Neuroendocrine characteristics of induced pluripotent stem cells from polycystic ovary syndrome women
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Polycystic ovary syndrome (PCOS) is a common female reproductive endocrinopathy that afflicts up to 10%–15% of women in reproductive age worldwide (Nestler, 2016). Women with PCOS exhibit hyperandrogenism, intermittent/absent menstrual cycles, and polycystic ovaries on ultrasound (Rotterdam, 2004). The pathophysiology of PCOS extends beyond infertility and hirsutism to hypothalamic neuroendocrine dysfunction (Goodarzi et al., 2011). Most women with PCOS exhibit increased luteinizing hormone (LH) levels, resulting from high-frequency gonadotropin-releasing hormone (GnRH) secretion (Cimino et al., 2016). Prenatal testosterone (T) treatment in sheep results in disrupted steroid feedback on gonadotropin release, which increases pituitary sensitivity to GnRH and subsequently leads to LH hypersecretion (Sullivan and Moenter, 2004; Cardoso et al., 2016). A recent study shows that GnRH-dependent LH pulsatility and secretion are elevated by anti-Müllerian hormone (AMH) in PCOS disease. The increased prenatal AMH reprograms fetus and induces PCOS in adults (Tata et al., 2018). Furthermore, the androgen receptor (AR) plays a role in hyperandrogenism and ovarian folliculogenesis in PCOS (Wang et al., 2015; Abbott, 2017). However, the disease mechanism behind PCOS remains unclear, and current management focuses on treating the symptoms but not the mechanism (Chen et al., 2016; Shi et al., 2018). A further understanding of this disease is necessary to uncover the pathology of PCOS and develop new potential therapeutic avenues and drugs.
Next, the total RNA was extracted from PCOS- and non-PCOS-derived iPSCs for RNA microarray analysis. The global transcriptional genes of PCOS-derived iPSCs were identified and enriched by gene ontology (GO). Filtering by P value less than 0.01 and fold-changes of more than 2, 2,904 differentially expressed genes (DEGs) were collected between PCOS- and non-PCOS-derived iPSCs. Among these DEGs, we were interested in genes related to neuroendocrine and metabolic processes in PCOS. The enriched top 40 up- and down-regulated genes were shown in heatmaps (Fig. 1D). The GO enrichment revealed that down-regulated transcripts were associated with neurogenesis, enteroendocrine cell differentiation and LDL particle binding process. Up-regulated transcripts were enriched in neural crest cell development, progesterone receptor pathway and cholesterol storage process (Fig. 1E). We focused on genes of GABA receptor, CYP family, TGF-β pathway and estrogen receptor pathway in neuroendocrine processes (Fig. 1F). Then seven significantly modulated genes (FBP1, PYGL, GAPDH, KDM1A, STAT5, GPI and UGP2) were verified by RT-qPCR, according to functional annotation and their fold change (Fig. 1G). Moreover, the protein levels of above genes were verified by Western blot in PCOS-derived iPSCs, demonstrating the similar changes with mRNA levels (Fig. 1H). These genes (FBP1, PYGL, GAPDH, GPI and UGP2) involved in glycolysis process were expressed abnormally, indicating deregulation of glucose metabolism (glycolysis and gluconeogenesis) in PCOS (Fig. 1J). In addition, we measured the expression levels of testosterone (T) and estradiol (E2) in the cultures of iPSCs via ELISA (enzyme-linked immunosorbent assay), to verify the neuroendocrine characteristics in PCOS. The results showed that PCOS-derived iPSCs secreted greater T significantly than non-PCOS-derived iPSCs, while there were no significant differences in E2 level between PCOS and non-PCOS-derived iPSCs (Fig. 1H). The change of T level was consistent with the clinical hyperandrogenic feature of PCOS (Haouzi et al., 2012). In order to compare the differences of metabolic function after reprogramming, we measured the mitochondrial respiration ability of ovarian granulosa cells (GCs) and iPSCs from PCOS and non-PCOS patients. The GCs were obtained from other three PCOS and non-PCOS patients. The results showed that the maximal respiration ability of GCs and iPSCs from PCOS patients were decreased (Fig. 1K and 1L), which suggested the metabolic ability of PCOS was deregulated. The attenuated mitochondrial ability corresponded to the deregulated endocrine and metabolism genes of functional annotation.
PCOS is a complicated and multifactorial metabolic syndrome. Elevated androgen levels, disturbed menstrual cycles and increased LH are the main features of PCOS disease. The accurate regulation of the hypothalamic-pituitary-gonadal axis involves some intrinsic factors including estrogen, progesterone, activin and inhibin, and extrinsic factors including neurotransmitters and stress. Any interference with or deregulation of these factors may result in reproductive endocrine irregularity. GnRH neurons play a role in the central regulation of fertility. A pulsatile signal of GnRH is necessary for the secretion of LH and FSH (follicle-stimulating hormone), which promotes steroidogenesis and follicular development in females (Chaudhari et al., 2018). In the altered hypothalamic-pituitary-gonadal axis, high levels of LH reflect increased GnRH release. GnRH receives input from GABAergic neurons, and GABA type A receptor activation may play a part in regulating steroids (Sullivan and Moenter, 2004) (Fig. 2F). Those deregulated genes enriched in glucose metabolic process, demonstrated depressed glycolysis process, decreased glucose transport and increased gluconeogenesis, which can attenuate metabolic ability and lead to glucose intolerance and insulin resistance in PCOS. Moreover, the differentially expressed genes were partially consistent with previously reported microarray profiles of cumulus cells of PCOS patients (Haouzi et al., 2012). However,the clinical cause and molecular mechanism of PCOS is still unclear. PCOS is considered a heredity disease with interaction of genomic variants and environmental factors. Three patients in each group were not enough to generalize all types of PCOS, although three iPSC lines showed the consistent change trends. Furthermore, it is necessary to be replicated and verified in other cohorts and clinical samples.
In this study, we identified neuroendocrine and metabolic characteristics of iPSCs from women with PCOS and non-PCOS. GO analysis on transcripts of PCOS-derived iPSCs reflected the neuroendocrine abnormalities of PCOS, and the mitochondrial respiration ability unfolded metabolic weaknesses in PCOS disease. The NSCs differentiated from PCOS-derived iPSCs showed decreased mitochondrial respiratory ability, similar to iPSCs and primary GCs from PCOS patients. This study provided a cell model demonstrating PCOS characteristics in vitro, which can be used to further understand the pathology and develop new potential therapeutic avenues of PCOS.
This work was supported in part by the National Key R&D Program of China (Nos. 2017YFC1001003 and 2016YFC1000601), the National Natural Science Funds (Grant Nos. 81671419, 81571400, 81771580, 81471427, 81570101, 81741006 and 81521002) and the Science and Technology Project of Guangzhou (201803010048), Major Science and Technology Projects of Yunnan Province (2017ZF028), the Interdisciplinary Medicine Seed Fund of Peking University (BMU2017MC007).
Zheying Min, Yue Zhao, Jing Hang, Yun Ren, Tao Tan, Yong Fan and Yang Yu declare that all authors have no conflicts of interest associated with the contents of this manuscript.
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the 1975 Declaration of Helsinki, as revised in 2000 (5). Informed consent was obtained from all patients prior to inclusion in the study.
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