control cells. Differences in the mutation frequency and apoptosis were ana- lyzed using Student's t-test (Sigma Stat package version 3). Statistical signifi-.
CONCLUSION: Consumption of the herbal beverage “Mate” was correlated with improved sperm motility and morphology in patients evaluated for infertility. This improvement in semen parameters appears to be mediated by increased seminal antioxidant activity in these men. Supported by: Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) P-849 COINCUBATION OF PERITONEAL FLUID FROM ENDOMETRIOSIS PATIENTS WITH SPERMATOZOA AND ITS CORRELATION WITH EXTENT OF DNA FRAGMENTATION. G. K. Mansour, R. K. Sharma, A. Agarwal, T. M. Said, J. M. Goldberg, T. Falcone. Cleveland Clinic, Cleveland, OH. OBJECTIVE: Oxidative stress and reactive oxygen species (ROS) in semen decrease sperm membrane integrity and consequently sperm function due to DNA damage. Evidence shows that endometriosis is a disease of oxidative stress. The objective of this study was to examine the effect of coincubation of peritoneal fluid of endometriosis patients on the extent of sperm DNA fragmentation. DESIGN: Prospective study. MATERIALS AND METHODS: Semen samples from 11 normal donors were prepared by density gradient separation. The samples were divided into 4 aliquots; group 1; control (bovine serum albumin (BSA 10%) ⫹ human tubal fluid media (HTF); groups 2 - 4: treated group (peritoneal fluid, 1:1 vol/vol). incubated with the sperm sample for 1.5, 4 and 24 h. DNA fragmentation of the spermatozoa was assessed using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). RESULTS: A significant difference was seen between the level of sperm DNA damage and the time of coincubation with the peritoneal fluid (Table). The extent of DNA fragmentation at 1.5 and at 4 hours was significantly different compared with both control and 24h incubation.
marker set of 12 tri- tetra- and pentanucleotide repeats on the Y chromosome (3) Multiplex containing markers with extended poly A repeats of 50-60bp. We also measured apoptosis using caspase-Glo3/7 assay (Promega). To test whether repeat markers were sensitive to oxidative stress a male human fibroblast cell line (AG01522) (Coriell cell repositories) was treated with 0.4, 0.8, 1.2, and 4 mM hydrogen peroxide diluted in PBS or PBS alone as control for 60 minutes. Following treatment, cells were placed in culture media and allowed to recover for 3 days. DNA was then extracted from both treated and control cells. Differences in the mutation frequency and apoptosis were analyzed using Student’s t-test (Sigma Stat package version 3). Statistical significance was defined as P ⬍ 0.05. RESULTS: We have identified three classes of microsatellite markers, which are sensitive to oxidative stress-induced mutations in human fibroblast cells. These include mononucleotide repeats with poly-A runs of over 40bp, A-rich pentanucleotide repeats and a variety of repeat types on the Y chromosome. We found that human fibroblast cells exposed to oxidative stress show a significant increase in mutation frequency (P ⬍ 0.05) when analyzed by either a multiplex assay of 5 extended mononucleotide repeats or one with 13 Y chromosome repeats. It is clear that some repeats are more sensitive to oxidative stress-induced mutations than others. Increased sensitivity may reflect differences in secondary structure between repeat sequences and/or differences in chromatin structure which could limit accessibility of the ROS molecules to the DNA. Results obtained from measuring apoptosis show that there is a significant increase in caspase 3/7 in the human fibroblast cells treated with 0.8 mM hydrogen peroxide (P ⬍ 0.05) and 4mM hydrogen peroxide (P ⬍ 0.001) compared to control cells. CONCLUSION: Our preliminary results demonstrate the efficacy of short tandem repeat markers for detection of mutations induced by oxidative stress. These markers may also be useful for monitoring potential health risks due to genetic damage. Supported by: National Aeronautics and Space Administration under Grant # NAG 9-1525 issued through the Office of Biological and Physical Research.
