EGF-like growth factors upregulate pentraxin 3 expression in human granulosa-lutein cells.

The epidermal growth factor (EGF)-like factors, comprising amphiregulin (AREG), betacellulin (BTC), and epiregulin (EREG), play a critical role in regulating the ovulatory process. Pentraxin 3 (PTX3), an essential ovulatory protein, is necessary for maintaining extracellular matrix (ECM) stability during cumulus expansion. The aim of this study was to investigate the impact of EGF-like factors, AREG, BTC, and EREG on the expression and production of PTX3 in human granulosa-lutein (hGL) cells and the molecular mechanisms involved. Our results demonstrated that AREG, BTC, and EREG could regulate follicular function by upregulating the expression and increasing the production of PTX3 in both primary (obtained from 20 consenting patients undergoing IVF treatment) and immortalized hGL cells. The upregulation of PTX3 expression was primarily facilitated by the activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway, induced by these EGF-like factors. In addition, we found that the upregulation of PTX3 expression triggered by the EGF-like factors was completely reversed by either pretreatment with the epidermal growth factor receptor (EGFR) inhibitor, AG1478, or knockdown of EGFR, suggesting that EGFR is crucial for activating the ERK1/2 signaling pathway in hGL cells. Overall, our findings indicate that AREG, BTC, and EREG may modulate human cumulus expansion during the periovulatory stage through the upregulation of PTX3.

Growth differentiation factor-11 upregulates matrix metalloproteinase 2 expression by inducing Snail in human extravillous trophoblast cells.

The human extravillous trophoblast (EVT) cell invasion is an important process during placentation. Although the placenta is normal tissue, the EVT cells exhibit some features common to cancer cells, including high migratory and invasive properties. Snail and Slug are transcription factors that mediate the epithelial-mesenchymal transition (EMT), a crucial event for cancer cell migration and invasion. It has been shown that GDF-11-induced matrix metalloproteinase 2 (MMP2) expression is required for EVT cell invasion. Whether GDF-11 can regulate Snail and Slug expression in human EVT cells remains unknown. If it does, the involvement of Snail and Slug in GDF-11-induced MMP2 expression and EVT cell invasion must also be defined. In the present study, using the immortalized human EVT cell line, HTR-8/SVneo, and primary cultures of human EVT cells as experimental models, our results show that GDF-11 upregulates Snail and Slug expression. ALK4 and ALK5 mediate the stimulatory effects of GDF-11 on Snail and Slug expression. In addition, we demonstrate that SMAD2 and SMAD3 are required for the GDF-11-upregulated Snail expression, while only SMAD3 is involved in GDF-11-induced Slug expression. Moreover, our results reveal that Snail mediates GDF-11-induced MMP2 expression and cell invasion but not Slug. This study increases our understanding of the biological function of GDF-11 in human EVT cells and provides a novel mechanism for regulating MMP2 and EVT cell invasion.

Effects of hepatitis B virus infection on the treatment outcomes following in vitro fertilization/intracytoplasmic sperm injection: An analysis of 21,999 first embryo transfer cycles.

