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Antioxidants induce apoptotic death of ovarian thecal-interstitial cells
cycles. For each participant, the results of the first pregnancy in a follow up period of 12 months after entering the study was analyzed. Early pregnancy loss was measured during 6 menstrual cycles using an ultra-sensitive urinary hCG assay, registering subtle hCG elevations in the peri-menstrual period, indicating early pregnancy loss. End points were early pregnancy loss, spontaneous abortion of a clinically recognized pregnancy, early delivery or delivery after 25 weeks of gestation. We estimated the effect of basal FSH concentrations towards these endpoints using stepwise logistic regression, taking into account possible confounders. Results: After correction for all known confounders, we observed no significant effect for basal FSH concentrations towards the incidence of early pregnancy loss, abortion, clinically recognized pregnancies, early delivery or the number of ongoing pregnancies. We confirmed the effect of smoking on pregnancy loss. Conclusions: We conclude that in this population of women without a history of subfertility and pursuing a spontaneous pregnancy, basal FSH concentrations are not related to the incidence of early pregnancy loss or abortions. This prospective study therefore fails to confirm a relation between signs of decreased ovarian reserve and aneuploid pregnancies, given the fact that the majority of cases of early pregnancy loss and abortion is known to be related to aneuploidies. Prospective studies investigating the relation between decreased ovarian reserve and pregnancy outcome or aneuploidies should therefore use more specific indicators for ovarian reserve than basal FSH, as for example the clomiphene challenge test, the EFOR test or antral follicle count. Supported by: Grant number 28 –2770 –1 of the Dutch PraeventiefondsZorg Onderzoek Nederland, The Hague, The Netherlands.
P-330 Baseline inhibin-B levels are associated with cycle cancellation or modification of gonadotropin dosages during ovarian stimulation for ART. Stephen M. Sanders, Janet Dye, Robin LeFever, Jose F. Pliego, Thomas J. Wincek, Thomas J. Kuehl. Scott & White Clin, Temple, TX. Objective: To measure inhibin-B concentrations in residual sera collected at baseline examinations prior to advanced ART procedures (IVF, ICSI, or ET of cryopreserved embryos) and evaluate the relationship between inhibin-B concentration and cycle outcomes, including cancellations for high and low ovarian responses to gonadotropins. Design: A cohort design matching ART cycle results with subsequently measured hormone levels. Materials/Methods: Serum samples from 102 ART cycles collected at the time of the baseline evaluation in each patient were assayed in duplicate using an inhibin-B-ELISA (Serotec, UK) kit specific for dimeric inhibin-B. Assay results were merged with a prospectively developed ART dataset including type of stimulation cycle, estradiol (E2) concentration at baseline, dosages and types of gonadotropins, E2 concentration at mid-cycle, cancellation status, total gonadotropin dose, gonadotropin dose adjustments, number of days of stimulation, number and status of ova recovered, number and quality of embryos produced, number and quality of embryos transferred, and cycle pregnancy result. Results: Inhibin-B concentrations did not vary significantly (p ⫽ 0.88) with the interval of time following the start of GnRH agonist suppression to the baseline examination. Inhibin-B concentrations differed (p ⬍0.001 by Kruskal-Wallis ANOVA) among ART cycles based on the nature of ovarian response, varying from a low of 32 ⫹/⫺ 24 (SD) pg/mL for those cancelled due to poor response (scored as 1) to a high of 137 ⫹/⫺ 132 pg/mL for those cancelled due to excessive response (scored as 5). Inhibin-B concentrations at baseline were also related (p ⫽ 0.033) to ovarian response as measured by the E2 level per ampule of gonadotropin used prior to the mid-cycle evaluation. Inhibin-B levels were not predictive of pregnancy outcome nor the final yield of ova or embryos. Using ROC methods, a level of 49 pg/mL of inhibin-B at the time of the baseline evaluation was associated with cancellation for poor ovarian response or the need to increase the daily gonadotropin dosage to complete the cycle. Conclusions: Baseline inhibin-B levels are associated with ovarian response to gonadotropin stimulation and may predict poor ovarian response in ART cycles. This information suggests that in patients with baseline inhibin-B values less than 49 pg/mL, increasing the gonadotropin dose over standard dose schedules should decrease ART cycle cancellations and optimize ovarian response. Supported by: Funding provided the Mary Jane Noble Foundation.
