利用废弃胚胎行人原核移植方法的初步建立

Human Pronuclear Transfer with Discarded Triploid Zygotes

【目的】探索利用废弃胚胎进行人原核移植的方案。【方法】收集废弃多原核受精卵117个。用显微操作-电融合的方法构建原核移植重构胚胎。分别比较钙离子浓度0.05mmol/L(n=39)和0.1mmol/L(n=58)的两种电融合液以及电压分别为1800V/cm(n=58)和2000V/cm(n=20)的两种电场条件对重构合子融合率和发育的影响。【结果】操作成功率70.1%,融合成功率59.8%,得到43个卵裂胚胎,其中23个(53.5%)6细胞以上胚胎。共14个重构胚发育至8细胞或以上,得到3个囊胚。将电压由2000V/cm下降到1800V/cm,重构胚胎融合率、卵裂率和囊胚率的差异没有统计学意义(P>0.05),但6-细胞率明显上升(22.2%vs.60.9%,P<0.05)。和钙离子浓度为0.05mmol/L的融合液比较,0.1mmol/L的融合液融合率明显上升(39.3%vs.69.0%,P<0.05),卵裂率、6-细胞率和囊胚率皆无统计学差异(P>0.05)。【结论】通过显微操作-电融合的方法,用废弃胚胎重构人核-质异质的胚胎模型,是可行的、经济的。

[ Objective] To establish a micromanipulation-electrofusion procedure for human pronuclear transfer with discarded triploid zygotes. [ Materials ] A total of 117 oocytes showing 3 pronuclears and 2 polar bodies 16-19 h after in vitro fertilization （IVF）, were considered as triploid zygotes and used after informed patient consents. Different fusion media protocols （containing 0.05 mmol/L and 0.1 mmol/L calcium chloride） and electrofusion conditions （ 1 800 V/cm and 2 000 V/cm voltage） were used to assess their influences on the reconstructed zygote development potential. [Result] The manipulation successful rate was 70.1%. The karyoplast fusion success rate was 59.8%. Forty-three reconstructed zygotes cleaved, among which 23 embryos reached 6-cell stage （53.5% of 2- cell embryos） and 14 developed to 8-cell stage with 3 blastocysts. When the voltage was decreased from 2 000 V/cm to 1 800 V/cm, the fusion rate, cleavage rate, and blastocyst rate between these two groups were not statistically different （P 〉 0.05）, but the 6-cell embryo rate significantly increased （22.2% vs. 60.9%, P=0.049）. The fusion rate was significantly increased by increasing the calcium concentration from 0.05 mmol/L to 0.1 mmol/L （39.3% vs. 69.0%, P= 0.014）. However, no statistical difference was observed in cleavage rate, the 6-cell rate and the blastocyst rate between the two groups（P 〉 0.05 ）. [ Conclusion ] Using discarded triploid zygote to reconstruct diploid zygote with micromanipulation-electrofusion method is a practicable and economical method to get cytoplasmic- nuclear heteroplasmic embryo model.