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REPRODUCTIVE ENDOCRINOLOGY AND INFERTILITY

James Segars, MD, Head, Unit on Reproductive Endocrinology and Infertility Phyllis Leppert, MD, PhD, Senior Staff Scientist Alicia Armstrong, MD, Staff Clinician William Catherino, MD, PhD, Clinical Fellow Adrienne Neithardt, MD, Clinical Fellow Jason Parker, DO, Clinical Fellow Mark Payson, MD, Clinical Fellow Kathleen McDaniel, BS, Predoctoral Fellow

Our objective is to provide clinician scientists with expertise in clinical and basic research relating to reproductive disorders and diseases. To meet this goal, we support the multi-institutional clinical training program in Reproductive Endocrinology and Infertility jointly sponsored by NICHD, the Uniformed Services University of the Health Sciences, and Walter Reed Army Medical Center. We conduct both basic and clinical research studies of importance to reproduction. Uterine fibroids Catherino, Leppert, Payson, Segars Uterine leiomyoma, also know as fibroids, represent a considerable public health issue. Due to their prevalence and their association with pre-term birth and reproductive loss, NICHD has been interested in and is actively pursuing research on these benign tumors of the uterus. For reasons that are not understood, fibroids are three times more likely to affect women of African American ethnicity. To understand the molecular events that lead to leiomyoma development, we used global expression profiling to compare normal myometrium with fibroid tumors. In the past year, we confirmed differences in gene expression by using RT-PCR, real-time RT-PCR, immunohistochemistry, and other approaches. Somewhat unexpectedly, these experiments revealed that genes involved in sex steroid action were not featured as differentially expressed genes. For example, estrogen receptors alpha and beta, SRC-1, and CBP were not over- or underexpressed in leiomyoma tissues as compared with normal uterine tissue. We had suspected that such genes might be differentially expressed because estrogen had been shown to promote fibroid growth. Instead, the arrays revealed differential expression of genes involved in formation of extracellular matrix (ECM), the connective substance between cells largely composed of collagens and proteoglycans. ECM accumulation is a feature of fibroids, and accumulation of ECM suggests an imbalance between synthesis and dissolution. We confirmed increases in the large proteoglycan versican and transforming growth factor-beta 3 (TGF-beta 3). TGFs have pleiotropic effects and have been shown to play critical roles in pathologic conditions involving fibrosis, and they are potent promoters of connective tissue formation. In addition, we observed reduced expression of dermatopontin, a 22kDa extracellular protein that had been associated with hypertrophic scar formation, a condition similar to keloid scars. Using reverse transcriptase real-time PCR, immunohistochemistry, and electron microscopy, we observed striking similarities between the features of keloid tissues and leiomyoma tissues. The studies represent an initial characterization of the leiomyofibroblast cells forming fibroids and revealed that dysregulation of genes involved in formation of the ECM may contribute to the abnormal formation of fibrosis observed in leiomyoma. In the coming year, we plan to begin characterizing the abnormally formed ECM in an effort to identify strategies that may lead to nonsurgical methods of the treatment for fibroids. Understanding the expression pattern of the genes leading to fibroid development might lead to improved understanding of tumor growth and development, possibly resulting in development of new treatment strategies. Catherino WH, Leppert PC, Stenmark MH, Payson M, Nieman LK, Segars JH. Reduced dermatopontin expression is a molecular link between leiomyomas and keloids. Genes Chrom Cancer 2004;40:204-217. Catherino WH, Salama A, Potlog-Nahari C, Leppert PC, Tsibris J, Segars JH. Gene expression studies in leiomyomata: new directions for research. Sem Reprod Med 2004;22:83-90. Tsibris JCM, Maas S, Segars JH, Nicosia SV, Enkemann SA, O’Brien WF, Spellacy WN. New potential regulators of uterine leiomyomata from DNA arrays: the ionotropic glutamate receptor GluR2. Biochem Biophys Res Comm 2003;312:249-254. Mechanism of estrogen action McDaniel, Segars; in collaboration with Driggers, Westphal In addition to its suspected role in breast and uterine cancer, estrogen is of fundamental importance to many reproductive processes in women. Our previous studies examined the role of the Brx protooncoprotein in estrogen-dependent studies in vitro. In collaboration with Heiner Westphal, we engineered a knockout mouse for the Brx protooncogene. Preliminary examination of Brx-deficient mice has revealed profound effects on development and that Brx is required for survival of mice. In the coming year and in collaboration with Dr. Westphal, we plan to examine in greater detail the role of Brx in development. In addition, in studies with Drs. Driggers and Kino, we plan to expand our analysis of the ability of Brx to influence gene activation by hormones other than estrogen. We expect that these studies will further clarify the important and essential role of Brx in reproduction and development. Driggers PH, Segars JH. Estrogen action and cytoplasmic signaling pathways. Part II: the role of growth factors and phosphorylation in estrogen signaling. Trends Endocrinol Metab 2002;13:422-427. McDaniel K, Mayers C, Venere M, Driggers P, Westphal H, Gorivodsky M, Segars J. Brx, a cytoplasmic protein capable of augmenting estrogen action is essential for murine development. Proceedings of the Society for Gynecologic Investigation, Houston, TX. March 2004. Segars JH, Driggers PH. Estrogen action and cytoplasmic signaling cascades. Part I: membrane-associated signaling complexes. Trends Endocrinol Metab 2002;13:349-354. Infertility Armstrong, Catherino, Neithardt, Parker, Segars Ten percent of U.S. couples are unable to begin a family. Treatments for infertility may be expensive and result in substantial costs to the U.S. health care system. In the past year, we continued to examine causes and evidence-based treatment of diseases causing infertility as an integral part of the clinical fellowship program in Reproductive Endocrinology. Our continuing objective is to develop strategies to reduce multiple births and identify factors that are positively or negatively associated with pregnancy in couples undergoing treatment. In addition, we continue to examine critical elements of graduate medical education in the field of reproduction. Armstrong AY, Neithardt AB, Alvero R, Sharara F, Bush M, Segars J. The role of fallopian tube anastomosis in training fellows: a survey of current reproductive endocrinology fellows and practitioners. Fertil Steril 2004;82:495-497. Hosid S, Naik D, Materia D, McKeeby J, Armstrong A. Impacting in vitro fertilization multiple gestation rates through patient education and counseling. Women’s Health Care 2003;2:19. Neithardt AB, Murdock CM, Segars JH, McKeeby JL. Pre-embryo transfer sham at the time of embryo transfer improves clinical pregnancy rates. Fertil Steril 2005, in press. Parker JD, Alvero RJ, Luterzo J, Segars J, Armstrong AY. Assessing resident competency in the performance of sonohysterography: does the level of training impact the accuracy? Am J Obstet Gynecol 2004;191:582-586. COLLABORATORS Paul Driggers, PhD, Uniformed Services University of the Health Sciences, Bethesda, MD Tomoshige Kino, MD, PhD, Pediatric and Reproductive Endocrinology Branch, NICHD, Bethesda, MD Lynnette Nieman, MD, Pediatric and Reproductive Endocrinology Branch, NICHD, Bethesda, MD John Tsibris, PhD, University of South Florida, Tampa, FL Heiner Westphal, MD, Laboratory of Mammalian Genes and Development, NICHD, Bethesda, MD For further information, contact segarsj@mail.nih.gov