- Email: [email protected]
A comparison of the effect of metformin and rosiglitazone on insulin action and secretion in women with polycystic ovary syndrome
r-hFSH␣ 12.4 ⫾ 0.5 days, p ⬍0.05) and gonadotropin dose (21.7 ⫾ 0.8 vs. 25.3 ⫾ 1.3 ampoules, p ⬍0.05) were lower and LH (13.9 ⫾ 1.0 vs. 10.8 ⫾ 0.8 IU/L.day, p ⬍0.05) and hCG (3.3 ⫾ 0.9 vs. 0.5 ⫾ 0.2 IU/L.day, p ⬍0.01) levels were higher in the hMG group. Unexpectedly, serum FSH was also higher in the hMG group (13.9 ⫾ 1.0 vs. 10.8 ⫾ 0.8 IU/L.day, p ⬍0.01), despite matched daily FSH activity administration. Serum P levels (5.2 ⫾ 0.6 hMG vs. 8.4 ⫾ .4 ng/mL.day r-hFSH, p ⬍0.001) and small preovulatory follicle number (2.4 ⫾ 0.5 vs. 6.5 ⫾ .8, p ⬍0.001) were higher in the r-hFSH␣ group. Serum T, and preovulatory E2 levels and large follicle number did not differ; a nonsignificant trend towards higher pregnancy rates was seen in the hMG group (28% vs. 16%). The abortion rate (4%) was the same in both groups. Conclusions: When hMG and r-hFSH␣ COS regimens were compared under conditions of matched daily FSH activity administration, hMG was associated with: 1. Higher peripheral FSH levels, despite comparable exogenous FSH activity administration; 2. Increased LH and hCG serum levels; 3. A more efficient response, as reflected by reduced treatment duration and exogenous gonadotropin requirements; 4. Fewer signs of premature luteinization; 5. A lower number of small preovulatory follicles.6. Clinical outcome in IUI comparable to r-hFSH. Supported by: Grant C50000224 from the University of Bologna.
Wednesday, October 16, 2002 2:45 P.M. O-276 Lower serum levels of inhibin A and pro-␣C during the luteal phase after triggering oocyte maturation with gonadotropin releasing hormone (GnRH) agonist in comparison to human chorionic gonadotropin (hCG): Possible relevance for the prevention of ovarian hyperstimulation (OHSS). Ori Nevo, Talia Eldar-Geva, Shahar Kol, Joseph ItskovitzEldor. Rambam Medical Ctr, Haifa, Israel; Shaare-Zedec Medical Ctr, Jerusalem, Israel. Objective: Ovarian hyperstimulation syndrome (OHSS) is a major complication of ovarian stimulation. Ovulation triggering with GnRH agonist instead of hCG may prevent the development of OHSS. In the current study we investigated the effect of triggering oocyte maturation by GnRH agonist in comparison to hCG on corpus luteum function by measuring luteal phase levels of inhibin A and pro-␣C, as non-steroidal markers of luteal function. Design: Prospective collection of serum samples from three study groups. Comparison of the first patients that completed the requirements of the study protocol. Materials/Methods: Serum samples were collected during the luteal phase of IVF-ET cycles in three different protocols: 1) GnRH antagonist for prevention of an LH surge and hCG for triggering of final oocyte maturation (antagonist-hCG group, n⫽8, 4/8 pregnant). 2) GnRH antagonist for prevention of an LH surge and GnRH agonist for triggering final oocyte maturation (antagonist-agonist group, n⫽8, 4/8 pregnant). 3) GnRH agonist in a long protocol and hCG for final oocyte maturation (agonist-hCG group, n⫽5, 2/5 pregnant). Samples were assayed for inhibin A, pro-␣C, progesterone and estradiol. Transvaginal ultrasound was performed in mid-luteal phase for estimation of corpora lutea number and volume. Results: Serum levels of inhibin A, pro-␣C, estrogen and progesterone were significantly lower from day 4 to day 14 after triggering final oocyte maturation by GnRH agonist, in comparison to hCG triggering. Corpora lutea volume in the mid-luteal phase was significantly higher in hCG groups. Pregnancy caused a late luteal elevation of inhibin A and pro-␣C in 3 out of 4 patients in the hCG groups but not in the antagonist-agonist group. Non-pregnant patients showed no late luteal elevation of inhibin forms in all groups. Conclusions: Final oocyte maturation triggering with GnRH agonist instead of hCG in IVF cycles causes significantly lower luteal levels of inhibins and steroids hormones, suggesting that corpora lutea may secrete lower levels of other non-steroidal substances with vasoactive properties that may be involved in OHSS. This may explain the mechanism of OHSS prevention by the use of GnRH agonist. Supported by: The study was supported by a research fund of the department of Obstetrics and Gynecology in Rambam Medical Center.
