- Email: [email protected]
Pup birth from vitrified split embryos
OBJECTIVE: We hypothesize that long term exposure of culture medium to 37 C in an incubator can cause evaporation and subsequent increase in osmolarity. The objectives of the current study are: - To measure the osmolarity due to evaporation in an incubator. - To evaluate the effect of osmotic stress on mouse embryos by scoring the development and growth rate. - To count the total cell number in blastocysts developed at different osmolarity. Abbreviations: BQ: Blastocyst, ICM: Inner Cell Mass, TE: Trophectoderm, ART: Assisted Reproduction Technology, PI: Propidium iodide DESIGN: Different types of dishes with different volumes were tested for osmolarity over different times in order to measure how evaporation of culture medium can affect in osmolarity stabilising inside the incubator. Measurements were repeated six times under similar conditions and mean SD were calculated. MATERIALS AND METHODS: Exp. I: Osmolarity Optimisation Different types of dishes used in an IVF laboratory was tested in this experiment in order to see which one is more likely to be impacted by changing of osmolarity values due to evaporation inside the incubator. Exp. II: Embryo Development 173 tow-cell mouse embryos were cultured in cleavage medium. Embryos were scoring every day until blastocyst day (5) then BQ were fixed by 3.7% PFA and 5% PVP/PBS Exp. III: Total Cell Count Embryo in Blastocyst stage was stained by PI in order to count the cell of inner cell mass (ICM) and trophectoderm (TE) by using fluorescent microscope. RESULTS: TCN in the control group (260 mOsm) was 65.7 5.1 and not significantly different to the 270 mOsm group (60.8 4.0). However, the number of cells in blastocysts formed in 280 mOsm was a significantly lower (42.3 6.6) than the control, and in contrast, embryos cultured in 290 mOsm had significantly more cells (83.7 5.4). The results clearly indicate that changes in osmolarity due to evaporation of culture medium can affect embryos negatively. CONCLUSIONS: The optimal range of osmolarity in culture medium for 2-cell mouse embryos was found to be between 260–290 mOsM. However, our study was undertaken using mouse embryos and it should be acknowledged that the optimal osmolarity of embryo culture medium might be different for different species and affected by difference in medium compositions. Supported by: None.
P-657 THE IMPORTANCE OF THE RATE AND PATTERN OF CLEAVAGE FOR PREDICTING THE IMPLANTATION POTENTIAL OF IVF EMBRYOS. T. Schwartz, A. Carmon, T. Cohen, R. Kapustiansky, A. Amit, D. Ben-Yosef. Racine IVF Unit, Tel-Aviv Medical Center, Tel-Aviv, Israel, Israel. OBJECTIVE: Improved selection criteria for top-quality embryos with the best implantation potential have now been developed for which embryos are grown separately and examined at several time points for various morphological structures. Cleavage rate and uniformity are usually evaluated for identifying the best-quality embryo, but their influence on IVF outcome is not well established. We aimed at evaluating the significance of embryo cleavage rate and uniformity at days 2/3 of development on their implantation potential. DESIGN: All patients’ %40 years old with %3 previous failed IVF cycles, undergoing IVF/ICSI between Oct 2004–June 2006, were included. A subgroup of all cycles with certain implantation (97 embryos) was also analyzed and compared to 96 embryos with the same patient’s indications but with no implantation. MATERIALS AND METHODS: Embryos were assessed at days 2/3 of development for their cleavage rates and patterns. A cleavage pattern was considered ‘‘good’’ when it was even (2, 4, 8 similar-sized blastomeres, or 3, 5, 6, 7 cells with proportional numbers of larger cells before cleavage). All parameters, individually and combined, were correlated to the clinical pregnancy rate (CP) and implantation rate (IR). RESULTS: A total of 289 patients underwent 395 cycles. Embryos with >7 blastomeres on day 3 were more likely to implant than less advanced ones (P<0.05). Embryos in cycles that ended with CP had significantly more cells than those with no CP (7.7 0.9 and 7.0 1.5 cells/embryo, respectively; P<0.01). In addition, 80% of embryos with certain implantation had a normal cleavage rate compared to 58% of embryos with no implantation (P<0.05). Stepwise logistic regression analysis for blastomere number,
FERTILITY & STERILITYÒ
cleavage pattern, fragmentation and compaction revealed blastomere number as the most significant prognostic parameter for implantation (P<0.01). Furthermore, each additional blastomere to day 3 embryos increased CP 3.8-fold (P<0.02). Cycles with embryos of good cleavage patterns yielded 35.4% CP compared to only 16.7% CP for embryos with uneven cleavage (P<0.05). Most (89%) embryos with certain implantation demonstrated a good cleaving pattern. CONCLUSIONS: Blastomere number and uniformity are apparently useful for predicting implantation potential. Comprehensive monitoring of embryo morphology increases our ability to choose top-quality embryos with the best implantation potential. Supported by: None.