To investigate the effects of hepatitis B virus (HBV) infection on the outcomes of Chinese couples undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and the clinical data of their neonates. A total of 21,999 first embryo transfer cycles were included. They were categorized into four groups based on the couple's hepatitis B surface antigen (HBsAg) result (Group A = female HBsAg- and male HBsAg- ; Group B = female HBsAg+ and male HBsAg- ; Group C = female HBsAg- and male HBsAg+ ; Group D = female HBsAg+ and male HBsAg+ ). The fertilization rate (FR), cleavage rate (CR), implantation rate (IPR), clinical pregnancy rate (CPR), live birth rate (LBR) and miscarriage rate (MCR) were analysed. Multilevel logistic regression was applied to evaluate the association. The total prevalence of HBV infection was 5.74% (2526/43998). There were no statistically significant differences in CRs (98.69%, 98.76%, 98.66%, 98.72%, p > .05), IPRs (45.86%, 47.33%, 45.19%, 39.61%, p > .05), CPRs (62.84%, 65.05%, 61.80%, 56.81%, p > .05), MCRs (12.70%, 11.99%, 12.58%, 4%, p > .05) and LBRs (53.43%, 55.38%, 52.70%, 54.54%, p > .05) among the four groups. However, there were significant differences in FRs (66.25%, 66.55%, 66.32%, 61.92%, p < .05). Group D had the lowest FR. After adjusting for confounders, the multilevel logistic regression showed that HBsAg+ had no impact on the LBR, CPR or MCR. We also analysed the data of 14,465 newborns, including 8593 singletons and 2936 twins. Among the four groups, no variables reached statistical significance, including neonatal birth weight (NBW), twin ratio, gestational age, premature birth, delivery type, fetal macrosomia or low birth weight (p > .05). Our study demonstrates that, although biparental HBV infection may affect the FR, neither single-parent infection nor biparental HBV infection affects IVF/ICSI outcomes or neonatal outcomes.

Generation of a human haploid neural stem cell line for genome-wide genetic screening.

BACKGROUND: Haploid embryonic stem cells (haESCs) have been established in many species. Differentiated haploid cell line types in mammals are lacking due to spontaneous diploidization during differentiation that compromises lineage-specific screens. AIM: To derive human haploid neural stem cells (haNSCs) to carry out lineage-specific screens. METHODS: Human haNSCs were differentiated from human extended haESCs with the help of Y27632 (ROCK signaling pathway inhibitor) and a series of cytokines to reduce diploidization. Neuronal differentiation of haNSCs was performed to examine their neural differentiation potency. Global gene expression analysis was con-ducted to compare haNSCs with diploid NSCs and haESCs. Fluorescence activated cell sorting was performed to assess the diploidization rate of extended haESCs and haNSCs. Genetic manipulation and screening were utilized to evaluate the significance of human haNSCs as genetic screening tools. RESULTS: Human haESCs in extended pluripotent culture medium showed more compact and smaller colonies, a higher efficiency in neural differentiation, a higher cell survival ratio and higher stability in haploidy maintenance. These characteristics effectively facilitated the derivation of human haNSCs. These human haNSCs can be generated by differentiation and maintain haploidy and multipotency to neurons and glia in the long term in vitro. After PiggyBac transfection, there were multiple insertion sites in the human haNSCs' genome, and the insertion sites were evenly spread across all chromosomes. In addition, after the cells were treated with manganese, we were able to generate a list of manganese-induced toxicity genes, demonstrating their utility as genetic screening tools. CONCLUSION: This is the first report of a generated human haploid somatic cell line with a complete genome, proliferative ability and neural differentiation potential that provides cell resources for recessive inheritance and drug targeted screening.

WNK1 mediates amphiregulin-induced MMP9 expression and cell invasion in human extravillous trophoblast cells.

The invasion of human extravillous trophoblast (EVT) cells is a critical event required for a successful pregnancy. Amphiregulin, a ligand of the epidermal growth factor receptor (EGFR), has been shown to stimulate cell invasion in an immortalized human EVT cell line, HTR-8/SVneo. The with-no-lysine kinase 1 (WNK1) is involved in regulating cell invasion. It is known that WNK1 is expressed in the human placenta, but its role in human EVT cells remains unknown. In the present study, we show that AREG treatment phosphorylated WNK1 at Thr60 in both HTR-8/SVneo and primary human EVT cells. The stimulatory effect of AREG on WNK1 phosphorylation was mediated by the activation of PI3K/AKT, but not the ERK1/2 signaling pathway. AREG upregulated matrix metalloproteinase 9 (MMP9) but not MMP2. In addition, cell invasiveness was increased in response to the treatment of AREG. Using the siRNA-mediated knockdown approach, our results showed that the knockdown of WNK1 attenuated the AREG-induced upregulation of MMP9 expression and cell invasion. Moreover, the expression of WNK1 was downregulated in the placentas with preeclampsia, a disease resulting from insufficiency of EVT cell invasion during pregnancy. This study discovers the physiological function of WNK1 in human EVT cells and provides important insights into the regulation of MMP9 and cell invasion in human EVT cells.

