150 IN VITRO AND IN VIVO DEVELOPMENT OF EMBRYO FOLLOWING BIOPSY WITH DIFFERENT TECHNIQUES IN MOUSE EMBRYOS

Abstract

In vitro development ratios, quality evaluation, in vivo implantation, and fetal development ratios were investigated following aspiration biopsy in 8-cell mouse embryos and trophectoderm biopsy in blastocyst developed from 8-cell stage embryos in vitro. Superovulated CB6F1 hybrid female mice (5–6 weeks) were sacrificed 68 to 72 hours after hCG administration. Eight-cell embryos were flushed from oviducts of the sacrificed mouse with HTF medium supplemented with HEPES and 3 mg mL^–1 BSA. Embryos were randomly divided into two groups. In the first group, embryos at 8-cell stage were used for a single cell blastomer aspiration; in the second group, embryos were cultured in vitro until blastocyst stage. Trophectoderm cells (15% of trophoblastic cells) were biopsied from developing blastocysts. There were also control groups for both groups. Biopsy procedures for both groups were applied in 50 µL drops of Ca2+/Mg2+ free HTF medium containing HEPES+3 mg mL^–1 BSA+5 µg mL^–1 cytochalasine B. After biopsy, embryos were cultured in Quinn’s blastocyst medium supplemented with 4 mg mL^–1 BSA and incubated in 5% CO₂ and 5% O₂ incubator at 37°C for 48 and 24 hours for blastomer aspiration and trophectoderm biopsy groups, respectively. While some developing blastocysts were used for determining total cell number, some of them were transferred to the recipients. Results were evaluated by independent t-test and ANOVA of SPSS 17.0 statistic program (SPSS Inc., Chicago, IL, USA). In blastomere biopsy and control groups, development rates were determined as 81.02% (121/152) and 96.37% (62/63), while the total cell numbers were determined as 50 and 50, respectively. There was no significant difference between groups in terms of development ratios and total cells. In blastomer biopsy and control groups, the implantation sites and fetal development rates were found as 25% (9/36) and 26% (8/30), and 19.44% (7/36) and 20% (6/30), respectively. No significant difference was observed between groups in terms of implantation sites and fetal development rates. In trophectoderm biopsy and control groups, while the development rates were found as 86.96% (69/79) and 93.33% (23/28), the total cell numbers were 26.66 and 55.33, respectively. Although there was not any significant difference between groups in terms of development rates, there was a significant difference between groups in terms of total cell numbers (P < 0.05). In trophectoderm biopsy and control groups, the implantation sites and fetal development rates were determined as 21.88% (7/32) and 59.09% (13/22), and 0% (0/32) and 18.18% (4/22), respectively. Although there was not any significant difference between groups in terms of implantation sites, there was a significant difference between groups in terms of fetal development rates (P < 0.05). Therefore, it was concluded that biopsy applied at early stage of embryonic development does not affect embryo development negatively and biopsy procedures applied at early developmental stages have more advantages especially in embryos developing faster with low total cell numbers such as mouse species.

Supported by TUBITAK KAMAG-107G027).