Oocyte-induced haploidization.

Abstract

This paper describes the technical approach to treatment of age-related oocyte aneuploidy. Although one solution can be oocyte/embryo selection, another is represented by the nuclear transplantation procedure. The efficiency of nuclear transplantation into immature oocytes is described as a way of generating embryos, and the possibility that viable female gametes can be constructed by transfer of diploid somatic cell nuclei into enucleated oocytes. Germinal vesicle (GV)-stage mouse oocytes were collected from unstimulated ovaries and somatic nuclei were obtained from mouse cumulus cells obtained after ovarian stimulation. Spare human GV-stage oocytes were donated from consenting patients undergoing intracytoplasmic sperm injection (ICSI) treatment, and human somatic cells were stromal cells coming from uterine biopsies performed on consenting patients undergoing endometrial cell co-culture. GV ooplasts, prepared by enucleation, were transplanted with either GV or somatic nuclei by micromanipulation. Grafted oocytes were electrofused and cultured to allow maturation, following which they were selected at random for insemination or cytogenetic analysis. GV transplantation was accomplished with an overall efficiency of approximately 80 and 70% in the mouse and the human respectively. The maturation rate of 96% (mouse) and 62% (human) following reconstitution was comparable to that of control oocytes, as was the incidence of aneuploidy among the reconstituted oocytes. The reconstituted human oocytes were successfully fertilized by ICSI at a rate of 52%. After the transfer of mouse cumulus or human endometrial cell nuclei into enucleated immature oocytes, a polar body was extruded in >40%. In a limited number of observations where the nucleus of an aged oocyte was transferred into a younger ooplasm, the chromosomes segregated normally at the time of polar body extrusion. The technique of nuclear transplantation itself did not increase the incidence of chromosomal anomalies in the mouse or human, since their oocytes reconstituted with homologous donor GV resumed meiosis to metaphase II and maintained a normal ploidy. In addition, immature mouse ooplasts induced haploidization of transplanted somatic cell nuclei. Although further evaluation of their genetic status is needed, the procedure may offer a realistic way of producing normal oocytes in cases of aged-related infertility. While the procedure is technically similar to cloning, it would generate a unique individual as a result of the contribution of both parental genomes.