FERTILITY & STERILITY威
Wednesday, October 16, 2002 3:00 P.M. O-277 Daily low dose recombinant hCG and low dose recombinant FSH: A novel treatment for hypogonadotropic hypogonadism. Kathy M. Doody, Anna C. Nackley, Kevin J. Doody. Ctr for Assisted Reproduction, Bedford, TX. Objective: LH in the follicular phase is known to be important to the production of C-19 steroids that serve as precursors for estrogen formation. Estrogen produced by the follicle has been speculated to have a major role in oocyte maturation. Treatment of hypogonadotropic hypogonadism with recombinantly derived FSH has previously met with poor response secondary to LH deficiency. The administration of recombinant FSH in the absence of significant endogenous LH can lead to slow follicle development associated with extremely low estradiol production. Because of this, patients with hypogonadotropic amenorrhea have classically been treated with hMG. Despite this approach, these patients may require high doses of gonadotropin and prolonged treatment. This is likely due to the short half-life of the LH component of hMG preparations. Recombinant HCG has recently become available. This drug may serve as a source of LH activity with a significantly longer half life and duration of action than the LH component of HMG. This study was undertaken to evaluate the potential benefit of using a daily low dose of recombinant hCG as an adjunct to recombinant FSH treatment for ovulation induction in a patient with severe LH deficiency. Design: Prospective – case report. Materials/Methods: S.L. is a 23 year old nulligravida who presented for ovulation induction. She reported the onset of menarche at age 16 with sporadic menses. She reported normal thelarche and adrenarche. She failed to experience a withdrawal menses to a progestin challenge. She denied a history of strenuous exercise or eating disorder. Her physical examination was significant for a BMI of 16.9. Pelvic examination was unremarkable. Her laboratory profile included a normal TSH and prolactin. Her FSH was 5.2 mIU/ml and LH ⬍0.7 mIU/ml. MRI of the pituitary gland was normal. In view of the patient’s history of a negative withdrawal bleed in response to medroxyprogesterone acetate (Provera), she was given a 4 week course of oral estrogen replacement (Estrace 4 mg/day) followed by progesterone. She experienced the onset of a normal menstrual period in response to this hormonal treatment. Following her second withdrawal bleed from the estrogen/progesterone replacement therapy, she was begun on recombinant FSH (Gonal-F) 75 IU daily and microdose Ovidrel 2.5 mcg daily. Her initial estradiol following 6 days of therapy was 58 pg/ml. She was continued on the same protocol with estradiol levels and sonograms performed to monitor ovarian response. Following 11 days of treatment, sonogram revealed a single follicle which was greater than 17 mm with a peak estradiol of 146 pg/ml. She was given 250 mcg of Ovidrel to induce ovulation. A postcoital test done the day following Ovidrel administration was poor. She underwent intrauterine insemination 36 hours following Ovidrel administration. She received progesterone supplementation following insemination. Results: Twelve days following insemination a serum bhCG assay demonstrated a level of 74 mIU/ml. Transvaginal sonography at 6 weeks 3 days demonstrated a viable intrauterine pregnancy. Conclusions: Very low dose daily recombinant hCG can be used in conjunction with recombinant FSH for successful ovulation induction in hypogonadotropic patients. Supported by: Not Applicable.
