Oct 23, 2001 - (MCP-1) expression in endometrial cells of women with endometriosis. A. Arici, M. O. Bahtiyar, E. Seli, L. Senturk. Yale Univ Sch of Medicine,.
Design: The role of oxidative stress in aging and declining fertility has been recently evaluated. Reactive oxygen species (ROS) are known products of oxidative metabolism and are continuously generated in vivo. The most frequent oxidative modification of DNA is transformation of guanosine to 8-hydroxy-2⬘ deoxyguanosine (8-OHdG), leading to use of 8-OHdG as a noninvasive quantitative biomarker of oxidative DNA damage. We propose that inflammation related to endometriosis may causes infertility via a direct effect on oocyte quality related to ROS. Materials/Methods: Granulosa cells were obtained during IVF cycles from 12 women diagnosed with endometriosis and 12 age matched controls. Samples were processed using Isolate (Sigma) density gradient centrifugation. DNA was isolated through ethanol precipitation, digested with nucleases. 8-OHdG was measured by high performance liquid chromatography (HPLC) with electrochemical detection. Nonparametric tests were used for comparing 8-OHdG levels (Mann-Whitney test for two categories, KruskalWallis test for multiples) with Bonferroni correction. Results: There was a significant increase in 8-OHdG levels in endometriosis patients (n ⫽ 12, Mean 26361.9 ⫾ 19895.7 SD) compared to age-matched controls with male factor infertility (n ⫽ 12, Mean 469.4 ⫾ 700.1 SD) (P ⬍ 0.001). Additional comparisons of 8-OHdG levels between diagnostic populations, using the Mann-Whitney test evaluated with an alpha of .007 (Bonferroni correction), indicated that patients with endometriosis had 8-OHdG levels significantly higher (Median 2721.3, range between 258.4 and 63757.7) than controls (Median 25.05, range between 7.1 and 55.9) (P ⬍ .001). These results persisted despite corrections for age (35.6 ⫾ 2.8) vs. (36.7 ⫾ 2.7) number of ampules required for stimulation (24.0 ⫾ 9.0) vs. (29.3 ⫾ 12.7) and number of oocytes retrieved (10.23 ⫾ 4.5) vs. (10.53 ⫾ 2.9). Conclusions: Our findings indicate that granulosa cells exhibit higher levels of 8-OHdG in patients with endometriosis than in controls. We propose that oxidative damage to RBCs, apoptotic endometrial cells or undigested endometrial tissue may signal the recruitment and activation of mononuclear phagocytes. These activated macrophages generate oxidative stress, which might lead to a sterile, inflammatory reaction with secretion of growth factors, cytokines, and chemokines potentially deleterious to successful reproduction. Further investigations required to understand the exact biologic and pathologic significance of oxidative damage to granulosa cell DNA and the possibility of using 8-OHdG for the assessment of granulosa cell quality as a predictor of ART success rate and pregnancy outcome. Supported By: Mount Sinai School of Medicine, Department of Obstetrics and Gynecology.
Tuesday, October 23, 2001 4:15 P.M. O-123 Estradiol fails to down-regulate monocyte chemotactic protein-1 (MCP-1) expression in endometrial cells of women with endometriosis. A. Arici, M. O. Bahtiyar, E. Seli, L. Senturk. Yale Univ Sch of Medicine, New Haven, CT. Objective: MCP-1 is a potent chemoattractant for monocyte/macrophages. It is found at elevated levels in the peritoneal environment of women with endometriosis and induces proliferation of endometrial cells in culture. We have previously shown that MCP-1 expression is down-regulated by estradiol in normal endometrial stromal cells. Since estrogen levels are not different among women with or without endometriosis but local MCP-1 levels are higher in women with endometriosis; we hypothesized that the regulation of MCP-1 expression by estradiol may be different in endometrial cells of women with endometriosis. Design: Northern analysis and ELISA in human endometrial stromal cell cultures to assess the regulation of MCP-1 expression by estradiol. Materials/Methods: Endometrial samples were obtained from reproductive age women (n ⫽ 18) undergoing hysterectomy. Diagnoses were as follows: leiomyomata (n ⫽ 7, one of them with endometriosis); endometriosis (n ⫽ 7, one of them with leiomyoma); pelvic relaxation (n ⫽ 2); cervical intraepithelial neoplasia (n ⫽ 1); dermoid cyst (n ⫽ 1); pelvic pain (n ⫽ 1). Endometrial stromal cells were cultured in phenol red-free Ham’s F12:DME medium in the presence of charcoal stripped 10% FBS. The effect of estradiol (10-8 M) on the expression of MCP-1 mRNA was assessed by Northern analysis after 8 h of treatment. The expression of MCP-1 protein was assessed by ELISA after 24 h of treatment.
