- Email: [email protected]
The ‘muffin test’ – a potential alternative to formal oral glucose tolerance test (OGTT) for the detection of impaired glucose tolerance (IGT)
TABLE 1. Live birth rate by serum mid-luteal progesterone concentration
Mid-luteal progesterone (ng/mL)
Live birth rate
R7.9 – %10 (N¼25) >10 – %15 (N¼76) >15 – %25 (N¼110) >25 – %40 (N¼49) >40 (N¼22)
8% 14% 21% 29% 32%
CONCLUSIONS: Serum mid-luteal progesterone concentration is positively correlated with live birth rate in WHO group II anovulatory patients undergoing ovulation induction with a low-dose protocol. Supported by: Ferring Pharmaceuticals.
P-39 DAY 28 b-HCG VALUES OF VIABLE PREGNANCIES FROM BIOPSIED EMBRYOS FOR PREIMPLANTATION GENETIC DIAGNOSIS AND SCREENING (PGDS) ARE LOWER THAN VIABLE PREGNANCIES FROM NON-BIOPSIED EMBRYOS. S. L. Marshall, M. Liu, S. Talebian, J. Salas, D. Keegan, J. Grifo. NYU Medical Center, New York, NY. OBJECTIVE: To determine the predictive value between early serum bhCG and pregnancy outcome on in vitro fertilization (IVF) cycles of biopsied embryos. DESIGN: Retrospective data analysis at a university based fertility center. MATERIALS AND METHODS: A retrospective analysis was performed on 51 IVFþPGDS cycles during the years of 2001- 2003. A positive b-hCG was defined as >5 mlU/ml. Oocyte retrieval was considered day 14 of the cycle. Embryo transfer occurred on either day 18 or day 19. The majority of serum b-hCG levels were measured on day 27 or 28 and day 34 or 35. However patients with an initial b-hCG level <50 mlU/ml had repeat measurements prior to day 34 or 35. Pregnancies considered viable were defined as those continuing into the second trimester, including all singleton, twin, and high order multiple deliveries, and stillbirths. Non-viable pregnancies included biochemical and ectopic pregnancies, as well as spontaneous and therapeutic abortions. An estimate of the b-hCG level which would likely predict outcome at a certain time point was calculated using Receiver operator characteristic (ROC) curves. RESULTS: The mean age of the patients was 33 (range: 25-48) years. Table 1 illustrates the number of b-hCG measurements available for each of the 5 early pregnancy time points (data sets 1-5), and the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the associated b-hCG concentrations. TABLE 1.
Cycle Day
Number of Observations
Cut-Off
Sensitivity
Specificity
PPV
NPV
27-28 29-30 31-32 33-35 36-38
49 6 3 20 3
52 105 157 1232 2465
79 67 100 80 100
93 100 100 40 100
88.2 100 100 80 100
87.5 75 100 40 100
CONCLUSIONS: Our data reveals that a b-hCG > 52 mIU/ml on day 2728 of an IVFþPGDS cycle predicts a viable pregnancy 88% of the time. If the b-hCG level is < 52, the pregnancy is non-viable 88% of the time. In comparison to previous analysis of fresh IVF cycles and donor egg (DE) IVF cycles the b-hCG level which resulted in viable pregnancy on day 27-28 of cycles using PGDS was significantly lower (133mIU/ml in DEIVF and 85 mIU/ml in fresh IVF cycle, p<0.001). A possible explanation for the lower initial b-hcg could be a later implantation that may occur in biopsied embryos. This data provides reassuring information that despite a lower b-hCG on days 27-28 for biopsied embryos, a viable pregnancy is still a strong possibility. Supported by: None.
