we found that SMAD2/3-SMAD4 and p38 MAPK, but not SMAD1/5/8 signaling, was involved in the BMP2-induced upregulation of SERPINE2 expression via activin receptor-like kinase 3. These findings deepen our understanding of the differential effect of BMPs in regulating follicular function and provide new insights of the molecular mechanisms by which BMP2 regulates the expression of SERPINE2 in human granulosa cells.

Outcomes comparison of IVF/ICSI among different trigger methods for final oocyte maturation: A systematic review and meta-analysis.

During the in vitro fertilization treatment, human chorionic gonadotrophin (hCG) is routinely used as a substitute for the natural endogenous LH surge during the final stage of oocyte maturation. However, it does not provide the FSH surge observed in the mid-cycle of the natural cycle. To date, whether the FSH surge can improve oocyte quality and pregnancy outcomes remains unknown. Randomized controlled trials comparing the following four trigger methods to conventional hCG were examined: GnRH agonist (GnRHa), kisspeptin, GnRHa plus hCG (dual trigger), and FSH plus hCG (FSH co-trigger). The results showed that the use of dual triggers was associated with a significantly higher number of retrieved cumulus-oocyte complexes (COCs) (weighted mean difference [WMD] 1.625, 95% CI 0.684-2.565), retrieved mature oocytes (WMD 0.986, 95% CI 0.426-1.545) and fertilized (2PN) oocytes (WMD 0.792, 95% CI 0.083-1.501), compared with the use of hCG. However, there was no significant difference between the two groups in terms of pregnancy rate. The FSH co-trigger resulted in significantly higher rates of 2PN oocytes retrieved than the hCG trigger (WMD 0.077, 95% CI 0.028-0.126). Notably, the risk of OHSS did not differ among the three treatment groups compared to that of the hCG group. This review protocol was registered with PROSPERO (CRD 42020194201).

The interleukin 6 trans-signaling increases prostaglandin E2 production in human granulosa cells†.

As a potent autocrine regulator, the proinflammatory cytokine interleukin 6 (IL6) is expressed in granulosa cells and is involved in the modulation of various follicular functions, including follicular development and ovulation. At present, the detailed molecular mechanisms by which IL6 regulates the event of ovulation remain to be elucidated. In the present study, primary and immortalized (SVOG) human granulosa-lutein (hGL) cells were used to investigate the effects of IL6 on the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and the subsequent synthesis of prostaglandin E2 (PGE2) and to investigate the underlying molecular mechanisms. We found that instead of classic signaling, IL6/soluble form of the IL6 receptor (sIL-6Ralpha) trans-signaling induced the expression of PTGS2 and production of PGE2 in both SVOG cells and primary hGL cells. Moreover, IL6/sIL-6Ralpha activated the phosphorylation of Janus-activated kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), which in turn induced STAT3 nuclear translocation. In addition, these effects were suppressed by the addition of inhibitors (AG490 for JAK2 and C188-9 for STAT3) and by the small interfering RNA-mediated knockdown of STAT3. In addition, suppressor of cytokine signaling 3 (SOCS3) acts as a negative-feedback regulator in IL6/sIL-6Ralpha-induced cellular activities, including the activation and nuclear translocation of STAT3, upregulation of PTGS2 expression, and increase in PGE2 production in SVOG cells. In conclusion, IL6 trans-signaling upregulates the expression of PTGS2 and increases the production of PGE2 via the JAK2/STAT3/SOCS3 signaling pathway in hGL cells. Our findings provide insights into the molecular mechanisms by which IL6 trans-signaling may potentially modulate the event of ovulation in human ovaries.

Bone morphogenetic protein 2 inhibits growth differentiation factor 8-induced cell signaling via upregulation of gremlin2 expression in human granulosa-lutein cells.