EMBRYO TRANSFER P-658 PUP BIRTH FROM VITRIFIED SPLIT EMBRYOS. H.-C. Liu, Z. He, Y. Tang, W. Wang, Z. Rosenwaks. The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY. OBJECTIVE: Embryo splitting can be applied clinically to improve efficiency of IVF treatment. This technique can also spare part of the split embryos for other usages such as PGD or stem cell study without reducing efficiency of IVF treatment. However, the split embryos are more sensitive to hypothermal injuries than the intact ones. The purpose of this study was to evaluate the efficiency of vitrified split mouse embryos from different cleavage stages. DESIGN: Mouse vitrified split embryos were evaluated their viability and developmental potential after in vitro co-culture and embryo transfer. MATERIALS AND METHODS: Two-cell mouse embryos collected from superovulated female mice were cultured in-vitro to the 4-cell and the 8-cell stages. Split embryos were constructed by removing blastmeres from the cultured embryos followed by inserting half amount of the removed blastmeres into empty zona pellucida recipients. After reconstruction, the split embryos were cryopreserved by standard OPS-vitrification method. The fresh and thawed split embryos at 2-cell or 4-cell stage were either cocultured with mouse endometrial cells or transferred into the oviducts of surrogate females at the first day of the pseudopregnancy. The blastocyst formation, hatching and live birth rates of these embryos were examined. RESULTS: After splitting embryos at 4-cell and 8-cell stages, both fresh and vitrified split embryos did not exhibit significant differences in blastocyst formation rates (76% vs. 61%, P¼0.1, split at 4-cell stage; and 87% vs. 67%, P¼0.01, at 8-cell stage), hatching rates (94% vs. 81%, P¼0.07, at 4-cell stage; and 85% vs. 73%, P¼0.15, at 8-cell stage), and live birth rates (48% vs. 41%, P¼0.44, at 4-cell stage; and 31% vs. 15%, P¼0.05, at 8-cell stage).
Figure
CONCLUSIONS: High developmental potentials and live birth rates of vitrified split embryos indicate that OPS-vitrification was an effective cryopreservation method and the detrimental factors of cryoinjury on split embryos were reduced to minimum by vitrification followed by in-vitro coculture or embryo transfer. Supported by: None.
