The Burden of Pelvic Pain Associated with Endometriosis among Women in Selected European Countries and the United States: A Restricted Systematic Review.

OBJECTIVE: To evaluate the burden of endometriosis-associated pelvic pain (EAPP) on health-related quality of life (HRQoL) among women living in similar socio-economic conditions. DATA SOURCES: Searches were performed in PubMed and Embase on September 26, 2022. The review was performed in conformity with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis protocol (PRISMA-P) and was registered on PROSPERO (ID: CRD42023370363). METHODS OF STUDY SELECTION: Due to the high volume of eligible publications following initial review, inclusion criteria were restricted to studies undertaken in France, Germany, Italy, Spain, the United Kingdom, and the United States. This restriction was applied before screening as these countries have broad social and economic similarities, and previous studies in the literature suggest pain reporting and experience are influenced by numerous socio-cultural factors. Eligible studies were those published between 2013 and 2022 and include a sample size of ≥50 participants. The search strategy identified all relevant publications relating to the burden of illness due to EAPP. A variety of terms are used in the literature to describe pain associated with endometriosis, and this was considered in the design of the search strategy and screening procedure. TABULATION, INTEGRATION, AND RESULTS: The database searches resulted in a total of 6139 records. After removal of duplicates, 3855 records were assessed further. A total of 27 publications were identified as eligible. Fourteen (52%) were from Italy, 5 (19%) were multinational studies, 4 (15%) were from the United States, 3 (11%) were from Spain, and 1 (4%) was from Germany. Most studies were cross-sectional (n = 15; 56%); 7 (26%) were case-control studies; 3 (11%) were cohort studies; and 2 (7%) were longitudinal studies. These publications collectively highlighted an association between EAPP and reduced HRQoL. Several studies showed that EAPP was associated with lower HRQoL when compared with endometriosis without pain and potentially with chronic pelvic pain caused by other conditions, although the evidence is limited in this case. Moreover, the studies reported detrimental effects on general HRQoL, mental health functioning, and sexual functioning, culminating in reduced work productivity and difficulties in performing everyday activities. The associations were generally similar across study populations, including adolescents, as well as younger and older women. Results were consistent across the range of different patient-reported outcome tools used to assess HRQoL. CONCLUSION: The existing literature suggests that, among women in selected European countries and the United States, EAPP is associated with reduced HRQoL, including impaired mental and sexual functioning, as well as reduced work performance and productivity; each of which may contribute to the societal burden of endometriosis.

Scholarly activity following National Institutes of Health Women's Reproductive Health Research K12 training-a cohort study.

BACKGROUND: National Institutes of Health funding to address basic reproductive health for common female conditions remains disproportionately low, in part because of low success rates of grant applications by obstetrician-gynecologists. OBJECTIVE: This study aimed to evaluate the scholarly productivity of individuals supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development Women's Reproductive Health Research K12 career development award, created to advance careers of obstetrician-gynecologist physician-scientists. STUDY DESIGN: We performed a cohort study of individuals who completed at least 2 years of Women's Reproductive Health Research training by June 30, 2015, and had at least 5-year follow-up. Earliest training start date was December 1, 1998. Primary outcomes from public data sources (National Institutes of Health RePORTER, PubMed, iCite) were (1) number of total and R01 National Institutes of Health grants as principal investigator; (2) numbers of total and first and last author publications; and (3) median and highest publication impact factor measured by the relative citation ratio. Secondary outcomes from an email survey subcohort were total number of research grants, federally funded grants, and number of National Institutes of Health grants as coinvestigator; institutional promotions and academic appointments, national and National Institutes of Health leadership roles; and career and mentorship satisfaction. Outcomes were recorded at 5, 10, and 15 years postgraduation, and aggregate anonymized data were divided into 3 groups using Women's Reproductive Health Research completion dates: June 30 of 2005, 2010, and 2015. Temporal trends were assessed. Results were stratified by gender, number of awarded grant cycles (1-2 vs 3-4), and specialty type. Analyses used Fisher exact or Pearson chi-square tests, and Mantel-Haenszel tests of trend. RESULTS: The distribution of the cohort (N=178) by graduation completion date was: on or before June 30, 2005 (57 [32%]); July 1, 2005 to June 30, 2010 (60 [34%]); and July 1, 2010 to June 30, 2015 (61 [34%]). Most participants were female (112 [64%]) and maternal-fetal medicine trained (53 [30%]), followed by no fellowship (50 [28%]). Of the 178 participants, 72 (40%) received additional National Institutes of Health funding as a principal investigator, 45 (25%) received at least 1 R01, and 23 (13%) received 2 to 5 R01s. There were 52 (31%) scholars with >10 first author publications, 66 (39%) with >10 last author publications, and 108 (63%) with ≥25 publications. The highest relative citation ratio was a median of 8.07 (interquartile range, 4.20-15.16). There were 121 (71%) scholars with relative citation ratio ≥5, indicating >5-fold greater publication impact than that of other National Institutes of Health-funded scientists in similar areas of research. No differences by gender, institution, or temporal trends were observed. Of the full cohort, 69 (45.7%) responded to the survey; most self-identified as women (50 [73%]) and White (51 [74%]). CONCLUSION: Our findings suggest that the infrastructure provided by an institutional K award is an advantageous career development award mechanism for obstetrician-gynecologists, a group of predominantly women surgeons. It may serve as a corrective for the known inequities in National Institutes of Health funding by gender.

