The long term objective of this research is to examine the possible role of locally produced complement components in reproduction both in normal and diseased states. The immediate goals are to examine the regulation of human uterine C3 and to determine if it can be related to the infertility associated with endometriosis. In order to accomplish these long-term goals, we plan to examine the occurrence, cellular origin, and biological activity of C3 in the normal endometrium and in patients with endometriosis. Endometrial biopsies from patients with normal laparoscopic findings at various days of the menstrual cycle will be obtained and the tissue will be incubated in the presence of radioactive methionine. The secretory and tissue proteins will then be subjected to immunoprecipitation with C3 antibodies. Results will be expressed as the percentage of total counts per minute incorporated into protein which are specifically immunoprecipitated with anti-human C3. Endometrial biopsies from patients with endometriosis will also be subjected to the same analysis. RNA will be isolated from the endometrial biopsies and analyzed using a polymerase chain reaction (PCR) technique. The synthesis and regulation of another key complement component, factor B, will also be examined by these techniques from the same endometrial samples. Immunohistochemistry as well as in situ hybridization will be utilized to determine the cellular location and synthesis of C3 and factor B. To determine if the C3 produced by the uterus is biologically active, the ability of this protein to bind to sheep erythrocytes will be tested. Antibody sensitized sheep red blood cells will be utilized in which the end point of the assay is cell lysis. The hormonal regulation of C3 will be further studied by using the rat model of endometriosis. In this model we will examine the effect of various treatments including estrogen, progesterone, GnRH agonist, dexamethasone, Tamoxifen, and indomethacin. C3 production will be analyzed as described above. Thus, the combination of studies on the human and rat model will establish the hormonal regulation of C3 gene expression and biologic activity in normal endometrium and endometriotic tissue. By undertaking this investigation, new insight into the immune and inflammatory response of endometriosis will be gained, and perhaps further the understanding of infertility associated with this disease.
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