FERTILITY & STERILITY威
Results: We grouped the samples according the presence (n ⫽ 8) or absence (n ⫽ 10) of endometriosis. Samples were equally distributed between proliferative and secretory phases among two groups. In 9 out of 10 samples obtained from women without endometriosis estradiol induced inhibition of MCP-1 expression at both mRNA and protein levels. On the other hand, in cells that originated from women with endometriosis estradiol not only failed to down-regulate MCP-1 expression in 5 out 8 samples, but in three of these samples up-regulated MCP-1 expression. The difference between these two diagnostic groups in their response to estradiol was significant by Fisher’s exact t-test (p ⬍ 0.05). Conclusions: We found that estradiol down-regulates MCP-1 in normal endometrial cells but fails to do so in eutopic endometrial cells of women with endometriosis, and we suggest that this may be an important step in the pathogenesis of endometriosis.
Tuesday, October 23, 2001 4:30 P.M. O-124 Transcriptional regulation of the CYP1A1 gene by dioxin in eutopic endometrial and endometriotic stromal cells. B. Gurates, S. Yang, Z. Fang, M. Tamura, S. Sebastian, S. E. Bulun. Div of Reproductive Endocrinology and Infertility, Dept of Obstetrics and Gynecology, Univ of Illinois at Chicago, Chicago, IL. Objective: TCDD, also known as dioxin, is the archetype of a family of related polychlorinated compounds found ubiquitously in the environment. Exposure of primates to TCDD may enhance the development of endometriosis. We recently demonstrated significantly increased expression of a TCDD target gene, namely CYP1A1 in endometriotic tissue compared with the eutopic endometrium. We also demonstrated the promoter activity of the CYP1A1 gene in an endometrial cancer cell line and a benign endometrial stromal cell line. We hypothesize that P4501A1 enzyme, the product of the CYP1A1 gene, may promote the development and growth of endometriotic tissue by either activating procarcinogens or catalyzing conversion of estrogens to catechol estrogens. The objective of this study is to define the distribution of P4501A1 protein in eutopic endometrial and endometriotic tissues and the transcriptional regulation of the CYP1A1 gene in eutopic endometrial and endometriotic stromal cells in primary culture. Design: We used two model systems: 1) paired biopsies of eutopic endometrium and endometriosis performed simultaneously; 2) eutopic endometrial and endometriotic stromal cells in primary culture. Materials/Methods: We performed immunohistochemical analysis of paired samples (n ⫽ 7) of eutopic endometrium and endometriosis using an antibody against P4501A1. We transfected serial deletion mutants of the CYP1A1 gene promoter fused to the Luciferase reporter gene into eutopic endometrial and endometriotic stromal cells. Cells were treated with TCDD (10 nM) for 24 h. HepG2 liver cancer cell line was used as a positive control. Results: 1) We localized P4501A1 protein to epithelial, stromal and endothelial cells in both eutopic endometrial and endometriotic cells. 2) P4501A1 immunoreactivity in secretory endometrial and endometriotic samples seemed more intense compared with tissues in the proliferative phase. 3) The ⫺371/⫺49 bp region of the CYP1A1 promoter was found to contain cis-acting elements critical for baseline activity, whereas ⫺1171/ ⫺835 bp region was found to be critical for TCDD-induction in both cell types. Conclusions: P4501A1, the product of the CYP1A1 gene is present in both epithelial and stromal cells of eutopic endometrial and endometriotic tissues. The induction of the CYP1A1 gene by TCDD is mediated via a specific regulatory genomic region in both eutopic endometrial and endometriotic stromal cells. Supported By: NIH grant HD38691.