TGF-ß1 upregulates secreted protein acidic and rich in cysteine expression in human granulosa-lutein cells: a potential mechanism for the pathogenesis of ovarian hyperstimulation syndrome.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a serious complication during in vitro fertilization (IVF) treatment. The upregulation of ovarian transforming growth factor-beta 1 (TGF-ß1) is involved in the development of OHSS. The secreted protein acidic and rich in cysteine (SPARC) is a secreted multifunctional matricellular glycoprotein. Although the regulatory effects of TGF-ß1 on SPARC expression have been reported, whether TGF-ß1 regulates SPARC expression in the human ovary remains unknown. In addition, the role of SPARC in the pathogenesis of OHSS is unclear. METHODS: A steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing IVF treatment were used as experimental models. OHSS was induced in rats, and ovaries were collected. Follicular fluid samples were collected from 39 OHSS and 35 non-OHSS patients during oocyte retrieval. The underlying molecular mechanisms mediating the effect of TGF-ß1 on SPARC expression were explored by a series of in vitro experiments. RESULTS: TGF-ß1 upregulated SPARC expression in both KGN and hGL cells. The stimulatory effect of TGF-ß1 on SPARC expression was mediated by SMAD3 but not SMAD2. The transcription factors, Snail and Slug, were induced in response to the TGF-ß1 treatment. However, only Slug was required for the TGF-ß1-induced SPARC expression. Conversely, we found that the knockdown of SPARC decreased Slug expression. Our results also revealed that SPARC was upregulated in the OHSS rat ovaries and in the follicular fluid of OHSS patients. Knockdown of SPARC attenuated the TGF-ß1-stimulated expression of vascular endothelial growth factor (VEGF) and aromatase, two markers of OHSS. Moreover, the knockdown of SPARC reduced TGF-ß1 signaling by downregulating SMAD4 expression. CONCLUSIONS: By illustrating the potential physiological and pathological roles of TGF-ß1 in the regulation of SPARC in hGL cells, our results may serve to improve current strategies used to treat clinical infertility and OHSS. Video Abstract.

Betacellulin regulates gap junction intercellular communication by inducing the phosphorylation of connexin 43 in human granulosa-lutein cells.

BACKGROUND: The gap junction protein, connexin 43 (Cx43) is highly expressed in human granulosa-lutein (hGL) cells. The phosphorylation of certain amino acid residues in the Cx43 protein has been shown to be related to a decline in gap junction intercellular communication (GJIC), which subsequently affects oocyte meiotic resumption. As a member of the epidermal growth factor (EGF) family, betacellulin (BTC) mediates luteinizing hormone (LH)-induced oocyte maturation and cumulus cell expansion in mammalian follicles. Whether BTC can regulate Cx43 phosphorylation, which further reduces Cx43-coupled GJIC activity in hGL cells remains to be determined. METHODS: Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing in vitro fertilization in an academic research center were used as the study models. The expression levels of Cx43 and phosphorylated Cx43 were examined following cell incubation with BTC at different time points. Several kinase inhibitors (sotrastaurin, AG1478, and U0126) and small interfering RNAs targeting EGF receptor (EGFR) and receptor tyrosine-protein kinase 4 (ErbB4) were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance. RESULTS: The results showed that BTC induced the rapid phosphorylation of Cx43 at serine368 without altering the expression of Cx43 in primary and immortalized hGL cells. Additionally, using a dual inhibition approach (kinase inhibitors and siRNA-based expression knockdown), we demonstrated that this effect was mainly mediated by the EGFR but not the ErbB4 receptor. Furthermore, using a protein kinase C (PKC) kinase assay and a scrape-loading and dye transfer assay, we revealed that PKC signaling is the downstream signaling pathway that mediates the increase in Cx43 phosphorylation and subsequent decrease in GJIC activity in response to BTC treatment in hGL cells. CONCLUSIONS: BTC promptly induced the phosphorylation of connexin 43 at Ser368, leading to decreased GJIC activity in hGL cells. The BTC-induced cellular activities were most likely driven by the EGFR-mediated PKC-dependent signaling pathway. Our findings shed light on the detailed molecular mechanisms by which BTC regulates the process of oocyte meiotic resumption.