S224
Abstracts
P-331 Antioxidants induce apoptotic death of ovarian thecal-interstitial cells. Nastaran Foyouzi-Yousefi, Mehmet Karaca, Ciler Celik-Ozenci, Antoni J. Duleba. Dept of OB/GYN, Yale Univ Sch of Medicine, New Haven, CT; Dept of Histology and Embryology, Akdeniz Univ, Antalya, Turkey. Objective: Regulation of growth and apoptosis of ovarian mesenchyme is essential for normal ovarian development and homeostasis. Hyperplasia of ovarian mesenchyme is encountered in pathological conditions, such as polycystic ovary syndrome (PCOS) and hyperthecosis. These conditions are associated with insulin resistance and hyperinsulinemia. Other hyperinsulinemic states, such as syndrome X and type 2 diabetes mellitus are characterized by excessive oxidative stress. We have recently reported that oxidative stress stimulates growth of ovarian thecal-interstitial cells raising the possibility that excessive oxidative stress may contribute to thecalinterstitial hyperplasia. This study tested the hypothesis that antioxidants extinguish reactive oxygen species and limit growth by inducing apoptosis. Design: In vitro study evaluating apoptosis. Materials/Methods: Purified rat thecal-interstitial cells were cultured for 24 hours in chemically defined media without (Control) or with the following antioxidants: vitamin E succinate (VES, 30 –100M), ebselen (EBS, 30 –100M) and superoxide dismutase (SOD, 100 –1000 U/ml). Apoptosis was assessed by evaluation of nuclear fragmentation using 4’,6’-diamidino2’-phenylindole dihydrochloride (DAPI) and by DNA strand break labelling (TUNEL). Results: All tested antioxidants induced dose-dependent apoptosis of thecal-interstitial cells. VES increased apoptosis by up to 6-fold as assessed by TUNEL and 8.2-fold by DAPI staining. EBS stimulated apoptosis by up to 7-fold as determined by TUNEL and by 11.5-fold by DAPI. Similarly, SOD also increased apoptosis by up to 5.1-fold by TUNEL and by 9.1-fold by DAPI. All the above effects were significant at p ⬍0.001. Conclusions: The pro-apoptotic actions of antioxidants suggest that regulation of oxidative stress may play an important role in the regulation of thecal-interstitial growth and death. These findings raise the possibility of potential therapeutic effects of antioxidants in conditions of excessive ovarian mesenchymal growth. Supported by: NIH-RO1-HD40207.
REPRODUCTIVE LABORATORY TECHNOLOGY P-332 Mechanical reduction of the blastocoelic cavity before slow cooling cryopreservation does not improve blastocyst survival rates. Maria C. Bastias, Jaime M. Vasquez, Anita M. Carrico. Ctr for Reproductive Health, Nashville, TN. Objective: Mechanical reduction of the blastocoelic cavity has been recently reported by Vanderzwalmen and co-workers (Fertil Steril O-125, S48; Vol. 74:3, Suppl. 1, 2000) to increase blastocyst viability after vitrification. The purpose of our study was to evaluate the effectiveness of this novel technique in our ART laboratory⬘s slow cooling protocol for blastocyst cryopreservation. Design: Mouse blastocysts (B6C3F1/B6D2F1) with or without mechanical removal of the blastocoelic cavity⬘s fluid were cryopreserved using a two-step slow cryopreservation protocol, followed by a rapid thawing and a four-step dilution at room temperature. Blastocysts were maintained in culture for 24 hours to assess viability and further development. Materials/Methods: One hundred forty-six mouse blastocysts (n⫽3) were randomly allocated into two groups. Blastocysts in group A (n⫽72) were transferred into micro-droplets containing freeze-thaw diluent (modified human tubal fluid with hepes and supplemented