Tuesday, October 23, 2001 4:45 P.M. O-125 Oral contraceptives treatment suppresses proliferation and enhances apoptosis of eutopic endometrial tissue from patients with endometri-
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osis. G. F. Meresman, L. Auge, R. I. Baran˜ao, E. Lombardi, M. Tesone, C. Sueldo. Inst de Biologı´a y Medicine Experimental (IBYME)—CONICET, Buenos Aires, Argentina; Inst de Ginecologı´a y Fertilidad (IFER), Buenos Aires, Argentina; IBYME-CONICET, Buenos Aires, Argentina; IFER, Buenos Aires, Argentina. Objective: In previous studies we found that patients with endometriosis (EDT) have a significant enhancement of cell proliferation and diminished apoptosis of eutopic endometrium, and both factors may contribute to its etiopathogenesis. The purpose of this study was to assess the effect of combination oral contraceptives (COC) treatment on proliferation, apoptosis and its regulation by the expression of Bcl-2 and BAX, of eutopic endometrial tissue from patients with EDT and control women (C). Design: Linear study (non-randomized) by endometrial biopsies of patients undergoing gynecologic surgery and voluntary consenting controls. Materials/Methods: 11 EDT patients and 9 C had an endometrial biopsy before and after (11 EDT and 3 C) 30 days of COC. The assessment of cell proliferation was done by the presence of nuclear protein Ki-67 before and after COC. In the same biopsies apoptosis were detected by TUNEL assay; and its regulation, by the expressions of Bcl-2 and Bax, assessed by using immunohistochemical techniques (Meresman et al. Fertil Steril 2000;74: 760 – 6). Proliferation was measured as the percentage of Ki-67 positive cells and the other parameters were quantified as the number of positive cells/field at 630x magnification. Results: Before COC, EDT patients had an increased cell proliferation at the epithelial (EP) and stromal (ST) fractions compared to C (EP: p ⬍ 7.9 10⫺5, ST: p ⬍ 7.4 10⫺6). Simultaneously, a significantly decreased apoptosis, assessed by TUNEL, in EP (p ⬍ 0.002) and in ST (p ⬍ 0.0007), was observed in these patients correlating with an increased Bcl-2 and decreased BAX expression. After COC we found that, in EDT, patients cell proliferation was significantly decreased both in EP (p ⬍ 0.0002) as in ST (p ⬍ 7.2 10⫺5) however in this last fraction proliferation remains augmented with respect to C before COC (p ⬍ 0.02). At the same time, apoptosis of EP and ST was significantly augmented (p ⬍ 0.018 and p ⬍ 0.02 respectively vs. EDT before COC) and particularly in ST was similar to C before treatment (N.S.). Concurrently, after COC, EDT patients showed a significant increase in BAX expression and a decrease in Bcl-2 expression. At the same time in C, after COC, there was a significant increase in ST apoptosis (p ⬍0.05), however no significant changes in cell proliferation was observed. Conclusions: According with our results COC significantly diminished cell proliferation, induced apoptosis and an increased expression of BAX in eutopic endometrial tissue from EDT patients. This data may have clinical relevance in the etiopathogenesis and treatment of endometriosis
MALE REPRODUCTION AND UROLOGY Tuesday, October 23, 2001 2:00 P.M. O-126 Prevention of male infertility— experience with more than 600 young men with varicocele. D. A. Paduch, J. Niedzielski, E. Fuchs. Div of Urology, Oregon Health Science Univ, Portland, OR; Dept of Pediatric Surg, Lodz Univ Sch of Medicine, Lodz, Poland. Objective: To evaluate the epidemiology, natural history and risk factors for fertility impairment in adolescents with varicocele using ultrasound (US) based parameters. Design: Prospective study based on population screening of 2100 Polish high school students and patients referred to a tertiary andrology clinic. Materials/Methods: The clinical data, results of ultrasound examinations (7.5 MHz, linear probe and continues wave Doppler-CW), and results of subsequent exams and semen analysis were stored in computer database designed in early 90’s and maintained until now. US exams were performed every 6-12 months measuring testicular size, pampiniform plexus vein diameter, (PPVD), and peak and base venous flow velocity. Forty three patients had incomplete US measurements and were excluded from US based analysis. Only sexually mature (Tanner stage IV and V) men were included. Results: Of 2100 students screened we found 603 pts with varicocele (497 on the left, 100 bilateral and 6 on the right side only). Mean age of the
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Abstracts
patients was 15.7 yrs. Boys with grade III varicocele were on average 1 year younger than boys with grade I. Complete results of 560 USG exams were available. Left testicular atrophy was more often seen in boys with grade III varicocele. This was statistically significant. The left testicular atrophy was positively correlated with high peak retrograde flow velocity, high base flow velocity and pampiniform plexus vein diameter (PPVD) over 1.7 mm. However stepwise factorial analysis supported only stage, age and peak blood flow volume as factors significant in multifactorial linear model for left testicular atrophy. Based on 497 measurements of right PPVD in boys without varicocele the upper 95% of PPVD can be set as 1.7 mm. The lower-upper limits (5%–95%) of left PPD in boys with varicocele were as follows: G1: 1.7–1.99, G2: 1.94 –2.2, G3: 2.76 –3.01 mm. Repeated USG at 6 to 12 months showed persistent left testicular atrophy in 72 nontreated patients and significant increase in left testicular volume to equal the right testicle in 221 patients who underwent varicocele repair. Conclusions: Varicocele can cause left testicular atrophy in adolescents. The testicular atrophy is time and grade dependent. The clinical grading based on physical exam alone has excellent correlation with left testicular atrophy. PPVD above 1.7 mm is diagnostic of varicocele. Repair of varicocele in young males restores left testicular volume and can prevent future infertility. This is the largest prospective study on adolescent varicocele with US evaluation and thus allowed us to establish precise cut-off points and perform advanced statistical analysis of results.
