A novel trial of nuclear transfer for repairing an aged oocyte

Abstract

OBJECTIVE: Nuclear transfer into the metaphase-II(M-II) oocytes shows promise as a means of repairing female infertility due to ooplasmic deficiency and abnormalities. We therefore conducted nuclear transfer of in vitro matured metaphase-II oocytes (recipient oocytes) into enucleated freshly ovulated metaphase-II oocytes(donor oocyte). DESIGN: The fertilization rate, cleavage rate and blastocyst formation rate were investigated following nuclear transfer. MATERIALS AND METHODS: Recipient and donor oocytes were placed in a microdrop containing 5 microgram/ml of cytochalasinB (CCB). The aspirated M-II karyoplast of recipient oocytes was transferred into the perivitelline space of an enucleated donor oocyte. The grafted oocyte was transferred in Zimmerman cell fusion medium. Membrane fusion was facilitated by electrical stimulation (10V for 1 second AC + 10V for 45 microsecond DC) with an electro cell fusion generater (LF 201). After fusion, the constructed oocytes were cultured in HTF medium for 2 hours and ICSI was performed. RESULTS: The percentage of identification of M-II chromosome was 91.1 % (41 out of 45) in freshly ovulated oocytes and 96.0% (48 out of 50) in vitro-matured oocytes. The M-II karyoplast was removed successfully in 35 of 41 (85.4%) of the donor oocytes and 40 of 48 (83.3%) of the recipient oocytes. All of 35 karyoplasts of recipient oocytes were replaced in the periviteleine space of enucleated donor oocytes and 28 of these 80.0% were fused to form a reconstituted oocyte.The fertilization rate, cleavage rate and blastocyst formation rate following ICSI for constructed oocytes and recipients oocytes were(77.1%(27/35), 65.7%(23/35), 25.7%(9/35)),(59.0%(58/98), 26.1%(25/98), 3.4%(3/98))respectively. Chromosomal analysis of 4 embryos following nuclear transfer indicated they were all diploid sets of 46 chromosome. CONCLUSIONS: These results demonstrate that this technique can be applied to the treatment of female infertility due to ooplasmic deficiency and abnormalities in aged oocytes