Gender bias in fetal malformations: A cross-sectional study in Asian populations.

Objectives: The aim of this study was to detect any gender bias in fetal malformation cases. Design: This study was a cross-sectional, quantitative survey. Subjects: Overall, 1,661 Asian fetal malformation cases involving induced abortions in the obstetrics department of the first Affiliated Hospital of Zhengzhou University from 2012 to 2021 were included. Main outcome measures: Measurements of ultrasound detectable structural malformations were classified into 13 subtypes. Karyotyping, single nucleotide polymorphism (SNP) array, or sequencing diagnosis of these fetus was also included in the outcome measures. Results: The sex ratio (male/female) of all malformation types was 1.446. Cardiopulmonary had the highest proportion of all malformation types with 28%. Diaphragmatic hernia, omphalocele, gastroschisis, nuchal translucency (NT), and Multy malformations had significantly higher proportions of males (p < 0.05). Digestive system malformations had a significantly higher proportion of females (p < 0.05). Maternal age was associated with genetic factors (r = 0.953, p < 0.001) and inversely associated with brain malformations (r = -0.570, p = 0.002). More males were found with trisomy 21, trisomy 18, and monogenetic diseases, while duplications, deletions, and uniparental disomy (UPD) had similar sex ratios between males and females, but not statistically significant. Conclusion: Sex differences are common with fetal malformations, with higher proportions of males. Genetic testing has been proposed to account for these differences.

Biomarkers identification in follicular fluid of women with OHSS by using UPLC-MS method.

To figure out the differentially changed metabolites and disturbed pathways in follicular fluid (FF) of patients with OHSS in comparison to the control group undergoing in vitro fertilization (IVF), we conducted this metabolomic analysis between two groups, the OHSS group included 30 patients treated with oocyte retrieval and developed OHSS in the next 7-14 days, while another 30 patients without OHSS tendency were selected as the control group. The FF samples were obtained during the process of oocyte retrieval. FF samples were analyzed using ultra-high liquid chromatography-tandem mass spectrometry (UPLC-MS). The results identified a total of 59 differentially changed metabolites, including 33 decreased metabolites (P < 0.01) and 26 increased metabolites (P < 0.01) in FF of OHSS compared with the control group. 12 metabolites could be the most valuable biomarkers for OHSS based on ROC results. Our correlation analyses showed that deoxyinosine levels were found positively correlated with serum estradiol (E2) levels in OHSS patients, while L-isoleucine, pyruvic acid, maleamate, and arachidonic acid were found to be positively correlated with the number of retrieved oocytes. Furthermore, 4-hydroxyphenylacetaldehyde, deoxycorticosterone, creatinine, and creatine were found to be negatively associated with serum E2 levels, while 4-hydroxyphenylacetaldehyde, L-carnitine, isovaleric acid and L-2-hydroxyglutaric acid were negatively related with the number of oocytes retrieved in OHSS patients. Taken together, our study provides better identification of OHSS FF metabolic dynamics, suggesting the metabolic compounds can be used as valuable predictors or treatment targets of OHSS.

The role of amphiregulin in ovarian function and disease.

