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P-683
overall disadvantage beause obesity has been shown to negatively impact pregnancy outcome in the general population as well as IVF patients. It has also been shown that an altered stimulation protocol can help obese patients succeed in IVF, due to their altered response to gonadotropins. It is therefore necessary to identify different stimulation protocols tailored to benefit all kinds of patients including those with an increased BMI. In 2005 the FCNE reduced the number of stimulations to 4 main protocols: long cycle lupron, short cycle lupron, microflare and antagon as well as reducing the required follicle size for administration of hCG (from 20 mm to 17 mm) which realized an increase in clinical pregnancy rates and implantation rates in the entire program. DESIGN: Retrospective analysis of patients undergoing IVF/ICSI from January 2004 - December 2005. Outcomes were analyzed among BMI groups and overall rates of 2004 and 2005 were compared. All PGD, donor egg, gestational carrier, patients with a BMI ⬎40.0 and age ⬎42 were excluded. MATERIALS AND METHODS: The final data set for January 2004 to December of 2005 consisted of 1273 patients. 2004 and 2005 were analyzed separately with 2004 consisting of 645 patients, and 2005 with 628 patients. BMI was defined as: underweight ⬍20, normal 20-24.9, overweight 2529.9, obese ⬎30 and severely obese ⬎40 (WHO criteria). Implantation was defined as the number of fetal heartbeats per number of embryos transferred and pregnancy rate was defined as a positive hCG per patient transfer. RESULTS: Some significant improvements were identified due to the altered stimulation protocols adopted in 2005, both overall and among the different BMI groups.The implantation rate increased from 19% in 2004 to 23% in 2005, as well as an increase in pregnancy rates from 41 % in 2004 to 46 % in 2005. In 2004 there was a steady decline in CPR and IR as BMI increased (see table). In 2005 CPR and IR was increased overall as well as in each BMI category compared with 2004. The increase in the groups with BMI in the overweight and obese range was greater than in the normal weight group, which are not significantly altered.
MATERIALS AND METHODS: Oocytes were obtained following ovarian hyperstimulation from 38 patients. Immediately after ovum pick up, oocyte-cumulus complexes were incubated in In Vitro Fertilization (IVF) medium and after 3 hours of incubation, cumuli oophori were completely removed from all oocytes. After 1 h, metaphase II-oocytes were randomly allocated to two treatment groups: fertilization by standard ICSI procedure or parthenogenetic activation through sequential exposition to 5 M ionomycin in IVF medium for 5 minutes at 37 °C, 6% CO2 followed by an incubation in 2 mM 6-dimethylaminopurine (DMAP) in culture medium for 3 hours. Fertilized and activated oocytes were cultured in the same conditions for up to 3 or 5 days respectively. RESULTS: Seventy-one out of 114 oocytes (62.3%) randomized to ICSI procedure developed normal fertilization. Seventy out of 104 (67.3%) sibling activated oocytes showed one enlarged pronucleus and one extruded polar body and were considered parthenotes. Comparison between fertilization rate and parthenogenetic activation rate showed no significant difference (p⫽0.44). On day 2, 70 embryos (61.4% out of the initial number of oocytes) with at least 2 cells were observed in the group of oocytes allocated to ICSI procedure while number of parthenotes with at least 2 cells was 64 (61.5%) (p⫽0.90). The mean ⫾ SD number of blastomeres in the two groups was 3.7 ⫾ 1.0 and 3.7 ⫾ 0.8 (range 2-6 for both) respectively (p⫽0.63), but a lower percentage of good morphology was observed. On day 3, in the group of ICSI fertilized oocytes 65 (58.7%) embryos had undergone further cellular division while in the group of parthenotes this event was documented in 47 (45.2%) cases (p⫽0.11). Also at this stage the mean ⫾ SD number of blastomeres in the two groups were comparable (6.3 ⫾ 1.5 and 6.3 ⫾ 1.4, respectively: p⫽0.93) while cleaved parthenotes still showed a lower percentage of good morphology. On day 5, 9 oocytes destined for parthenogenetic activation (8.7%) developed into good quality activated blastocysts. In vitro blastocyst formation rate could not be evaluated for ICSI oocytes since all viable embryos were transferred on day 3; indirect evidence can be figured out from ICSI cycle outcome. Since single pregnancy occurred in 9 women, it can indeed be inferred that at least 7.9% of initially inseminated oocytes have developed into good quality blastocysts. CONCLUSION: This protocol allows the activation of human oocytes and their development in vitro to a rate comparable with that of embryos fertilized by ICSI. Parthenotes can form good quality blastocysts. We propose that this protocol can eliminate the requirement to produce or disaggregate normal competent human embryos for assessing oocyte developmental competence or for deriving embryonic stem cells. Supported by: None.