S325
P-657 THE IMPORTANCE OF THE RATE AND PATTERN OF CLEAVAGE FOR PREDICTING THE IMPLANTATION POTENTIAL OF IVF EMBRYOS. T. Schwartz, A. Carmon, T. Cohen, R. Kapustiansky, A. Amit, D. Ben-Yosef. Racine IVF Unit, Tel-Aviv Medical Center, Tel-Aviv, Israel, Israel. OBJECTIVE: Improved selection criteria for top-quality embryos with the best implantation potential have now been developed for which embryos are grown separately and examined at several time points for various morphological structures. Cleavage rate and uniformity are usually evaluated for identifying the best-quality embryo, but their influence on IVF outcome is not well established. We aimed at evaluating the significance of embryo cleavage rate and uniformity at days 2/3 of development on their implantation potential. DESIGN: All patients’ %40 years old with %3 previous failed IVF cycles, undergoing IVF/ICSI between Oct 2004–June 2006, were included. A subgroup of all cycles with certain implantation (97 embryos) was also analyzed and compared to 96 embryos with the same patient’s indications but with no implantation. MATERIALS AND METHODS: Embryos were assessed at days 2/3 of development for their cleavage rates and patterns. A cleavage pattern was considered ‘‘good’’ when it was even (2, 4, 8 similar-sized blastomeres, or 3, 5, 6, 7 cells with proportional numbers of larger cells before cleavage). All parameters, individually and combined, were correlated to the clinical pregnancy rate (CP) and implantation rate (IR). RESULTS: A total of 289 patients underwent 395 cycles. Embryos with >7 blastomeres on day 3 were more likely to implant than less advanced ones (P<0.05). Embryos in cycles that ended with CP had significantly more cells than those with no CP (7.7 0.9 and 7.0 1.5 cells/embryo, respectively; P<0.01). In addition, 80% of embryos with certain implantation had a normal cleavage rate compared to 58% of embryos with no implantation (P<0.05). Stepwise logistic regression analysis for blastomere number,
FERTILITY & STERILITYÒ
cleavage pattern, fragmentation and compaction revealed blastomere number as the most significant prognostic parameter for implantation (P<0.01). Furthermore, each additional blastomere to day 3 embryos increased CP 3.8-fold (P<0.02). Cycles with embryos of good cleavage patterns yielded 35.4% CP compared to only 16.7% CP for embryos with uneven cleavage (P<0.05). Most (89%) embryos with certain implantation demonstrated a good cleaving pattern. CONCLUSIONS: Blastomere number and uniformity are apparently useful for predicting implantation potential. Comprehensive monitoring of embryo morphology increases our ability to choose top-quality embryos with the best implantation potential. Supported by: None.
EMBRYO TRANSFER P-658 PUP BIRTH FROM VITRIFIED SPLIT EMBRYOS. H.-C. Liu, Z. He, Y. Tang, W. Wang, Z. Rosenwaks. The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY. OBJECTIVE: Embryo splitting can be applied clinically to improve efficiency of IVF treatment. This technique can also spare part of the split embryos for other usages such as PGD or stem cell study without reducing efficiency of IVF treatment. However, the split embryos are more sensitive to hypothermal injuries than the intact ones. The purpose of this study was to evaluate the efficiency of vitrified split mouse embryos from different cleavage stages. DESIGN: Mouse vitrified split embryos were evaluated their viability and developmental potential after in vitro co-culture and embryo transfer. MATERIALS AND METHODS: Two-cell mouse embryos collected from superovulated female mice were cultured in-vitro to the 4-cell and the 8-cell stages. Split embryos were constructed by removing blastmeres from the cultured embryos followed by inserting half amount of the removed blastmeres into empty zona pellucida recipients. After reconstruction, the split embryos were cryopreserved by standard OPS-vitrification method. The fresh and thawed split embryos at 2-cell or 4-cell stage were either cocultured with mouse endometrial cells or transferred into the oviducts of surrogate females at the first day of the pseudopregnancy. The blastocyst formation, hatching and live birth rates of these embryos were examined. RESULTS: After splitting embryos at 4-cell and 8-cell stages, both fresh and vitrified split embryos did not exhibit significant differences in blastocyst formation rates (76% vs. 61%, P¼0.1, split at 4-cell stage; and 87% vs. 67%, P¼0.01, at 8-cell stage), hatching rates (94% vs. 81%, P¼0.07, at 4-cell stage; and 85% vs. 73%, P¼0.15, at 8-cell stage), and live birth rates (48% vs. 41%, P¼0.44, at 4-cell stage; and 31% vs. 15%, P¼0.05, at 8-cell stage).
Figure
CONCLUSIONS: High developmental potentials and live birth rates of vitrified split embryos indicate that OPS-vitrification was an effective cryopreservation method and the detrimental factors of cryoinjury on split embryos were reduced to minimum by vitrification followed by in-vitro coculture or embryo transfer. Supported by: None.
S325