S124
Abstracts
P-40 EXPRESSION OF AROMATASE INCREASES IN BOTH THE MYOCARDIUM AND THE AORTIC ENDOTHELIUM DURING ACUTE ILLNESS. D. A. Pennington, J. Connerney, D. I. Spratt. Obstetrics and Gynecology, Maine Medical Center, Portland, ME; Maine Medical Center Research Institute, Scarborough, ME; Obstetrics and Gynecology and Endocrine Research, Maine Medical Center and MMC Research Institute, Portland, ME. OBJECTIVE: Serum estrogen levels rise in humans during acute illness due to increased aromatase expression in adipose tissue. We have also demonstrated increased expression of aromatase in adipose and myocardial tissue in a mouse model of sepsis. Because estrogen appears beneficial to the cardiovascular system, we determined if vascular endothelium as well as myocardial tissue can locally express aromatase and if that expression increases during acute illness. DESIGN: Aromatase expression was assessed in both aortic endothelial tissue and myocardial in acutely ill male black 6 mice and compared to healthy male black 6 mice. MATERIALS AND METHODS: Acute illness was produced by cecal ligation and puncture (CLP) in 24 male black 6 mice. 12 mice undergoing sham surgery (SS) served as controls. Animals were scored to determine severity of illness. Aortic and cardiac tissues were harvested at the time of sacrifice 36-72 hours after surgery. The presence of aromatase was assessed by immunohistochemistry in cardiac and aortic tissue using a rabbit anti-mouse aromatase antibody (BioVision, Ca). RESULTS: Mice undergoing SS recovered promptly after surgery and demonstrated no evidence of illness at the time of sacrifice. Mice undergoing CLP were markedly ill at the time of surgery as assessed by ruffled fur, lethargy and weight loss. Increased expression of aromatase was clearly evident by immunohistochemistry in both the aortic endothelium and myocardium by immunohistochemistry staining in the CLP mice compared to the SS mice. CONCLUSIONS: We conclude that both myocardial and aortic endothelial tissues can express aromatase and that aromatase expression increases during acute illness. Thus, in addition to increased systemic estrogen production by enhanced aromatase expression in adipose tissue, cardiovascular tissues (that may benefit from estrogen effects during acute illness) appear capable of producing their own estrogen. The physiology of aromatase expression in cardiovascular tissues during illness and its potential impact on cardiovascular function and survival can be further evaluated in this mouse model of sepsis. Supported by: Maine Medical Center Research Strategic Plan and Maine Medical Center Research Institute. P-41 THE ‘MUFFIN TEST’ – A POTENTIAL ALTERNATIVE TO FORMAL ORAL GLUCOSE TOLERANCE TEST (OGTT) FOR THE DETECTION OF IMPAIRED GLUCOSE TOLERANCE (IGT). M. L. Traub, A. Jain, B.-S. Maslow, L. Pal, D. T. Stein, R. Freeman. Obstetrics & Gynecology & Women’s Health, Albert Einstein College of Medicine, Bronx, NY; Albert Einstein College of Medicine, Bronx, NY; Yale University School of Medicine, New Haven, CT. OBJECTIVE: For better tolerability and reflecting more physiologic postmeal glucose excursions, a muffin containing glucose, lipid and protein may help identify women at risk of metabolic syndrome. We reported preliminary results from the KEEPS (Kronos Early Estrogen Prevention Study) showing the ‘‘Muffin Test’’ (MT) compared favorably to OGTT. Here we compare the effectiveness of the MT versus OGTT for diagnosing IGT in different patient populations. DESIGN: Prospective cohort study. MATERIALS AND METHODS: Subjects: 2 Populations of participants. Group 1 consisted of 73 women aged 42-58, <36 months post-menopause, recruited for the KEEPS, of which a subset (n¼10) underwent metabolic testing. Group 2 consisted of 13 women age 26-38 being followed clinically for polycystic ovarian syndrome (PCOS). After a 10-hour fasting blood draw, participants were given a muffin and beverage. 2-Hour finger stick glucose, height, weight, and waist circumference were assessed. A subset in each population underwent OGTT at a later visit. RESULTS: The patients in Group 1 were older with higher LDL levels than Group 2. Although both populations were given a muffin and beverage, the total CHO load was higher in Group 2, reflective of a different brand of muffin. The prevalence of IGT in Group 1 was 11% (8/71) and FPG would have missed 63% (5/8); the prevalence of IGT in Group 2 was 15% (2/13) and FPG would have missed 50% (1/2). Fasting and 2-hour MT glucose values
