Rescue of in-vitro matured oocytes following metaphase-II nuclear transfer in humans

Abstract

OBJECTIVE: Nuclear transfer into the metaphase-II (M-II) oocytes shows promise as a means of repairing either mitochondrial diseases or female infertility due to ooplasmic deficiency and abnormalities. We therefore conducted nuclear transfer between in vitro matured metaphase-II oocytes.DESIGN: The fertilzation rate, cleavage rare and blastocyst formation rate were investigated following nuclear transfer.MATERIALS AND METHODS: Recipient oocytes were derived from immature oocytes in ICSI treatment and cultured in vitro until the first polar body extrusion was observed. Donor oocytes were prepared from IVF or ICSI patients who had consented to participate in these experiments. Two oocytes were placed in a microdrop containing 5 microgram/ml of cytochalasinB (CCB). A 10-12 micrometer innerdiameter glass pipette was inserted into the perivitelline space and the cytoplasm contatining M-II chromosome was aspirated. The karyoplast of donor oocytes was removed using the same procedure that was used for recipient oocytes and transferred into the perivitelline space of an enucleated donor oocyte. Insertion of the karyoplast, the grafted oocyte was washed for 60 minutes in HTF medium to remove CCB, and then transferred in Zimmerman cell fusion medium. Membrane fusion between oocyte and karyoplast was facilitated by electrical stimulation (10V/sec AC + 15V/45 μsec DC) with an electro cell fusion generater (LF 101). 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 in 42 of the 44 freshly ovulated oocytes (95.5%) and in 38 of the 40 in vitro-matured oocytes [92.7% (55/55)]. The M-II karyoplast was removed successfully in 38 of 42 (90.5%) of the donor oocytes and 45 of 51 (88.2%) of the recipient oocytes. All of 38 karyoplasts of recipient oocytes were replaced in periviteleine space of enucleated donor oocytes and 30 of these 78.9% 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 (76.7% (23/30), 66.7% (20/30), 33.3% (10/30))respectively. Chromosomal analysis of 4 embryos following nuclear transfer were diploid sets of 46 chromosome.CONCLUSIONS: These results suggest that normal embryonic development can occur after the transfer of karyoplast between in vitro-matured metaphase-II oocytes. OBJECTIVE: Nuclear transfer into the metaphase-II (M-II) oocytes shows promise as a means of repairing either mitochondrial diseases or female infertility due to ooplasmic deficiency and abnormalities. We therefore conducted nuclear transfer between in vitro matured metaphase-II oocytes. DESIGN: The fertilzation rate, cleavage rare and blastocyst formation rate were investigated following nuclear transfer. MATERIALS AND METHODS: Recipient oocytes were derived from immature oocytes in ICSI treatment and cultured in vitro until the first polar body extrusion was observed. Donor oocytes were prepared from IVF or ICSI patients who had consented to participate in these experiments. Two oocytes were placed in a microdrop containing 5 microgram/ml of cytochalasinB (CCB). A 10-12 micrometer innerdiameter glass pipette was inserted into the perivitelline space and the cytoplasm contatining M-II chromosome was aspirated. The karyoplast of donor oocytes was removed using the same procedure that was used for recipient oocytes and transferred into the perivitelline space of an enucleated donor oocyte. Insertion of the karyoplast, the grafted oocyte was washed for 60 minutes in HTF medium to remove CCB, and then transferred in Zimmerman cell fusion medium. Membrane fusion between oocyte and karyoplast was facilitated by electrical stimulation (10V/sec AC + 15V/45 μsec DC) with an electro cell fusion generater (LF 101). 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 in 42 of the 44 freshly ovulated oocytes (95.5%) and in 38 of the 40 in vitro-matured oocytes [92.7% (55/55)]. The M-II karyoplast was removed successfully in 38 of 42 (90.5%) of the donor oocytes and 45 of 51 (88.2%) of the recipient oocytes. All of 38 karyoplasts of recipient oocytes were replaced in periviteleine space of enucleated donor oocytes and 30 of these 78.9% 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 (76.7% (23/30), 66.7% (20/30), 33.3% (10/30))respectively. Chromosomal analysis of 4 embryos following nuclear transfer were diploid sets of 46 chromosome. CONCLUSIONS: These results suggest that normal embryonic development can occur after the transfer of karyoplast between in vitro-matured metaphase-II oocytes.