Wednesday, October 16, 2002 3:45 P.M. O-278 A comparison of the effect of metformin and rosiglitazone on insulin action and secretion in women with polycystic ovary syndrome. Angela C. Thyer, Robert Brzyski, Carann Easton, Robert Schenken. Univ of Washington, Seattle, WA; Univ of Texas at San Antonio, San Antonio, TX. Objective: To compare the effect of metformin and rosiglitazone on insulin action and secretion in women with polycystic ovary syndrome (PCOS).
S105
Design: Randomized, double-blinded, controlled trial. Materials/Methods: Forty-two women diagnosed with PCOS according to the 1990 NICHD consensus trial (oligomenorrhea, clinical or laboratory hyperandrogenism, absence of other causes of anovulation) were randomized to receive metformin 1000 mg bid, rosiglitazone 4 mg bid, or placebo. Diabetes was excluded using a 2 hour glucose tolerance test. Subjects underwent a frequently sampled intravenous glucose tolerance test (FSIGT) pre-treatment, and after 3 completed months of therapy. Results were analyzed using the minimal model, MINMOD. Insulin levels were measured using a specific and sensitive double antibody radioimmunoassay. Data was analyzed using ANOVA. Post hoc differences were analyzed using Tukey’s Honestly Significantly Different (HSD) test. Results: Seventeen subjects were randomized to metformin, 18 to rosiglitazone, and 7 to placebo. There was no difference in baseline BMI, hirsutism score, total testosterone, free testosterone, DHEAS, FSH, or LH among groups. The prevalence of impaired glucose tolerance at baseline was not significantly different among groups: 35% in the metformin group (6 of 17), 44% in the rosiglitazone group (8 of 18), and 29% in the placebo group (2 of 7). Post treatment, insulin sensitivity (Si) was significantly improved in the rosiglitazone group compared to the other groups (p ⬍0.05). Si increased from 2.27 ⫾ 0.69 to 3.63 ⫾ 0.89 min-1/(pmol/L) in the rosiglitazone group. Si did not improve in the metformin group, 2.41 ⫾ 0.60 to 2.36 ⫾ 0.82 min-1/(pmol/L), or the placebo group, 2.46 ⫾ 1.18 to 1.97 ⫾ 0.89 min-1/(pmol/L). First phase insulin secretion (AIRg) improved in the metformin group 393 ⫾ 69 to 690 ⫾ 157 pmol/L (p ⫽ 0.05), but not in the rosiglitazone 668 ⫾ 123 to 629 ⫾ 129 pmol/L or placebo groups 763 ⫾ 257 to 541 ⫾ 157 pmol/L. The disposition index (DI, the product of Si and AIRg) improved in the metformin and rosiglitazone groups (733 ⫾ 150 to 846 ⫾ 191, and 846 ⫾ 152 to 1261 ⫾ 273, respectively). DI did not improve in the placebo group (1531 ⫾ 898 to 768 ⫾ 277). Mean BMI was 36 ⫾ 1.4 (range 21– 63). Women lost an average of 2.7 ⫾ 1.2 lbs in the metformin group and 0.7 ⫾ 0.5 pounds in the placebo group. Women in the rosiglitazone group gained an average of 5.1 ⫾ 2.0 lbs (p ⫽ 0.001 for metformin and rosiglitazone). There was no significant changes in any other parameters including fasting glucose and insulin, total and free testosterone, DHEAS, FSH and LH among the groups after 3 months of treatment. Conclusions: Rosiglitazone improves insulin sensitivity and metformin improves the first phase insulin secretion in women with PCOS. Supported by: General Clinical Research Center (GCRC), supported by NIH Grant M01-RR-01346.
