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PCOS Independent Hyperandrogenemia in Recurrent Pregnancy Loss
P-112
P-113
PCOS Independent Hyperandrogenemia in Recurrent Pregnancy Loss. 1 G. W. Bates, 2A. E. Taylor, 2J. M. Adams, 1J. A. Hill. Department of OB/GYN, 1Brigham & Women’s Hospital, 2Massachusetts General Hospital, Harvard Medical School, Boston, MA.
Embryogenesis by Cryopreserved Germinal Vesicles via Sequential Oocyte and Zygote Reconstruction. Z. Y. He, H. C. Liu, Z. Rosenwaks. The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY.
Objectives: Many potential etiologies have been proposed for recurrent pregnancy loss (RPL) including elevated levels of androgens and LH. Furthermore, the Polycystic Ovarian Syndrome, defined as ovulatory dysfunction in the setting of clinical or laboratory signs of androgen excess, has been linked to RPL. The objective of this study was to determine whether inherent dysregulation in steroid dynamics, independent of PCOS, exist in women with recurrent pregnancy loss. Design: Prospective observational study. Materials and Methods: Fourteen patients with a history of two or more consecutive, unexplained pregnancy losses before 12 weeks of gestation, with regular ovulatory cycles, luteal phases of at least 10 days, and no clinical evidence of hyperandrogenism were assessed. Genetic, anatomic, infectious, and immunologic etiologies were excluded. Regularly cycling, healthy women (N 5 118) with no history of pregnancy loss served as controls. Morning blood samples were obtained throughout the cycle and assayed for LH, FSH, estradiol, and progesterone. Testosterone, DHEAS, 17-OH progesterone, and SHBG were obtained in the early follicular, mid-luteal and ovulatory phases. The data were centered on the day of the LH surge. The LH surge was determined by a combination of the LH, FSH, and estradiol peak, as well as a doubling or exceeding 0.6 ng/ml of the progesterone level. Baseline ultrasounds were performed to identify polycystic ovarian morphology by a single examiner (JMA).
Objective: The aim of this study was to establish a reliable method for the cryopreservation of germinal vesicles (GVs) which can ultimately be used to generate embryos through sequential oocyte and zygote reconstruction. Design: Randomized prospective study to evaluate the potential of zygote reconstruction by cryopreserved germinal vesicles. Materials and Methods: Both immature oocytes and donor zygotes were collected from PMSG stimulated and PMCG/hCG superovulated B6D2F1 mice, respectively. The isolated GVs were first packed in a donor zona pellucida and then cryopreserved by open pulled straw (OPS) verification. After thawing, the GVs were either assessed for their survival by propidium iodide (PI) and Hoechst 33342 dual staining (Group A, n548) or transferred into enucleared GV-stage cytoplasts for oocyte reconstruction (Group B, n556). These reconstructed oocytes were matured in vitro to MII stage oocytes and then induced to form the female pronuclei by treatment with 7% ethanol. The female karyoplasts prepared from the induced female pronuclei were transferred to the donor zygotes containing male karyoplasts for zygote reconstruction. Results: The survival rates of the cryopreserved GVs were 77% and 82% in groups A and B, respectively. Of the 46 survived GVs in group B used for oocyte reconstruction, 70% (32/46) were fused, and 75% (24/32) were matured. After ethanol treatment, 82% (20/24) of these matured oocytes were induced to form female pronuclei. Nineteen of them were used to prepare female karyoplast for zygote reconstruction. Results are shown in the following table: No. of embryos (%) Total of F-karyoplast Electrofused (%) Survival (%) 2-cell stage (%) 4-cell stage (%) Blastocyst (%)
Results: Patients with a history of RPL demonstrated a decreased SHBG, and an increased Free Testosterone Index. No difference in serum LH, FSH, estradiol, progesterone, 17-OH progesterone or DHEAS were noted throughout the cycle. Nine of the 13 patients had PCO morphology. No differences in the hormonal profile were noted among study subjects with and without PCO.
