NFkB and other markers of chronic inflammation in the prediction of ovarian aging and infertility

p38 MAPK phosphorylation, suggesting a possible non-genomic estrogenic response through p38 MAPK pathway in these cells. ICI did not have a significant effect on p38 MAPK phosphorylation by itself, but it completely reversed the effect of E2. The observation that ICI together with E2 induced an inhibition of p38 MAPK phosphorylation to levels below the control levels may have further implications that need to be investigated. Supported by: None. P-515 NFkB and other markers of chronic inflammation in the prediction of ovarian aging and infertility. A. Zimon, A. Erat, R. Reindollar, A. Usheva. Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA. OBJECTIVE: Modulation of inflammation and responses to oxidative stress are leading theories of cellular aging. Whether changes in these pathways occur with aging in the ovary is not known. To search for aging markers and pathways in the mammalian ovary, we aimed to profile agedependent changes of ovarian gene expression at the level of RNA and protein in reproductively young and old mice. DESIGN: Laboratory investigation. MATERIALS AND METHODS: Whole ovaries were harvested from synchronized young (6 – 8 weeks) and old (9 –10 months) C57BL/6 mice using a protocol approved by the institutional animal research facilities. RNA was extracted from homogenized whole ovaries and cDNA was prepared, radiolabeled and hybridized to a domestic oligonucleotide membrane array containing 430 oligonucleotides corresponding to potential aging-related genes. Differential gene expression was confirmed via virtual Northern analysis. Immunohistochemistry was performed on fixed whole ovary microsections using specific primary antibodies directed against proteins of the differentially expressed genes. Western analysis of protein extracted from tissue lysate was performed to examine age-related changes in protein content. All experiments were repeated in triplicate. RESULTS: With aging, we observed an increase in nuclear factor kB (NFkB) in association with an increase in heme oxygenase-1 (HO-1), which is known to be transcriptionally regulated by NFkB. HO-1 is a stress induced enzyme involved in the conversion of heme to biliverdin. Bilirubin and biliverdin have been shown to be important in the regulation of MAP kinase p38 and transcription factors such as Yin Yang 1 (YY1). High levels of HO-1 are therefore expected to be associated with higher levels of bilirubin and biliverdin, likely explaining the lower levels of YY1 observed with aging. In systems with less YY1 activity, minimization of cellular proliferative capacity is expected. Increases in MAP kinase p38 and phosphorylated p38 were seen with aging, as well as changes in the Jak and Erk pathways, suggesting that changes in phosphorylation rates of important cell cycle regulators may modulate cellular proliferation with age. Retinoic acid receptor-␣, a nuclear factor which modulates NFkB, was upregulated with aging. Changes in ubiquitin and A20, additional modulators of NFkB were also observed. Together, higher levels of NFkB and HO-1 and lower levels of YY1 may contribute to an age-dependent rate of cellular proliferation in the mammalian ovary. CONCLUSION: NFkB-dependent inflammatory pathways are likely involved in aging in the mammalian ovary whereby aging coincides with upregulation of NFkB and HO-1 and down regulation of YY1. These findings implicate a chronic compensatory inflammatory response to increased oxidative stress with ovarian aging and suggest that changes in chronic inflammatory markers may predict ovarian age and infertility. Supported by: Educational Research Fund, Department of Obstetrics & Gynecology, Beth Israel Deaconess Medical Center, Boston

REPRODUCTIVE IMMUNOLOGY P-516 Interleukin-1 system mRNA and protein expression at the endometrial myometrial interface (EMI) of human uterine adenomyosis: Possible pathophysiologic implications of endometriosis. H. Y. Huang, S. H. Chan, J. H. Wu, Y. C. Wu, H. S. Wang, Y. K. Soong. Chang Gung Memorial Hospital, Tao-Yuan, Taiwan Republic of China. OBJECTIVE: Adenomyosis is a disease specifically characterized by deep invasion of the inner myometrium by endometrial glands and stroma