Anatomic nomenclature and 3-dimensional regional model of the human ovary: call for a new paradigm.

The ovaries are the female gonads that are crucial for reproduction, steroid production, and overall health. Historically, the ovary was broadly divided into regions defined as the cortex, medulla, and hilum. This current nomenclature lacks specificity and fails to consider the significant anatomic variations in the ovary. Recent technological advances in imaging modalities and high-resolution omic analyses have brought about the need for revision of the existing definitions, which will facilitate the integration of generated data and enable the characterization of organ subanatomy and function at the cellular level. The creation of these high-resolution multimodal maps of the ovary will enhance collaboration and communication among disciplines and between clinicians and researchers. Beginning in March 2021, the Pediatric and Adolescent Gynecology Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development invited subject-matter experts to participate in a series of workshops and meetings to standardize ovarian nomenclature and define the organ's features. The goal was to develop a spatially defined and semantically consistent terminology of the ovary to support collaborative, team science-based endeavors aimed at generating reference atlases of the human ovary. The group recommended a standardized, 3-dimensional description of the ovary and an ontological approach to the subanatomy of the ovary and definition of follicles. This new greater precision in nomenclature and mapping will better reflect the ovary's heterogeneous composition and function, support the standardization of tissue collection, facilitate functional analyses, and enable clinical and research collaborations. The conceptualization process and outcomes of the effort, which spanned the better part of 2021 and early 2022, are introduced in this article. The institute and the workshop participants encourage researchers and clinicians to adopt the new systems in their everyday work to advance the overarching goal of improving human reproductive health.

Uterine Artery Embolization for Symptomatic Adenomyosis: Proceedings from a Society of Interventional Radiology Foundation Research Consensus Panel.

Adenomyosis poses an important diagnostic and therapeutic challenge in women's health because of a variety of clinical/imaging presentations and frequent coexistence with other benign gynecologic conditions. In recent years, uterine artery embolization (UAE) for the treatment of adenomyosis has shown encouraging and favorable outcomes and long-term symptom improvement. To expand the current understanding of adenomyosis pathophysiology, imaging diagnostic criteria, and treatment outcomes, the Society of Interventional Radiology Foundation gathered a multidisciplinary Research Consensus Panel with experts from diverse backgrounds. The topics addressed were centered around the following: (i) the clinical presentation and imaging findings to diagnose adenomyosis; (ii) the currently available medical, interventional, and surgical treatment options; and (iii) existing literature for and experiences with UAE in symptomatic disease. The panel acknowledged that before the pursuit of a clinical trial, it would be necessary to first evaluate the imaging criteria for adenomyosis and correlate them with pathology and symptoms to establish a noninvasive imaging classification system. Second priority was given to the development of a quality of life questionnaire to assess patient outcomes following treatment. The third priority was the performance of a prospective clinical trial comparing UAE with medical therapy, which would help establish UAE in the treatment algorithm and societal guidelines for symptomatic adenomyosis.

Report of the National Cancer Institute and the Eunice Kennedy Shriver National Institute of Child Health and Human Development-sponsored workshop: gynecology and women's health-benign conditions and cancer.