** p ⬍0.01 * p ⬍ 0.05
P-684
CONCLUSION: Although an overall positive result was realized in 2005 in all BMI groups, obesity still negatively impacted success rates, but the data show an altered stimulation can positively effect rates in increasing BMI populations. Supported by: None
OOCYTE BIOLOGY P-683 ASSESSMENT OF HUMAN OOCYTES DEVELOPMENTAL COMPETENCE BY IN VITRO CULTURE FOLLOWING PARTHENOGENETIC ACTIVATION. A. Paffoni, T. A. Brevini, E. Somigliana, L. Restelli, F. Gandolfi, G. Ragni. Fondazione Policlinico Mangiagalli, Milan, Italy; Univ. of Milan, Dept. of Anatomy of Domestic Animals, Milan, Italy. OBJECTIVE: In mammals, parthenotes, the embryos obtained by parthenogenesis, can develop to different stages after oocyte activation, but never to term. The development of human parthenotes to the blastocyst stage was recently reported. Aim of the present work was to establish an activation protocol for the generation of parthenotes and to determine if their developmental competence would reflect that of zygotes obtained by fertilization. In this hypothesis, activation protocol could represent an ethical tool for the objective assessment of oocyte developmental competence following experimental procedures in the area of reproductive biology. DESIGN: In vitro study.
S386
Abstracts
THE DECLINE IN OVARIAN NON-GROWING FOLLICLE NUMBER FROM BIRTH TO MENOPAUSE: A NEW MODEL OF REPRODUCTIVE AGING. K. R. Hansen, A. C. Thyer, N. S. Knowlton, J. M. Buckner, M. R. Soules, N. A. Klein. Univ of Oklahoma, Oklahoma City, OK; Seattle Reproductive Medicine, Seattle, WA; NSK Statistical Solutions, Oklahoma City, OK. OBJECTIVE: The primary determinant of reproductive age in women is ovarian non-growing follicle (primordial, intermediate and primary) number. Our understanding of the initial endowment of ovarian follicles and the rate of follicle loss in the human is based upon data from three independent studies that reported estimates of non-growing follicle (NGF) number in a pooled total of 103 ovarian pairs from females aged 6-55. These previous studies have employed model-based methods for number estimates. Modelbased methods require correction factors and assumptions regarding cell orientation, shape, size and volume which ultimately limit their accuracy. Furthermore, model-based methods are less efficient than modern stereology methods. To better define the decline in NGF number associated with aging, we have employed modern, unbiased stereology methods based upon the optical fractionator technique to determine ovarian NGF number in human ovaries from birth to menopause. DESIGN: Prospective study, university setting. MATERIALS AND METHODS: Normal ovaries were collected from 111 women undergoing oophorectomy, organ donation, or autopsy. After fixation, each ovary was cut into 1 mm “slabs.” Using systematic random sampling rules, eight slabs were selected. The slabs were embedded in large (2” x 3”) glycomethacrylate blocks and exhaustively sectioned at a thickness of 25 m. Every 10th section was collected, stained, and mounted.
