The mitochondrial dysfunction in the granulosa cells of women with PCOS is caused by alterations in oxidative phosphorylation (OXPHOS) and the unfolded protein response, and leads to follicular dysfunction, particularly in insulin-resistant women.
Polycystic ovarian syndrome (PCOS) is a prevalent benign gyneacological disorder, affecting approximately 4-20% women worldwide and causing infertility. To achieve pregnancy and a live birth, women with PCOS require assisted reproduction techniques 8-10x more than healthy women. PCOS is a heterogeneous disorder, with elusive etiologies and different clinical presentations. Women with PCOS may present with amenorrhea, oligomenorrhea, or hypomenorrhea; enlarged and/or cystic ovaries (the polycystic ovarian morphology [PCOM]); hirsutism, acne or oily skin, and androgenic alopecia associated with clinical hyperandrogenism; infertility; and several dermatological conditions. In 2012, the NIH consensus panel established four phenotypes of PCOS, based on different combinations of these clinical and metabolic criteria, and these provided the foundation for the most recent international guidelines (2018), which additionally included insulin resistance (IR) and hyperinsulinemia as key PCOS characteristics. The PCOM is characterized by a higher number of follicles arrested at all developmental stages, often containing immature oocytes with compromised quality. This phenotype results from the relative deficiency in FSH, which impedes follicle growth and synthesis of estrogen from the granulosa cells (GCs), and excess in LH and LH receptor, that lead to the terminal differentiation of the GCs. Collectively, these processes contribute to the anovulation and elevated circulating androgen levels experienced by patients with PCOS. Specifically, the aberrant neuroendocrine signaling causes the theca cells to produce more androgens and secrete higher levels of dehydroepiandrosterone and 17-hydroxyprogesterone in response to LH, compared to normoresponder patients. While augmented circulating androgen levels are observed in approximately 60–80% of patients with PCOS, biochemical hyperandrogenism does not always correlate with manifestations of clinical hyperandrogenism, thus it is immediately to establish focused evaluation criteria and maintain detailed patient histories to be able to differentiate possible etiologies. On the other hand, the bioavailability of testosterone is enhanced by IR, via reduction of sex hormone binding globulin levels in serum, and hyperinsulinaemia has been associated with the antral follicle dysfunction and anovulation in PCOS, though the exact mechanisms remain unclear. Clinically, almost 75% of PCOS patients present varying levels of IR, which is a major determinant of PCOS pathogenesis, independent of obesity. Insulin signaling through the insulin-like growth factor receptors (IGFRs), which are overexpressed in the ovaries of obese women with PCOS, is postulated to stimulate the theca production of androgens, lead to hyperplasia of the thecal/stromal compartment, promote the premature acquisition of the LH receptors, and ultimately, leads to early follicular luteinization. Despite the clinical implications of IR in women with PCOS, IR testing is not routinely performed for these patients, and there is currently no agreement regarding the best methodology for diagnosing IR and characterizing its severity in PCOS. There is increasing interest for elucidating the connection between oxidative stress and PCOS pathogenesis. For Study I, we conducted a literature review to compile evidence on how mitochondrial dysfunction in women with PCOS is related to IR, diminished oocyte competence and metabolic abnormalities in leukocytes. Mitochondrial dysfunction can be driven by the presence of one or several factors simultaneously: mutations in the mitochondrial DNA of GCs; cellular inability to respond to oxidative stress, specifically a detrimental accumulation of reactive oxygen species (ROS); and a poor response to the accumulation of unfolded proteins (UPRmt). Likewise, another response to unfolded proteins occurs in the endoplasmic reticulum (UPRre) and may influence GCs competency. Failure to restore cellular homeostasis by both responses triggers apoptosis processes in GCs. However, further studies are needed to unravel the specific mechanisms of PCOS pathogenesis in relation to mitochondrial dysfunction, and the response to unfolded proteins in IR and non-IR patients. As part of Study II, and with the aim of characterizing the transcriptomic profiles of granulosa cells and peripheral blood cells among PCOS patients, young poor responders (YPRs) and normoresponders (YNRs) patients, we recruited a total of 60 patients (nºPCOS = 20; nºYPRs = 20; nºYNRs = 20) undergoing controlled ovarian stimulation. After RNA extraction followed by sequencing and bioinformatics analysis to reveal differentially expressed genes and enriched functions between groups, we uncovered several genes with potential effect on PCOS pathogenesis. Among granulosa cells from PCOS and YNRs patients, we revealed dysregulation of MBE2M and MTMR9 both associated with apoptotic processes, while among PCOS and YPRs patients, we determined an overexpression of oxidative phosphorylation-associated genes in mitochondria, which may be related to decreased mtDNA and oxidative stress. Among peripheral blood cells in PCOS and YNRs patients, we found over-expression of the RNA component of telomerase in PCOS patients, which may be due to oxidative stress; and down-regulation of the RAB4B gene, whose deficiency has been associated with the development of insulin resistance. In the comparison between PCOS and YPRs, we highlighted ZPF57 and PXDN, both downregulated in the PCOS group and related to extracellular matrix development and DNA methylation, respectively. In Study III, we recruited 60 patients: 40 of them with PCOS (20 with IR and 20 without IR) and 20 YNRs to compare the expression of genes involved in the responses to proteins unfolded in the mitochondria and endoplasmic reticulum of granulosa cells. After a reverse transcription protocol from RNA extracted from granulosa cells and subsequent statistical analysis between groups, we revealed the overexpression, especially significant in the group of PCOS patients with IR, of the chaperones HSP10 and HSP40 of the UPRmt response. These chaperones promote cell survival under stress due to misfolded proteins that we found overexpressed in GCs of patients, which would explain an attempt of GCs to respond to high stress conditions leading to apoptosis. HSP10, furthermore, suppresses IGF1R polyubiquitination, preventing its degradation, and potentially contributing to the pathogenesis of PCOS in IR patients by increasing androgen biogenesis in theca cells, due to an increase in LH receptor acquisition. Similarly, PCOS women, especially with IR, had higher expression of IRE1, ATF4 and XBP1 than controls, being chaperone regulators (such as Bip) that promote stress-relieving mechanisms due to accumulation of unfolded proteins in the ER. For both IR and non-IR patients, overexpression of CHOP, a pro-apoptotic factor, indicates that homeostasis could not be recovered in GCs, probably leading to an apoptotic burst. In view of the results obtained in Studies II and III, and in line with the literature review of Study I, we demonstrate that PCOS-related follicular dysfunction is mediated by alterations in different molecular mechanisms in the mitochondria and endoplasmic reticulum of the granulosa cells. These complex factors linked to the proper development of follicles reaffirm the need for further studies to develop therapeutic strategies aimed at alleviating cellular stress, especially in patients with insulin resistance, and ultimately improving the developmental competence of oocytes in PCOS patients.
Tesis Doctoral leída en la Universidad Rey Juan Carlos de Madrid en 2023. Supervisors: Juan Antonio Garcia Velasco y Emre Seli
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