- Email: [email protected]
P-56
was 28% vs. 30%, the abort rate was 10.1 vs. 12.1 in A and B groups respectively. Using Chi square statistical analysis there were found no significance difference between groups. CONCLUSION: we can conclude that the presence of SERC in oocytes as an unique abnormality, does not affect the embryo development nor implantation, pregnancy or abortion rates. Therefore, in the selection of the best embryo to transfer the SERC in the oocyte should be the last parameter to keep in mind. Further studies must be performed with bigger number of cycles to corroborate this results. Supported by: Instituto Valenciano de Infertilidad.
P-56 VALIDATION OF VITRIFICATION FOR CLINICAL USE: POSTWARMING IN VITRO MATURATION AND BLASTOCYST OUTGROWTH. P. M. Villines, A. L. Clifford, M. A. Henry, C. C. Wegner. Center for Reproductive Biology of Indiana, LLC., Indianapolis, IN. OBJECTIVE: The effectiveness of vitrification as a method for cryopreservation of oocytes and day 5 blastocysts was studied prior to introduction of vitrification for routine clinical use. DESIGN: Extranumerary oocytes and blastocysts were donated for research with informed consent by infertility patients after conclusion of treatments. Vitrified immature oocytes at Germinal Vesicle (GV) or Metaphase I (MI) stage were warmed, placed into maturation medium for 24-72 hours and observed for progression to Metaphase II (MII) stage. Oocytes that reached MII stage in vitro were activated with calcium ionophore to demonstrate functional integrity. Vitrified MII oocytes were activated as controls. Previously vitrified day 5 blastocysts were warmed and cultured for an additional 8 days in vitro. Blastocyst outgrowth size and hCG production was analyzed at 48 hour intervals. MATERIALS AND METHODS: Oocytes and day 5 blastocysts were vitrified in hemistraws using 10% dimethylsulfoxide (DMSO), 10% ethylene glycol in modified Human Tubal Fluid medium (mHTF) with 10% human serum albumin (HSA) for 2 minutes followed by brief exposure to 20% DMSO, 20% ethylene glycol, Ficoll, 0.65M sucrose in mHTF with 10 % HSA before loading hemistraw and plunging into liquid nitrogen. Vitrified oocytes and blastocysts were warmed in 37oC 0.5M sucrose in mHTF, then immediately placed in 0.25M sucrose (5 min), 0.125M sucrose (5 min) and final wash in mHTF. Warmed oocytes were cultured in maturation medium (G2 medium with HSA, 1 IU/mL FSH, 1IU/mL LH, 9 IU/mL hCG, 1 g E2). Artificial activation of MII oocytes was performed by exposure to 5M calcium ionophore A23187 and 10 g/ml puromycin as previously described by Nakagawa et al., (Fertility and Sterility 76:1, 2001). Blastocysts were cultured in 0.5 ml G2-plus medium with 10% heat inactivated FBS in an organ culture dish for 8 days. At 48 hrs intervals, conditioned medium was stored at -20C until analysis and replaced with 0.5 ml fresh medium. Outgrowth size was measured and photographed at 48 hour intervals. Conditioned medium was analyzed for hCG levels using a solid-phase two-site chemiluminescent immunometric assay (DPC quantitative hCG kit LKCG1). Control medium consisted of medium cultured for the same interval without blastocyst exposure. RESULTS: Vitrified immature oocytes were able to undergo in vitro maturation and artificial activation by calcium ionophore upon warming and continued culture. Of 5 vitrified GVs, 4 (80%) survived warming, 75% (3) of these progressed to MII in culture and 100% (3) activated when challenged with calcium ionophore. Activation was demonstrated by the formation of pronuclei. Of 12 vitrified MI stage oocytes, 75% (9) survived warming, and 66% (6) progressed to the MII stage and 50% (3) were activated by calcium ionophore. Vitrified MII oocytes were activated as positive controls. Warmed day 5 blastocysts exhibited attachment and a mean 5-fold increase in outgrowth diameter (221 microns at 48 hrs to 1085 microns at 192 hours). Increasing concentration of hCG per attached blastocyst (15 ng/ml at 48 hours to 11,942 ng/ml at 192 hours) was also detected in the conditioned medium. CONCLUSION: Vitrification and warming of oocytes and blastocysts preserved functional integrity of both oocytes and blastocysts as demonstrated by in vitro maturation to MII stage with artificial activation and blastocyst outgrowth with hCG production. Supported by: Research was financially supported by Clarian Values Fund VFR 185 to CCW.
