ANGPTL4 Expression in Ovarian Granulosa Cells Is Associated With Polycystic Ovary Syndrome.

This study aims to characterize the expression of ANGPTL4 in ovarian granulosa cells (GCs) and its association with polycystic ovary syndrome (PCOS). This study included 104 PCOS patients and 112 women in control group undergoing in vitro fertilization-embryo transfer (IVF-ET) from the reproductive hospital affiliated with Shandong University from 2019 to 2021. By reverse transcription and real-time quantitative (RT-q) PCR, the mRNA expression of ANGPTL4 in GCs was assessed, and clinical information for these patients were then reviewed and analyzed. The RT-qPCR results showed that ANGPTL4 expression in the control group was significantly lower than that in the PCOS group (p = 0.000) and had positive association with AMH (r = 0.211), HOMA-IR (r = 0.174), LDL/HDL (r = 0.176), ApoB/ApoAI (r = 0.155), and TC/HDL (r = 0.189). Additionally, the high expression of ANGPTL4 in the ovarian granulosa cells might be an independent predictor in PCOS (OR: 3.345; 95% CI: 1.951-5.734) with a close contact with incidence of PCOS (AUC: 0.704; 95% CI: 0.633-0.774, p < 0.001). Our study revealed higher ANGPTL4 expression in ovarian GCs with PCOS. Its association with glucose and lipid metabolism showed that ANGPTL4 might play an important role in PCOS metabolism and pathogenesis.

Jiang Q ，Pan Y ，Li P ，Zheng Y ，Bian Y ，Wang W ，Wu G ，Song T ，Shi Y ... -  《Frontiers in Endocrinology》

The regulation and signalling of anti-Müllerian hormone in human granulosa cells: relevance to polycystic ovary syndrome.

What prevents the fall in anti-Müllerian hormone (AMH) levels in polycystic ovary syndrome (PCOS) and what are the consequences of this for follicle progression in these ovaries? Exposure of granulosa cells (GCs) to high levels of androgens, equivalent to that found in PCOS, prevented the fall in AMH and was associated with dysregulated AMH-SMAD signalling leading to stalled follicle progression in PCOS. In normal ovaries, AMH exerts an inhibitory role on antral follicle development and a fall in AMH levels is a prerequisite for ovulation. Levels of AMH are high in PCOS, contributing to the dysregulated follicle growth that is a common cause of anovulatory infertility in these women. Human KGN-GC (the cell line that corresponds to immature GC from smaller antral follicles (AF)) were cultured with a range of doses of various androgens to determine the effects on AMH production. KGN-GC were also treated with PHTPP (an oestrogen receptor β (ERβ) antagonist) to examine the relationship between AMH expression and the ratio of ERα:ERβ. The differential dose-related effect of AMH on gene expression and SMAD signalling was investigated in human granulosa-luteal cells (hGLC) from women with normal ovaries, with polycystic ovarian morphology (PCOM) and with PCOS. KGN-GC were also cultured for a prolonged period with AMH at different doses to assess the effect on cell proliferation and viability. AMH protein production by cells exposed to androgens was measured by ELISA. The effect of PHTPP on the mRNA expression levels of AMH, ERα and ERβ was assessed by real-time quantitative PCR (qPCR). The influence of AMH on the relative mRNA expression levels of aromatase, AMH and its receptor AMHRII, and the FSH and LH receptor (FSHR and LHR) in control, PCOM and PCOS hGLCs was quantified by qPCR. Western blotting was used to assess changes in levels of SMAD proteins (pSMAD-1/5/8; SMAD-4; SMAD-6 and SMAD-7) after exposure of hGLCs from healthy women and women with PCOS to AMH. The ApoTox-Glo Triplex assay was used to evaluate the effect of AMH on cell viability, cytotoxicity and apoptosis. Testosterone reduced AMH protein secreted from KGN-GC at 10-9-10-7 M (P < 0.05; P < 0.005, multiple uncorrected comparisons Fishers least squares difference), but at equivalent hyperandrogenemic levels no change was seen in AMH levels. 5α-DHT produced a significant dose-related increase in AMH protein secreted into the media (P = 0.022, ANOVA). Increasing the mRNA ratio of ERα:ERβ produced a corresponding increase in AMH mRNA expression (P = 0.015, two-way ANOVA). AMH increased mRNA levels of aromatase (P < 0.05, one-way ANOVA) and FSHR (P < 0.0001, one-way ANOVA) in hGLCs from women with PCOM, but not from normal cells or PCOS (normal n = 7, PCOM n = 5, PCOS n = 4). In contrast to hGLCs from ovulatory ovaries, in PCOS AMH reduced protein levels (cell content) of stimulatory pSMAD-1/5/8 and SMAD-4 but increased inhibitory SMAD-6 and -7 (P < 0.05, normal n = 6, PCOS n = 3). AMH at 20 and 50 ng/ml decreased KGN-GC cell proliferation but not viability after 8 days of treatment (P < 0.005, two-way ANOVA). N/A. Luteinised GC from women undergoing IVF have a relatively low expression of AMH/AMHRII but advantageously continue to display responses inherent to the ovarian morphology from which they are collected. To compensate, we also utilised the KGN cell line which has been characterised to be at a developmental stage close to that of immature GC. The lack of flutamide influence on testosterone effects is not in itself sufficient evidence to conclude that the effect on AMH is mediated via conversion to oestrogen, and the effect of aromatase inhibitors or oestrogen-specific inhibitors should be tested. The effect of flutamide was tested on testosterone but not DHT. Normal folliculogenesis and ovulation are dependent on the timely reduction in AMH production from GC at the time of follicle selection. Our findings reveal for the first time that theca-derived androgens may play a role in this model but that this inhibitory action is lost at levels of androgens equivalent to those seen in PCOS. The AMH decline may either be a direct effect of androgens or an indirect one via conversion to oestradiol and acting through the upregulation of ERα, which is known to stimulate the AMH promoter. Interestingly, the ability of GCs to respond to this continually elevated AMH level appears to be reduced in cells from women with PCOS due to an adaptive alteration in the SMAD signalling pathway and lower expression of AMHRII, indicating a form of 'AMH resistance'. This study was funded by the Thomas Addison Scholarship, St Georges Hospital Trust. The authors report no conflict of interest in this work and have nothing to disclose. N/A.