Vol. 90, Suppl 1, September 2008
were related. On multivariable linear regression, adjusting for BMI and CHO ingested, an increase in FPG by 1 mg/dL was related to a 1.15 mg/dl increase in 2-hour post MT glucose in Group 1 (p<0.001) and a similar 1.23 mg/dl increase in Group 2 (p¼0.022). This association was exaggerated in overweight (BMI >25 kg/m2) women in both Group 1 (coefficient 1.38, p<0.001) and Group 2 (coefficient 1.54, p¼0.007). A total of 10 KEEPS and 8 PCOS patients had both the MT and OGTT. Within each group, both fasting and 2-hr glucose values were comparable between MT and OGTT (p>0.05). CONCLUSIONS: FPG was inadequate for diagnosing IGT in both populations. FPG was related to 2-hour glucose levels; the post MT excursion in glucose values was exaggerated in heavier women. In this limited study, the MT demonstrated 100% sensitivity and specificity for diagnosing IGT in comparison to the gold standard OGTT. The Muffin Test needs to be validated in a larger population prior to consideration for routine clinical use. Supported by: Kronos Longevity Research Institute (NS, RF), HD041978 (NS) and RR95261064. P-42 REGULATION OF ADULT HUMAN SPERMATOGONIAL STEM CELLS PROLIFERATION BY ESTRADIOL IN VITRO. L. Fan, K. Tao, Y. Sun, J. Tu, W. Zhu. Institute of Reproductive & Stem Cell Engineering, Changsha, China. OBJECTIVE: Spermatogonial stem cells (SSCs), which are the only germline stem cells in adults, self-renew and produce a large number of differentiating germ cells. In the niche on the basement membrane of seminiferous tubules, SSCs are secluded from the outside world and affected by all kinds of nutrients, growth factors and hormones. Estrogens are now known to have profound effects on male reproductive systems and estrogen receptors (ERs) have been detected in adult human germ cells, thus the aim of the present study was to evaluate the role of 17b-estradiol, the main physiological estrogen, in SSCs proliferation in vitro. DESIGN: Employing a defined culture system for SSCs, which were characterized positive for its potential specific markers by detecting AP activity and immunofluorescence staining, we evaluated the role of estradiol in SSCs proliferation in vitro by co-labelling for c-Kit and BrdU positive cells. MATERIALS AND METHODS: Testis tissue was obtained from nine healthy and fertile donors in the Reproductive and Genetic Hospital of CITIC-Xiangya. Testis cells were collected by two-step enzymatic digestion and Ficoll fractionation. Dissociated cells were aliquoted into tissue culture plates and cultivated with a modified system composed of serum-free defined medium on mouse embryonic fibroblasts (MEF) feeders. To investigate the effect of estrogen, 17b-estradiol was added at 100ng/ml in the defined culture medium. Alkaline phosphatase (AP) activity and immunofluorescence staining of oct3/4 and c-Kit was analyzed by following the manufacturer’s instructions. Additionally, Co-labelling for c-Kit and 5-Bromodeoxyuridine (BrdU) were used to determine SSCs proliferation in the presence or absence of estradiol. RESULTS: The cultured cells defined as SSCs were characterized positive for AP activity, oct3/4 and c-Kit. SSCs could expand in serum-free defined conditions on MEF. By contrast, the number of BrdU-labelled SSCs was markedly enhanced by treatment with 17b-estradiol for 2 days. CONCLUSIONS: Our studies indicating that estradiol appears to be a potent regulator in the adult human SSCs proliferation in vitro. The result obtained in the present study could be used when optimizing in vitro culture conditions of adult human SSCs. These findings suggest that estradiol are involved in physiological paracrine signalling during the spermatogenesis. Supported by: National Natural Science Foundation of China (No. 30170480 and No. 30470884). P-43 EFFECT OF CELL CYCLE SPECIFICITY OF CHEMOTHERAPEUTIC AGENTS ON THE GONADAL FUNCTIONS OF A PEDIATRIC CANCER POPULATION: ROLE OF ANTI-MULLERIAN HORMONE (AMH). M. A. Bedaiwy, R. Mahfouz, H. Kubaney, G. Plautz, R. Peterson, T. Falcone. Obstetrics and Gynecology, University Hospitals of Cleveland, Cleveland, OH; The Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: The objectives of the study are to 1) determine whether AMH levels could be used to quantify gonadal damage inflicted by gonadotoxic chemotherapy and 2) to examine the effect of cell cycle specificity of the chemotherapeutic agents on the gonadal reserve as evidenced by the post-treatment AMH assay. DESIGN: Cohort study.