The Division of Cancer Prevention and the Division of Cancer Biology at the National Cancer Institute and the Gynecologic Health and Disease Branch in the National Institute of Child Health and Human Development organized a workshop in April 2019 to explore current insights into the progression of gynecologic cancers from benign conditions. Working groups were formed based on 3 gynecologic disease types: (1) Endometriosis or Endometrial Cancer and Endometrial-Associated Ovarian Cancer, (2) Uterine Fibroids (Leiomyoma) or Leiomyosarcoma, and (3) Adenomyosis or Adenocarcinoma. In this report, we highlight the key questions and current challenges that emerged from the working group discussions and present potential research opportunities that may advance our understanding of the progression of gynecologic benign conditions to cancer.

Antithyroid antibodies and parity: further evidence for microchimerism in autoimmune thyroid disease.

OBJECTIVE: Fetal microchimerism may have a role in development of autoimmune thyroid disorders. Using parity as a surrogate for increasing fetal cell exposure, we analyzed its association with thyroid peroxidase antibody levels. STUDY DESIGN: Secondary analysis of serum thyroid analytes determined in 17,298 women from a population-based prospective study between 2001 and 2003. Sera were assayed for thyrotropin, free thyroxine, and antithyroid peroxidase antibodies. We analyzed the relationship between thyroid peroxidase antibodies and increasing parity. RESULTS: The incidence of abnormally elevated thyroid peroxidase antibody levels (>50 IU/mL) increased with advancing parity, but was not significant after adjustment for maternal characteristics. However, at higher thyroid peroxidase antibody levels (>500 IU/mL), a significant relationship with advancing parity persisted after adjustments (P = .002). CONCLUSION: Advancing parity is associated with an increased risk for high serum concentrations of antithyroid peroxidase antibodies. This suggests fetal microchimerism may play a role in development of autoimmune thyroid disorders.

Eye to the Future in Adenomyosis Research.

Adenomyosis is a poorly understood and clinically underappreciated gynecologic disorder associated with substantial morbidity including dysmenorrhea, pelvic pain, heavy menstrual bleeding, infertility, and poor pregnancy outcomes. Substantial gaps persist in our understanding of essentially all aspects of this disorder - epidemiology, risk factors, pathogenesis, pathophysiology, diagnosis, and treatment. In this article, we summarize current thoughts on future directions in basic, translational, and clinical adenomyosis research.

Effect of preconception low dose aspirin on pregnancy and live birth according to socioeconomic status: A secondary analysis of a randomized clinical trial.

Low socioeconomic status (SES) is associated with adverse pregnancy outcomes and infertility. Low-dose aspirin (LDA) was shown to improve livebirth rates in certain subsets of women, and therefore, may impact pregnancy rates differentially by SES status. Therefore, the aim of the current study was to examine whether daily preconception-initiated LDA affects rates of pregnancy, livebirth, and pregnancy loss differently across strata of socioeconomic status (SES). This is a secondary analysis of The Effects of Aspirin in Gestation and Reproduction (EAGeR) Trial, a multisite, block- randomized, placebo-controlled trial conducted at four U.S. medical centers (n = 1,228, 2007-2012). Women attempting spontaneous conception with a history of pregnancy loss were randomly allocated preconception to 81mg of aspirin + 400mcg of folic acid (n = 615) or placebo + 400mcg of folic acid (n = 613). Study medication was administered for six menstrual cycles or until 36 weeks' gestation if pregnancy was achieved. For this analysis, women were stratified by SES, which included income (low, mid, high) and a combined grouping of education and income (low-low, low-high, high-low, high-high). Log binomial models with robust variance estimated risks of pregnancy, livebirth, and pregnancy loss for LDA versus placebo. LDA increased pregnancy and livebirth rates (RR 1.23, 95% CI: 1.03, 1.45) in the high-income, but not mid- or low-income groups. LDA increased pregnancy rates in both the low education-low income group (RR 1.22, 95% CI: 1.02, 1.46) and the high education-high income group (RR 1.23, 95%CI: 1.06, 1.42), with no effect observed in mid-SES groupings. LDA, a low-cost and widely available treatment, may be particularly beneficial to women at the highest and lowest ends of the socioeconomic spectrum, though underlying mechanisms of this disparity are unclear. Confirming these findings and identifying factors which may modulate the effectiveness of LDA will ultimately facilitate personalized clinical care and improvements in population-level reproductive health. Trial registration number: ClinicalTrials.gov, NCT00467363.

