A novel computational approach to the genetics of polycystic ovarian syndrome (PCOS)

Wednesday, October 17, 2007 3:15 pm O-193 LYMPHANGIOGENESIS IN THE OVARY: IMPORTANCE IN FOLLICLE MATURATION AND MURINE PREGNANCY. J. Ihm, J. M. Rutkowski, S. Lee, J. A. Hubbell, M. A. Swartz. Institute of Bioengineering, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland. OBJECTIVE: Our objective was to determine the role of lymphatics in follicle maturation and pregnancy, as well as the necessity of lymphangiogenesis for these processes. DESIGN: Lymphangiogenesis was blocked in female mice by injecting a specific mouse antibody (mF4–31C1) to block VEGFR-3 signaling, a critical regulator of lymphangiogenesis. The treated female mice were monitored to their pregnancy by examination of uteruses and fetuses, live births, and immunohistochemistry on ovarian tissue. Retrieved follicles and 2-cell embryos were cultured in vitro to determine maturation potential outside of the lymphatic-deficient uterine environment. MATERIALS AND METHODS: F1 hybride mice were injected with 0.2 ml of 2.5mg/ml mF4–31C1 or saline every 2 days for 2–4 weeks. Lymphatic and blood vessels were labeled immunohistochemically on ovarian cryosections to quantify the vasculature of follicles. Preantral follicles were retrieved, cultured, and matured using in vitro drop culture. Two-cell embryos were retrieved and cultured to blastocysts in vitro, the uteruses examined for fetuses and implantation sites, and birth. RESULTS: Ovarian lymphangiogenesis was inhibited in mF4–31C1treated mice, which displayed significantly decreased lymphatic densities (up to a 93% decrease) in all stages of follicle maturation without affecting concurrent and essential blood angiogenesis. VEGFR-3 neutralized before fertilization, led to failed pregnancies, despite the number of ovulated follicles, fertilization, and embryonic implantation in the uterus being identical to control animals. Embryonic culture revealed a markedly reduced blastocyst cell density in treated animals, and examination of pregnancies found subsequent developmental defects of the fetuses and miscarriage. However, secondary follicles extracted from these ovaries were able to mature normally in vitro through the MII phase and did not negatively respond to direct neutralization of VEGFR-3 in culture. CONCLUSIONS: Our results suggest that the lymphatic vasculature is important in maintaining a environment for follicle maturation and that insufficient lymphatic drainage retards normal follicle development. We propose that VEGFR-3 signaling and proper lymphatic function in the ovary are critical in follicle development and are therefore inherently necessary for successful reproduction. Supported by: Bronislaw Pytowski and ImClone Systems, Inc, for the VEGFR-3 neutralizing antibodies, and to the NIH (HL075217) and the Swiss National Science Foundation (107602), and EPFL for funding.

Wednesday, October 17, 2007 3:30 pm O-194 A NOVEL COMPUTATIONAL APPROACH TO THE GENETICS OF POLYCYSTIC OVARIAN SYNDROME (PCOS). M. Nam Menke, N. B. Menke, D. G. Bonchev, T. M. Witten, J. R. Wood, J. F. Strauss. Department of Obstetrics and Gynecology, Virginia Commonwealth University Medical Center, Richmond, VA; Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, VA; Department of Emergency Medicine and VCU Reanimation Engineering Shock Center, Virginia Commonwealth University Medical Center, Richmond, VA; Department of Animal Science, University of Nebraska-Lincoln, Lincoln, NE. OBJECTIVE: To narrow the list of potential PCOS genes through advanced computational techniques and network analysis. DESIGN: Using a graph theoretic structure composed of nodes and links, a PCOS gene network was constructed using the PathWay Studio software (Ariadne Genomics). Nodes were defined as genes or gene products; links were defined as molecular interactions between nodes. The PCOS network combined information gleaned from the literature with experimentally derived microarray data obtained from oocytes.

FERTILITY & STERILITYÒ

Figure 1. PCOS network (large figure) with expanded modular tree diagram of the PCOS intersection set Lines between genes dence corrections only without hierarchal reference.

MATERIALS AND METHODS: The PCOS network was decomposed into modules in order to simplify the large initial network. The Newman algorithm was used to mathematically organize the network into modules based on connectivity. Functional modules were created by using the clinical characteristics of PCOS. The Newman modules were integrated with the functional modules in order to find the smallest set of genes that could lead to the PCOS phenotype. RESULTS: The PCOS network is composed of 680 genes/proteins and 2017 interactions. It has scale-free and small-world properties (network radius of 3.75) typical for dynamic evolutionary networks. The PCOS intersection set (Figure 1) includes genes involved in satiety and obesity (GH1, GHRH, GHRL, GHSR, HRCT, LEP, NPY, and POMC), gonadotropin function (GNRH1), and insulin resistance (IGF1, SLC2A4, and SST). The genes also affect a wide variety of functions such as vasoconstriction (EDN1, VIP) and cell growth (EGF). CONCLUSIONS: Network analysis provides a new method for understanding the genetic basis of complex diseases such as PCOS. This novel, polygenic model accounts for interactions that may occur between multiple, previously unconnected genes with small contributions to the phenotype. Further experimental testing of the PCOS intersection set will allow refinement of this technique. Through computational analysis, PCOS may be redefined as the end result of a dysfunctional gene/protein network which may lead to novel therapeutic strategies. Supported by: This work was supported by the National Institutes of Health (NIH) Grant U54 HD034449.

Wednesday, October 17, 2007 3:45 pm O-195 CO-TREATMENT WITH METFORMIN XR DOES NOT REDUCE THE THRESHOLD DOSE OF CLOMIPHENE TO INDUCE OVULATION IN POLYCYSTIC OVARY SYNDROME: RESULTS FROM THE PREGNANCY IN POLYCYSTIC OVARY SYNDROME (PPCOS) STUDY. N. A. Cataldo, R. S. Legro, E. R. Myers, H. X. Barnhart, Cooperative Reproductive Medicine Network. None, Birmingham, AL; OB/GYN, Pennsylvania State Univ, Hershey, PA; OB/GYN, Duke Univ Med Ctr, Durham, NC. OBJECTIVE: To determine if co-treatment with metformin can lower the threshold dose of clomiphene citrate (CC) needed to induce ovulation in women with polycystic ovary syndrome (PCOS). DESIGN: Secondary analysis of data from prospective, double-blind placebo-controlled multicenter clinical trial. MATERIALS AND METHODS: Women with PCOS by NICHD criteria who participated in the PPCOS study (ref. 1) were randomized to receive in placebo-controlled fashion for up to 6 cycles/30 weeks or until pregnancy occurred: (a) metformin XR at a target dose of 1000mg BID plus placebo for CC; (b) placebo for metformin XR plus CC for 5 days at doses starting at 50 mg in the first cycle and increasing to 100 mg

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