FERTILITY & STERILITYÒ
MATERIALS AND METHODS: Baseline blood samples were obtained from 34 cancer patients (group I: 27 males and 7 females) and 10 normal controls (group II: 7 males and 3 females). Group I was composed of patients treated with chemotherapy for hematological, musculoskeletal and soft tissue cancers in 18,6 and 10 patients, respectively. Patients were followed until the completion of chemotherapy (range1-3 years). A second blood sample was collected at the completion of chemotherapy. AMH assay was completed using a commercially available kit. RESULTS: Using Spearman correlation, baseline AMH levels were significantly correlated with age of the study population (R¼0.41, P¼0.007). Patients and controls were comparable regarding their age at the initiation of chemotherapy. Baseline AMH levels were comparable between patients and controls irrespective of gender, age, pubertal status and type of malignancy (Boys7.834.75ng/ml vs 17.247.77 ng/ml; P¼0.056) and (Girls 1.531.39 ng/ml vs 1.860.55 ng/ml; P¼0.12)}. Post-chemotherapy AMH levels were not significantly different from baseline in boys (9.867.30 ng/ml vs 7.834.75 ng/ml, P¼0.28). On the other side, postchemotherapy AMH levels were significantly lower from baseline in girls (0.811.11 ng/ml vs 1.531.39 ng/ml, P¼0.02). In boys and girls there was no significant reduction of post-chemotherapy AMH levels in subjects who received cell cycle specific chemotherapy. Similarly, there was no significant reduction of post-chemotherapy AMH levels in male subjects who received cell cycle non specific chemotherapy, P¼0.06. On the contrary, girls who received cell cycle non specific chemotherapy showed significantly lower AMH levels compared to baseline (0.22 0.35 ng/ml vs 1.28 1.43 ng/ml; P¼0.01). CONCLUSIONS: AMH assay is a sensitive marker for ovarian functions before and after puberty in patients at risk of gonadal damage secondary to chemotherapy. Ovarian reserve appears to be more sensitive to the insult of cell cycle non specific chemotherapeutic agents than the testicular reserve. AMH assay could be added to the initial work up of all patients undergoing chemotherapy to assess the damage to their reproductive potential. Supported by: None. P-44 2-METHOXY ESTRADIOL INHIBITS ANGIOGENESIS IN SHEEP UTERINE ARTERY ENDOTHELIAL CELLS. S. Salih, A. Kapur, R. Magness. University of Wisconsin, Madison, WI. OBJECTIVE: Estrogen plays a key role in priming the endometrium for luteal phase development, embryo implantation, and early placentation. The effects of estrogen metabolites and estrogen metabolizing enzymes in endometrial vasulature are not well studied. Here we investigate the angiogenic and vasodilatory effect of 17b estradiol and 2-methoxy estradiol using the sheep model. DESIGN: Angiogenic activity of 17b estradiol and 2-methoxy estradiol on uterine artery endothelial cells (UAEC) from luteal phase, follicular phase, and pregnant sheep was investigated. MATERIALS AND METHODS: UAEC from luteal phase, follicular phase, and pregnant sheep were cultured and plated 1105 cells per well in 12 well plates on a matrigel with vehicle, 17b estradiol, and 2-methoxy estradiol. Capillary-like structure formation was checked 6 hours. Protein assay for ERK, AKT and eNOS expression was performed using Western Blot analysis. RESULTS: 17b estradiol induced capillary tube formation on UAEC from all phases; luteal, follicular, and pregnant sheep. The most pronounced effect was in UAEC from pregnant sheep. 2-methoxy estradiol on the other hand completely inhibited capillary tube formation in UAECs. In follicular phase UAEC there was no significant change in pAKT/tAKT with 17b estradiol or 2-methoxy estradiol. However there was a significant decrease in pERK/tERK with 2-methoxy estradiol but no change with 17b estradiol. There was no difference in pENOS/tENOS ratio with either 17b or 2-methoxy estradiol. In UAEC from pregnant sheep there was a significant decrease in pERK/tERK with 2-methoxy estradiol. There was no significant difference in pAKT/tAKT or pENOS/tENOS with 2-methoxy estradiol; however there was a significant increase in pAKT/tAKT and pERK/tERK with 17b estradiol. In luteal phase UAEC there was a significant decrease in pAKT/tAKT, pERK/tERK as well as pENOS/tENOS with 2-methoxy estradiol as compared to control. Similar decrease was observed with 17b estradiol, but the decrease was not significant. CONCLUSIONS: The anti-angiogenic effect of 2-methoxy estradiol appears to be mediated in part via ERK signaling pathway. There was significant decrease in phosphorylation of ERK1 and ERK2 in UAEC from luteal, follicular, and pregnant sheep. 2-methoxy estradiol inhibits
S125
Mid-luteal progesterone (ng/mL)
Live birth rate
R7.9 – %10 (N¼25) >10 – %15 (N¼76) >15 – %25 (N¼110) >25 – %40 (N¼49) >40 (N¼22)
8% 14% 21% 29% 32%
CONCLUSIONS: Serum mid-luteal progesterone concentration is positively correlated with live birth rate in WHO group II anovulatory patients undergoing ovulation induction with a low-dose protocol. Supported by: Ferring Pharmaceuticals.