with 20 mg/ml human serum albumin) for mechanical reduction of the blastocoelic cavity. For the procedure, each blastocyst was placed with the inner cell mass at either 12 or 6 o⬘clock and firmly secured with a holding pipet. A micro-needle positioned at 3 o⬘clock, was slowly inserted through the cellular junction of the trophectoderm into the blastocoele cavity until complete blastocyst compaction was observed. Control blastocysts in group B (n⫽74) were incubated in freeze-thaw diluent for 15 min. before cryopreservation. The freezing solutions for both groups of embryos were 5% glycerol (10 min.), followed by 9% glycerol and 0.2 M sucrose (10min.). Blastocysts were transferred into cryovials and placed into the freezing machine at 20 °C,
Vol. 78, No. 3, Suppl. 1, September 2002
P-330 Baseline inhibin-B levels are associated with cycle cancellation or modification of gonadotropin dosages during ovarian stimulation for ART. Stephen M. Sanders, Janet Dye, Robin LeFever, Jose F. Pliego, Thomas J. Wincek, Thomas J. Kuehl. Scott & White Clin, Temple, TX. Objective: To measure inhibin-B concentrations in residual sera collected at baseline examinations prior to advanced ART procedures (IVF, ICSI, or ET of cryopreserved embryos) and evaluate the relationship between inhibin-B concentration and cycle outcomes, including cancellations for high and low ovarian responses to gonadotropins. Design: A cohort design matching ART cycle results with subsequently measured hormone levels. Materials/Methods: Serum samples from 102 ART cycles collected at the time of the baseline evaluation in each patient were assayed in duplicate using an inhibin-B-ELISA (Serotec, UK) kit specific for dimeric inhibin-B. Assay results were merged with a prospectively developed ART dataset including type of stimulation cycle, estradiol (E2) concentration at baseline, dosages and types of gonadotropins, E2 concentration at mid-cycle, cancellation status, total gonadotropin dose, gonadotropin dose adjustments, number of days of stimulation, number and status of ova recovered, number and quality of embryos produced, number and quality of embryos transferred, and cycle pregnancy result. Results: Inhibin-B concentrations did not vary significantly (p ⫽ 0.88) with the interval of time following the start of GnRH agonist suppression to the baseline examination. Inhibin-B concentrations differed (p ⬍0.001 by Kruskal-Wallis ANOVA) among ART cycles based on the nature of ovarian response, varying from a low of 32 ⫹/⫺ 24 (SD) pg/mL for those cancelled due to poor response (scored as 1) to a high of 137 ⫹/⫺ 132 pg/mL for those cancelled due to excessive response (scored as 5). Inhibin-B concentrations at baseline were also related (p ⫽ 0.033) to ovarian response as measured by the E2 level per ampule of gonadotropin used prior to the mid-cycle evaluation. Inhibin-B levels were not predictive of pregnancy outcome nor the final yield of ova or embryos. Using ROC methods, a level of 49 pg/mL of inhibin-B at the time of the baseline evaluation was associated with cancellation for poor ovarian response or the need to increase the daily gonadotropin dosage to complete the cycle. Conclusions: Baseline inhibin-B levels are associated with ovarian response to gonadotropin stimulation and may predict poor ovarian response in ART cycles. This information suggests that in patients with baseline inhibin-B values less than 49 pg/mL, increasing the gonadotropin dose over standard dose schedules should decrease ART cycle cancellations and optimize ovarian response. Supported by: Funding provided the Mary Jane Noble Foundation.
