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P-081 Human blastocyst development in vitro without co-culture using a sequential culture system
used for insemination of oocytes were found to have decreased pH values following fertilization check and zygote transfer to culture wells (7.063+/- 0.019SD). These pH values are below our excepted standards (7.30-7.50). We do not know whether these conditions adversely effect the long term results of embryo development within our systern. The overall delivered/ongoing pregnancy rate per stimulation for 1996 in our program was 31%. Conclusion: Culture medium pH is an important parameter for healthy embryo development in-vitro. Small volume microdrop culture conditions make accurate pH assessment important to the health of cultured oocytes and developing embryos. High numbers of cumulus cells in microdrops may alter the pH conditions of the medium. Further studies are in progress to determine the impact of different mediums on microdrop culture pH and pregnancy rates.
P-081 H u m a n Blastocyst Development In Vitro Without Co-Culture Using a Sequential Culture System. B. Behr, D. Moore, J. Gebhardt, S. Hall, D. Dasig. Department GYN/OB Stanford University Medical Center, Stanford, CA. Objective: This preliminary analysis was designed to measure the percent blastocyst development of supernumerary embryos without the use of feeder cells or conditioned medium. Embryos derived from IVF that were not transferred or cryopreserved were included for this study. Design: A retrospective analysis of embryonic development from IVF or GIFT/IVF patients who's embryos did not meet criteria for cryopreservation or embryo transfer, were cultured in a sequential fashion to asses percent blastocyst development after 120hrs of culture. Materials and Methods: Ova were harvested from patients undergoing IVF with a standard ovarian stimulation with GnRHa/hMG. Ova were collected and cultured in 150ul droplets of P1 (Irvine Scientific) under mineral oil, in groups at 37°C under 5%CO2, 5%02 and 90%N2 environment. Embryo transfer was performed 72hrs post harvest. Viable embryos not transferred or cryopreserved were placed into a modified Hams F-10 (Irvine Scientific) and cultured for an additional 48hrs. Embryos that exhibited an expanded blastocele cavity and well defined inner cell mass at 120hrs were counted. Results: 109 embryos from 17 consecutive patients with supernumerary embryos were cultured. 51% (56/109) reached the expanded blastocyst stage by 120hrs of culture and patients were given the option of cryopreservation. The embryos were cryopreserved using a standard serial addition of glycerol protocol. Several patients had blastocysts develop after 120hrs of culture that were not included Conclusion: This report demonstrates that implementing a simple system of sequential culture generated acceptable blastocyst development (51%) with "left over" embryos without the use of feeder cells or conditioned medium. Recognizing the deferential metabolic requirements of early and late cleavage stage embryos has enabled the implementation of a glucose/phosphate-free sire-
ple culture medium (P1) for up to 72hrs of culture and a complex glucose containing medium (Mod. Hams F-10) for subsequent blastocyst development. Five patients have had thawed blastocyststransferred with 3 resulting in clinical pregnancy. The role of specific medium components and their relevancy to preimplantation embryo metabolism and the implications of blastocyst cryopreservation will be discussed.
P-082 Effects of TEST-Yolk Buffer and Glycerol Cryopreservation o n H u m a n Spermatozoa Morphology and Function. J. Hallak, R.S. Sidhu, A.J. Thomas Jr., A. Agarwal. Andrology Research and Clinical Laboratories, Department of Urology, Cleveland Clinic Foundation, Cleveland, OH. Objective: Cryoprotective medium is important to the survival of h u m a n sperm during long-term freezing in liquid nitrogen. Glycerol and Test-yolk buffer (TYB) are two commonly used cryopreservative media. We compared the effects of these two media to determine which better preserves sperm function and morphology. Design: A prospective clinical study. Materials and Methods: Semen was obtained by masturbation from 10 healthy donors after 2 to 3 days of sexual abstinence. After liquefaction, each sample was divided in two aliquots. Each aliquot was cryopreserved by either mixing glycerol only (6% v/v with the ejaculate) or TYB, which contains glycerol and other buffers (1:1 with semen, final glycerol content 6% v/v). Prefreeze and post-thaw total sperm count, percent motility, morphology (Kruger's strict criteria and WHO method) and the hypo-osmotic swelling, bovine cervical mucus penetration, and viability tests were evaluated. Motility was analyzed at 0, 60, 120, and 180 min after thawing and washing with human tubal fluid medium. Results: Percent motility decreased significantly in postthaw samples compared to prefreeze values in both media. Postthaw motility was greater in TYB specimens compared to glycerol (17.2 +_ 18.7; 10.2 ___ 15.5; P <0.004). TYB aliquots also had higher motility for up 180 min compared to glycerol aliquots (P = 0.05). Percent viability was significantly greater with TYB than glycerol (8.7 _+ 8.6; 5.7 -+ 5.9; P <0.02). Sperm morphology scored by Kruger's and WHO criteria declined significantly in post-thaw specimens frozen with either medium (P <0.003). Comparing prefreeze and postthaw morphology by WHO method showed TYB better preserved morphology (P <0.005). The postthaw bovine cervical mucus test value was significantly lower in glycerol preserved specimens (P <0.005). Postthaw hypo-osmotic swelling did not significantly differ from prefreeze results nor between the two media. Conclusions: Sperm motility decreased less over time in specimens cryopreserved in TYB compared to glycerol. The TYB specimens had significantly better functional and morphological characteristics compared to the glycerolonly specimens. This may be due to the addition of egg yolk and zwitter ion buffers to glycerol in the TYB. We, recommend using Test-yolk buffer for long-term storage of h u m a n spermatozoa. Abstracts