BACKGROUND: Bone morphogenetic protein 2 (BMP2), growth differentiation factor 8 (GDF8) and their functional receptors are expressed in human ovarian follicles, and these two intrafollicular factors play essential roles in regulating follicle development and luteal function. As BMP antagonists, gremlin1 (GREM1) and gremlin2 (GREM2) suppress BMP signaling through blockage of ligand-receptor binding. However, whether BMP2 regulates the expression of GREM1 and GREM2 in follicular development remains to be determined. METHODS: In the present study, we investigated the effect of BMP2 on the expression of GREM1 and GREM2 and the underlying mechanisms in human granulosa-lutein (hGL) cells. An established immortalized human granulosa cell line (SVOG) and primary hGL cells were used as study models. The expression of GREM1 and GREM2 were examined following cell incubation with BMP2 at different concentrations and time courses. The TGF-ß type I inhibitors (dorsomorphin, DMH-1 and SB431542) and small interfering RNAs targeting ALK2, ALK3, SMAD2/3, SMAD1/5/8 and SMAD4 were used to investigate the involvement of the SMAD-dependent pathway. RESULTS: Our results showed that BMP2 significantly increased the expression of GREM2 (but not GREM1) in a dose- and time-dependent manner. Using a dual inhibition approach combining kinase inhibitors and siRNA-mediated knockdown, we found that the BMP2-induced upregulation of GREM2 expression was mediated by the ALK2/3-SMAD1/5-SMAD4 signaling pathway. Moreover, we demonstrated that BMP2 pretreatment significantly attenuated the GDF8-induced phosphorylation of SMAD2 and SMAD3, and this suppressive effect was reversed by knocking down GREM2 expression. CONCLUSIONS: Our findings provide new insight into the molecular mechanisms by which BMP2 modulates the cellular activity induced by GDF8 through the upregulated expression of their antagonist (GREM2).

The p38 signaling pathway mediates the TGF-ß1-induced increase in type I collagen deposition in human granulosa cells.

Type I collagen, which is mainly composed of collagen type I alpha 1 chain (COL1A1), is the most abundant extracellular matrix (ECM) protein in the mammalian ovary; and the cyclical remodeling of the ECM plays an essential role in the regulation of corpus luteum formation. Our previous studies have demonstrated that TGF-ß1 is a potent inhibitor of luteinization in human granulosa-lutein (hGL) cells. Whether TGF-ß1 can regulate the expression of COL1A1 during the luteal phase remains to be elucidated. The aim of this study was to investigate the effect of TGF-ß1 on the regulation of COL1A1 expression and the underlying molecular mechanisms using an immortalized hGL cell line (SVOG cells) and primary hGL cells (obtained from 20 consenting patients undergoing IVF treatment). The results showed that TGF-ß1 significantly upregulated the expression of COL1A1. Using inhibition approaches, including pharmacological inhibition (a specific p38 inhibitor, SB203580, and a specific ERK1/2 inhibitor, U0126) and specific siRNA-mediated knockdown inhibition, we demonstrated that TGF-ß1 promoted the expression and production of COL1A1 in hGL cells, most likely via the ALK5-mediated p38 signaling pathway. Our findings provide insights into the molecular mechanisms by which TGF-ß1 promotes the deposition of type I collagen during the late follicular phase in humans.

The regulation of IGFBP3 by BMP2 has a role in human endometrial remodeling.