Gynecologic Health and Disease Research at the Eunice Kennedy Shriver National Institute of Child Health and Human Development: A Scientific Vision.

In May 2016, the newly formed Gynecologic Health and Disease Branch in the Eunice Kennedy Shriver National Institute of Child Health and Human Development invited experts to a 2-day meeting aimed at identification of emerging opportunities in gynecologic investigation. Four primary disorders were chosen for emphasis because they represent the majority of the current Gynecologic Health and Disease Branch portfolio: uterine leiomyomas, endometriosis, pelvic floor disorders, and gynecologic pain conditions. Discussions generated a set of seven cross-cutting themes, which encompass both gaps in our current knowledge and potential directions for further research. These themes formed a continuum for understanding these disorders beginning with the need for classification systems, improved understanding of the natural history and etiology of these disorders, development of novel diagnostics, identification of opportunities for prevention, and the generation of new treatments using cutting-edge approaches. Along with these themes, three broad strategies were proposed to facilitate future research. First, investigators should improve utilization of existing research resources and focus on developing new resources to include databases, biospecimen repositories, animal models, and patient cohorts. Second, multidisciplinary scientific partnerships should be strengthened to bring new insights and approaches to gynecologic research. Third, patient and health care provider education must be promoted to ensure timely and accurate diagnosis and optimize treatment of gynecologic disorders. This article provides a summary of the workshop themes and suggestions, several of which have already been implemented through the development of program priorities and funding opportunity announcements aimed at improving women's reproductive health.

Liver receptor homologue-1 regulates gonadotrope function.

Over the past decade, substantial advances have been made in our understanding of the transcription factors which regulate gene expression in gonadotropes. One of the most important of these factors, steroidogenic factor-1 (SF-1; NR5A1) is critical for gonadotropin and GnRH-receptor expression. Interestingly, a closely related nuclear hormone receptor, liver receptor homologue-1 (LRH-1; NR5A2) has recently been detected in the anterior pituitary gland; however, its functional significance in this tissue has not been investigated. For the experiments reported here, we hypothesized that LRH-1 plays a previously unrecognized role in gonadotrope physiology. Towards this end, we first demonstrate LRH-1 mRNA and protein expression in both primary pituitary cells and gonadotrope-derived cell lines. We next show that LRH-1 stimulates promoter activity of the GnRH-receptor and gonadotropin subunit genes. Within the LHbeta gene, this response appears to be mediated by DNA-binding and transactivation through previously characterized SF-1 cis-elements. To our knowledge, this is the first report demonstrating a functional role for LRH-1 in the gonadotrope population of the anterior pituitary gland.

Pituitary tumors: diagnosis, management, and implications for reproduction.

Pituitary tumors are the most common intracranial neoplasms. They are commonly encountered by the gynecologist during an evaluation for galactorrhea, menstrual disturbances, or infertility. Although the majority of these tumors are benign, their impact on the endocrine and nervous system can be striking. The availability of neuroimaging techniques has allowed for more rapid diagnosis, affording earlier treatment. This review is intended to describe the common pituitary tumors seen by the gynecologist, and their impact on reproduction and fertility in the female patient.

Pituitary homeobox 1 (Pitx1) stimulates rat LHbeta gene expression via two functional DNA-regulatory regions.

Luteinizing hormone (LH) plays a central role in the reproductive axis, stimulating both gonadal steroid biosynthesis and the development of mature gametes. Over the past decade, significant progress has been made in characterizing the transcription factors and associated DNA-regulatory sites which mediate expression of the LH beta-subunit gene (LHbeta). One of these factors, pituitary homeobox 1 (Pitx1), has been shown to stimulate LHbeta gene promoter activity, both alone and in synergy with the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response gene 1 (Egr-1). Prior reports have attributed the Pitx1 response to a cis-element located at position -101 in the rat LHbeta gene promoter. While investigating the role of Pitx1 in regulating rat LHbeta gene expression, we observed a small, but significant, residual Pitx1 response despite mutation or deletion of this site. In the studies presented here, we identify the presence of a second functional Pitx1 region spanning positions -73 to -52 in the rat LHbeta gene promoter. Based on electrophoretic mobility shift assay, Pitx1 binds to both the initially described 5'Pitx1 site as well as this putative 3'Pitx1 region. In transient transfection analysis, mutation of the LHbeta-3'Pitx1 site significantly blunted Pitx1 responsiveness, with elimination of the Pitx1 response in a construct containing mutations in both Pitx1 cis-elements. We also analyzed the importance of each of these Pitx1 sites for providing functional synergy with SF-1 and with Egr-1. We observed a markedly decreased synergistic response with mutation of the 5'Pitx1 site with further loss following mutation of the 3'Pitx1 site. In contrast, functional interaction between Pitx1 and Egr-1 persisted with mutation of both Pitx1 regions. We conclude that Pitx1 stimulates the rat LHbeta gene promoter via two Pitx1 DNA-regulatory regions. These results further our understanding of the molecular mechanisms that regulate expression of this critical reproductive gene promoter.