Dilaver N ，Pellatt L ，Jameson E ，Ogunjimi M ，Bano G ，Homburg R ，D Mason H ，Rice S ... -  《-》

Loss of LH-induced down-regulation of anti-Müllerian hormone receptor expression may contribute to anovulation in women with polycystic ovary syndrome.

Pierre A ，Peigné M ，Grynberg M ，Arouche N ，Taieb J ，Hesters L ，Gonzalès J ，Picard JY ，Dewailly D ，Fanchin R ，Catteau-Jonard S ，di Clemente N ... -  《-》

ANGPTL4 inhibits granulosa cell proliferation in polycystic ovary syndrome by EGFR/JAK1/STAT3-mediated induction of p21.

Polycystic ovary syndrome (PCOS) is one of the most common, heterogenous endocrine disorders and is the leading cause of ovulatory obstacle associated with abnormal folliculogenesis. Dysfunction of ovarian granulosa cells (GCs) is recognized as a major factor that underlies abnormal follicle maturation. Angiopoietin-like 4 (ANGPTL4) expression in GCs differs between patients with and without PCOS. However, the role and mechanism of ANGPTL4 in impaired follicular development are still poorly understood. Here, the case-control study was designed to investigate the predictive value of ANGPTL4 in PCOS while cell experiments in vitro were set for mechanism research. Results found that ANGPTL4 levels in serum and in follicular fluid, and its expression in GCs, were upregulated in patients with PCOS. In KGN and SVOG cells, upregulation of ANGPTL4 inhibited the proliferation of GCs by blocking G1/S cell cycle progression, as well as the molecular activation of the EGFR/JAK1/STAT3 cascade. Moreover, the STAT3-dependent CDKN1A(p21) promoter increased CDKN1A transcription, resulting in remarkable suppression effect on GCs. Together, our results demonstrated that overexpression of ANGPTL4 inhibited the proliferation of GCs through EGFR/JAK1/STAT3-mediated induction of p21, thus providing a novel epigenetic mechanism for the pathogenesis of PCOS.

Jiang Q ，Miao R ，Wang Y ，Wang W ，Zhao D ，Niu Y ，Ding Q ，Li Y ，Leung PCK ，Wei D ，Chen ZJ ... -  《-》

Comprehensive assessment the expression of core elements related to IGFIR/PI3K pathway in granulosa cells of women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common multisystem endocrinopathy in women, characterized by chronic hyperandrogenism, ovulatory dysfunction, and polycystic ovaries. But its etiology remains elusive. A plethora of information suggests phosphatidylinositol-3-kinase (PI3K) pathway is key to the pathogenesis of PCOS but little is known about the expression pattern and possible role of insulin like growth factor 1 receptor (IGFIR)/PI3K pathway in PCOS. The goal of this study was to determine whether the core elements of the IGF1R/PI3K pathway were differentially expressed in GCs isolated from PCOS. Western blot (WB) and reverse transcription-polymerase chain reaction (RT-PCR) for IGF1R, insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2) and phosphatase and tensin homolog (PTEN) related to IGFIR/PI3K pathway were performed in GCs isolated from 60 PCOS patients and 60 controls. Compared to controls, body mass index (BMI), the levels of fasting plasma glucose (FPG), fasting insulin (FINS), anti-Mullerian hormone (AMH), testosterone (T), luteotropic hormone (LH), homeostasis model assessment of insulin resistance (HOMA-IR), antral follicle count (AFC) were markedly elevated while follicle stimulating hormone (FSH) decreased (p < 0.05). Furthermore, at both mRNA and protein levels, the expression of IGF1R, IRS1, IRS2 were significantly increased whereas PTEN was dramatically decreased in PCOS patients (p <  0.05). Our findings indicate that IGFIR/PI3K pathway is differently expressed in PCOS GCs compared with controls, with IGFIR, IRS1, IRS2 significantly increased while PTEN decreased. Thus, our study probably provides new evidences about the pathogenesis of PCOS in term of molecular mechanism.

He T ，Liu Y ，Zhao S ，Liu H ，Wang Z ，Shi Y ... -  《-》