P-39 DAY 28 b-HCG VALUES OF VIABLE PREGNANCIES FROM BIOPSIED EMBRYOS FOR PREIMPLANTATION GENETIC DIAGNOSIS AND SCREENING (PGDS) ARE LOWER THAN VIABLE PREGNANCIES FROM NON-BIOPSIED EMBRYOS. S. L. Marshall, M. Liu, S. Talebian, J. Salas, D. Keegan, J. Grifo. NYU Medical Center, New York, NY. OBJECTIVE: To determine the predictive value between early serum bhCG and pregnancy outcome on in vitro fertilization (IVF) cycles of biopsied embryos. DESIGN: Retrospective data analysis at a university based fertility center. MATERIALS AND METHODS: A retrospective analysis was performed on 51 IVFþPGDS cycles during the years of 2001- 2003. A positive b-hCG was defined as >5 mlU/ml. Oocyte retrieval was considered day 14 of the cycle. Embryo transfer occurred on either day 18 or day 19. The majority of serum b-hCG levels were measured on day 27 or 28 and day 34 or 35. However patients with an initial b-hCG level <50 mlU/ml had repeat measurements prior to day 34 or 35. Pregnancies considered viable were defined as those continuing into the second trimester, including all singleton, twin, and high order multiple deliveries, and stillbirths. Non-viable pregnancies included biochemical and ectopic pregnancies, as well as spontaneous and therapeutic abortions. An estimate of the b-hCG level which would likely predict outcome at a certain time point was calculated using Receiver operator characteristic (ROC) curves. RESULTS: The mean age of the patients was 33 (range: 25-48) years. Table 1 illustrates the number of b-hCG measurements available for each of the 5 early pregnancy time points (data sets 1-5), and the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the associated b-hCG concentrations. TABLE 1.
Cycle Day
Number of Observations
Cut-Off
Sensitivity
Specificity
PPV
NPV
27-28 29-30 31-32 33-35 36-38
49 6 3 20 3
52 105 157 1232 2465
79 67 100 80 100
93 100 100 40 100
88.2 100 100 80 100
87.5 75 100 40 100
CONCLUSIONS: Our data reveals that a b-hCG > 52 mIU/ml on day 2728 of an IVFþPGDS cycle predicts a viable pregnancy 88% of the time. If the b-hCG level is < 52, the pregnancy is non-viable 88% of the time. In comparison to previous analysis of fresh IVF cycles and donor egg (DE) IVF cycles the b-hCG level which resulted in viable pregnancy on day 27-28 of cycles using PGDS was significantly lower (133mIU/ml in DEIVF and 85 mIU/ml in fresh IVF cycle, p<0.001). A possible explanation for the lower initial b-hcg could be a later implantation that may occur in biopsied embryos. This data provides reassuring information that despite a lower b-hCG on days 27-28 for biopsied embryos, a viable pregnancy is still a strong possibility. Supported by: None.