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thereby disrupting the endometrial myometrial interface (EMI). Adenomyosis is also complicated by prominent clinical manifestations, especially for women in reproductive age. Understanding the molecular factors involved in the level of EMI is critical to comprehending the mechanisms of endometriosis and the Interleukin-1 (IL-1) system is a major candidate for these molecular regulators. The purpose of this study is to investigate the role of the IL-1 system including IL-1␤, IL-1 receptor antagonist (IL-1ra) and IL-1 receptor type I (IL-1RtI), expression at the level of EMI in human eutopic endometrium and corresponding ectopic endometriotic tissue in uterine adenomyosis. DESIGN: Quantitative competitive PCR (QC PCR) determined the steady-amount of IL-1 system mRNA levels in human uterine adenomyosis and IL-1 protein levels of EMI were determined with immunohistochemistry. MATERIALS AND METHODS: Pairs of human uterine adenomyosis tissue with corresponding eutopic endometrium and myometrium (n⫽4) were obtained from surgical specimens of normally cycling women undergoing hysterectomy for benign uterine tumors after informed consent and IRB approval. The uterine tissue samples used for this study were histologically shown to contain adenomyosis. To demonstrate the IL-1 system mRNA expression, total extracted RNA was reverse transcribed and amplified by PCR using specific primers for IL-1␤ (548 bp), IL-1ra (179 bp), and IL-1R tI (284 bp). To further determine the IL-1 system expression in endometrium, myometrium and adenomyosis at the level of EMI, the steady state amount of IL-1␤, IL-1ra and IL-1R tI mRNA expression were determined by QC PCR. To determine the presence of IL-1 proteins, pairs of tissues were fixed and processed for immunohistochemical staining using the avidin-biotin alkaline phosphatase method. Human luteal phase endometrium was used as a positive control. Deletion of the primary antibody was used as a negative control. Data analysis was done with ANOVA and Pearson’s correlation. RESULTS: A complete IL-1 system including IL-1␤, IL-1ra and IL-1 R tI mRNA was detected in all samples. QC PCR showed sequential decreased expression in the order of endometrium, myometrium and ectopic endometriotic tissue in adenomyosis. A significant down-regulation of IL-1R tI mRNA expression was demonstrated in ectopic endometriotic tissue in comparison to corresponding endometrium (p⬍0.05), but not to the myometrium. There is no significant difference in IL-1␤ and IL-1ra in all three groups of samples. Pearson’s correlation also showed a significant correlation between IL-1␤ and IL-1 ra in endometrium, but not in ectopic endometriotic tissue and normal myometrium. Immunoreactive IL-1 system proteins were also present in the level of EMI. CONCLUSION: These results suggest that IL-1 expression may play a crucial role in EMI during the process of endometriosis. This co-expression and sequential decreased expression of IL-1 agonist and receptor antagonist in uterine adenomyosis may mediate a molecular dialogue at the level of EMI, then initiate the process of the eutopic endometrium breaking through the EMI, resulting the pathological process of endometriosis complicated with uterine adenomyosis. Supported by: This work was supported in part by Chang Gung University Research Grant No. CMRP 1191 (to H.Y.H).

P-517 Withdrawn P-518 Detectable levels of interleukin-18 in uterine luminal secretions at oocyte retrieval predict failure of the embryo transfer. N. LedeeBataille, F. Olivennes, J. Kadoch, S. Dubanchet, G. Chaouat, R. Frydman. Centre hospitalier intercommunal de Poissy, Poissy, France; hopital de Baudelocque, Paris, France; Hopiatl Be´ cle`re, Clamart, France; Hopital Be´ cle`re, Clamart, France. OBJECTIVE: Most implantation failures after successful in vitro fertilization-embryo transfer (IVF-ET) result from inadequate uterine receptivity. There is currently no way to predict this receptivity. DESIGN: We investigated whether the detection of interleukin-18 by ELISA in uterine luminal secretions might predict implantation failure. MATERIALS AND METHODS: Secretions of 133 patients enrolled in our IVF-ET program were sampled by uterine flushing immediately before oocyte retrieval. We assessed the following outcomes: pregnancy rate, multiple pregnancy rate, and implantation rate per embryo transferred.

Vol. 82, Suppl. 2, September 2004