Amphiregulin (AREG) is an epidermal growth factor (EGF)-like growth factor that binds exclusively to the EGF receptor (EGFR). Treatment with luteinizing hormone (LH) and/or human chorionic gonadotropin dramatically induces the expression of AREG in the granulosa cells of the preovulatory follicle. In addition, AREG is the most abundant EGFR ligand in human follicular fluid. Therefore, AREG is considered a predominant propagator that mediates LH surge-regulated ovarian functions in an autocrine and/or paracrine manner. In addition to the well-characterized stimulatory effect of LH on AREG expression, recent studies discovered that several local factors and epigenetic modifications participate in the regulation of ovarian AREG expression. Moreover, aberrant expression of AREG has recently been reported to contribute to the pathogenesis of several ovarian diseases, such as ovarian hyperstimulation syndrome, polycystic ovary syndrome, and epithelial ovarian cancer. Furthermore, increasing evidence has elucidated new applications of AREG in assisted reproductive technology. Collectively, these studies highlight the importance of AREG in female reproductive health and disease. Understanding the normal and pathological roles of AREG and elucidating the molecular and cellular mechanisms of AREG regulation of ovarian functions will inform innovative approaches for fertility regulation and the prevention and treatment of ovarian diseases. Therefore, this review summarizes the functional roles of AREG in ovarian function and disease.

scm6A-seq reveals single-cell landscapes of the dynamic m6A during oocyte maturation and early embryonic development.

N6-methyladenosine (m6A) has been demonstrated to regulate RNA metabolism and various biological processes, including gametogenesis and embryogenesis. However, the landscape and function of m6A at single cell resolution have not been extensively studied in mammalian oocytes or during pre-implantation. In this study, we developed a single-cell m6A sequencing (scm6A-seq) method to simultaneously profile the m6A methylome and transcriptome in single oocytes/blastomeres of cleavage-stage embryos. We found that m6A deficiency leads to aberrant RNA clearance and consequent low quality of Mettl3Gdf9 conditional knockout (cKO) oocytes. We further revealed that m6A regulates the translation and stability of modified RNAs in metaphase II (MII) oocytes and during oocyte-to-embryo transition, respectively. Moreover, we observed m6A-dependent asymmetries in the epi-transcriptome between the blastomeres of two-cell embryo. scm6A-seq thus allows in-depth investigation into m6A characteristics and functions, and the findings provide invaluable single-cell resolution resources for delineating the underlying mechanism for gametogenesis and early embryonic development.

AREG upregulates secreted protein acidic and rich in cysteine expression in human granulosa cells.

The secreted protein acidic and rich in cysteine (SPARC) is a secreted glycoprotein and the expression of ovarian SPARC peaks during ovulation and luteinization. Besides, SPARC expression was induced by human chorionic gonadotropin (hCG) in rat granulosa cells. Amphiregulin (AREG) is the most abundant epidermal growth factor receptor (EGFR) ligand expressed in human granulosa cells and follicular fluid. AREG mediates the physiological functions of luteinizing hormone (LH)/hCG in the ovary. However, to date, the biological function of SPARC in the human ovary remains undetermined, and whether AREG regulates SPARC expression in human granulosa cells is unknown. In this study, we show that AREG upregulated SPARC expression via EGFR in a human granulosa-like tumor cell line, KGN. Treatment of AREG activated ERK1/2, JNK, p38 MAPK, and PI3K/AKT signaling pathways and all of them were required for the AREG-induced SPARC expression. Using RNA-sequencing, we identified that steroidogenic acute regulatory protein (StAR) was a downstream target gene of SPARC. In addition, we demonstrated that SPARC mRNA levels were positively correlated with the levels of StAR mRNA in the primary culture of human granulosa cells. Moreover, SPARC protein levels were positively correlated with progesterone levels in follicular fluid of in vitro fertilization patients. This study provides the regulatory role of AREG on the expression of SPARC and reveals the novel function of SPARC in progesterone production in granulosa cells.

Comparison of embryo implantation potential between time-lapse incubators and standard incubators: a randomized controlled study.