In mammals, bone morphogenetic protein 2 (BMP2) is a critical regulator of endometrial decidualization and early implantation. Insulin-like growth factor-binding protein 3 (IGFBP3) is highly expressed in the endometrium and at the maternal-fetal interface in multiple species, including humans. BMP2-induced IGFBP3 signaling has been confirmed to have a role in trophoblast cell invasion; however, the involvement of this signaling pathway in endometrial remodeling remains poorly understood. To determine the roles of BMP2 in regulating IGFBP3 expression during the transformation of endometrial stromal cells, we employed immortalized human endometrial stromal cells (HESCs) and primary human decidual stromal cells (HDSCs) as study models. We showed that BMP2 significantly increased the expression of IGFBP3 in a dose- and time-dependent manner in both HESCs and primary HDSCs. Additionally, the BMP2-induced upregulation of IGFBP3 is mediated by the inhibitor of DNA-binding 1 (ID1), and knockdown of ALK3 completely abolished BMP2-induced upregulation of ID1. Moreover, BMP2 increased the expression of matrix metalloproteinases 2 (MMP2) and promoted cell migration in HESCs and primary HDSCs. Knockdown of either IGFBP3 or ID1 significantly suppressed the basal and the BMP2-induced increase in MMP2 expression as well as the cell migration in both cell models. These data demonstrated that BMP2 upregulated the expression of ID1, which in turn induced the expression of IGFBP3, and these BMP2-induced cell activities were most likely mediated by the ALK3 type I receptor. The increased expression of IGFBP3 promoted the MMP2 expression and cell migration in both HESCs and HDSCs. These findings deepen our understanding of a newly identified mechanism by which BMP2 and IGFBP3 regulate endometrial remodeling in humans, which provides insight into potential therapies for endometrium-related diseases and pregnancy-induced complications.

BMP2 increases the production of BDNF through the upregulation of proBDNF and furin expression in human granulosa-lutein cells.

Locally produced in human granulosa cells of the developing follicle, bone morphogenetic protein 2 (BMP2) plays a crucial role in the regulation of ovarian folliculogenesis and luteal formation. Brain-derived neurotrophic factor (BDNF) is an intraovarian neurotrophic factor that has been shown to promote oocyte maturation and subsequent fertilization competency. At present, little is known regarding the intracellular regulation, assembly and secretion of endogenous BDNF in human granulosa cells. The aim of this study was to explore the effect of BMP2 on the expression and production of BDNF in human granulosa cells and the molecular mechanisms underlying this effect. An immortalized human granulosa cell line (SVOG) and primary human granulosa-lutein (hGL) cells were utilized as in vitro study models. Our results showed that BMP2 significantly increased the mRNA and secreted levels of BDNF. Additionally, BMP2 upregulated the expression of furin at the transcriptional and translational levels. Knockdown of endogenous furin partially attenuated the BMP2-induced increase in BDNF production, indicating that furin is involved in the maturation process of BDNF. Using pharmacological (kinase receptor inhibitors) and siRNA-mediated inhibition approaches, we demonstrated that BMP2-induced upregulation of BDNF and furin expression is most likely mediated by the activin receptor-like kinase (ALK)2/ALK3-SMAD4 signaling pathway. Notably, analysis using clinical samples revealed that there was a positive correlation between follicular fluid concentrations of BMP2 and those of BDNF. These results indicate that BMP2 increases the production of mature BDNF by upregulating the precursor BDNF and promoting the proteolytic processing of mature BDNF. Finally, we also investigated the effects of BMP2 on ovarian steroidogenesis and the results showed that BMP2 treatment significantly increased the accumulated level of estradiol (by upregulating the expression of FSH receptor and cytochrome P450 aromatase), whereas it decreased the accumulated level of progesterone (by downregulating the expression of LH receptors and steroidogenic acute regulatory protein) in primary hGL cells. Our findings provide a novel paracrine mechanism underlying the regulation of an intraovarian growth factor in human granulosa cells.

Bone morphogenetic protein 2 promotes human trophoblast cell invasion and endothelial-like tube formation through ID1-mediated upregulation of IGF binding protein-3.

