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Apoptosis of human peritoneal and adhesion fibroblasts after hypoxia: Role of inducible nitric oxide synthase
baseline LH were ⬍0.5 mIU/mL. An additional amp of hMG was given daily upon starting ganirelix. HCG 10,000 U IM was given when at least two follicles reached 18-20 mm diameter. Each donor donated oocytes to 2-3 recipients. Recipients received 1.875 mg depot leuprolide acetate in the late luteal phase prior to endometrial preparation. Oral estradiol 6 mg/d and estradiol patch 0.1 mg/d were given until transfer. Progesterone in oil was given IM starting 3 days before transfer. ICSI was done on all mature oocytes. All transfers were done 3 days after ICSI. Results: Group A had 17 donors and 36 recipients. Group B had 12 donors and 26 recipients. Donor age, recipient age, and donor BMI were similar in both groups. There were no premature LH surges and no cancellations in either group. Peak E2, LH, and P4 levels were similar in both groups. The number of oocytes retrieved, mature oocytes, fertilized oocytes, embryos transferred, implantation rates, and clinical pregnancy rates were not statistically different between groups. (Table 1)
mRNA (45%) and protein (59%) levels of the iNOS gene in normal peritoneal fibroblasts and adhesion fibroblasts. As previously shown, baseline apoptosis is significantly less in adhesion fibroblasts (2% apoptotic cells p ⬍ 0.005) as compared to normal peritoneal fibroblasts (30% apoptotic cells; p ⬍ 0.005). Augmentation of NO by SNAP treatment increased apoptosis in adhesion fibroblasts (27% apoptotic cells; p ⬍ 0.001); in contrast SNAP had no effects on normal fibroblasts. Conclusion: Our data suggests that restoration of NO to normal levels may be a mechanism by which to reduce/eliminate adhesions during peritoneal healing.
Tuesday, October 14, 2003 11:30 A.M. O-100 Comparison of ionophore induced Ca2ⴙ release in fresh and cryopreserved oocytes. Amy E. Jones, Jonathan Van Blerkom, Andrew A. Toledo, Patrick Davis. Reproductive Biology Assoc, Atlanta, GA; Univ of Colorado, Boulder, CO.
Conclusion: The GnRH antagonist ganirelix can achieve similar clinical efficacy compared to a long protocol with a depot GnRH agonist in donor oocyte cycles, suggesting no effect on embryo quality. Dissociating the GnRH antagonist from the recipient endometrium may account for the lack of a trend toward lower implantation and pregnancy rates in this study.
Tuesday, October 14, 2003 11:15 A.M. O-99 Apoptosis of human peritoneal and adhesion fibroblasts after hypoxia: Role of inducible nitric oxide synthase. Ghassan M. Saed, Husam M. Abu-Soud, Michael P. Diamond. Wayne State University/Detroit Medical Ctr, Detroit, MI. Introduction: Fibroblasts beneath the mesothelium in the human peritoneum play an important role in the healing process of the peritoneum. We have previously characterized differences between fibroblasts isolated from normal peritoneum and adhesions, and have identified phenotype differences between these two cell types from the same patients. Specifically, adhesion fibroblasts have a lower apoptosis than normal fibroblasts, which may contribute to the formation of postoperative adhesion. The role of nitric oxide in apoptosis is not well defined as in some cells it is reported to increase apoptosis while in others it decreases apoptosis. Objective: The objective of this study is to determine baseline levels of nitric oxide in fibroblasts isolated from normal peritoneum and from adhesions, whether inducible nitric oxide synthase (iNOS) is expressed in human fibroblasts isolated from normal peritoneal and adhesion tissues, and whether this expression is modulated by hypoxia. Materials and Methods: We have obtained fibroblast primary cultures from normal peritoneum and adhesion tissues of the same patients. NO synthesis by these fibroblasts was estimated by measuring nitrite accumulated in the culture medium over time by the Griess assay. Fibroblasts were cultured under normal and hypoxic condition with and without 400 M SNAP treatment for 24 h before evaluating apoptosis by the Tunel assay. Northern and Western blots were used to measure mRNA and protein levels of iNOS in these fibroblasts under normal and hypoxic conditions respectively. Results: The studies herein define normal fibroblasts to have higher NO (30 %;p ⬍ 0.001) levels than adhesion fibroblasts although there were no differences in mRNA and protein levels of the iNOS gene. Hypoxia decreased NO (45 %; p ⬍ 0.001) in normal peritoneal fibroblasts to levels observed for adhesion fibroblasts. In addition hypoxia increased both
