Investigating the role of androgens in myometrial biology during pregnancy

Abstract

Understanding the physiology of pregnancy enables effective management of pregnancy complications that could otherwise be life threatening for both mother and fetus. A functional uterus (a) retains the fetus in utero during pregnancy without initiating stretch-induced contractions and (b) is able to dilate the cervix and contract the myometrium at term to deliver the fetus. The onset of labour is associated with successful cervical remodelling and contraction of myometrium, arising from concomitant activation of uterine immune and endocrine systems. A large body of evidence suggest that the action of local sex hormones may drive changes occurring in the uterine microenvironment at term. Although there have been a number of studies considering the potential role(s) played by progesterone and estrogens at the time of parturition, the role of androgens has received less scrutiny. The overarching aim of this thesis was to investigate the potential roles of androgens in myometrial biology at the time of pregnancy. We examined both the genetranscription dependent (genomic) and independent (non-genomic) action of androgens on the uterine smooth muscle, employing in vitro, ex vivo, and in vivo approaches. We found that the androgen receptor (AR) mRNA was significantly increased in the myometrium during labour when compared to the term non-labouring myometrium. Our gene expression studies revealed that ligand-dependent AR signalling in the myometrium might play a role in regulation of uterine smooth muscle cell contractility. We explored the effect of androgens on contraction of uterine smooth muscle strips obtained from both human myometrial biopsies collected at term and murine uterine horns. We found that testosterone (T) and dihydrotestosterone (DHT) in a range of 10-100 μM concentrations rapidly relaxed spontaneous and oxytocin-initiated contractions. The relaxant effect was not mediated by the classical intracellular AR nor was cell-surface initiated as shown by experiments employing a specific AR antagonist (flutamide) and a cell-surface impermeable androgen (TBSA). We investigated whether the relaxant effect was specific to androgens or a generic effect of sex hormones. We demonstrated that both estradiol (E2) and progesterone (P4) were also capable of relaxing the human and murine myometrium at the same dose range. In addition, a sex hormone “cocktail” (all four sex hormones combined at 10 μM dose each) mimicked the relaxant effect that each individual sex hormone elicited at a 40 μM dose, implying that the effect was possibly attributable to the steroid structure of the sex hormones. To study the underlying molecular events that mediate the relaxant effect of sex hormones observed ex vivo, we employed two human myometrial cell lines namely PHM1-41s and UtSMCs. We demonstrated that the androgen-induced relaxation in vitro was not induced by cell death but was mediated by a physiological mechanism whereby incubation with the androgen impaired the stimulated-Ca2+ entry into the uterine myocytes, which in turn resulted in poor phosphorylation of myosin light chain protein. Finally, we conducted a pilot study to explore the hypothesis that administration of androgen could relax the uterine muscle in vivo. We utilised a mouse model of infection-induced preterm labour, where infection was induced by intrauterine administration of liposaccharide (LPS) on day 17 of murine pregnancy. Our preliminary data showed that intrauterine administration of DHT on day 17 did not significantly reduce the rate of LPS-induced preterm birth in the doses tested in this study. In conclusion, the androgen-induced in vitro tocolysis appears to be sex hormone-specific rather than androgen-specific. Therefore, sex hormones might have the potential to be used for effective in vivo tocolysis to inhibit premature-initiated contractions. Our investigation of the androgen-dependent signalling in the myometrium contributed to the development of novel hypotheses regarding the role of androgens in the regulation of the phenotypic transition of MSMCs during pregnancy. These hypotheses remain to be confirmed in future studies.