Vol. 86, Suppl 2, September 2006
MATERIALS AND METHODS: Oocytes were obtained following ovarian hyperstimulation from 38 patients. Immediately after ovum pick up, oocyte-cumulus complexes were incubated in In Vitro Fertilization (IVF) medium and after 3 hours of incubation, cumuli oophori were completely removed from all oocytes. After 1 h, metaphase II-oocytes were randomly allocated to two treatment groups: fertilization by standard ICSI procedure or parthenogenetic activation through sequential exposition to 5 M ionomycin in IVF medium for 5 minutes at 37 °C, 6% CO2 followed by an incubation in 2 mM 6-dimethylaminopurine (DMAP) in culture medium for 3 hours. Fertilized and activated oocytes were cultured in the same conditions for up to 3 or 5 days respectively. RESULTS: Seventy-one out of 114 oocytes (62.3%) randomized to ICSI procedure developed normal fertilization. Seventy out of 104 (67.3%) sibling activated oocytes showed one enlarged pronucleus and one extruded polar body and were considered parthenotes. Comparison between fertilization rate and parthenogenetic activation rate showed no significant difference (p⫽0.44). On day 2, 70 embryos (61.4% out of the initial number of oocytes) with at least 2 cells were observed in the group of oocytes allocated to ICSI procedure while number of parthenotes with at least 2 cells was 64 (61.5%) (p⫽0.90). The mean ⫾ SD number of blastomeres in the two groups was 3.7 ⫾ 1.0 and 3.7 ⫾ 0.8 (range 2-6 for both) respectively (p⫽0.63), but a lower percentage of good morphology was observed. On day 3, in the group of ICSI fertilized oocytes 65 (58.7%) embryos had undergone further cellular division while in the group of parthenotes this event was documented in 47 (45.2%) cases (p⫽0.11). Also at this stage the mean ⫾ SD number of blastomeres in the two groups were comparable (6.3 ⫾ 1.5 and 6.3 ⫾ 1.4, respectively: p⫽0.93) while cleaved parthenotes still showed a lower percentage of good morphology. On day 5, 9 oocytes destined for parthenogenetic activation (8.7%) developed into good quality activated blastocysts. In vitro blastocyst formation rate could not be evaluated for ICSI oocytes since all viable embryos were transferred on day 3; indirect evidence can be figured out from ICSI cycle outcome. Since single pregnancy occurred in 9 women, it can indeed be inferred that at least 7.9% of initially inseminated oocytes have developed into good quality blastocysts. CONCLUSION: This protocol allows the activation of human oocytes and their development in vitro to a rate comparable with that of embryos fertilized by ICSI. Parthenotes can form good quality blastocysts. We propose that this protocol can eliminate the requirement to produce or disaggregate normal competent human embryos for assessing oocyte developmental competence or for deriving embryonic stem cells. Supported by: None.
** p ⬍0.01 * p ⬍ 0.05
P-684
CONCLUSION: Although an overall positive result was realized in 2005 in all BMI groups, obesity still negatively impacted success rates, but the data show an altered stimulation can positively effect rates in increasing BMI populations. Supported by: None
OOCYTE BIOLOGY P-683 ASSESSMENT OF HUMAN OOCYTES DEVELOPMENTAL COMPETENCE BY IN VITRO CULTURE FOLLOWING PARTHENOGENETIC ACTIVATION. A. Paffoni, T. A. Brevini, E. Somigliana, L. Restelli, F. Gandolfi, G. Ragni. Fondazione Policlinico Mangiagalli, Milan, Italy; Univ. of Milan, Dept. of Anatomy of Domestic Animals, Milan, Italy. OBJECTIVE: In mammals, parthenotes, the embryos obtained by parthenogenesis, can develop to different stages after oocyte activation, but never to term. The development of human parthenotes to the blastocyst stage was recently reported. Aim of the present work was to establish an activation protocol for the generation of parthenotes and to determine if their developmental competence would reflect that of zygotes obtained by fertilization. In this hypothesis, activation protocol could represent an ethical tool for the objective assessment of oocyte developmental competence following experimental procedures in the area of reproductive biology. DESIGN: In vitro study.
S386
Abstracts
THE DECLINE IN OVARIAN NON-GROWING FOLLICLE NUMBER FROM BIRTH TO MENOPAUSE: A NEW MODEL OF REPRODUCTIVE AGING. K. R. Hansen, A. C. Thyer, N. S. Knowlton, J. M. Buckner, M. R. Soules, N. A. Klein. Univ of Oklahoma, Oklahoma City, OK; Seattle Reproductive Medicine, Seattle, WA; NSK Statistical Solutions, Oklahoma City, OK. OBJECTIVE: The primary determinant of reproductive age in women is ovarian non-growing follicle (primordial, intermediate and primary) number. Our understanding of the initial endowment of ovarian follicles and the rate of follicle loss in the human is based upon data from three independent studies that reported estimates of non-growing follicle (NGF) number in a pooled total of 103 ovarian pairs from females aged 6-55. These previous studies have employed model-based methods for number estimates. Modelbased methods require correction factors and assumptions regarding cell orientation, shape, size and volume which ultimately limit their accuracy. Furthermore, model-based methods are less efficient than modern stereology methods. To better define the decline in NGF number associated with aging, we have employed modern, unbiased stereology methods based upon the optical fractionator technique to determine ovarian NGF number in human ovaries from birth to menopause. DESIGN: Prospective study, university setting. MATERIALS AND METHODS: Normal ovaries were collected from 111 women undergoing oophorectomy, organ donation, or autopsy. After fixation, each ovary was cut into 1 mm “slabs.” Using systematic random sampling rules, eight slabs were selected. The slabs were embedded in large (2” x 3”) glycomethacrylate blocks and exhaustively sectioned at a thickness of 25 m. Every 10th section was collected, stained, and mounted.
Vol. 86, Suppl 2, September 2006