S38
Abstracts
Objective: Differences in outcome between embryos derived from thawed oocytes or fertilized eggs have been largely attributed to unique properties of the zygote that may be labile to freezing-associated disruption. We have previously shown the apparent depolarization of pericortical mitochondria in MII human oocytes after thawing. While the level of mitochondrial influence on initiation and propagation of Ca2⫹ waves in the human oocyte is unknown, the role of calcium as a critical epigenetic factor at the earliest stages of mammalian oocyte activation has been demonstrated in several species. Here, we compared levels of ionophore induced Ca2⫹ release in fresh and thawed metaphase II oocytes. Design: A prospective, randomized study of the changes in intracellular free calcium levels in fresh and thawed MII human oocytes after exposure to a calcium ionophore. Materials and Methods: Fresh Day 1 oocytes which failed to fertilize were used in this study as well as Day 0 and Day 1 cryopreserved human oocytes. Frozen oocytes were stained at two time intervals (i) immediately after thawing and (ii) after 4 hours of culture. Fresh oocytes were stained at approximately 24 hours post egg retrieval. Oocytes were preloaded with the calcium-sensitive fluorescent probe Fluo-4-AM (5M, 60 min., 37oC) followed by washing in normal medium for 30 min and placement in ⌬_upper;T dishes with temperature maintained precisely at 37oC with a ⌬_upper;T controller. Oocytes were examined by scanning laser confocal microscopy at 488 nm excitation to determine baseline levels of fluorescence. The calcium ionophore A23187 was added to achieve a final concentration of 10uM and changes in relative fluorescent intensity (RFI) indicative of a rise in intracellular free calcium levels were measured at 5 sec intervals until maximum fluorescence was observed. Results: Fresh oocytes generated a significantly higher RFI than their thawed counterparts. Day 1 thawed oocytes an average RFI of while the average intensity increase in the day 1 post ICSI fresh oocytes was 65.4 (P ⫽ 0.0058). The average intensity increase in oocytes frozen on Day 0 was 22. Within that group, the RFI of oocytes stained at 0h hours was 17.6 while those stained after 4 hours of culture was 32.7. This difference was not significant (P ⫽ 0.1091). Conclusion: At fertilization, human oocytes undergo a series of Ca2⫹ transients that initiate the completion of meiosis and regulate both the first mitotic division and the release of cortical granules. Recent experimental evidence indicates that relatively small changes in calcium fluxes at the earliest stage of activation have profound downstream consequences for development (Ozil and Huneau, 2001). Previously, we demonstrated that cryopreserved oocytes show a decrease in mitochondrial membrane potential when compared to their fresh counterparts. The current findings suggest that mitochondrial depolarization and reduced Ca2⫹ response in thawed oocytes could have downstream consequences resulting from an influence on the amplitude of the initial Ca2⫹ transient(s) that may contribute to implantation failure or early pregnancy loss.
Vol. 80, Suppl. 3, September 2003
mRNA (45%) and protein (59%) levels of the iNOS gene in normal peritoneal fibroblasts and adhesion fibroblasts. As previously shown, baseline apoptosis is significantly less in adhesion fibroblasts (2% apoptotic cells p ⬍ 0.005) as compared to normal peritoneal fibroblasts (30% apoptotic cells; p ⬍ 0.005). Augmentation of NO by SNAP treatment increased apoptosis in adhesion fibroblasts (27% apoptotic cells; p ⬍ 0.001); in contrast SNAP had no effects on normal fibroblasts. Conclusion: Our data suggests that restoration of NO to normal levels may be a mechanism by which to reduce/eliminate adhesions during peritoneal healing.
Tuesday, October 14, 2003 11:30 A.M. O-100 Comparison of ionophore induced Ca2ⴙ release in fresh and cryopreserved oocytes. Amy E. Jones, Jonathan Van Blerkom, Andrew A. Toledo, Patrick Davis. Reproductive Biology Assoc, Atlanta, GA; Univ of Colorado, Boulder, CO.
Conclusion: The GnRH antagonist ganirelix can achieve similar clinical efficacy compared to a long protocol with a depot GnRH agonist in donor oocyte cycles, suggesting no effect on embryo quality. Dissociating the GnRH antagonist from the recipient endometrium may account for the lack of a trend toward lower implantation and pregnancy rates in this study.