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Abstracts
P-57 YOUNG WOMEN AND ASSISTED REPRODUCTION OUTCOME: A CONUNDRUM. N. Esfandiari, M. Subido, M. Javed, H. Burjaq, L. Gotlieb, R. F. Casper. Toronto Centre for Advanced Reproductive Technology, Toronto, ON, Canada. OBJECTIVE: Maternal age has been considered a major determinant for the success of assisted reproduction treatment. Women experience a sharp decline in fecundity after the age of 40 due to decreasing oocyte competency and ovarian function. While the correlation between fecundity and age in women in their 30s and early 40s has been extensively studied and well established, there is no clear data on younger patients’ conception through the assisted reproduction. The objective of this study was to determine whether young age (less than 25 years) has an impact on outcome where the cause of infertility is male factor or in egg donation cycles. DESIGN: A retrospective study in a university affiliated tertiary care facility. MATERIALS AND METHODS: The study was conducted with patients undergoing IVF-ET in our clinic from 1994 to 2005. Seventy-four women less than 25 years of age with 80 cycles (group I) underwent IVF-ET. The control groups consisted of patients between 25 and 34 (n ⫽ 115, group II) and 35 - 39 (n ⫽ 83, group III) enrolled for IVF-ET treatment at the same time. Controlled ovarian stimulation was performed using the long luteal phase GnRH-agonist protocol, and 10,000 IU hCG was given for the final stage of follicular maturation. Oocytes were retrieved 34-36 hours after administration of hCG and underwent either insemination or ICSI. Normal fertilization (2PN) and embryo development were assessed and embryos that had cleaved to the two-cell stage or further were identified and graded using criteria based on blastomere number, symmetry, and degree of fragmentation. Embryo transfer (ET) was performed 72 hours post retrieval. When available, only embryos of the highest quality at the 6-8-cell stage were transferred. A pregnancy test was performed 14 days after ET. Clinical pregnancy was defined as a gestational sac within the uterus on ultrasound. RESULTS: Data were obtained from 360 cycles of IVF / ICSI (Table I). Each group was compared in terms of embryo development, embryo quality, average number of embryos transferred, and chemical or clinical pregnancy. For each patient there was at least one excellent grade embryo to transfer. There were no significant differences between the number of mature oocytes (MII), fertilization rate, and the quality of the embryos transferred. However the number of oocytes retrieved, fertilization rate following IVF and ICSI, and positive -hCG were lower, although not significantly so, in younger patients. The clinical pregnancy rate was significantly lower in the young patients compared to the other two groups.
CONCLUSION: Our results demonstrating lower pregnancy rates in young patients compared to the patients in their thirties were surprising.
Vol. 86, Suppl 2, September 2006
P-56 VALIDATION OF VITRIFICATION FOR CLINICAL USE: POSTWARMING IN VITRO MATURATION AND BLASTOCYST OUTGROWTH. P. M. Villines, A. L. Clifford, M. A. Henry, C. C. Wegner. Center for Reproductive Biology of Indiana, LLC., Indianapolis, IN. OBJECTIVE: The effectiveness of vitrification as a method for cryopreservation of oocytes and day 5 blastocysts was studied prior to introduction of vitrification for routine clinical use. DESIGN: Extranumerary oocytes and blastocysts were donated for research with informed consent by infertility patients after conclusion of treatments. Vitrified immature oocytes at Germinal Vesicle (GV) or Metaphase I (MI) stage were warmed, placed into maturation medium for 24-72 hours and observed for progression to Metaphase II (MII) stage. Oocytes that reached MII stage in vitro were activated with calcium ionophore to demonstrate functional integrity. Vitrified MII oocytes were activated as controls. Previously vitrified day 5 blastocysts were warmed and cultured for an additional 8 days in vitro. Blastocyst outgrowth size and hCG production was analyzed at 48 hour intervals. MATERIALS AND METHODS: Oocytes and day 5 blastocysts were vitrified in hemistraws using 10% dimethylsulfoxide (DMSO), 10% ethylene glycol in modified Human Tubal Fluid medium (mHTF) with 10% human serum albumin (HSA) for 2 minutes followed by brief exposure to 20% DMSO, 20% ethylene glycol, Ficoll, 0.65M sucrose in mHTF with 10 % HSA before loading hemistraw and plunging into liquid nitrogen. Vitrified oocytes and blastocysts were warmed in 37oC 0.5M sucrose in mHTF, then immediately placed in 0.25M sucrose (5 min), 0.125M sucrose (5 min) and final wash in mHTF. Warmed oocytes were cultured in maturation medium (G2 medium with HSA, 1 IU/mL FSH, 1IU/mL LH, 9 IU/mL hCG, 1 g E2). Artificial activation of MII oocytes was performed by