POLYCYSTIC OVARY SYNDROME P-851
CONCLUSION: Peritoneal fluid from women with endometriosis causes significant DNA damage of the spermatozoa. This may be one of the many factor(s) contributing to infertility in these patients. Supported by: None.
P-850 MONITORING OF GENETIC DAMAGE INDUCED BY OXIDATIVE STRESS. W. Abdel Megid, M. Kent-First, R. Halberg, M. Ensenberger, J. Bacher. Univ. of Wisconsin Madison, Madison, WI; Dept. of Biological Sciences, Mississippi State Univ., Starkville MS. Dept. of Obstetrics and Gynecology, Univ. of WI, Madison, WI; Promega Corporation, Madison, WI. OBJECTIVE: Reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion and hydroxyl radical can damage a wide variety of cellular components causing lipid peroxidation, protein oxidation, apoptosis and genetic damage through oxidation of DNA. Such damage has been implicated in various disease processes including cancer, aging and infertility. We hypothesize that repetitive DNA sequences are highly sensitive to oxidative stress, resulting in increased mutation frequency. DESIGN: To test this hypothesis, we exposed human fibroblast cells to variable doses of hydrogen peroxide to induce oxidative stress. We then screened repetitive DNA sequences to identify the genetic loci most sensitive to oxidative stress-induced mutations. MATERIALS AND METHODS: Microsatellite analysis was performed using small-pool PCR with 1-2 genome equivalents of DNA to detect mutant alleles present at low frequencies. A number of different microsatellite marker panels were used including (1) Microsatellite Instability Analysis System (Promega), a multiplexed marker set of five mononucleotide repeats and two A-rich pentanucleotide repeat markers (2) PowerPlex-Y (Promega), a multiplexed
FERTILITY & STERILITY威
IN VITRO EVIDENCE THAT HYPERGLYCEMIA STIMULATES INTERLEUKIN-6 (IL-6) RELEASE FROM MONONUCLEAR CELLS OF OBESE WOMEN WITH POLYCYSTIC OVARY SYNDROME. F. Gonzalez, N. S. Rote, J. Minium, J. P. Kirwan. Case Western Reserve Univ. School of Medicine, Cleveland, OH. OBJECTIVE: Hyperglycemia promotes inflammation in Polycystic Ovary Syndrome (PCOS), and circulating interleukin-6 (IL-6), a proinflammatory, proatherogenic cytokine is elevated in obese women with PCOS. We examined the effect of in vitro exposure to hyperglycemia on IL-6 release from mononuclear cells (MNC) of women with PCOS compared to those of ovulatory controls, and the relationship of this effect with body composition. DESIGN: Quantitative analysis within a cell culture system. MATERIALS AND METHODS: Twelve women with PCOS (6 lean, 6 obese) between ages 18-40, diagnosed on the basis of oligo- or amenorrhea and hyperandrogenemia, and 11 ovulatory controls (5 lean, 6 obese) of similar age were selected for study. Subjects with diabetes, inflammatory illnesses or other endocrinopathies were excluded. IL-6 release was measured from MNC cultured under euglycemic (5mM) and hyperglycemic (10mM and 15mM) conditions following isolation from fasting blood samples obtained within 5-8 days of menses. Body composition was assessed by DEXA. RESULTS: Body mass index (BMI), % total body fat and % truncal fat were similar for women with PCOS and control subjects. The mean % change in the concentration of IL-6 (% ⌬ IL-6) released from MNC of obese women with PCOS during hyperglycemic conditions in vitro increased progressively and was significantly higher compared to obese controls at glucose concentrations of 10mM (113.7%⫾8.3 vs. 100.4%⫾1.2, p⫽0.05) and 15mM (263.1%⫾76.3 vs. 95.5%⫾7.3, p⫽ 0.02). The mean % ⌬ IL-6 release from MNC of lean women with PCOS increased modestly in response to these hyperglycemic conditions but did not achieve significance. In contrast, the mean % ⌬ IL-6 release from MNC of lean and obese controls remained unchanged in response to hyperglycemia compared to the euglycemic baseline. There was no correlation of the in vitro IL-6 response to hyperglycemia with BMI, % total body fat or % truncal fat.
