Diagnosis of antiovarian autoantibodies in infertile women help in approaches for assisted reproduction

Studies on the ovarian antigens and its corresponding specific antibodies have showed the relationship between the incidences of antiovarian autoantibodies and the clinical mani-festation of ovarian dysfunction and infertility in women.

The human ovary can be the target for autoimmune attack and clinically the ensuing ovarian dysfunction often results in premature ovarian failure, but other pathologies involving the ovaries, such as unexplained infertility, polycystic ovarian syndrome and endometriosis have been associated with antiovarian autoimmunity, too. The diagnosis of an autoimmune mechanism in these pathologies has relied on the testing of antiovarian autoantibodies, which are detected earlier before the clinical manifestation of disturbed ovarian function.

The antiovarian autoantibodies are generated against the ovarian cellular structures as Zona Pellucida (ZP), Granullosa cells (GCs), Teca cells (TCs) and cells of the Corpus Luteum (CL). The variety of potential autoimmune target molecules of these ovarian structures relies with different mechanisms of disturbances of the ova-rian function that are the object of further investigations. Studies on the ovarian antigens and its corresponding specific antibodies have showed the relationship between the incidences of antiovarian autoantibodies and the clinical mani-festation of ovarian dysfunction and infertility in women.

In series of systemic studies on the ZP antigens (the extracellular translucent envelop of the oocyte, that play the important role in fertilization) and on the antigens of GCs(follicular cells situated around the oocyte and involved in its development and maturation, GCs are the main cell type of the CL) carried out at the Department of Immunobiology of Reproduction, Institute of Biology and Immunology of Reproduction "Acad. K. Bratanov" under the guidance of assoc. prof. Dr. Maria D. Ivanova, the occurrence and the role of these autoantibodies were studed in infertile women attending the programs of assisted reproduction. The prevalence of autoantibodies against ZP (19,26%) and autoantibodies against GCs (21,55%) was demonstrated in infertile women attending the program of in vitro fertilization and embryo transfer (IVF-ET), compared to the fertile women (5,71% and 3,85%), respectively. Our studies on the biological role of these antiovarian autoantibodies showed their negative relationship with the response to the hormonale stimulation (anti-GCS autoantibodies), as well as with the IVF -ET outcome (both types of autoantibodies) in infertile women. On the basis of the results obtained and the results from other authors for the clinical signifificance of antiovarian autoantibodies in the diagnosis of the causes for infertility in women and for the specifying the approaches in assisted reproduction, the immunological methods (ELISA, enzyme linked immunosorbent assay and IIF, indirect immunofluorescence) were elaborated for the screening of autoantibodies against ZP and against GCs in sera and follicular fluids of women:

ELISA - for the screening of autoantibodies against Zona pellucida in sera and follicular fluids. On the basis of the interspecies specificity and homology of ZP antigens (73-80% homology between the human and porcine ZP-antigens was demonstrated), as antigen in this method the sollubilized porcine ZP in concentration 5μg/ml was used. The biological fluids tested were diluted as follows: 1:100 (v/v) in physiological saline for sera and 1:50 - for follicular fluids.

ELISA - for the detection of autoantibodies against GCs in sera and follicular fluids. As antigen, the NP-40 lysate of GCs, in concentration 5 μg/ml proteins was used. By Immunoblot of GCs NP-40 lysate with sera from infertile women, the proteins with approximately MM of 110, 70-80, 47 and 37 kDa against which the prevalence of antiovarian autoantibodies were detected. The biological fluids tested were diluted as follows: 1:100 (v/v) in physiological saline for sera and 1:50 - for follicular fluids.

IIF - Indirect immunofluorescence method for the screening of autoantibodies against ZP. In this method the porcine oocytes isolated from the ovaries, obtained from the slaughterhouse, were used. The porcine oocytes (15-20 per sample) were incubated in the sera and the follicular fluids tested in dilution 1:10 for serum samples and 1:4, for follicular fluid samples.

The screening of sera from infertile women for antiovarian autoantibodies takes place in the clinical practice of the Consulting rooms of infertility and in the Medical centers of assisted reproduction. The Institute of biology and immunology of reproduction "Acad. K. Bratanov" have the joint research programs on the antiovarian autoantibodies with the Medical center "ReproBioMed", Sofia, as well as projects for the diagnosis of antiovarian autoantibodies with Medical A&G Center "Viara" and with the Instiutute of Reproductive Health, Sofia. The shared opinion of the A&G doctors showed that the diagnosis of the antiovarian autoantibodies in women with disturbances of the ovarian function and in infertile women with unexplained infertility, help them in the explanation of the causes for the ovarian disturbances and infertility in women, as well as in the finding of exact approaches for assisted reproduction.