Wednesday, October 16, 2002 4:00 P.M. O-279 Extracellular matrix-activin interactions in granulosa cell growth and apoptosis. Erkan Buyuk, Maja Oktay, Filippo Giancotti, Zev Rosenwaks, Kutluk H. Oktay. Ctr for Reproductive Medicine and Infertility, Weill Medical Coll of Cornell Univ, New York, NY; Montefiore Medical Ctr, Bronx, NY; Memorial Sloan Kettering Cancer Ctr, New York, NY. Objective: The precise mechanism that regulates the granulosa cell growth in preantral follicles is unknown. We have previously shown in mouse ovarian organ culture studies that extracellular matrix (ECM) affects preantral follicle growth, in possible interaction with activin-a (1). The objective of this study was to determine the role of ECM and activin-a in preantral/undifferentiated granulosa cell growth and survival. Design: Controlled in vitro study. Materials/Methods: A spontaneously immortalized rat granulosa cell line (SIGC) was used. SIGC carry the characteristics of preantral/undifferentiated granulosa cells(2) and express integrins that bind collagen(C), laminin(L), and fibronectin(F)(3). They are responsive to activin-a as determined by luciferase assay using p3TP-lux reporter gene containing activin-responsive PAI-1 promoter(4). SIGC were cultured in triplicates in serum free media on C, L, F or polylysine(P) coated (negative control) coverslips for 24 – 48 hours with or without 30ng/ml activin-a. Cell proliferation was determined by BrdU uptake after 24 hours, and the apoptosis was determined by TUNEL assay at 24 and 48 hours using immunofluorescence microscopy in a blinded fashion. Results: All three types of ECM molecules increased granulosa cell proliferation significantly compared to polylysine (p ⫽ 0.0001)(Figure). However, the proliferation rate was higher if cells were grown on C and L
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Abstracts
than on F (p ⫽ 0.0085). Activin-a suppressed cell proliferation on C (82%) more significantly than L (42%) (p ⫽ 0.05) but did not significantly influence cell proliferation on polylysine. Likewise, all three types of ECM molecules prevented apoptosis compared to polylysine (p ⫽ 0.0001)(Table). Among the three ECM tested, C seemed to have the strongest antiapoptotic effect. Cells grown on C, regardless of activin-a treatment, showed significantly less apoptosis compared to F at 24 hours (p ⫽ 0.03) or to L&F in the absence of activin-a at 48 hours (p ⫽ 0.045, p ⫽ 0.04, respectively). However addition of activin-a increased apoptosis of cells grown on C after 48 hours (p ⫽ 0.006).
Effects of ECM and Activin-A on mean percentage of apoptosis in SIGC
C L F P
24 h
Activin48 h
P
24 h
Activin⫹ 48 h
P
2.5 3.3 4.7 51.3
4.5 10.9 11.4 67
n.s. 0.0001 0.04 n.s
1.7 2.3 4.3 62.9
7.5 12.3 9.5 61
0.006 0.002 0.02 n.s
Conclusions: 1) ECM, especially C and L stimulate granulosa cell proliferation; 2) Activin-a suppresses granulosa cell proliferation especially on C, indicating an interaction between ECM and activin-a; 3) ECM, especially C prevents apoptosis but this protective effect may be reduced in the presence of activin-a. Overall, these results indicate that ECM and activin-a regulate survival and growth of granulosa cells of preantral follicles through complex interactions. The mechanism of these interactions is being investigated at the signaling level in both SIGC and primary granulosa cells. 1. Oktay et al. Biol Reprod 2000;63:457– 61. 2. Stein et al. Cancer Res 1991;51:696 –706. 3. Oktay et al. J Soc Gynecol Investig 2002;9(Supp): 338A. 4. Oktay et al. ASRM 2002 abstract. Supported by: ASRM-Serono Research Grant to K.O., and by the Center for Reproductive Medicine & Infertility.
