PMID- 35459945 OWN - NLM STAT- MEDLINE DCOM- 20220602 LR - 20240324 IS - 1460-2350 (Electronic) IS - 0268-1161 (Linking) VI - 37 IP - 6 DP - 2022 May 30 TI - At the crossroads of fertility and metabolism: the importance of AMPK-dependent signaling in female infertility associated with hyperandrogenism. PG - 1207-1228 LID - 10.1093/humrep/deac067 [doi] AB - STUDY QUESTION: What biological processes are linked to the signaling of the energy sensor 5'-AMP-activated protein kinase (AMPK) in mouse and human granulosa cells (GCs)? SUMMARY ANSWER: The lack of alpha1AMPK in GCs impacted cell cycle, adhesion, lipid metabolism and induced a hyperandrogenic response. WHAT IS KNOWN ALREADY: AMPK is expressed in the ovarian follicle, and its activation by pharmacological medications, such as metformin, inhibits the production of steroids. Polycystic ovary syndrome (PCOS) is responsible for infertility in approximately 5-20% of women of childbearing age and possible treatments include reducing body weight, improving lifestyle and the administration of a combination of drugs to improve insulin resistance, such as metformin. STUDY DESIGN, SIZE, DURATION: AMPK signaling was evaluated by analyzing differential gene expression in immortalized human granulosa cells (KGNs) with and without silencing alpha1AMPK using CRISPR/Cas9. In vivo studies included the use of a alpha1AMPK knock-out mouse model to evaluate the role of alpha1AMPK in folliculogenesis and fertility. Expression of alpha1AMPK was evaluated in primary human granulosa-luteal cells retrieved from women undergoing IVF with and without a lean PCOS phenotype (i.e. BMI: 18-25 kg/m2). PARTICIPANTS/MATERIALS, SETTING, METHODS: alpha1AMPK was disrupted in KGN cells and a transgenic mouse model. Cell viability, proliferation and metabolism were evaluated. Androgen production was evaluated by analyzing protein levels of relevant enzymes in the steroid pathway by western blots, and steroid levels obtained from in vitro and in vivo models by mass spectrometry. Differential gene expression in human GC was obtained by RNA sequencing. Analysis of in vivo murine folliculogenesis was performed by histology and immunochemistry, including evaluation of the anti-Mullerian hormone (AMH) marker. The alpha1AMPK gene expression was evaluated by quantitative RT-PCR in primary GCs obtained from women with the lean PCOS phenotype (n = 8) and without PCOS (n = 9). MAIN RESULTS AND THE ROLE OF CHANCE: Silencing of alpha1AMPK in KGN increased cell proliferation (P < 0.05 versus control, n = 4), promoted the use of fatty acids over glucose, and induced a hyperandrogenic response resulting from upregulation of two of the enzymes involved in steroid production, namely 3beta-hydroxysteroid dehydrogenase (3betaHSD) and P450 side-chain cleavage enzyme (P450scc) (P < 0.05, n = 3). Female mice deficient in alpha1AMPK had a 30% decrease in their ovulation rate (P < 0.05, n = 7) and litter size, a hyperandrogenic response (P < 0.05, n = 7) with higher levels of 3betaHSD and p450scc levels in the ovaries, and an increase in the population of antral follicles (P < 0.01, n = 10) compared to controls. Primary GCs from lean women with PCOS had lower alpha1AMPK mRNA expression levels than the control group (P < 0.05, n = 8-9). LARGE SCALE DATA: The FastQ files and metadata were submitted to the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB46048. LIMITATIONS, REASONS FOR CAUTION: The human KGN is a not fully differentiated, transformed cell line. As such, to confirm the role of AMPK in GC and the PCOS phenotype, this model was compared to two others: an alpha1AMPK transgenic mouse model and primary differentiated granulosa-lutein cells from non-obese women undergoing IVF (with and without PCOS). A clear limitation is the small number of patients with PCOS utilized in this study and that the collection of human GCs was performed after hormonal stimulation. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal that AMPK is directly involved in steroid production in human GCs. In addition, AMPK signaling was associated with other processes frequently reported as dysfunctional in PCOS models, such as cell adhesion, lipid metabolism and inflammation. Silencing of alpha1AMPK in KGN promoted folliculogenesis, with increases in AMH. Evaluating the expression of the alpha1AMPK subunit could be considered as a marker of interest in infertility cases related to hormonal imbalances and metabolic disorders, including PCOS. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by the Institut National de la Recherche Agronomique (INRA) and the national programme << FERTiNERGY >> funded by the French National Research Agency (ANR). The authors report no intellectual or financial conflicts of interest related to this work. R.K. is identified as personnel of the International Agency for Research on Cancer/World Health Organization. R.K. alone is responsible for the views expressed in this article and she does not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization. TRIAL REGISTRATION NUMBER: N/A. CI - (c) The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com. FAU - Froment, Pascal AU - Froment P AUID- ORCID: 0000-0002-7388-9598 AD - CNRS, IFCE, INRAE, Universite de Tours, PRC, Nouzilly, France. FAU - Plotton, Ingrid AU - Plotton I AUID- ORCID: 0000-0003-2539-0087 AD - Molecular Endocrinology and Rare Diseases, University Hospital, Claude Bernard Lyon 1 University, Bron, France. FAU - Giulivi, Cecilia AU - Giulivi C AD - Department of Molecular Biosciences, University of California Davis, School of Veterinary Medicine, Davis, CA, USA. AD - The MIND Institute, University of California Davis Medical Center, Sacramento, CA, USA. FAU - Fabre, Stephane AU - Fabre S AD - GenPhySE, Universite de Toulouse, INRAE, ENVT, Castanet-Tolosan, France. FAU - Khoueiry, Rita AU - Khoueiry R AD - Epigenetics Group, International Agency for Research on Cancer (IARC), Lyon, France. FAU - Mourad, Nizar I AU - Mourad NI AD - Pole de Chirurgie Experimentale et Transplantation, Universite Catholique de Louvain, Brussels, Belgium. FAU - Horman, Sandrine AU - Horman S AD - Pole of Cardiovascular Research, Institut de Recherche Experimentale et Clinique, Universite catholique de Louvain, Brussels, Belgium. FAU - Rame, Christelle AU - Rame C AD - CNRS, IFCE, INRAE, Universite de Tours, PRC, Nouzilly, France. FAU - Rouillon, Charlene AU - Rouillon C AD - CNRS, IFCE, INRAE, Universite de Tours, PRC, Nouzilly, France. FAU - Grandhaye, Jeremy AU - Grandhaye J AD - CNRS, IFCE, INRAE, Universite de Tours, PRC, Nouzilly, France. FAU - Bigot, Yves AU - Bigot Y AD - CNRS, IFCE, INRAE, Universite de Tours, PRC, Nouzilly, France. FAU - Chevaleyre, Claire AU - Chevaleyre C AD - CNRS, IFCE, INRAE, Universite de Tours, PRC, Nouzilly, France. FAU - Le Guevel, Remy AU - Le Guevel R AD - Plate-forme ImPACcell, Universite de Rennes 1, Rennes, France. FAU - Mallegol, Patricia AU - Mallegol P AD - SOPAM, U1063, INSERM, UNIV Angers, Angers, France. AD - Federative Structure of Research Cellular Interactions and Therapeutic Applications, SFR 4208 ICAT, Univ Angers, Angers, France. FAU - Andriantsitohaina, Ramaroson AU - Andriantsitohaina R AD - SOPAM, U1063, INSERM, UNIV Angers, Angers, France. AD - Federative Structure of Research Cellular Interactions and Therapeutic Applications, SFR 4208 ICAT, Univ Angers, Angers, France. FAU - Guerif, Fabrice AU - Guerif F AD - CECOS, Hopital Bretonneau, Tours, France. FAU - Tamburini, Jerome AU - Tamburini J AD - Universite de Paris, Institut Cochin, CNRS UMR8104, INSERM U1016, Paris, France. FAU - Viollet, Benoit AU - Viollet B AD - Universite de Paris, Institut Cochin, CNRS UMR8104, INSERM U1016, Paris, France. FAU - Foretz, Marc AU - Foretz M AD - Universite de Paris, Institut Cochin, CNRS UMR8104, INSERM U1016, Paris, France. FAU - Dupont, Joelle AU - Dupont J AD - CNRS, IFCE, INRAE, Universite de Tours, PRC, Nouzilly, France. LA - eng GR - 001/WHO_/World Health Organization/International PT - Journal Article PT - Research Support, Non-U.S. Gov't PL - England TA - Hum Reprod JT - Human reproduction (Oxford, England) JID - 8701199 RN - 80497-65-0 (Anti-Mullerian Hormone) RN - 9100L32L2N (Metformin) RN - EC 2.7.11.31 (AMP-Activated Protein Kinases) SB - IM MH - AMP-Activated Protein Kinases MH - Animals MH - Anti-Mullerian Hormone/metabolism MH - *Biological Phenomena MH - Female MH - Fertility MH - Humans MH - *Hyperandrogenism/complications MH - *Infertility, Female MH - *Metformin/pharmacology MH - Mice MH - *Polycystic Ovary Syndrome/metabolism OTO - NOTNLM OT - AMP-activated protein kinases OT - AMPK OT - androgens OT - anti-Mullerian hormone OT - fertility OT - granulosa cells OT - ovary OT - polycystic ovary syndrome OT - testosterone EDAT- 2022/04/24 06:00 MHDA- 2022/06/03 06:00 CRDT- 2022/04/23 05:24 PHST- 2021/07/26 00:00 [received] PHST- 2022/03/01 00:00 [revised] PHST- 2022/04/24 06:00 [pubmed] PHST- 2022/06/03 06:00 [medline] PHST- 2022/04/23 05:24 [entrez] AID - 6572684 [pii] AID - 10.1093/humrep/deac067 [doi] PST - ppublish SO - Hum Reprod. 2022 May 30;37(6):1207-1228. doi: 10.1093/humrep/deac067.