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P-081 H u m a n Blastocyst Development In Vitro Without Co-Culture Using a Sequential Culture System. B. Behr, D. Moore, J. Gebhardt, S. Hall, D. Dasig. Department GYN/OB Stanford University Medical Center, Stanford, CA. Objective: This preliminary analysis was designed to measure the percent blastocyst development of supernumerary embryos without the use of feeder cells or conditioned medium. Embryos derived from IVF that were not transferred or cryopreserved were included for this study. Design: A retrospective analysis of embryonic development from IVF or GIFT/IVF patients who's embryos did not meet criteria for cryopreservation or embryo transfer, were cultured in a sequential fashion to asses percent blastocyst development after 120hrs of culture. Materials and Methods: Ova were harvested from patients undergoing IVF with a standard ovarian stimulation with GnRHa/hMG. Ova were collected and cultured in 150ul droplets of P1 (Irvine Scientific) under mineral oil, in groups at 37°C under 5%CO2, 5%02 and 90%N2 environment. Embryo transfer was performed 72hrs post harvest. Viable embryos not transferred or cryopreserved were placed into a modified Hams F-10 (Irvine Scientific) and cultured for an additional 48hrs. Embryos that exhibited an expanded blastocele cavity and well defined inner cell mass at 120hrs were counted. Results: 109 embryos from 17 consecutive patients with supernumerary embryos were cultured. 51% (56/109) reached the expanded blastocyst stage by 120hrs of culture and patients were given the option of cryopreservation. The embryos were cryopreserved using a standard serial addition of glycerol protocol. Several patients had blastocysts develop after 120hrs of culture that were not included Conclusion: This report demonstrates that implementing a simple system of sequential culture generated acceptable blastocyst development (51%) with "left over" embryos without the use of feeder cells or conditioned medium. Recognizing the deferential metabolic requirements of early and late cleavage stage embryos has enabled the implementation of a glucose/phosphate-free sire-
ple culture medium (P1) for up to 72hrs of culture and a complex glucose containing medium (Mod. Hams F-10) for subsequent blastocyst development. Five patients have had thawed blastocyststransferred with 3 resulting in clinical pregnancy. The role of specific medium components and their relevancy to preimplantation embryo metabolism and the implications of blastocyst cryopreservation will be discussed.
P-082 Effects of TEST-Yolk Buffer and Glycerol Cryopreservation o n H u m a n Spermatozoa Morphology and Function. J. Hallak, R.S. Sidhu, A.J. Thomas Jr., A. Agarwal. Andrology Research and Clinical Laboratories, Department of Urology, Cleveland Clinic Foundation, Cleveland, OH. Objective: Cryoprotective medium is important to the survival of h u m a n sperm during long-term freezing in liquid nitrogen. Glycerol and Test-yolk buffer (TYB) are two commonly used cryopreservative media. We compared the effects of these two media to determine which better preserves sperm function and morphology. Design: A prospective clinical study. Materials and Methods: Semen was obtained by masturbation from 10 healthy donors after 2 to 3 days of sexual abstinence. After liquefaction, each sample was divided in two aliquots. Each aliquot was cryopreserved by either mixing glycerol only (6% v/v with the ejaculate) or TYB, which contains glycerol and other buffers (1:1 with semen, final glycerol content 6% v/v). Prefreeze and post-thaw total sperm count, percent motility, morphology (Kruger's strict criteria and WHO method) and the hypo-osmotic swelling, bovine cervical mucus penetration, and viability tests were evaluated. Motility was analyzed at 0, 60, 120, and 180 min after thawing and washing with human tubal fluid medium. Results: Percent motility decreased significantly in postthaw samples compared to prefreeze values in both media. Postthaw motility was greater in TYB specimens compared to glycerol (17.2 +_ 18.7; 10.2 ___ 15.5; P <0.004). TYB aliquots also had higher motility for up 180 min compared to glycerol aliquots (P = 0.05). Percent viability was significantly greater with TYB than glycerol (8.7 _+ 8.6; 5.7 -+ 5.9; P <0.02). Sperm morphology scored by Kruger's and WHO criteria declined significantly in post-thaw specimens frozen with either medium (P <0.003). Comparing prefreeze and postthaw morphology by WHO method showed TYB better preserved morphology (P <0.005). The postthaw bovine cervical mucus test value was significantly lower in glycerol preserved specimens (P <0.005). Postthaw hypo-osmotic swelling did not significantly differ from prefreeze results nor between the two media. Conclusions: Sperm motility decreased less over time in specimens cryopreserved in TYB compared to glycerol. The TYB specimens had significantly better functional and morphological characteristics compared to the glycerolonly specimens. This may be due to the addition of egg yolk and zwitter ion buffers to glycerol in the TYB. We, recommend using Test-yolk buffer for long-term storage of h u m a n spermatozoa. Abstracts
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