RPL
Control
p value
78.7 6 11.2 83.8 6 13.8 79.3 6 12.6
130.8 6 12.5 129.4 6 15.4 137.8 6 13.4
0.005 0.04 0.005
Free Testosterone Index
Follicular Ovulation Luteal
RPL
Control
p value
1.46 6 0.38 2.02 6 0.46 1.75 6 0.36
0.63 6 0.08 0.92 6 0.09 0.67 6 0.08
0.05 0.04 0.01
Conclusions: Patients with Recurrent Pregnancy Loss display hyperandrogenemia throughout the menstrual cycle independent of PCO morphology. No dysregulation in cycle hormone dynamics or elevation in LH was observed in the RPL study population.
S130
Abstracts
Conclusion: Our data clearly demonstrates that maternal genome materials at the GV stage can be successfully cryopreserved by OPS vitrification. Using this technique, the detrimental effect of cryoinjury on the cytoplasts and other subcellular components of immature oocytes could be avoided. Therefore, cryopreservation of GVs can serve as an alternative method to preserve immature oocytes. More importantly, these preserved GVs can be used to reconstruct oocytes and zygotes with high potential for embryo development. Clinically, it may offer IVF-ET patients an additional option to effectively utilize their disposable immature oocytes for embryo generation.
P-114
SHBG
Follicular Ovulation Luteal
19 16 (84) 15 (93) 15 (100) 11 (73) 6 (40)
Variation in Oviductin mRNA Expression Throughout the Ovulatory Cycle. C. M. Briton-Jones, I. H. Lok, T. T. Chiu, P. M. Yuen, L. P. Cheung, C. J. Haines. The Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Hong Kong SAR China. Objectives: Oviductin is a glycoprotein that is produced exclusively in the oviduct. Current evidence suggests that oviductin facilitates sperm-zona pellucida binding and it may also have embryotrophic qualities. Little is known about the regulation of oviductin mRNA in humans. The objective of this study was to compare the level of oviductin mRNA expression in different phases of the ovulatory cycle. Design: A semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used, with specific oligonucleotide primers for oviductin and the internal control gene b-actin. Materials and Methods: Oviducts (N59) were obtained from women undergoing tubal sterilization. The cycle phase when the tissue was collected was determined by last menstrual period (LMP) and confirmed by serum concentrations of estradiol (E2), luteinizing hormone (LH) and progesterone (P). Each sample was analyzed separately to enable comparisons
Vol. 74, No. 3, Suppl. 1, September 2000
P-113
PCOS Independent Hyperandrogenemia in Recurrent Pregnancy Loss. 1 G. W. Bates, 2A. E. Taylor, 2J. M. Adams, 1J. A. Hill. Department of OB/GYN, 1Brigham & Women’s Hospital, 2Massachusetts General Hospital, Harvard Medical School, Boston, MA.
Embryogenesis by Cryopreserved Germinal Vesicles via Sequential Oocyte and Zygote Reconstruction. Z. Y. He, H. C. Liu, Z. Rosenwaks. The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY.
Objectives: Many potential etiologies have been proposed for recurrent pregnancy loss (RPL) including elevated levels of androgens and LH. Furthermore, the Polycystic Ovarian Syndrome, defined as ovulatory dysfunction in the setting of clinical or laboratory signs of androgen excess, has been linked to RPL. The objective of this study was to determine whether inherent dysregulation in steroid dynamics, independent of PCOS, exist in women with recurrent pregnancy loss. Design: Prospective observational study. Materials and Methods: Fourteen patients with a history of two or more consecutive, unexplained pregnancy losses before 12 weeks of gestation, with regular ovulatory cycles, luteal phases of at least 10 days, and no clinical evidence of hyperandrogenism were assessed. Genetic, anatomic, infectious, and immunologic etiologies were excluded. Regularly cycling, healthy women (N 5 118) with no history of pregnancy loss served as controls. Morning blood samples were obtained throughout the cycle and assayed for LH, FSH, estradiol, and progesterone. Testosterone, DHEAS, 17-OH progesterone, and SHBG were obtained in the early follicular, mid-luteal and ovulatory phases. The data were centered on the day of the LH surge. The LH surge was determined by a combination of the LH, FSH, and estradiol peak, as well as a doubling or exceeding 0.6 ng/ml of the progesterone level. Baseline ultrasounds were performed to identify polycystic ovarian morphology by a single examiner (JMA).