Wednesday, October 16, 2002 4:15 P.M. O-280 High levels of apoptosis in ejaculated spermatozoa from infertile men. Mohamed H. Moustafa, Ramadan A. Saleh, Mehmet Oder, Anthony J. Thomas Jr., Mohammed A. Abdel-Hafez, Ashok Agarwal. Cleveland Clin Fdn, Cleveland, OH; Cairo Univ, Cairo, Egypt. Objective: Animal studies have suggested that apoptosis, i.e. programmed cell death, is a key regulator of spermatogenesis in normal and pathological states. In this study we compared levels of apoptosis in ejaculated spermatozoa from a group of infertile men and normal sperm donors. Design: Prospective study in a male infertility clinic. Materials/Methods: Our study included a randomly selected group of infertile men (n ⫽ 28) with a history of infertility of more than one year. Twenty-two donors with normal standard semen parameters served as a control. Semen samples were obtained after 2 to 3 days of sexual abstinence
Vol. 78, No. 3, Suppl. 1, September 2002
Wednesday, October 16, 2002 2:45 P.M. O-276 Lower serum levels of inhibin A and pro-␣C during the luteal phase after triggering oocyte maturation with gonadotropin releasing hormone (GnRH) agonist in comparison to human chorionic gonadotropin (hCG): Possible relevance for the prevention of ovarian hyperstimulation (OHSS). Ori Nevo, Talia Eldar-Geva, Shahar Kol, Joseph ItskovitzEldor. Rambam Medical Ctr, Haifa, Israel; Shaare-Zedec Medical Ctr, Jerusalem, Israel. Objective: Ovarian hyperstimulation syndrome (OHSS) is a major complication of ovarian stimulation. Ovulation triggering with GnRH agonist instead of hCG may prevent the development of OHSS. In the current study we investigated the effect of triggering oocyte maturation by GnRH agonist in comparison to hCG on corpus luteum function by measuring luteal phase levels of inhibin A and pro-␣C, as non-steroidal markers of luteal function. Design: Prospective collection of serum samples from three study groups. Comparison of the first patients that completed the requirements of the study protocol. Materials/Methods: Serum samples were collected during the luteal phase of IVF-ET cycles in three different protocols: 1) GnRH antagonist for prevention of an LH surge and hCG for triggering of final oocyte maturation (antagonist-hCG group, n⫽8, 4/8 pregnant). 2) GnRH antagonist for prevention of an LH surge and GnRH agonist for triggering final oocyte maturation (antagonist-agonist group, n⫽8, 4/8 pregnant). 3) GnRH agonist in a long protocol and hCG for final oocyte maturation (agonist-hCG group, n⫽5, 2/5 pregnant). Samples were assayed for inhibin A, pro-␣C, progesterone and estradiol. Transvaginal ultrasound was performed in mid-luteal phase for estimation of corpora lutea number and volume. Results: Serum levels of inhibin A, pro-␣C, estrogen and progesterone were significantly lower from day 4 to day 14 after triggering final oocyte maturation by GnRH agonist, in comparison to hCG triggering. Corpora lutea volume in the mid-luteal phase was significantly higher in hCG groups. Pregnancy caused a late luteal elevation of inhibin A and pro-␣C in 3 out of 4 patients in the hCG groups but not in the antagonist-agonist group. Non-pregnant patients showed no late luteal elevation of inhibin forms in all groups. Conclusions: Final oocyte maturation triggering with GnRH agonist instead of hCG in IVF cycles causes significantly lower luteal levels of inhibins and steroids hormones, suggesting that corpora lutea may secrete lower levels of other non-steroidal substances with vasoactive properties that may be involved in OHSS. This may explain the mechanism of OHSS prevention by the use of GnRH agonist. Supported by: The study was supported by a research fund of the department of Obstetrics and Gynecology in Rambam Medical Center.
