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

Dilaver, N; Pellatt, L; Jameson, E; Ogunjimi, M; Bano, G; Homburg, R; D Mason, H; Rice, S (2019) The regulation and signalling of anti-Müllerian hormone in human granulosa cells: relevance to polycystic ovary syndrome. Hum Reprod, 34 (12). pp. 2467-2479. ISSN 1460-2350 https://doi.org/10.1093/humrep/dez214
SGUL Authors: Rice, Suman

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Abstract

STUDY QUESTION: 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? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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'. STUDY FUNDING/COMPETING INTEREST(S): 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. TRIAL REGISTRATION NUMBER: N/A.

Item Type: Article
Additional Information: This is a pre-copyedited, author-produced version of an article accepted for publication in Human Reproduction following peer review. The version of record Nafi Dilaver, Laura Pellatt, Ella Jameson, Michael Ogunjimi, Gul Bano, Roy Homburg, Helen D Mason, Suman Rice, The regulation and signalling of anti-Müllerian hormone in human granulosa cells: relevance to polycystic ovary syndrome, Human Reproduction, Volume 34, Issue 12, December 2019, Pages 2467–2479 is available online at: https://doi.org/10.1093/humrep/dez214
Keywords: FSH receptor, SMAD, androgens, anti-Müllerian hormone, aromatase, folliculogenesis, oestradiol, polycystic ovary syndrome, FSH receptor, SMAD, androgens, anti-Müllerian hormone, aromatase, folliculogenesis, oestradiol, polycystic ovary syndrome, 16 Studies In Human Society, 11 Medical And Health Sciences, Obstetrics & Reproductive Medicine
SGUL Research Institute / Research Centre: Academic Structure > Institute of Medical & Biomedical Education (IMBE)
Academic Structure > Institute of Medical & Biomedical Education (IMBE) > Centre for Biomedical Education (INMEBE)
Journal or Publication Title: Hum Reprod
ISSN: 1460-2350
Language: eng
Dates:
DateEvent
December 2019Published
18 November 2019Published Online
23 September 2019Accepted
Publisher License: Publisher's own licence
PubMed ID: 31735954
Go to PubMed abstract
URI: https://openaccess.sgul.ac.uk/id/eprint/111402
Publisher's version: https://doi.org/10.1093/humrep/dez214

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