Article
Expression of genes that regulate follicle development and maturation during ovarian stimulation in poor responders

https://doi.org/10.1016/j.rbmo.2020.05.012Get rights and content

Abstract

Research question

Sex hormone-binding globulin (SHBG), androgen receptor (AR), LH beta polypeptide (LHB), progesterone receptor membrane component 1 (PGRMC1) and progesterone receptor membrane component 2 (PGRMC2) regulate follicle development and maturation. Their mRNA expression was assessed in peripheral blood mononuclear cells (PBMC) of normal and poor responders, during ovarian stimulation.

Design

Fifty-two normal responders and 15 poor responders according to the Bologna criteria were enrolled for IVF and intracytoplasmic sperm injection and stimulated with 200 IU of follitrophin alpha and gonadotrophin-releasing hormone antagonist. HCG was administered for final oocyte maturation. On days 1, 6 and 10 of stimulation, blood samples were obtained, serum hormone levels were measured, RNA was extracted from PBMC and real-time polymerase chain reaction was carried out to identify the mRNA levels. Relative mRNA expression of each gene was calculated by the comparative 2−DDCt method.

Results

Differences between mRNA levels of each gene on the same time point between the two groups were not significant. PGRMC1 and PGRMC2 mRNA levels were downregulated, adjusted for ovarian response and age. Positive correlations between PGRMC1 and AR (standardized beta = 0.890, P < 0.001) from day 1 to 6 and PGRMC1 and LHB (standardized beta = 0.806, P < 0.001) from day 1 to 10 were found in poor responders. PGRMC1 and PGRMC2 were positively correlated on days 6 and 10 in normal responders.

Conclusions

PGRMC1 and PGRMC2 mRNA are significantly decreased during ovarian stimulation, with some potential differences between normal and poor responders.

Introduction

Poor responders constitute a challenging group of patients to treat in reproductive medicine (Alviggi et al., 2018). The difficulties in their management and the disappointing results obtained in clinical practice are caused by the high heterogeneity for definition of poor ovarian response, as well as to the limited knowledge of its pathophysiology (Polyzos and Devroey, 2011). The need to decrease this heterogeneity led to the establishment of the Bologna criteria (Ferraretti et al., 2011). So far, limited gene expression studies have been conducted on poor responders (Zhao et al., 2014), targeting the mRNA expression levels of genes, which interact to regulate follicle development and maturation. These are interrelated as they affect the production, secretion and function of the reproductive hormones. Such genes are the sex hormone-binding globulin (SHBG), androgen receptor LH beta polypeptide (LHB), progesterone receptor membrane component 1 (PGRMC1) and progesterone receptor membrane component 2 (PGRMC2).

Sex hormone-binding globulin is regulated by FSH (Munell et al., 2002) to transport active steroids in peripheral blood cells (Hammond, 2011) and to regulate the actions of androgens on target tissues (Botwood et al., 1995). It interacts with androgen receptors, sustains the access of androgens to the specific receptor and enhances the activity of androgen receptors by promoting androgen receptor retention in the nucleus (Hong et al., 2011). Androgen receptor expression is decreased by progesterone and is upregulated by androgens and oestrogens, i.e. oestradiol (Hulchiy et al., 2016). Androgen receptors bind testosterone and 5a-dihydrotestosterone, mediates the biological effects of androgens (Heinlein and Chang, 2002) and enhances the expression of the FSH receptor in granulosa cells (Oudendijk et al., 2012). Furthermore, androgen receptor signalling significantly affects the neuroendocrine pathways that regulate the gonadotrophin-releasing hormone (GnRH), FSH and LH release (Walters et al., 2009). GnRH regulates LHB expression (Andrade et al., 2013; Riccetti et al., 2017). Particularly, LHB mRNA levels are increased by rapid and high GnRH pulses (Miller et al., 2012), as well as by oestradiol and progesterone in gonadotrophic cells (Kanasaki et al., 2012). Progesterone is bound to PGRMC1 with high affinity (Petersen et al., 2013). In ovarian cells, PGRMC1 regulates progesterone's anti-mitotic and anti-apoptotic actions (Friel et al., 2015) and induces steroidogenesis (Li et al., 2016). PGRMC1 interacts with PGRMC2 (Peluso et al., 2014), and their expression significantly affects the number of the follicles developed after administration of exogenous gonadotrophins during ovarian stimulation (Sueldo et al., 2015).