FERTILITY & STERILITY威
Wednesday, October 16, 2002 3:00 P.M. O-277 Daily low dose recombinant hCG and low dose recombinant FSH: A novel treatment for hypogonadotropic hypogonadism. Kathy M. Doody, Anna C. Nackley, Kevin J. Doody. Ctr for Assisted Reproduction, Bedford, TX. Objective: LH in the follicular phase is known to be important to the production of C-19 steroids that serve as precursors for estrogen formation. Estrogen produced by the follicle has been speculated to have a major role in oocyte maturation. Treatment of hypogonadotropic hypogonadism with recombinantly derived FSH has previously met with poor response secondary to LH deficiency. The administration of recombinant FSH in the absence of significant endogenous LH can lead to slow follicle development associated with extremely low estradiol production. Because of this, patients with hypogonadotropic amenorrhea have classically been treated with hMG. Despite this approach, these patients may require high doses of gonadotropin and prolonged treatment. This is likely due to the short half-life of the LH component of hMG preparations. Recombinant HCG has recently become available. This drug may serve as a source of LH activity with a significantly longer half life and duration of action than the LH component of HMG. This study was undertaken to evaluate the potential benefit of using a daily low dose of recombinant hCG as an adjunct to recombinant FSH treatment for ovulation induction in a patient with severe LH deficiency. Design: Prospective – case report. Materials/Methods: S.L. is a 23 year old nulligravida who presented for ovulation induction. She reported the onset of menarche at age 16 with sporadic menses. She reported normal thelarche and adrenarche. She failed to experience a withdrawal menses to a progestin challenge. She denied a history of strenuous exercise or eating disorder. Her physical examination was significant for a BMI of 16.9. Pelvic examination was unremarkable. Her laboratory profile included a normal TSH and prolactin. Her FSH was 5.2 mIU/ml and LH ⬍0.7 mIU/ml. MRI of the pituitary gland was normal. In view of the patient’s history of a negative withdrawal bleed in response to medroxyprogesterone acetate (Provera), she was given a 4 week course of oral estrogen replacement (Estrace 4 mg/day) followed by progesterone. She experienced the onset of a normal menstrual period in response to this hormonal treatment. Following her second withdrawal bleed from the estrogen/progesterone replacement therapy, she was begun on recombinant FSH (Gonal-F) 75 IU daily and microdose Ovidrel 2.5 mcg daily. Her initial estradiol following 6 days of therapy was 58 pg/ml. She was continued on the same protocol with estradiol levels and sonograms performed to monitor ovarian response. Following 11 days of treatment, sonogram revealed a single follicle which was greater than 17 mm with a peak estradiol of 146 pg/ml. She was given 250 mcg of Ovidrel to induce ovulation. A postcoital test done the day following Ovidrel administration was poor. She underwent intrauterine insemination 36 hours following Ovidrel administration. She received progesterone supplementation following insemination. Results: Twelve days following insemination a serum bhCG assay demonstrated a level of 74 mIU/ml. Transvaginal sonography at 6 weeks 3 days demonstrated a viable intrauterine pregnancy. Conclusions: Very low dose daily recombinant hCG can be used in conjunction with recombinant FSH for successful ovulation induction in hypogonadotropic patients. Supported by: Not Applicable.
Wednesday, October 16, 2002 3:45 P.M. O-278 A comparison of the effect of metformin and rosiglitazone on insulin action and secretion in women with polycystic ovary syndrome. Angela C. Thyer, Robert Brzyski, Carann Easton, Robert Schenken. Univ of Washington, Seattle, WA; Univ of Texas at San Antonio, San Antonio, TX. Objective: To compare the effect of metformin and rosiglitazone on insulin action and secretion in women with polycystic ovary syndrome (PCOS).
