Persistent genital hyperinnervation following progesterone administration to adolescent female rats1 Running title: Adolescent progesterone & vaginal hyperinnervation Summary sentence: High dose progesterone administration during adolescence, which strongly predisposes women to genital hyperinnervation and pelvic pain (vulvar

Provoked vestibulodynia, a female pelvic pain syndrome affecting substantial numbers of women, is characterized by genital hypersensitivity and sensory hyperinnervation. Previous studies have shown that the risk of developing provoked vestibulodynia is markedly elevated following adolescent use of oral contraceptives with high progesterone content. We hypothesized that progesterone, a steroid hormone with known neurotropic properties, may alter genital innervation through direct or indirect actions. Female Sprague Dawley rats received progesterone (20 mg/kg sc) from days 20-27; tissue was removed for analysis in some rats on Day 28, while others were ovariectomized on Day 43 and infused for 7 days with vehicle or 17beta estradiol. Progesterone resulted in overall increases in vaginal innervation at both Day 28 and 50 due to proliferation of peptidergic sensory and sympathetic (but not parasympathetic) axons. Estradiol reduced innervation in progesterone-treated and untreated groups. To assess mechanisms of sensory hyperinnervation, we cultured dissociated dorsal root ganglion neurons and found that progesterone increases neurite outgrowth by small unmyelinated (but not myelinated) sensory neurons, was receptor-mediated, and was non-additive with NGF. Pre-treatment of ganglion with progesterone also increased neurite outgrowth in response to vaginal target explants. However, pre-treatment of BOR Papers in Press. Published on October 30, 2014 as DOI:10.1095/biolreprod.114.121103 Copyright 2014 by The Society for the Study of Reproduction. 2 vaginal target with progesterone did not improve outgrowth. We conclude that adolescent progesterone exposure may contribute to provoked vestibulodynia by eliciting persistent genital hyperinnervation via a direct effect on unmyelinated sensory nociceptor neurons, and that estradiol, a well-documented therapeutic, may alleviate symptoms in part by reducing progesterone-induced sensory hyperinnervation. INTRODUCTION Provoked vestibulodynia (PVD), formerly called vulvar vestibulitis syndrome, is a vulvodynial pain syndrome with a reported lifetime prevalence of approximately 16%, concurrently affecting up to 6% of adult females [1]. PVD can affect women of all ages and markedly compromises quality of life. PVD is characterized histologically by the presence of inflammatory cells and marked proliferation of vestibular epithelial and subepithelial axons [2-8]. While some pain relief is afforded by topical estrogen as well as neuroleptics and other centrally acting drugs [9], surgical excision may provide better outcomes; some 80% of patients are reported to experience relief when the hyperinnervated region is surgically excised [10], consistent with a peripheral origin of the pain. PVD is a major female health problem that demands a better understanding of its etiology and strategies for prevention. Mechanisms responsible for PVD are unknown and multiple factors are implicated. These include repeated vaginal infections [11], co-morbid painful conditions [12], and childhood physical or sexual abuse [13]. Some studies have linked PVD risk to use of oral contraceptives. Oral contraceptive use is reported to increase overall relative risk of developing PVD; however risk was most pronounced in females receiving formulations containing high amounts of the female gonadal steroid hormone, progesterone. Relative risk was greatest when first use occurred between 10-15 years of age, with these individuals showing more than a 9-fold increase [1, 14-16], strongly implicating adolescent progesterone exposure as a factor in some cases of PVD. Although the role of oral contraceptives in PVD remains controversial [17], progesterone should be given careful consideration given its potent effects on the nervous system. Progesterone is reported to be neuroprotective, to promote myelination, and to enhance axon outgrowth [18-23]. Further, vaginal tissues are well imbued with abundant progesterone receptors [24]. Therefore, progesterone acting directly on neurons or indirectly via vaginally derived neuroactive proteins could contribute to neural changes observed in PVD. In the present study, we assessed progesterone’s ability to induce genital neuroplasticity. We treated sexually immature adolescent rats with progesterone and quantified vaginal innervation immediately after treatment and at sexual maturity. Because another gonadal steroid, estrogen (17β-estradiol, E2), is known to alter vaginal innervation [25-28] we assessed progesterone’s effects under lowE2 conditions (i.e in the adolescent rat at d28 prior to reproductive hormonal cycling [29] and after ovariectomy [OVX] in the sexually mature rat at 50d [30]). We also examined E2’s ability to modulate innervation after progesterone treatment in the sexually mature rat following OVX to eliminate the suppressive effect of this hormone on vaginal innervation density [25, 28]. In addition, we investigated progesterone’s mechanism of action in dissociated dorsal root ganglion (DRG) sensory neuron cultures and in vaginal and DRG explant co-culture experiments.