S124
Abstracts
P-40 EXPRESSION OF AROMATASE INCREASES IN BOTH THE MYOCARDIUM AND THE AORTIC ENDOTHELIUM DURING ACUTE ILLNESS. D. A. Pennington, J. Connerney, D. I. Spratt. Obstetrics and Gynecology, Maine Medical Center, Portland, ME; Maine Medical Center Research Institute, Scarborough, ME; Obstetrics and Gynecology and Endocrine Research, Maine Medical Center and MMC Research Institute, Portland, ME. OBJECTIVE: Serum estrogen levels rise in humans during acute illness due to increased aromatase expression in adipose tissue. We have also demonstrated increased expression of aromatase in adipose and myocardial tissue in a mouse model of sepsis. Because estrogen appears beneficial to the cardiovascular system, we determined if vascular endothelium as well as myocardial tissue can locally express aromatase and if that expression increases during acute illness. DESIGN: Aromatase expression was assessed in both aortic endothelial tissue and myocardial in acutely ill male black 6 mice and compared to healthy male black 6 mice. MATERIALS AND METHODS: Acute illness was produced by cecal ligation and puncture (CLP) in 24 male black 6 mice. 12 mice undergoing sham surgery (SS) served as controls. Animals were scored to determine severity of illness. Aortic and cardiac tissues were harvested at the time of sacrifice 36-72 hours after surgery. The presence of aromatase was assessed by immunohistochemistry in cardiac and aortic tissue using a rabbit anti-mouse aromatase antibody (BioVision, Ca). RESULTS: Mice undergoing SS recovered promptly after surgery and demonstrated no evidence of illness at the time of sacrifice. Mice undergoing CLP were markedly ill at the time of surgery as assessed by ruffled fur, lethargy and weight loss. Increased expression of aromatase was clearly evident by immunohistochemistry in both the aortic endothelium and myocardium by immunohistochemistry staining in the CLP mice compared to the SS mice. CONCLUSIONS: We conclude that both myocardial and aortic endothelial tissues can express aromatase and that aromatase expression increases during acute illness. Thus, in addition to increased systemic estrogen production by enhanced aromatase expression in adipose tissue, cardiovascular tissues (that may benefit from estrogen effects during acute illness) appear capable of producing their own estrogen. The physiology of aromatase expression in cardiovascular tissues during illness and its potential impact on cardiovascular function and survival can be further evaluated in this mouse model of sepsis. Supported by: Maine Medical Center Research Strategic Plan and Maine Medical Center Research Institute. P-41 THE ‘MUFFIN TEST’ – A POTENTIAL ALTERNATIVE TO FORMAL ORAL GLUCOSE TOLERANCE TEST (OGTT) FOR THE DETECTION OF IMPAIRED GLUCOSE TOLERANCE (IGT). M. L. Traub, A. Jain, B.-S. Maslow, L. Pal, D. T. Stein, R. Freeman. Obstetrics & Gynecology & Women’s Health, Albert Einstein College of Medicine, Bronx, NY; Albert Einstein College of Medicine, Bronx, NY; Yale University School of Medicine, New Haven, CT. OBJECTIVE: For better tolerability and reflecting more physiologic postmeal glucose excursions, a muffin containing glucose, lipid and protein may help identify women at risk of metabolic syndrome. We reported preliminary results from the KEEPS (Kronos Early Estrogen Prevention Study) showing the ‘‘Muffin Test’’ (MT) compared favorably to OGTT. Here we compare the effectiveness of the MT versus OGTT for diagnosing IGT in different patient populations. DESIGN: Prospective cohort study. MATERIALS AND METHODS: Subjects: 2 Populations of participants. Group 1 consisted of 73 women aged 42-58, <36 months post-menopause, recruited for the KEEPS, of which a subset (n¼10) underwent metabolic testing. Group 2 consisted of 13 women age 26-38 being followed clinically for polycystic ovarian syndrome (PCOS). After a 10-hour fasting blood draw, participants were given a muffin and beverage. 2-Hour finger stick glucose, height, weight, and waist circumference were assessed. A subset in each population underwent OGTT at a later visit. RESULTS: The patients in Group 1 were older with higher LDL levels than Group 2. Although both populations were given a muffin and beverage, the total CHO load was higher in Group 2, reflective of a different brand of muffin. The prevalence of IGT in Group 1 was 11% (8/71) and FPG would have missed 63% (5/8); the prevalence of IGT in Group 2 was 15% (2/13) and FPG would have missed 50% (1/2). Fasting and 2-hour MT glucose values