S105
Design: Randomized, double-blinded, controlled trial. Materials/Methods: Forty-two women diagnosed with PCOS according to the 1990 NICHD consensus trial (oligomenorrhea, clinical or laboratory hyperandrogenism, absence of other causes of anovulation) were randomized to receive metformin 1000 mg bid, rosiglitazone 4 mg bid, or placebo. Diabetes was excluded using a 2 hour glucose tolerance test. Subjects underwent a frequently sampled intravenous glucose tolerance test (FSIGT) pre-treatment, and after 3 completed months of therapy. Results were analyzed using the minimal model, MINMOD. Insulin levels were measured using a specific and sensitive double antibody radioimmunoassay. Data was analyzed using ANOVA. Post hoc differences were analyzed using Tukey’s Honestly Significantly Different (HSD) test. Results: Seventeen subjects were randomized to metformin, 18 to rosiglitazone, and 7 to placebo. There was no difference in baseline BMI, hirsutism score, total testosterone, free testosterone, DHEAS, FSH, or LH among groups. The prevalence of impaired glucose tolerance at baseline was not significantly different among groups: 35% in the metformin group (6 of 17), 44% in the rosiglitazone group (8 of 18), and 29% in the placebo group (2 of 7). Post treatment, insulin sensitivity (Si) was significantly improved in the rosiglitazone group compared to the other groups (p ⬍0.05). Si increased from 2.27 ⫾ 0.69 to 3.63 ⫾ 0.89 min-1/(pmol/L) in the rosiglitazone group. Si did not improve in the metformin group, 2.41 ⫾ 0.60 to 2.36 ⫾ 0.82 min-1/(pmol/L), or the placebo group, 2.46 ⫾ 1.18 to 1.97 ⫾ 0.89 min-1/(pmol/L). First phase insulin secretion (AIRg) improved in the metformin group 393 ⫾ 69 to 690 ⫾ 157 pmol/L (p ⫽ 0.05), but not in the rosiglitazone 668 ⫾ 123 to 629 ⫾ 129 pmol/L or placebo groups 763 ⫾ 257 to 541 ⫾ 157 pmol/L. The disposition index (DI, the product of Si and AIRg) improved in the metformin and rosiglitazone groups (733 ⫾ 150 to 846 ⫾ 191, and 846 ⫾ 152 to 1261 ⫾ 273, respectively). DI did not improve in the placebo group (1531 ⫾ 898 to 768 ⫾ 277). Mean BMI was 36 ⫾ 1.4 (range 21– 63). Women lost an average of 2.7 ⫾ 1.2 lbs in the metformin group and 0.7 ⫾ 0.5 pounds in the placebo group. Women in the rosiglitazone group gained an average of 5.1 ⫾ 2.0 lbs (p ⫽ 0.001 for metformin and rosiglitazone). There was no significant changes in any other parameters including fasting glucose and insulin, total and free testosterone, DHEAS, FSH and LH among the groups after 3 months of treatment. Conclusions: Rosiglitazone improves insulin sensitivity and metformin improves the first phase insulin secretion in women with PCOS. Supported by: General Clinical Research Center (GCRC), supported by NIH Grant M01-RR-01346.