RESEARCH QUESTION: What are the potential clinical benefits of embryo culture and assessment in a time-lapse incubator compared with a standard incubator using static assessment? DESIGN: This large multicentre, single-blinded, randomized controlled study included 1224 participants randomly assigned (1:1) to the time-lapse or standard incubator group. In all patients one or two embryos were transferred on day 3. The primary outcome was the implantation rate in the first embryo transfer cycle. Secondary outcomes included the cumulative implantation rate, live birth rate in the first embryo transfer cycle and cumulative live birth rate. RESULTS: Among 1224 participants recruited, 1182 underwent embryo transfer. The number of successfully implanted embryos in the first transfer cycle was significantly higher in the time-lapse incubator group (time-lapse group: 52.35%, standard incubator group: 47.11%, P = 0.014). The implantation rate in the first embryo transfer cycle was still significantly higher in the time-lapse group than the standard incubator group after adjusting for age, body mass index, medical centre and embryo status (relative risk 1.11, 95% confidence interval 1.02-1.20, P = 0.020). However, the cumulative implantation rate, live birth rate in the first embryo transfer cycle and cumulative live birth rate were not statistically different between the groups. CONCLUSIONS: The implantation rate in the first embryo transfer cycle was significantly improved in the time-lapse group, but the effect of the time-lapse system on the cumulative implantation rate or cumulative live birth rate was not significant. The embryo assessment method offered by time-lapse systems rather than an undisturbed environment may play an important role in improving the implantation rate in the first embryo transfer cycle. These results are only applicable to young patients.

HB-EGF upregulates StAR expression and stimulates progesterone production through ERK1/2 signaling in human granulosa-lutein cells.

BACKGROUND: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) belongs to the epidermal growth factor (EGF) family of growth factors. HB-EGF and its receptors, epidermal growth factor receptor (EGFR) and HER4, are expressed in the human corpus luteum. HB-EGF has been shown to regulate luteal function by preventing cell apoptosis. Steroidogenesis is the primary function of the human corpus luteum. Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis. StAR expression and progesterone (P4) production in human granulosa-lutein (hGL) cells have been shown to be upregulated by a ligand of EGFR, amphiregulin. However, whether HB-EGF can achieve the same effects remains unknown. METHODS: A steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of hGL cells obtained from patients undergoing in vitro fertilization treatment were used as experimental models. The underlying molecular mechanisms mediating the effects of HB-EGF on StAR expression and P4 production were explored by a series of in vitro experiments. RESULTS: Western blot showed that EGFR, HER2, and HER4 were expressed in both KGN and hGL cells. Treatment with HB-EGF for 24 h induced StAR expression but did not affect the expression of steroidogenesis-related enzymes, P450 side chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase, and aromatase. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we showed that EGFR, HER4, but not HER2, were required for HB-EGF-stimulated StAR expression and P4 production. In addition, HB-EGF-induced upregulations of StAR expression and P4 production were mediated by the activation of the ERK1/2 signaling pathway. CONCLUSION: This study increases the understanding of the physiological role of HB-EGF in human luteal functions. Video Abstract.

GDF-11 promotes human trophoblast cell invasion by increasing ID2-mediated MMP2 expression.

BACKGROUND: Growth differentiation factor-11 (GDF-11), also known as bone morphogenetic protein-11, belongs to the transforming growth factor-beta superfamily. GDF-11 was first identified as an important regulator during embryonic development. Increasing evidence has demonstrated that GDF-11 regulates the development of various organs and its aberrant expressions are associated with the risk of cardiovascular diseases and cancers. Extravillous trophoblast (EVT) cells invasion is a critical event for placenta development and needs to be finely regulated. However, to date, the biological function of GDF-11 in the human EVT cells remains unknown. METHODS: HTR-8/SVneo, a human EVT cell line, and primary cultures of human EVT cells were used to examine the effect of GDF-11 on matrix metalloproteinase 2 (MMP2) expression. Matrigel-coated transwell invasion assay was used to examine cell invasiveness. A series of in vitro experiments were applied to explore the underlying mechanisms that mediate the effect of GDF-11 on MMP2 expression and cell invasion. RESULTS: Treatment with GDF-11 stimulates MMP2 expression, in the HTR-8/SVneo and primary human EVT cells. Using a pharmacological inhibitor and siRNA-mediated knockdown approaches, our results demonstrated that the stimulatory effect of GDF-11 on MMP2 expression was mediated by the ALK4/5-SMAD2/3 signaling pathways. In addition, the expression of inhibitor of DNA-binding protein 2 (ID2) was upregulated by GDF-11 and that was required for the GDF-11-stimulated MMP2 expression and EVT cell invasion. CONCLUSIONS: These findings discover a new biological function and underlying molecular mechanisms of GDF-11 in the regulation of human EVT cell invasion. Video Abstract.