S224
Abstracts
P-331 Antioxidants induce apoptotic death of ovarian thecal-interstitial cells. Nastaran Foyouzi-Yousefi, Mehmet Karaca, Ciler Celik-Ozenci, Antoni J. Duleba. Dept of OB/GYN, Yale Univ Sch of Medicine, New Haven, CT; Dept of Histology and Embryology, Akdeniz Univ, Antalya, Turkey. Objective: Regulation of growth and apoptosis of ovarian mesenchyme is essential for normal ovarian development and homeostasis. Hyperplasia of ovarian mesenchyme is encountered in pathological conditions, such as polycystic ovary syndrome (PCOS) and hyperthecosis. These conditions are associated with insulin resistance and hyperinsulinemia. Other hyperinsulinemic states, such as syndrome X and type 2 diabetes mellitus are characterized by excessive oxidative stress. We have recently reported that oxidative stress stimulates growth of ovarian thecal-interstitial cells raising the possibility that excessive oxidative stress may contribute to thecalinterstitial hyperplasia. This study tested the hypothesis that antioxidants extinguish reactive oxygen species and limit growth by inducing apoptosis. Design: In vitro study evaluating apoptosis. Materials/Methods: Purified rat thecal-interstitial cells were cultured for 24 hours in chemically defined media without (Control) or with the following antioxidants: vitamin E succinate (VES, 30 –100M), ebselen (EBS, 30 –100M) and superoxide dismutase (SOD, 100 –1000 U/ml). Apoptosis was assessed by evaluation of nuclear fragmentation using 4’,6’-diamidino2’-phenylindole dihydrochloride (DAPI) and by DNA strand break labelling (TUNEL). Results: All tested antioxidants induced dose-dependent apoptosis of thecal-interstitial cells. VES increased apoptosis by up to 6-fold as assessed by TUNEL and 8.2-fold by DAPI staining. EBS stimulated apoptosis by up to 7-fold as determined by TUNEL and by 11.5-fold by DAPI. Similarly, SOD also increased apoptosis by up to 5.1-fold by TUNEL and by 9.1-fold by DAPI. All the above effects were significant at p ⬍0.001. Conclusions: The pro-apoptotic actions of antioxidants suggest that regulation of oxidative stress may play an important role in the regulation of thecal-interstitial growth and death. These findings raise the possibility of potential therapeutic effects of antioxidants in conditions of excessive ovarian mesenchymal growth. Supported by: NIH-RO1-HD40207.
REPRODUCTIVE LABORATORY TECHNOLOGY P-332 Mechanical reduction of the blastocoelic cavity before slow cooling cryopreservation does not improve blastocyst survival rates. Maria C. Bastias, Jaime M. Vasquez, Anita M. Carrico. Ctr for Reproductive Health, Nashville, TN. Objective: Mechanical reduction of the blastocoelic cavity has been recently reported by Vanderzwalmen and co-workers (Fertil Steril O-125, S48; Vol. 74:3, Suppl. 1, 2000) to increase blastocyst viability after vitrification. The purpose of our study was to evaluate the effectiveness of this novel technique in our ART laboratory⬘s slow cooling protocol for blastocyst cryopreservation. Design: Mouse blastocysts (B6C3F1/B6D2F1) with or without mechanical removal of the blastocoelic cavity⬘s fluid were cryopreserved using a two-step slow cryopreservation protocol, followed by a rapid thawing and a four-step dilution at room temperature. Blastocysts were maintained in culture for 24 hours to assess viability and further development. Materials/Methods: One hundred forty-six mouse blastocysts (n⫽3) were randomly allocated into two groups. Blastocysts in group A (n⫽72) were transferred into micro-droplets containing freeze-thaw diluent (modified human tubal fluid with hepes and supplemented with 20 mg/ml human serum albumin) for mechanical reduction of the blastocoelic cavity. For the procedure, each blastocyst was placed with the inner cell mass at either 12 or 6 o⬘clock and firmly secured with a holding pipet. A micro-needle positioned at 3 o⬘clock, was slowly inserted through the cellular junction of the trophectoderm into the blastocoele cavity until complete blastocyst compaction was observed. Control blastocysts in group B (n⫽74) were incubated in freeze-thaw diluent for 15 min. before cryopreservation. The freezing solutions for both groups of embryos were 5% glycerol (10 min.), followed by 9% glycerol and 0.2 M sucrose (10min.). Blastocysts were transferred into cryovials and placed into the freezing machine at 20 °C,
Vol. 78, No. 3, Suppl. 1, September 2002