Increased uterine androgen receptor protein abundance results in implantation and mitochondrial defects in pregnant rats with hyperandrogenism and insulin resistance

Abstract

In this study, we show that during normal rat pregnancy, there is a gestational stage-dependent decrease in androgen receptor (AR) abundance in the gravid uterus and that this is correlated with the differential expression of endometrial receptivity and decidualization genes during early and mid-gestation. In contrast, exposure to 5α-dihydrotestosterone (DHT) and insulin (INS) or DHT alone significantly increased AR protein levels in the uterus in association with the aberrant expression of endometrial receptivity and decidualization genes, as well as disrupted implantation. Next, we assessed the functional relevance of the androgen-AR axis in the uterus for reproductive outcomes by treating normal pregnant rats and pregnant rats exposed to DHT and INS with the anti-androgen flutamide. We found that AR blockage using flutamide largely attenuated the DHT and INS-induced maternal endocrine, metabolic, and fertility impairments in pregnant rats in association with suppressed induction of uterine AR protein abundance and androgen-regulated response protein and normalized expression of several endometrial receptivity and decidualization genes. Further, blockade of AR normalized the expression of the mitochondrial biogenesis marker Nrf1 and the mitochondrial functional proteins Complexes I and II, VDAC, and PHB1. However, flutamide treatment did not rescue the compromised mitochondrial structure resulting from co-exposure to DHT and INS. These results demonstrate that functional AR protein is an important factor for gravid uterine function. Impairments in the uterine androgen-AR axis are accompanied by decreased endometrial receptivity, decidualization, and mitochondrial dysfunction, which might contribute to abnormal implantation in pregnant PCOS patients with compromised pregnancy outcomes and subfertility. Key messages The proper regulation of uterine androgen receptor (AR) contributes to a normal pregnancy process, whereas the aberrant regulation of uterine AR might be linked to polycystic ovary syndrome (PCOS)-induced pregnancy-related complications. In the current study, we found that during normal rat pregnancy there is a stage-dependent decrease in AR abundance in the gravid uterus and that this is correlated with the differential expression of the endometrial receptivity and decidualization genes Spp1, Prl, Igfbp1, and Hbegf. Pregnant rats exposed to 5α-dihydrotestosterone (DHT) and insulin (INS) or to DHT alone show elevated uterine AR protein abundance and implantation failure related to the aberrant expression of genes involved in endometrial receptivity and decidualization in early to mid-gestation. Treatment with the anti-androgen flutamide, starting from pre-implantation, effectively prevents DHT + INS-induced defects in endometrial receptivity and decidualization gene expression, restores uterine mitochondrial homeostasis, and increases the pregnancy rate and the numbers of viable fetuses. This study adds to our understanding of the mechanisms underlying poor pregnancy outcomes in PCOS patients and the possible therapeutic use of anti-androgens, including flutamide, after spontaneous conception.

Overactivation of the androgen receptor exacerbates gravid uterine ferroptosis via interaction with and suppression of the NRF2 defense signaling pathway

“…S2A−C). In agreement with our previous report [25], similar high levels of immunoreactivity of AR were found between DHT+INS–exposed pregnant rats treated with sesame oil (Fig. S1B) and with flutamide (without fetuses) (Fig.…”

“…We previously demonstrated that mid‐gestational co‐exposure to DHT and INS (which mimics the typical PCOS features) resulted in decreased levels of GSH and GPX4, increased iron deposition, and aberrant expression of ferroptosis‐associated genes [transferrin receptor ( Tfrc ), Slc7a11 , heme oxygenase‐1 ( Ho1 ), and metallothionein 1G ( Mt1g )] in the gravid uterus [23,31]. Pregnant rats co‐exposed to DHT and INS from early‐to‐mid gestation used in this study showed increased maternal androgen levels and exhibited a greater increase in glucose tolerance than control pregnant rats [25]. The maternal endocrine and metabolic alterations in this pregnant rat model were similar to those observed in pregnant women with PCOS [15–17].…”

“…In this study, we investigated a potential mechanism through which AR regulates gravid uterine ferroptosis, which is proposed to be involved in fetal loss in cases of PCOS [31]. This new information will help us better understand the therapeutic efficacy and mechanisms through which anti‐androgens can increase the chances of childbirth in pregnant PCOS patients [22] and PCOS‐like pregnant rats [25]. We show that as an anti‐androgen, flutamide inhibits DHT+INS–triggered uterine ferroptosis through the upregulation of SLC7A11, GSH, and GPX4 protein expression; the normalization of iron homeostasis and metabolism; and the suppression of 4‐HNE production; as well as the upregulation of NRF2 protein levels in pregnant rats with fetuses.…”

“…Based on our previous publication [25], the pregnant rats ( n = 55) were randomly divided into the control group (co‐treatment with saline and sesame oil, n = 26) or the experimental group (hyperandrogenism alone, n = 12; hyperandrogenism and insulin resistance, n = 17). The control group was treated intraperitoneally (i.p.)…”

“…The dose of INS was chosen because it induces metabolic disturbances, including peripheral and uterine insulin resistance in rats [36,40]. The body weight, circulating levels of androgens (testosterone, androstenedione, DHT, and dehydroepiandrosterone), and oral glucose tolerance were measured to confirm that hyperandrogenism/insulin resistance (HAIR) was induced by exposure to DHT and INS, as reported previously [25].…”