MANUFACTURING OOCYTES THROUGH SOMATIC CELL NUCLEAR TRANSFER

Abstract

Somatic cell haploidization in a project of neogametogenesis is normally monitored by the sole extrusion of a pseudo-polar body. Here we determined whether an earlier identification of a meiotic-like spindle after somatic cell nuclear transfer (SCNT) enhances the likelihood of successful haploidization. Constructs were observed for meiotic-like spindle identification at least 2 hours after SCNT. Oocytes with either negative (NS) or positive (PS) spindle were inseminated, together with an unmanipulated control. Embryo development and molecular karyotype were assessed between the two cohorts. Metaphase II oocytes from B6D2F1 mice were exposed to cytochalasin B, and under Oosight™ visualization, meiotic spindles and polar bodies were removed. A single cumulus cell was fused with the resulting ooplast using Sendai virus. Reconstituted oocytes were assessed for the presence of a newly formed meiotic-like spindle and inseminated by piezo-ICSI. Piezo-ICSI of unmanipulated oocytes served as control. Successful haploidization was confirmed by the extrusion of a pseudo-polar body at the time fertilization, and time-lapse imaging was used to monitor preimplantation development. Whole genome sequencing of resulting blastocysts determined ploidy. Out of 320 oocytes that underwent SCNT, 186 (58%) displayed a meiotic-like spindle at least 2 hours later, while the remaining did not (42%). Control oocytes (n=49), 92 NS oocytes, and all 186 PS oocytes underwent piezo-ICSI insemination. NS and PS groups, although at a lower rate than the control (86%, P<0.0001), survived at a comparable rate of 63% and 52%, respectively. Of the surviving NS oocytes, only 10% showed successful haploidization by extrusion of a pseudo-polar body, while 55% of PS oocytes haploidized (P<0.00001). The control oocytes fertilized at a superior rate of 74% compared to the experimental groups (P<0.00001); however, between the two experimental cohorts, NS oocytes fertilized at a rate of 7%, while PS oocytes fertilized at a significantly higher rate of 53% (P<0.00001). Lastly, although at lower rates than the control (81%, P<0.0001), NS and PS oocytes developed to blastocysts at comparable rates of 50% and 27%, respectively. DNA sequencing of trophectoderm biopsies (4-8 cells) from 2 NS blastocysts and 6 PS blastocysts showed that both NS blastocysts (100%) and 4 PS (67%) blastocysts were euploid. The assessment of a meiotic-like spindle after SCNT demonstrated a higher proportion of haploidized and fertilized oocytes. In both cohorts, we were able to observe a full preimplantation development morphokinetically comparable to the control and proved to be euploid. Once the reproducibility and safety of this technique are demonstrated, it may be applied in humans to generate oocytes for women of advanced maternal age or with exhausted ovarian reserve.