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CONCLUSION: These preliminary data indicate that hyperglycemia promotes an increased risk of atherosclerotic plaque rupture in women with PCOS that is independent of obesity. However, abdominal adiposity may be an additional perpetuator of inflammation that increases the risk of thrombosis in obese women with PCOS. Supported by: Grants HD048535 (FG) and HD01273-03 (WRHR) from NIH/NICHD
P-853 HYPERPROINSULINEMIA, INSULIN RESISTANCE AND BETACELL FUNCTION IN WOMEN WITH POLYCYSTIC OVARY SYNDROME. A. A. Rouzi, M. S. Ardawi. King Abdulaziz Univ., Jeddah, Saudi Arabia. CONCLUSION: These preliminary data indicate that MNC of obese women with PCOS exhibit increased IL-6 release under physiologic hyperglycemic conditions in vitro that may represent a proinflammatory trigger for atherogenesis. Although this phenomenon is not related to adiposity per se, it is most evident when the combination of PCOS and increased adiposity is present. Supported by: Grants HD048535 (FG) and HD01273-03 (WRHR) from NIH/NICHD P-852 HYPERGLYCEMIA STIMULATES PROATHEROGENIC INFLAMMATION PATHWAYS IN POLYCYSTIC OVARY SYNDROME. F. Gonzalez, N. S. Rote, J. Minium, J. P. Kirwan. Case Western Reserve Univ. School of Medicine, Cleveland, OH. OBJECTIVE: Activation of the proinflammatory transcription factors activator protein-1 (AP-1) and early growth response-1 (EGR-1) culminate in the transcription of tissue factor (TF) and metalloproteinase-2 (MMP-2), respectively. Atherosclerotic plaque rupture and thrombosis are promoted in the process. We examined the effect of glucose challenge on AP-1 activation and proinflammatory, proatherogenic protein expression in mononuclear cells (MNC) of women with Polycystic Ovary Syndrome (PCOS) compared to ovulatory controls, and the relationship of this effect with body composition. DESIGN: Prospective, controlled study MATERIALS AND METHODS: Sixteen women with PCOS (8 lean, 8 obese) between ages 18-40, diagnosed on the basis of oligomenorrhea and hyperandrogenemia, and 16 ovulatory controls (8 lean, 8 obese) of similar age were selected for study. Subjects with diabetes, inflammatory illnesses or other endocrinopathies were excluded. A 2-hour glucose tolerance test was performed within 5-8 days of menses. AP-1 activation was quantified by ELISA, and the protein expression of EGR-1, TF and MMP-2 were quantified by Western blotting in MNC isolated from blood samples drawn fasting (pre) and 2 hours after (post) glucose ingestion. Body composition was assessed by DEXA. RESULTS: Percent (%) total body fat and % truncal fat were similar for women with PCOS and control subjects. The mean percent change (% ⌬) in activated AP-1 in lean and obese women with PCOS (4.0⫾6.0%, 8.1⫾6.0%) increased (p⬍0.03) compared to lean controls which decreased (-13.5⫾5.0%). The mean % ⌬ in MMP-2 expression in lean and obese women with PCOS (10.8⫾6.5%, 20.9⫾11.4%) also increased (p⬍0.05) compared to lean controls which decreased (-13.0⫾7.5%). In contrast, the mean % ⌬ in the expression of EGR-1 and TF increased (p⬍0.03) in obese women with PCOS (31.7⫾8.4%, 39.2⫾12.5%), but was similar in lean women with PCOS (-3.9⫾8.4%, -2.7⫾2.7%) compared to lean controls (-0.8⫾10.9%, -12.1⫾7.2%). % truncal fat correlated positively with the % ⌬ in AP-1 (r⫽0.48, p⬍0.009), MMP-2 (r⫽0.41, p⬍0.04), EGR-1 (r⫽0.63, p⬍0.004) and TF (r⫽0.62, p⬍0.0008).