Tuesday, October 14, 2003 11:15 A.M. O-99 Apoptosis of human peritoneal and adhesion fibroblasts after hypoxia: Role of inducible nitric oxide synthase. Ghassan M. Saed, Husam M. Abu-Soud, Michael P. Diamond. Wayne State University/Detroit Medical Ctr, Detroit, MI. Introduction: Fibroblasts beneath the mesothelium in the human peritoneum play an important role in the healing process of the peritoneum. We have previously characterized differences between fibroblasts isolated from normal peritoneum and adhesions, and have identified phenotype differences between these two cell types from the same patients. Specifically, adhesion fibroblasts have a lower apoptosis than normal fibroblasts, which may contribute to the formation of postoperative adhesion. The role of nitric oxide in apoptosis is not well defined as in some cells it is reported to increase apoptosis while in others it decreases apoptosis. Objective: The objective of this study is to determine baseline levels of nitric oxide in fibroblasts isolated from normal peritoneum and from adhesions, whether inducible nitric oxide synthase (iNOS) is expressed in human fibroblasts isolated from normal peritoneal and adhesion tissues, and whether this expression is modulated by hypoxia. Materials and Methods: We have obtained fibroblast primary cultures from normal peritoneum and adhesion tissues of the same patients. NO synthesis by these fibroblasts was estimated by measuring nitrite accumulated in the culture medium over time by the Griess assay. Fibroblasts were cultured under normal and hypoxic condition with and without 400 M SNAP treatment for 24 h before evaluating apoptosis by the Tunel assay. Northern and Western blots were used to measure mRNA and protein levels of iNOS in these fibroblasts under normal and hypoxic conditions respectively. Results: The studies herein define normal fibroblasts to have higher NO (30 %;p ⬍ 0.001) levels than adhesion fibroblasts although there were no differences in mRNA and protein levels of the iNOS gene. Hypoxia decreased NO (45 %; p ⬍ 0.001) in normal peritoneal fibroblasts to levels observed for adhesion fibroblasts. In addition hypoxia increased both
S38
Abstracts
Objective: Differences in outcome between embryos derived from thawed oocytes or fertilized eggs have been largely attributed to unique properties of the zygote that may be labile to freezing-associated disruption. We have previously shown the apparent depolarization of pericortical mitochondria in MII human oocytes after thawing. While the level of mitochondrial influence on initiation and propagation of Ca2⫹ waves in the human oocyte is unknown, the role of calcium as a critical epigenetic factor at the earliest stages of mammalian oocyte activation has been demonstrated in several species. Here, we compared levels of ionophore induced Ca2⫹ release in fresh and thawed metaphase II oocytes. Design: A prospective, randomized study of the changes in intracellular free calcium levels in fresh and thawed MII human oocytes after exposure to a calcium ionophore. Materials and Methods: Fresh Day 1 oocytes which failed to fertilize were used in this study as well as Day 0 and Day 1 cryopreserved human oocytes. Frozen oocytes were stained at two time intervals (i) immediately after thawing and (ii) after 4 hours of culture. Fresh oocytes were stained at approximately 24 hours post egg retrieval. Oocytes were preloaded with the calcium-sensitive fluorescent probe Fluo-4-AM (5M, 60 min., 37oC) followed by washing in normal medium for 30 min and placement in ⌬_upper;T dishes with temperature maintained precisely at 37oC with a ⌬_upper;T controller. Oocytes were examined by scanning laser confocal microscopy at 488 nm excitation to determine baseline levels of fluorescence. The calcium ionophore A23187 was added to achieve a final concentration of 10uM and changes in relative fluorescent intensity (RFI) indicative of a rise in intracellular free calcium levels were measured at 5 sec intervals until maximum fluorescence was observed. Results: Fresh oocytes generated a significantly higher RFI than their thawed counterparts. Day 1 thawed oocytes an average RFI of while the average intensity increase in the day 1 post ICSI fresh oocytes was 65.4 (P ⫽ 0.0058). The average intensity increase in oocytes frozen on Day 0 was 22. Within that group, the RFI of oocytes stained at 0h hours was 17.6 while those stained after 4 hours of culture was 32.7. This difference was not significant (P ⫽ 0.1091). Conclusion: At fertilization, human oocytes undergo a series of Ca2⫹ transients that initiate the completion of meiosis and regulate both the first mitotic division and the release of cortical granules. Recent experimental evidence indicates that relatively small changes in calcium fluxes at the earliest stage of activation have profound downstream consequences for development (Ozil and Huneau, 2001). Previously, we demonstrated that cryopreserved oocytes show a decrease in mitochondrial membrane potential when compared to their fresh counterparts. The current findings suggest that mitochondrial depolarization and reduced Ca2⫹ response in thawed oocytes could have downstream consequences resulting from an influence on the amplitude of the initial Ca2⫹ transient(s) that may contribute to implantation failure or early pregnancy loss.
Vol. 80, Suppl. 3, September 2003