Vol. 90, Suppl 1, September 2008
were related. On multivariable linear regression, adjusting for BMI and CHO ingested, an increase in FPG by 1 mg/dL was related to a 1.15 mg/dl increase in 2-hour post MT glucose in Group 1 (p<0.001) and a similar 1.23 mg/dl increase in Group 2 (p¼0.022). This association was exaggerated in overweight (BMI >25 kg/m2) women in both Group 1 (coefficient 1.38, p<0.001) and Group 2 (coefficient 1.54, p¼0.007). A total of 10 KEEPS and 8 PCOS patients had both the MT and OGTT. Within each group, both fasting and 2-hr glucose values were comparable between MT and OGTT (p>0.05). CONCLUSIONS: FPG was inadequate for diagnosing IGT in both populations. FPG was related to 2-hour glucose levels; the post MT excursion in glucose values was exaggerated in heavier women. In this limited study, the MT demonstrated 100% sensitivity and specificity for diagnosing IGT in comparison to the gold standard OGTT. The Muffin Test needs to be validated in a larger population prior to consideration for routine clinical use. Supported by: Kronos Longevity Research Institute (NS, RF), HD041978 (NS) and RR95261064. P-42 REGULATION OF ADULT HUMAN SPERMATOGONIAL STEM CELLS PROLIFERATION BY ESTRADIOL IN VITRO. L. Fan, K. Tao, Y. Sun, J. Tu, W. Zhu. Institute of Reproductive & Stem Cell Engineering, Changsha, China. OBJECTIVE: Spermatogonial stem cells (SSCs), which are the only germline stem cells in adults, self-renew and produce a large number of differentiating germ cells. In the niche on the basement membrane of seminiferous tubules, SSCs are secluded from the outside world and affected by all kinds of nutrients, growth factors and hormones. Estrogens are now known to have profound effects on male reproductive systems and estrogen receptors (ERs) have been detected in adult human germ cells, thus the aim of the present study was to evaluate the role of 17b-estradiol, the main physiological estrogen, in SSCs proliferation in vitro. DESIGN: Employing a defined culture system for SSCs, which were characterized positive for its potential specific markers by detecting AP activity and immunofluorescence staining, we evaluated the role of estradiol in SSCs proliferation in vitro by co-labelling for c-Kit and BrdU positive cells. MATERIALS AND METHODS: Testis tissue was obtained from nine healthy and fertile donors in the Reproductive and Genetic Hospital of CITIC-Xiangya. Testis cells were collected by two-step enzymatic digestion and Ficoll fractionation. Dissociated cells were aliquoted into tissue culture plates and cultivated with a modified system composed of serum-free defined medium on mouse embryonic fibroblasts (MEF) feeders. To investigate the effect of estrogen, 17b-estradiol was added at 100ng/ml in the defined culture medium. Alkaline phosphatase (AP) activity and immunofluorescence staining of oct3/4 and c-Kit was analyzed by following the manufacturer’s instructions. Additionally, Co-labelling for c-Kit and 5-Bromodeoxyuridine (BrdU) were used to determine SSCs proliferation in the presence or absence of estradiol. RESULTS: The cultured cells defined as SSCs were characterized positive for AP activity, oct3/4 and c-Kit. SSCs could expand in serum-free defined conditions on MEF. By contrast, the number of BrdU-labelled SSCs was markedly enhanced by treatment with 17b-estradiol for 2 days. CONCLUSIONS: Our studies indicating that estradiol appears to be a potent regulator in the adult human SSCs proliferation in vitro. The result obtained in the present study could be used when optimizing in vitro culture conditions of adult human SSCs. These findings suggest that estradiol are involved in physiological paracrine signalling during the spermatogenesis. Supported by: National Natural Science Foundation of China (No. 30170480 and No. 30470884). P-43 EFFECT OF CELL CYCLE SPECIFICITY OF CHEMOTHERAPEUTIC AGENTS ON THE GONADAL FUNCTIONS OF A PEDIATRIC CANCER POPULATION: ROLE OF ANTI-MULLERIAN HORMONE (AMH). M. A. Bedaiwy, R. Mahfouz, H. Kubaney, G. Plautz, R. Peterson, T. Falcone. Obstetrics and Gynecology, University Hospitals of Cleveland, Cleveland, OH; The Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: The objectives of the study are to 1) determine whether AMH levels could be used to quantify gonadal damage inflicted by gonadotoxic chemotherapy and 2) to examine the effect of cell cycle specificity of the chemotherapeutic agents on the gonadal reserve as evidenced by the post-treatment AMH assay. DESIGN: Cohort study.