TGF-ß1 inhibits human trophoblast cell invasion by upregulating kisspeptin expression through ERK1/2 but not SMAD signaling pathway.

BACKGROUND: Tightly regulation of extravillous cytotrophoblast (EVT) cell invasion is critical for the placentation and establishment of a successful pregnancy. Insufficient EVT cell invasion leads to the development of preeclampsia (PE) which is a leading cause of maternal and perinatal mortality and morbidity. Transforming growth factor-beta1 (TGF-ß1) and kisspeptin are expressed in the human placenta and have been shown to inhibit EVT cell invasion. Kisspeptin is a downstream target of TGF-ß1 in human breast cancer cells. However, whether kisspeptin is regulated by TGF-ß1 and mediates TGF-ß1-suppressed human EVT cell invasion remains unclear. METHODS: The effect of TGF-ß1 on kisspeptin expression and the underlying mechanisms were explored by a series of in vitro experiments in a human EVT cell line, HTR-8/SVneo, and primary cultures of human EVT cells. Serum levels of TGF-ß1 and kisspeptin in patients with or without PE were measured by ELISA. RESULTS: TGF-ß1 upregulates kisspeptin expression in HTR-8/SVneo cells and primary cultures of human EVT cells. Using pharmacological inhibitor and siRNA, we demonstrate that the stimulatory effect of TGF-ß1 on kisspeptin expression is mediated via the ALK5 receptor. Treatment with TGF-ß1 activates SMAD2/3 canonical pathways as well as ERK1/2 and PI3K/AKT non-canonical pathways. However, only inhibition of ERK1/2 activation attenuates the stimulatory effect of TGF-ß1 on kisspeptin expression. In addition, siRNA-mediated knockdown of kisspeptin attenuated TGF-ß1-suppressed EVT cell invasion. Moreover, we report that serum levels of TGF-ß1 and kisspeptin are significantly upregulated in patients with PE. CONCLUSIONS: By illustrating the potential physiological role of TGF-ß1 in the regulation of kisspeptin expression, our results may serve to improve current strategies used to treat placental diseases.

A meta-analysis of serum lipid profiles in premature ovarian insufficiency.

Premature ovarian insufficiency (POI), defined as loss of normal ovarian functions before the age of 40 years, occurs in at least 1% of all women. It affects the reproductive system and causes many health problems and psychological stress. Abnormal serum lipid profile leads to cardiovascular diseases, which are strongly associated with high mortality in patients with POI. To date, several studies have examined the levels of different serum lipids in patients with POI. The results, however, are either inconclusive or inconsistent. Therefore, the aim of this meta-analysis was to measure whether serum levels of total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglyceride are varied in patients with POI. Ten studies in total were included in this meta-analysis involving 1009 individuals: 458 patients with POI and 551 controls. Our analysis results showed that serum total cholesterol (P < 0.00001), LDL-C (P < 0.0001), and triglyceride (P = 0.01) levels were significantly higher in patients with POI compared with healthy controls. Serum HDL-C levels, however, did not vary significantly between controls and patients with POI. These results suggest that elevations in unfavourable lipids may contribute to the high risk of cardiovascular diseases that are observed in patients with POI.

Epigallocatechin-3-gallate stimulates StAR expression and progesterone production in human granulosa cells through the 67-kDa laminin receptor-mediated CREB signaling pathway.

Epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active catechins extracted from green tea. The health benefits of EGCG have been extendedly studied. Ovarian steroidogenesis plays a pivotal role in maintaining normal reproductive function. Granulosa cells in the ovary are essential for steroid hormone production. To date, the effect of EGCG on steroidogenesis in human granulosa cells remains unclear. In the present study, we examine the physiological concentrations of EGCG on steroidogenesis in a steroidogenic human granulosa-like tumor cell line, KGN. Our results demonstrate that treatment with EGCG upregulates steroidogenic acute regulatory protein (StAR) expression and increases progesterone (P4) production. EGCG does not affect the expression levels of other steroidogenesis-related enzymes, such as P450 side-chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase, and aromatase. In addition, we identify the expression of 67-kDa laminin receptor (67LR) in KGN cells. Moreover, EGCG-induced StAR expression and P4 production require the 67LR-mediated activation of the PKA-CREB signaling pathway. These results provide a better understanding of the function of EGCG on ovarian steroidogenesis, which may lead to the development of alternative therapeutic approaches for reproductive disorders.

G protein-coupled estrogen receptor stimulates human trophoblast cell invasion via YAP-mediated ANGPTL4 expression.

Insufficient invasion of trophoblast cells into the uterine decidua is associated with preeclampsia (PE). G protein-coupled estrogen receptor (GPER) is a membrane estrogen receptor involved in non-genomic estrogen signaling. GPER is expressed in human trophoblast cells and downregulated GPER levels are noted in PE. However, to date, the role of GPER in trophoblast cells remains largely unknown. Here, we applied RNA sequencing (RNA-seq) to HTR-8/SVneo human trophoblast cells in response to G1, an agonist of GPER, and identified angiopoietin-like 4 (ANGPTL4) as a target gene of GPER. Treatment of trophoblast cells with G1 or 17ß-estradiol (E2) activated Yes-associated protein (YAP), the major downstream effector of the Hippo pathway, via GPER but in a mammalian STE20-like protein kinase 1 (MST1)-independent manner. Using pharmacological inhibitors as well as loss- and gain-of-function approaches, our results revealed that YAP activation was required for GPER-stimulated ANGPTL4 expression. Transwell invasion assays demonstrated that activation of GPER-induced ANGPTL4 promoted cell invasion. In addition, the expression levels of GPER, YAP, and ANGPTL4 were downregulated in the placenta of patients with PE. Our findings reveal a mechanism by which GPER exerts its stimulatory effect on human trophoblast cell invasion by upregulating YAP-mediated ANGPTL4 expression.

EGF stimulates human trophoblast cell invasion by downregulating ID3-mediated KISS1 expression.

BACKGROUND: During pregnancy, trophoblast cell invasion needs to be finely controlled. Aberrant trophoblast cell invasion is associated with placental diseases. Epidermal growth factor (EGF) and its receptor, EGFR, are expressed in trophoblast cells. Although the pro-invasive effect of EGF on trophoblast cells has been reported, the underlying mechanism remains largely unknown. RESULTS: In the present study, we conducted an RNA sequencing (RNA-seq) to HTR-8/SVneo human trophoblast cells in response to EGF and identified KISS1 as a target gene of EGF. The human KISS1 gene encodes kisspeptin, also known as metastin, which can suppress tumor metastasis. Our results showed that EGF treatment downregulated KISS1 expression and secretion by activating the EGFR-mediated PI3K/AKT signaling pathway. In addition, the expression of inhibitor of DNA-binding protein 3 (ID3) was downregulated by EGF and that was required for the EGF-suppressed KISS1 expression. Functionally, transwell invasion assays demonstrated that EGF stimulated human trophoblast cell invasion by downregulating KISS1 expression. Preeclampsia (PE) is a placental disease characterized by insufficient trophoblast cell invasion. Our clinical results revealed that serum levels of EGF were downregulated while serum and placental levels of KISS1 were upregulated in PE patients. CONCLUSIONS: This study demonstrates that downregulation of EGF can lead to poor trophoblast cell invasion by increasing KISS1 expression which subsequently contributes to the pathogenesis of PE. Video Abstract.