Changes in factors that affect the regulation of gonadotrophin receptors and ovarian function can lead to poor ovarian response (POR) (Oudendijk et al., 2012), which is the precursor of ovarian insufficiency, in which the amount of existing oocytes is decreased and this cannot be reversed (Ferraretti et al., 2014). Inefficient or loss of androgen receptor function has been associated with elevated apoptosis of granulosa cells and with reduced ovulation caused by dysfunctional regulation of gonadotrophin production (Shiina et al., 2006; Walters et al., 2009; Walters et al., 2019), leading to primary ovarian insufficiency (POI) (Shiina et al., 2006; Kimura et al., 2007). Decreased mRNA expression of PGRMC1 in ovarian cells has also been associated with POI (Mansouri et al., 2008; Guo et al., 2016). Poor responders to ovarian stimulation may be characterized by diminished ovarian reserve (DOR) (Ferraretti et al., 2011). Women with DOR have been characterized by elevated PGRMC2 mRNA levels in granulosa cells compared with women with normal ovarian reserve (Skiadas et al., 2012). Noticeably, in low responders, SHBG levels are lower in blood and follicular fluid compared with high responders (Ben-Rafael et al., 1986).

The ovarian response to gonadotrophin stimulation is not easy to predict even when women have similar endocrine profiles (Cus et al., 2019). The tests used to assess ovarian reserve status have not been reliable (Ferraretti et al., 2011; Guo et al., 2014). A previously published study using microarray analysis of peripheral blood leukocytes revealed potential RNA signatures of neuropsychiatric disorders (Middleton et al., 2005). Therefore, using peripheral blood mononuclear cells (PBMC) may give an indication of gene expression changes in ovarian cells. Data on the expression of the above-mentioned genes in PBMC during ovarian stimulation are lacking. Therefore, the aim of this study was to assess the mRNA expression levels of SHBG, AR, LHB, PGRMC1 and PGRMC2 genes and to investigate any correlations among them in PBMC of normal and poor responders, during ovarian stimulation.

Section snippets

Eligibility criteria

This prospective study was conducted at the Unit for Human Reproduction and at the Laboratory of Genetics of the 1st Department of Obstetrics and Gynaecology, School of Medicine, Aristotle University of Thessaloniki, Papageorgiou General Hospital. The study was approved by the hospital's Institutional Review Board (561; 13 July 2015), as well as by the Bioethics Committee (104; 8 August 2014) of Aristotle University of Thessaloniki. All patients provided written informed consent before

Patient population

In total, 67 patients were included in the study and were categorized into normal (n = 52) or poor (n = 15) responders. Their baseline and ovarian stimulation characteristics are presented in Table 1. Age (P < 0.001), basal FSH (P = 0.004) oestradiol levels (P < 0.001), AMH levels (P = 0.004) before ovarian stimulation, AFC (P < 0.001), duration of stimulation (P = 0.020), total rFSH units administered (P = 0.043), and numbers of follicles measuring 15–20 mm (P < 0.001) of the mature MII

Discussion

The present study showes significant downregulation of PGRMC1 and PGRMC2 mRNA expression in PBMC adjusted for the type of ovarian response and age, as well as for the number of follicles measuring 15–20 mm, during ovarian stimulation with GnRH antagonists and gonadotrophins for IVF/ICSI. Positive correlations between PGRMC1 and AR on day 6 and PGRMC1 and LHB on day 10 were found in poor responders, in whom PGRMC1 mRNA was downregulated from day 1 to 10. In normal responders, PGRMC2 was

Acknowledgements

The authors would like to thank the staff of the 1st Department of Obstetrics and Gynaecology at Papageorgiou General Hospital, Thessaloniki, Greece, for their help in this project regarding women's enrolment and briefing, as well all women who gave their written consent to participate in the study.

Christine Vaitsopoulou is a PhD candidate at Aristotle University of Thessaloniki, with a master's degree in applications of molecular biology, molecular genetics and diagnostic markers. She works at the Unit of Assisted Reproduction, Aretaieion Hospital, 2nd Department of Obstetrics and Gynecology, and National and Kapodistrian University of Athens.

Key message

PGRMC1 and PGRMC2 are downregulated at the mRNA level during ovarian stimulation in peripheral blood mononuclear cells.

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  • Cited by (0)

    Christine Vaitsopoulou is a PhD candidate at Aristotle University of Thessaloniki, with a master's degree in applications of molecular biology, molecular genetics and diagnostic markers. She works at the Unit of Assisted Reproduction, Aretaieion Hospital, 2nd Department of Obstetrics and Gynecology, and National and Kapodistrian University of Athens.

    Key message

    PGRMC1 and PGRMC2 are downregulated at the mRNA level during ovarian stimulation in peripheral blood mononuclear cells. PGRMC1 is highly and positively correlated with androgen receptors and LHB in poor responders, on days 6 and 10, respectively. The mRNA expression of these genes may be used to study ovarian response.

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