FERTILITY & STERILITYÒ
MATERIALS AND METHODS: Baseline blood samples were obtained from 34 cancer patients (group I: 27 males and 7 females) and 10 normal controls (group II: 7 males and 3 females). Group I was composed of patients treated with chemotherapy for hematological, musculoskeletal and soft tissue cancers in 18,6 and 10 patients, respectively. Patients were followed until the completion of chemotherapy (range1-3 years). A second blood sample was collected at the completion of chemotherapy. AMH assay was completed using a commercially available kit. RESULTS: Using Spearman correlation, baseline AMH levels were significantly correlated with age of the study population (R¼0.41, P¼0.007). Patients and controls were comparable regarding their age at the initiation of chemotherapy. Baseline AMH levels were comparable between patients and controls irrespective of gender, age, pubertal status and type of malignancy (Boys7.834.75ng/ml vs 17.247.77 ng/ml; P¼0.056) and (Girls 1.531.39 ng/ml vs 1.860.55 ng/ml; P¼0.12)}. Post-chemotherapy AMH levels were not significantly different from baseline in boys (9.867.30 ng/ml vs 7.834.75 ng/ml, P¼0.28). On the other side, postchemotherapy AMH levels were significantly lower from baseline in girls (0.811.11 ng/ml vs 1.531.39 ng/ml, P¼0.02). In boys and girls there was no significant reduction of post-chemotherapy AMH levels in subjects who received cell cycle specific chemotherapy. Similarly, there was no significant reduction of post-chemotherapy AMH levels in male subjects who received cell cycle non specific chemotherapy, P¼0.06. On the contrary, girls who received cell cycle non specific chemotherapy showed significantly lower AMH levels compared to baseline (0.22 0.35 ng/ml vs 1.28 1.43 ng/ml; P¼0.01). CONCLUSIONS: AMH assay is a sensitive marker for ovarian functions before and after puberty in patients at risk of gonadal damage secondary to chemotherapy. Ovarian reserve appears to be more sensitive to the insult of cell cycle non specific chemotherapeutic agents than the testicular reserve. AMH assay could be added to the initial work up of all patients undergoing chemotherapy to assess the damage to their reproductive potential. Supported by: None. P-44 2-METHOXY ESTRADIOL INHIBITS ANGIOGENESIS IN SHEEP UTERINE ARTERY ENDOTHELIAL CELLS. S. Salih, A. Kapur, R. Magness. University of Wisconsin, Madison, WI. OBJECTIVE: Estrogen plays a key role in priming the endometrium for luteal phase development, embryo implantation, and early placentation. The effects of estrogen metabolites and estrogen metabolizing enzymes in endometrial vasulature are not well studied. Here we investigate the angiogenic and vasodilatory effect of 17b estradiol and 2-methoxy estradiol using the sheep model. DESIGN: Angiogenic activity of 17b estradiol and 2-methoxy estradiol on uterine artery endothelial cells (UAEC) from luteal phase, follicular phase, and pregnant sheep was investigated. MATERIALS AND METHODS: UAEC from luteal phase, follicular phase, and pregnant sheep were cultured and plated 1105 cells per well in 12 well plates on a matrigel with vehicle, 17b estradiol, and 2-methoxy estradiol. Capillary-like structure formation was checked 6 hours. Protein assay for ERK, AKT and eNOS expression was performed using Western Blot analysis. RESULTS: 17b estradiol induced capillary tube formation on UAEC from all phases; luteal, follicular, and pregnant sheep. The most pronounced effect was in UAEC from pregnant sheep. 2-methoxy estradiol on the other hand completely inhibited capillary tube formation in UAECs. In follicular phase UAEC there was no significant change in pAKT/tAKT with 17b estradiol or 2-methoxy estradiol. However there was a significant decrease in pERK/tERK with 2-methoxy estradiol but no change with 17b estradiol. There was no difference in pENOS/tENOS ratio with either 17b or 2-methoxy estradiol. In UAEC from pregnant sheep there was a significant decrease in pERK/tERK with 2-methoxy estradiol. There was no significant difference in pAKT/tAKT or pENOS/tENOS with 2-methoxy estradiol; however there was a significant increase in pAKT/tAKT and pERK/tERK with 17b estradiol. In luteal phase UAEC there was a significant decrease in pAKT/tAKT, pERK/tERK as well as pENOS/tENOS with 2-methoxy estradiol as compared to control. Similar decrease was observed with 17b estradiol, but the decrease was not significant. CONCLUSIONS: The anti-angiogenic effect of 2-methoxy estradiol appears to be mediated in part via ERK signaling pathway. There was significant decrease in phosphorylation of ERK1 and ERK2 in UAEC from luteal, follicular, and pregnant sheep. 2-methoxy estradiol inhibits
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