Wednesday, October 16, 2002 4:00 P.M. O-279 Extracellular matrix-activin interactions in granulosa cell growth and apoptosis. Erkan Buyuk, Maja Oktay, Filippo Giancotti, Zev Rosenwaks, Kutluk H. Oktay. Ctr for Reproductive Medicine and Infertility, Weill Medical Coll of Cornell Univ, New York, NY; Montefiore Medical Ctr, Bronx, NY; Memorial Sloan Kettering Cancer Ctr, New York, NY. Objective: The precise mechanism that regulates the granulosa cell growth in preantral follicles is unknown. We have previously shown in mouse ovarian organ culture studies that extracellular matrix (ECM) affects preantral follicle growth, in possible interaction with activin-a (1). The objective of this study was to determine the role of ECM and activin-a in preantral/undifferentiated granulosa cell growth and survival. Design: Controlled in vitro study. Materials/Methods: A spontaneously immortalized rat granulosa cell line (SIGC) was used. SIGC carry the characteristics of preantral/undifferentiated granulosa cells(2) and express integrins that bind collagen(C), laminin(L), and fibronectin(F)(3). They are responsive to activin-a as determined by luciferase assay using p3TP-lux reporter gene containing activin-responsive PAI-1 promoter(4). SIGC were cultured in triplicates in serum free media on C, L, F or polylysine(P) coated (negative control) coverslips for 24 – 48 hours with or without 30ng/ml activin-a. Cell proliferation was determined by BrdU uptake after 24 hours, and the apoptosis was determined by TUNEL assay at 24 and 48 hours using immunofluorescence microscopy in a blinded fashion. Results: All three types of ECM molecules increased granulosa cell proliferation significantly compared to polylysine (p ⫽ 0.0001)(Figure). However, the proliferation rate was higher if cells were grown on C and L
S106
Abstracts
than on F (p ⫽ 0.0085). Activin-a suppressed cell proliferation on C (82%) more significantly than L (42%) (p ⫽ 0.05) but did not significantly influence cell proliferation on polylysine. Likewise, all three types of ECM molecules prevented apoptosis compared to polylysine (p ⫽ 0.0001)(Table). Among the three ECM tested, C seemed to have the strongest antiapoptotic effect. Cells grown on C, regardless of activin-a treatment, showed significantly less apoptosis compared to F at 24 hours (p ⫽ 0.03) or to L&F in the absence of activin-a at 48 hours (p ⫽ 0.045, p ⫽ 0.04, respectively). However addition of activin-a increased apoptosis of cells grown on C after 48 hours (p ⫽ 0.006).
Effects of ECM and Activin-A on mean percentage of apoptosis in SIGC
C L F P
24 h
Activin48 h
P
24 h
Activin⫹ 48 h
P
2.5 3.3 4.7 51.3
4.5 10.9 11.4 67
n.s. 0.0001 0.04 n.s
1.7 2.3 4.3 62.9
7.5 12.3 9.5 61
0.006 0.002 0.02 n.s
Conclusions: 1) ECM, especially C and L stimulate granulosa cell proliferation; 2) Activin-a suppresses granulosa cell proliferation especially on C, indicating an interaction between ECM and activin-a; 3) ECM, especially C prevents apoptosis but this protective effect may be reduced in the presence of activin-a. Overall, these results indicate that ECM and activin-a regulate survival and growth of granulosa cells of preantral follicles through complex interactions. The mechanism of these interactions is being investigated at the signaling level in both SIGC and primary granulosa cells. 1. Oktay et al. Biol Reprod 2000;63:457– 61. 2. Stein et al. Cancer Res 1991;51:696 –706. 3. Oktay et al. J Soc Gynecol Investig 2002;9(Supp): 338A. 4. Oktay et al. ASRM 2002 abstract. Supported by: ASRM-Serono Research Grant to K.O., and by the Center for Reproductive Medicine & Infertility.
Wednesday, October 16, 2002 4:15 P.M. O-280 High levels of apoptosis in ejaculated spermatozoa from infertile men. Mohamed H. Moustafa, Ramadan A. Saleh, Mehmet Oder, Anthony J. Thomas Jr., Mohammed A. Abdel-Hafez, Ashok Agarwal. Cleveland Clin Fdn, Cleveland, OH; Cairo Univ, Cairo, Egypt. Objective: Animal studies have suggested that apoptosis, i.e. programmed cell death, is a key regulator of spermatogenesis in normal and pathological states. In this study we compared levels of apoptosis in ejaculated spermatozoa from a group of infertile men and normal sperm donors. Design: Prospective study in a male infertility clinic. Materials/Methods: Our study included a randomly selected group of infertile men (n ⫽ 28) with a history of infertility of more than one year. Twenty-two donors with normal standard semen parameters served as a control. Semen samples were obtained after 2 to 3 days of sexual abstinence
Vol. 78, No. 3, Suppl. 1, September 2002