THE MECHANISM OF 1PN FORMATION AFTER IVF INCLUDING FEMALE AND MALE GENOMES.

Normal fertilization after in vitro fertilization (IVF) occurs in metaphase II oocytes by the fusion of a single sperm with the oocyte membrane. A sperm which has fused with the oocyte membrane sometimes forms an ooplasmic protrusion (FC: fertilization cone) and then forms a male pronucleus (PN). Shortly after PN formation, a female PN develops beneath the 2nd polar body (PB). However, 2.7-5.6% of fertilized zygotes are reported to exhibit only one PN (1PN). Diploid 1PN formation has been considered to be the result of the fusion of paternal and maternal PNs. Likewise, the combining of the female and male genomes when sperm entry was in proximity to the spindle has been deemed a probable cause of diploid 1PN formation. In this study we aim to explore the mechanisms of 1PN formations, which include both maternal and paternal genomes, by focusing on cytoplasmic wave/flare (CW), the FC and the position of the 2nd PB extrusion. Zygotes in which both the 2nd PB extrusions and the FCs, and /or initial CWs were observed by time-lapse system (iBIS, Astec, Japan) from January to July 2020, were examined. Time-lapse data for 24 1PN and 453 2PN zygotes from 599 patients was compared. The distance between the position of the 2nd PB extrusion and the FC and/or starting position of the CW was measured. The 1PN formation rate after IVF, the time from the initiation of FC protrusion to its depression, and the time from FC protrusion to the initiation of CW were measured. The threshold for the distance between the 2nd PB extrusion and the FC /starting position was calculated by ROC curve analysis. This study has been approved by the Institutional Review Board (IRB) of Hanabusa Women’s Clinic (approval number: 2020-11) and Okayama University (approval number: K2101-009). The 1PN formation rate after IVF was 4.3% (145/3337) of the fertilized oocytes. The average time from the beginning of protrusion to the depression of the FC was 100 (±37) minutes. The CW occurred within 15-30 minutes after the depression of the FC. The cut-off value for the difference in distance between the 2nd PB extrusion and the FC, or the starting position of the CW, was 18.0μm (AUC: 0.972, 95%CI: 0.955-0.988). Consequently, we calculated that the theoretical risk of 1PN formation which included both female and male genomes was approximately 2.7%. In this study, it was found with a high degree of accuracy that 1PNs are formed when a fusion of the sperm with the oocyte membrane occurs within 18 μm from the point of the 2nd PB extrusion. The theoretical chance of 1PN occurrence after IVF was calculated to be 2.7%, in cases where the sperm is considered to have fused with oocyte, having entered the oocyte from any point. As in humans both the fusion of sperm with the oocyte membrane and the entry of sperm into an oocyte might occur anywhere on the surface of the oocyte, the logical conclusion is that babies derived from 1PNs during natural conception may result in live birth.