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Abstracts
OBJECTIVE: To determine serum pro-insulin levels in relation to insulin resistance (IR), Beta-cell function (BF) and hyperandrogenemia in women with polycystic ovary syndrome ( PCOS ). DESIGN: Prospective study. MATERIALS AND METHODS: One hundred and eighty selected women were classified as follows: 45 obese (BMI ⬎ 30 kg/m2) with PCOS; 45 lean (BMI⬍25 kg/m2) with PCOS, 45 obese ( BMI ⬎ 30 kg/m2) without PCOS and 45 lean ( BMI ⬍25 kg/m2) without PCOS. The diagnosis of PCOS was based on the presence of oligomenorrhea or amenorrhea or hirsutism, enlarged ovaries with multiple follicles (ⱖ10 measuring 2-8mm in diameter) arranged peripherally and scattered throughout the dense core of stroma on transvaginal ultrasonography, and/or elevated serum testosterone. Blood samples were collected from all women with or without PCOS between 8:00-10:00 am, after an overnight fast, and during a 75g-oral glucose tolerance test (OGTT). Serum levels of LH, FSH, TSH, FT4, T, 17-OHP, ⌬4-A, DHEA, DHEAS, and SHBG were measured. Serum insulin, pro-insulin and glucose levels during OGTT were also measured and BF was determined. Measures of IR included: fasting serum insulin, GIR and HOMA. RESULTS: Pro-insulin levels were found to be significantly increased in women with PCOS as compared to lean women without PCOS. The measures of pro-insulin and insulin secretion together with the area under the curves (AUC) for pro-insulin were increased in obese women with or without PCOS. The AUC of pro-insulin correlated positively with IR, BF and the extent of hyperandrogenaemia. PCOS was associated with higher T, ⌬4-A, DHEA-S, and lower SHBG levels. Stepwise linear multiple regression analysis showed that AUC for insulin, serum ⌬4-A were the most significant independent determinants of AUC for pro-insulin. Measures of IR and ⌬4-A were the most significant independent determinants of proinsulin- to -insulin ratio. CONCLUSION: Hyperpro-insulinaemia together with IR were evident in women with PCOS. IR could contribute to the changes in BF, and in that of both serum pro-insulin and androgens in women with PCOS . Supported by: NONE
P-854 PREVALENCE AND PREDICTORS OF THE METABOLIC SYNDROME IN SAUDI WOMEN WITH POLYCYSTIC OVARY SYNDROME. M. S. Ardawi, A. A. Rouzi. King Abdulaziz Univ., Jeddah, Saudi Arabia. OBJECTIVE: To determine the prevalence and predictors of the metabolic syndrome (MBS) in women with polycystic ovary syndrome (PCOS) as compared with women without PCOS and to assess the role of insulin resistance (IR) and androgens in the development of MBS. DESIGN: A prospective Case Control Study. MATERIALS AND METHODS: Four hundred and fifty Saudi women living in the Jeddah area were classified as follows: 225 with PCOS and 225 age-matched women without PCOS. The diagnosis of PCOS was based on the presence of oligomenorrhea or amenorrhea or hirsutism, enlarged ovaries with multiple follicles (ⱖ10 measuring 2-8mm in diameter) arranged peripherally and scattered throughout the dense core of stroma on transvaginal ultrasonography, and/or elevated serum testosterone. Blood samples were collected from all women with or without PCOS between 8:00-11:00, after an overnight fast. Serum levels of LH, FSH, TSH, FT4, 17-OHP, ⌬4-A, DHEAs, total T, free T, SHBG, insulin, HDL-c, triglycerides and plasma levels of glucose were determined.
Vol. 86, Suppl 2, September 2006