Extravillous cytotrophoblasts (EVTs) invade into the uterine wall and remodel spiral arteries for proper placentation. Studies from us and others have demonstrated that the transforming growth factor-ß superfamily member bone morphogenetic protein 2 (BMP2) plays important roles in endometrial decidualization and trophoblast cell invasion. However, BMP2 has also been shown to regulate endothelial cell migration and capillary-like tube formation, as has its downstream signaling molecule inhibitor of DNA binding 1 (ID1). Interestingly, insulin-like growth factor binding protein 3 (IGFBP3) also promotes cell migration and angiogenesis in endothelial precursor cell. Moreover, Id1 has a regulatory effect on Igfbp3 expression in rat prostate epithelial cells. However, whether ID1 and IGFBP3 are integrated in BMP2 signaling and involved in the regulation of trophoblast invasive and endovascular differentiation remains unknown. The objective of our study was to examine the effects of BMP2 on ID1 and IGFBP3 expression and their roles in BMP2-regulated human trophoblast invasion and endothelial-like tube formation. Primary and immortalized (HTR8/SVneo) cultures of human trophoblast cells were employed as study models. BMP2 treatment increased ID1 and IGFBP3 mRNA and protein levels in HTR8/SVneo and primary human EVT cells. Intriguingly, ID1 was essential for BMP2-induced IGFBP3 upregulation in both study models, and BMP2-induced trophoblast invasion was attenuated by knockdown of either ID1 or IGFBP3. In addition, BMP2 significantly increased endothelial-like tube formation and knockdown of ID1 and IGFBP3 reduced basal and BMP2-induced tube formation in HTR8/SVneo cells. Similarly, BMP2 increased placenta growth factor (PlGF) production in HTR8/SVneo cells and these effects were attenuated by knockdown of ID1 or IGFBP3. Our results reveal that BMP2 promotes trophoblast cell invasion and endothelial-like tube formation by ID1-mediated IGFBP3 upregulation.

Melatonin improves the effect of cryopreservation on human oocytes by suppressing oxidative stress and maintaining the permeability of the oolemma.

Cryopreservation causes cryoinjury to oocytes and impairs their developmental competence. Melatonin (MLT) can improve the effect of cryopreservation in animal oocytes. However, no such studies on human oocytes have been reported. In this study, collected in vitro-matured human oocytes were randomly divided into the following groups: fresh group, MLT-treated cryopreservation (MC) group, and no-MLT-treated cryopreservation (NC) group. After vitrification and warming, viable oocytes from these three groups were assessed for their mitochondrial function, ultrastructure, permeability of oolemma, early apoptosis, developmental competence, and cryotolerance-related gene expression. First, fluorescence staining results revealed that oocytes from the 10-9  M subgroup showed the lowest intracellular reactive oxygen species and Ca2+ levels and highest mitochondrial membrane potential among the MC subgroups (10-11 , 10-9 , 10-7 , and 10-5  M). In subsequent experiments, oocytes from the 10-9  M-MC group were observed to maintain the normal ultrastructural features and the permeability of the oolemma. Compared with those of the oocytes in the NC group, the early apoptosis rate significantly decreased (P < .01), whereas both the high-quality cleavage embryo and blastocyst rates significantly increased (both P < .05) in the oocytes of the 10-9  M-MC group. Finally, single-cell RNA sequencing and immunofluorescence results revealed that aquaporin (AQP) 1/2/11 gene expression and AQP1 protein expression were upregulated in the MC group. Therefore, these results suggest that MLT can improve the effect of cryopreservation on human oocytes by suppressing oxidative stress and maintaining the permeability of the oolemma.

TGF-ß1 promotes vitamin D-induced prostaglandin E2 synthesis by upregulating vitamin D receptor expression in human granulosa-lutein cells.