Objective: The aim of this study was to establish a reliable method for the cryopreservation of germinal vesicles (GVs) which can ultimately be used to generate embryos through sequential oocyte and zygote reconstruction. Design: Randomized prospective study to evaluate the potential of zygote reconstruction by cryopreserved germinal vesicles. Materials and Methods: Both immature oocytes and donor zygotes were collected from PMSG stimulated and PMCG/hCG superovulated B6D2F1 mice, respectively. The isolated GVs were first packed in a donor zona pellucida and then cryopreserved by open pulled straw (OPS) verification. After thawing, the GVs were either assessed for their survival by propidium iodide (PI) and Hoechst 33342 dual staining (Group A, n548) or transferred into enucleared GV-stage cytoplasts for oocyte reconstruction (Group B, n556). These reconstructed oocytes were matured in vitro to MII stage oocytes and then induced to form the female pronuclei by treatment with 7% ethanol. The female karyoplasts prepared from the induced female pronuclei were transferred to the donor zygotes containing male karyoplasts for zygote reconstruction. Results: The survival rates of the cryopreserved GVs were 77% and 82% in groups A and B, respectively. Of the 46 survived GVs in group B used for oocyte reconstruction, 70% (32/46) were fused, and 75% (24/32) were matured. After ethanol treatment, 82% (20/24) of these matured oocytes were induced to form female pronuclei. Nineteen of them were used to prepare female karyoplast for zygote reconstruction. Results are shown in the following table: No. of embryos (%) Total of F-karyoplast Electrofused (%) Survival (%) 2-cell stage (%) 4-cell stage (%) Blastocyst (%)
Results: Patients with a history of RPL demonstrated a decreased SHBG, and an increased Free Testosterone Index. No difference in serum LH, FSH, estradiol, progesterone, 17-OH progesterone or DHEAS were noted throughout the cycle. Nine of the 13 patients had PCO morphology. No differences in the hormonal profile were noted among study subjects with and without PCO.
RPL
Control
p value
78.7 6 11.2 83.8 6 13.8 79.3 6 12.6
130.8 6 12.5 129.4 6 15.4 137.8 6 13.4
0.005 0.04 0.005
Free Testosterone Index
Follicular Ovulation Luteal
RPL
Control
p value
1.46 6 0.38 2.02 6 0.46 1.75 6 0.36
0.63 6 0.08 0.92 6 0.09 0.67 6 0.08
0.05 0.04 0.01
Conclusions: Patients with Recurrent Pregnancy Loss display hyperandrogenemia throughout the menstrual cycle independent of PCO morphology. No dysregulation in cycle hormone dynamics or elevation in LH was observed in the RPL study population.
S130
Abstracts
Conclusion: Our data clearly demonstrates that maternal genome materials at the GV stage can be successfully cryopreserved by OPS vitrification. Using this technique, the detrimental effect of cryoinjury on the cytoplasts and other subcellular components of immature oocytes could be avoided. Therefore, cryopreservation of GVs can serve as an alternative method to preserve immature oocytes. More importantly, these preserved GVs can be used to reconstruct oocytes and zygotes with high potential for embryo development. Clinically, it may offer IVF-ET patients an additional option to effectively utilize their disposable immature oocytes for embryo generation.
P-114
SHBG
Follicular Ovulation Luteal
19 16 (84) 15 (93) 15 (100) 11 (73) 6 (40)
Variation in Oviductin mRNA Expression Throughout the Ovulatory Cycle. C. M. Briton-Jones, I. H. Lok, T. T. Chiu, P. M. Yuen, L. P. Cheung, C. J. Haines. The Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Hong Kong SAR China. Objectives: Oviductin is a glycoprotein that is produced exclusively in the oviduct. Current evidence suggests that oviductin facilitates sperm-zona pellucida binding and it may also have embryotrophic qualities. Little is known about the regulation of oviductin mRNA in humans. The objective of this study was to compare the level of oviductin mRNA expression in different phases of the ovulatory cycle. Design: A semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used, with specific oligonucleotide primers for oviductin and the internal control gene b-actin. Materials and Methods: Oviducts (N59) were obtained from women undergoing tubal sterilization. The cycle phase when the tissue was collected was determined by last menstrual period (LMP) and confirmed by serum concentrations of estradiol (E2), luteinizing hormone (LH) and progesterone (P). Each sample was analyzed separately to enable comparisons
Vol. 74, No. 3, Suppl. 1, September 2000