Pituitary adenylate cyclase-activating polypeptide (PACAP) mimics neuroendocrine and behavioral manifestations of stress: Evidence for PKA-mediated expression of the corticotropin-releasing hormone (CRH) gene.

The physiologic response to stress is highly dependent on the activation of corticotropin-releasing hormone (CRH) neurons by various neurotransmitters. A particularly rich innervation of hypophysiotropic CRH neurons has been detected by nerve fibers containing the neuropeptide PACAP, a potent activator of the cAMP-protein kinase A (PKA) system. Intracerebroventricular (icv) injections of PACAP also elevate steady-state CRH mRNA levels in the paraventricular nucleus (PVN), but it is not known whether PACAP effects can be associated with acute stress responses. Likewise, in cell culture studies, pharmacologic activation of the PKA system has stimulated CRH gene promoter activity through an identified cAMP response element (CRE); however, a direct link between PACAP and CRH promoter activity has not been established. In our present study, icv injection of 150 or 300 pmol PACAP resulted in robust phosphorylation of the transcription factor CREB in the majority of PVN CRH neurons at 15 to 30 min post-injection and induced nuclear Fos labeling at 90 min. Simultaneously, plasma corticosterone concentrations were elevated in PACAP-injected animals, and significant increases were observed in face washing, body grooming, rearing and wet-dog shakes behaviors. We investigated the effect of PACAP on human CRH promoter activity in alphaT3-1 cells, a PACAP-receptor expressing cell line. Cells were transiently transfected with a chloramphenicol acetyltransferase (CAT) reporter vector containing region - 663/+124 of the human CRH gene promoter then treated for with PACAP (100 nM) or with the adenylate cyclase activating agent, forskolin (2.5 muM). Both PACAP and forskolin significantly increased wild-type hCRH promoter activity relative to vehicle controls. The PACAP response was abolished in the CRE-mutant construct. Pretreatment of transfected cells with the PKA blocker, H-89, completely prevented both PACAP- and forskolin-induced increases in CRH promoter activity. Furthermore, CREB overexpression strongly enhanced PACAP-mediated stimulation of hCRH promoter activity, an effect which was also lost with mutation of the CRE. Thus, we demonstrate that icv PACAP administration to rats under non-stressed handling conditions leads to cellular, hormonal and behavioral responses recapitulating manifestations of the acute stress response. Both in vivo and in vitro data point to the importance of PACAP-mediated activation of the cAMP/PKA signaling pathway for stimulation of CRH gene transcription, likely via the CRE.

Gonadotropin-releasing hormone and gonadal steroids regulate transcription factor mRNA expression in primary pituitary and immortalized gonadotrope cells.

Hormonal regulation of pituitary gonadotropin gene expression has been attributed to gonadotropin-releasing hormone (GnRH)-mediated stimulation of immediate early gene expression and gonadal steroid interactions with their respective nuclear receptors. A number of orphan nuclear receptors including steroidogenic factor 1, liver receptor homologue 1, dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1, and chicken ovalbumin upstream promoter-transcription factors I/II as well as the GATA family members, GATA2 and GATA4, have also been implicated in transcriptional regulation of the gonadotropin genes. We hypothesized that hormonally mediated changes in these latter transcription factors may provide an additional mechanism for mediating hormonal effects beyond the more classically appreciated pathways. In these studies, we demonstrate significant regulation of orphan nuclear receptor and GATA messenger RNA levels by GnRH, dihydrotestosterone, estradiol, and progesterone in both cultured primary pituitary cells and gonadotrope-derived cell line, LßT2. These results advance our understanding of the complex mechanisms by which GnRH and steroid hormones achieve precise regulation of anterior pituitary function.