There is increasing evidence showing the importance of vitamin D (Vit D) and its nuclear receptor, the Vit D receptor (VDR), in female reproductive health. Transforming growth factor-ß1 (TGF-ß1) and its functional receptors are expressed in human oocytes and granulosa cells that participate in follicular development and ovulation. Recently, Sma- and Mad-related protein 3 (SMAD3; a downstream effector of TGF-ß1) has been proposed to mediate crosstalk between the Vit D and TGF-ß1 signaling pathways, but this relationship has not been fully explored and has yet to be tested in human granulosa-lutein (hGL) cells. In this study, we showed that TGF-ß1 significantly promoted the expression of VDR, and this stimulatory effect occurred through the activin receptor-like kinase 5 type I receptor-mediated SMAD3 and ERK1/2 signaling pathways in hGL cells. Additionally, we showed that Vit D increased the expression of cyclooxygenase 2 (COX-2) and the synthesis of prostaglandin E2 (PGE2) in a time- and dose-dependent manner. Furthermore, we demonstrated a synergistic effect of TGF-ß1 and Vit D on the expression of COX-2 and synthesis of PGE2, and this effect could be attenuated by silencing the expression of VDR. Our findings indicate that TGF-ß1 upregulates the expression of VDR, which promotes Vit D-induced COX-2 expression and subsequent PGE2 production by activating the SMAD3 and ERK1/2 signaling pathways in hGL cells.

Regulation of LH secretion by RFRP-3 - From the hypothalamus to the pituitary.

RFamide-related peptides (RFRPs) have long been identified as inhibitors of the hypothalamus-pituitary-gonad axis in mammals. However, less progress has been made in the detailed roles of RFRPs in the control of LH secretion. Recent studies have suggested that RFRP-3 neurons in the hypothalamus can regulate the secretion of LH at different levels, including kisspeptin neurons, GnRH neurons, and the pituitary. Additionally, conflicting results regarding the effects of RFRP-3 on these levels exist. In this review, we collect the latest evidence related to the effects of RFRP-3 neurons in regulating LH secretion by acting on kisspeptin neurons, GnRH neurons, and the pituitary and discuss the potential role of the timely reduction of RFRP-3 signaling in the modulation of the preovulatory LH surge.

Transvaginal ovarian drilling followed by controlled ovarian stimulation from the next day improves ovarian response for the poor responders with polycystic ovary syndrome during IVF treatment: a pilot study.

BACKGROUND: Poor response patients with PCOS who are not susceptible to gonadotropin stimulation are more likely to have canceled cycles or poor clinical outcomes during IVF treatment. However, some limitations exist in the present therapies. In this study, we evaluated the effects of using the transvaginal ovarian drilling (TVOD) followed by controlled ovarian stimulation (COS) from the second day of these poor responders. METHODS: During IVF, 7 poor responders with PCOS and 28 PCOS patients (14 normal and 14 high responders) were recruited. All patients received COS with the gonadotropin-releasing hormone antagonist protocol. For the poor responders, after undergoing 10 to 14 days of ovulation induction with no response, the TVOD was applied and then ovarian stimulation was performed from the next day at the same gonadotropin dose. Serum samples during COS and follicular fluid samples from the dominant follicles on the oocyte pick-up (OPU) day in all three groups were collected. Besides, follicular fluid from small follicles (diameter < 1 cm) in the normal and high responders on the OPU day and those in the poor responders on the TVOD day were gathered. Hormonal levels were examined in all samples using immunometric assays. RESULTS: All the poor responders restored ovary response after receiving TVOD. There was no significant difference in the stimulation duration, total gonadotrophin dose used and the clinical outcomes among the three groups. The body mass index, serum and follicular levels of anti-Müllerian hormone (AMH) and testosterone in poor responders were higher than those in the other two groups, and the application of TVOD significantly decreased the levels of AMH and testosterone in both serum and follicular fluid. CONCLUSIONS: TVOD followed by ovulation induction from the next day is effective and convenient for poor responders with PCOS. The decline of AMH and testosterone resulted from TVOD may be the main reason resulting in the recovery of ovary sensitivity to gonadotropins. The small sample size is the primary limitation of this study, future studies using a large population cohort and monitoring the long-term outcomes of this strategy will be required. TRIAL REGISTRATION: ChiCTR1900023612. Registered 04 June 2019-Retrospectively registered.