exposure to 5M calcium ionophore A23187 and 10 g/ml puromycin as previously described by Nakagawa et al., (Fertility and Sterility 76:1, 2001). Blastocysts were cultured in 0.5 ml G2-plus medium with 10% heat inactivated FBS in an organ culture dish for 8 days. At 48 hrs intervals, conditioned medium was stored at -20C until analysis and replaced with 0.5 ml fresh medium. Outgrowth size was measured and photographed at 48 hour intervals. Conditioned medium was analyzed for hCG levels using a solid-phase two-site chemiluminescent immunometric assay (DPC quantitative hCG kit LKCG1). Control medium consisted of medium cultured for the same interval without blastocyst exposure. RESULTS: Vitrified immature oocytes were able to undergo in vitro maturation and artificial activation by calcium ionophore upon warming and continued culture. Of 5 vitrified GVs, 4 (80%) survived warming, 75% (3) of these progressed to MII in culture and 100% (3) activated when challenged with calcium ionophore. Activation was demonstrated by the formation of pronuclei. Of 12 vitrified MI stage oocytes, 75% (9) survived warming, and 66% (6) progressed to the MII stage and 50% (3) were activated by calcium ionophore. Vitrified MII oocytes were activated as positive controls. Warmed day 5 blastocysts exhibited attachment and a mean 5-fold increase in outgrowth diameter (221 microns at 48 hrs to 1085 microns at 192 hours). Increasing concentration of hCG per attached blastocyst (15 ng/ml at 48 hours to 11,942 ng/ml at 192 hours) was also detected in the conditioned medium. CONCLUSION: Vitrification and warming of oocytes and blastocysts preserved functional integrity of both oocytes and blastocysts as demonstrated by in vitro maturation to MII stage with artificial activation and blastocyst outgrowth with hCG production. Supported by: Research was financially supported by Clarian Values Fund VFR 185 to CCW.
S150
Abstracts
P-57 YOUNG WOMEN AND ASSISTED REPRODUCTION OUTCOME: A CONUNDRUM. N. Esfandiari, M. Subido, M. Javed, H. Burjaq, L. Gotlieb, R. F. Casper. Toronto Centre for Advanced Reproductive Technology, Toronto, ON, Canada. OBJECTIVE: Maternal age has been considered a major determinant for the success of assisted reproduction treatment. Women experience a sharp decline in fecundity after the age of 40 due to decreasing oocyte competency and ovarian function. While the correlation between fecundity and age in women in their 30s and early 40s has been extensively studied and well established, there is no clear data on younger patients’ conception through the assisted reproduction. The objective of this study was to determine whether young age (less than 25 years) has an impact on outcome where the cause of infertility is male factor or in egg donation cycles. DESIGN: A retrospective study in a university affiliated tertiary care facility. MATERIALS AND METHODS: The study was conducted with patients undergoing IVF-ET in our clinic from 1994 to 2005. Seventy-four women less than 25 years of age with 80 cycles (group I) underwent IVF-ET. The control groups consisted of patients between 25 and 34 (n ⫽ 115, group II) and 35 - 39 (n ⫽ 83, group III) enrolled for IVF-ET treatment at the same time. Controlled ovarian stimulation was performed using the long luteal phase GnRH-agonist protocol, and 10,000 IU hCG was given for the final stage of follicular maturation. Oocytes were retrieved 34-36 hours after administration of hCG and underwent either insemination or ICSI. Normal fertilization (2PN) and embryo development were assessed and embryos that had cleaved to the two-cell stage or further were identified and graded using criteria based on blastomere number, symmetry, and degree of fragmentation. Embryo transfer (ET) was performed 72 hours post retrieval. When available, only embryos of the highest quality at the 6-8-cell stage were transferred. A pregnancy test was performed 14 days after ET. Clinical pregnancy was defined as a gestational sac within the uterus on ultrasound. RESULTS: Data were obtained from 360 cycles of IVF / ICSI (Table I). Each group was compared in terms of embryo development, embryo quality, average number of embryos transferred, and chemical or clinical pregnancy. For each patient there was at least one excellent grade embryo to transfer. There were no significant differences between the number of mature oocytes (MII), fertilization rate, and the quality of the embryos transferred. However the number of oocytes retrieved, fertilization rate following IVF and ICSI, and positive -hCG were lower, although not significantly so, in younger patients. The clinical pregnancy rate was significantly lower in the young patients compared to the other two groups.
CONCLUSION: Our results demonstrating lower pregnancy rates in young patients compared to the patients in their thirties were surprising.
Vol. 86, Suppl 2, September 2006