PACAP modulates GnRH signaling in gonadotropes.

Hypothalamic gonadotropin-releasing hormone is known to be critical for normal gonadotropin biosynthesis and secretion by the gonadotrope cells of the anterior pituitary gland. Additional regulation is provided by gonadal steroid feedback as well as by intrapituitary factors, such as activin and follistatin. Less well-appreciated is the role of pituitary adenylate-cyclase activating polypeptide (PACAP) as both a hypothalamic-pituitary releasing factor as well as an autocrine-paracrine factor within the pituitary. PACAP regulates gonadotropin expression alone and through modulation of GnRH responsiveness achieved by increases in GnRH receptor expression and interactions at the level of intracellular signaling pathways. In addition to direct effects on the gonadotrope, PACAP stimulates follistatin secretion by the folliculostellate cells and thereby contributes to differential expression of the gonadotropin subunits. Conversely, GnRH augments the ability of PACAP to regulate gonadotrope function by increasing pituitary PACAP and PACAP receptor expression. This review will summarize the current understanding of the mechanisms by which PACAP modulates gonadotrope function, with a focus on interactions with GnRH.

Interaction of gonadal steroids and gonadotropin-releasing hormone on pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP receptor expression in cultured rat anterior pituitary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are expressed in the hypothalamus, the gonadotrope cells of the anterior pituitary gland, and the gonads, forming an autocrine-paracrine system in these tissues. Within the pituitary, PACAP functions either alone or synergistically with gonadotropin-releasing hormone (GnRH) to stimulate gonadotropin gene expression and secretion. Our goal was to define the hormonal regulation of pituitary PACAP and PACAP receptor (PAC1) gene expression by dihydrotestosterone (DHT), estradiol, and progesterone alone or in conjunction with GnRH. Treatment of adult male rat pituitary cell cultures with DHT or progesterone augmented GnRH-mediated increase in PACAP messenger RNA (mRNA) levels, but neither had an effect when present alone. Conversely, estradiol treatment blunted PACAP gene expression but did not alter GnRH effects on PACAP expression. Expression of PACAP receptor mRNA was decreased by GnRH treatment, minimally increased by DHT treatment, but not altered by the addition of estradiol or progesterone. DHT and GnRH together blunted PACAP receptor gene expression. Taken together, these results suggest that the activity of the intrapituitary PACAP-PAC1 system is regulated via the complex interaction of gonadal steroids and hypothalamic GnRH.

Androgen receptor drives transcription of rat PACAP in gonadotrope cells.

Gonadotropin expression is precisely regulated within the hypothalamic-pituitary-gonadal axis through the complex interaction of neuropeptides, gonadal steroids. and both gonadal- and pituitary-derived peptides. In the anterior pituitary gland, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) modulates gonadotropin biosynthesis and secretion, acting both alone and in conjunction with GnRH. Steroid hormone feedback also influences gonadotropin expression via both direct and indirect mechanisms. Evidence from nonpituitary tissues suggests that PACAP may be a target for gonadal steroid regulation. In the present study, we show that androgen markedly stimulates rat (r) PACAP promoter-reporter activity in the LßT2 mature mouse gonadotrope cell line. 5'-Serial deletion analysis of reporter constructs identifies 2 regions of androgen responsiveness located at (-915 to -818) and (-308 to -242) of the rPACAP promoter. Androgen receptor (AR) binds directly to DNA cis-elements in each of these regions in vitro. Site-directed mutagenesis of 3 conserved hormone response element half-sites straddling the (-308 to -242) region dramatically blunts androgen-dependent PACAP promoter activity and prevents AR binding at the mutated promoter element. Chromatin immunoprecipitation demonstrates that endogenous AR binds the homologous region on mouse chromatin in LßT2 cells in both the presence and absence of androgen. These data demonstrate that androgen stimulates PACAP gene expression in the pituitary gonadotrope via direct binding of AR to a specific cluster of evolutionarily conserved hormone response elements in the proximal rPACAP gene promoter. Thus, androgen regulation of pituitary PACAP expression may provide an additional layer of control over gonadotropin expression within the hypothalamic-pituitary-gonadal axis.