Tuesday, October 23, 2001 2:15 P.M. O-127 The effect of varicocele repair on optimized sperm penetration assay. D. A. Ohl, J. D. McCarthy, T. G. Schuster, L. M. Keller, A. C. Menge, G. D. Smith. Univ of Michigan, Ann Arbor, MI. Objective: To determine the effect of varicocele repair on sperm function as measured by sperm penetration assay (SPA) of hamster oocytes. Design: Prospective case series examined before and after varicocele repair. Materials/Methods: Ninety-one men undergoing varicocele repair produced semen samples both prior to and ⬎ 4 months after varicocele repair. Optimized hamster oocyte SPA was performed on each sample as described by Johnson et al. (1984). The percentage of men with a normal pre-operative SPA was compared to the number with a normal post-operative SPA by the Chi-square analysis. Pre and post-operative differences in percentage of oocytes penetrated and penetrations per oocyte were compared with paired t-test. Assay results and changes were also correlated to pregnancy success or failure. Results: Of the 91 men enrolled in the study, only 19 had a normal SPA (100% oocyte penetration/5 or more penetrations per oocyte) pre-operatively. Post-operatively, 42/91 had a normal SPA (p ⬍ 0.0001). Average oocyte penetration rate was 55 ⫾ 37% (mean ⫾ SD) pre-operatively vs. 74 ⫾ 35% after surgery (p ⬍ 0.0001). Sperm penetrations per oocyte rose from 3.6 ⫾ 5.9 to 6.5 ⫾ 7.3 after surgery (p ⫽ 0.0004). Of the 72 men with a sub-standard preoperative SPA, 60 saw improvement post-operatively in either penetrations per oocyte or percentage of oocytes penetrated. In the group with an abnormal SPA prior to surgery, percentage of oocytes penetrated rose from 43 ⫾ 35% to 68 ⫾ 36% (p ⬍ 0.0001, paired t-test), and penetrations per oocyte rose from 1.0 ⫾ 1.2 to 5.1 ⫾ 6.9 (p ⬍ 0.0001, paired t-test). Sixty-one percent of couples with complete follow-up data achieved a natural pregnancy. Men initiating a natural pregnancy had a significant increase in oocyte penetration rate (58 ⫾ 37% to 82 ⫾ 28%, p ⫽ 0.001) and penetrations per oocyte (3.6 ⫾ 5.3 to 8.0 ⫾ 9.1, p ⫽ 0.018). Men unable to produce a pregnancy after surgery did not exhibit significantly increased oocyte penetration rate (61 ⫾ 44% to 80 ⫾ 32%, p⬎0.05) or penetrations per oocyte (6.4 ⫾ 9.7 to 6.3 ⫾ 6.3, p 0.05). Despite these differences, no obvious level of pre-operative SPA scores was found that would be predictive of post-operative success. Conclusions: A high percentage of men with varicocele exhibit abnormal SPA parameters, which are improved by varicocele repair. These results suggest that part of the pathophysiology of varicocele-related infertility is decreased fertilizing capacity. Improvement in sperm function, as opposed to standard semen analysis parameters, may be a major factor in successful varicocele repair. However, a pre-operative SPA cannot prospectively discriminate those who would benefit from surgery. Supported By: Internal funding.
Vol. 76, No. 3, Suppl. 1, September 2001
