The timing of the release of the first polar body predicts the cleavage rate after parthenogenetic activation for human oocytes obtained by in vitro maturation

Abstract

Human parthenogenetic blastocysts (HPB) are important materials for making parthenogenetic human embryonic stem cells (pHESC) and so on. Though, the efficient method for making HPB considering the progress of time-lapse imaging (TLI) during in vitro maturation (IVM) has not been established. The objectives were to clarify the behavior of oocytes obtained by IVM during parthenogenetic activtion (PA) and to characterize the features of TLI of cleaved embryos by focusing on the timing of the first polar body (fPB) releases during IVM. Basic research study. 55 immature oocytes were collected from resected ovaries of the 5 patients with endometrioid adenocarcinoma. These oocytes were assigned to either early or late groups based on the results of 24 hour IVM. Oocytes that released fPB within 24 hours were defined as the early group, and underwent PA. On the other hand, oocytes that did not release fPB after 24 hour IVM were subjected to an additional 24 hours of IVM, and the oocytes that subsequently released the fPB were defined as the late group and underwent PA. PA was carried out using calcium ionophore and 6-dimethylaminopurine, and subsequently oocytes were cultured in single step medium with observation by TLI. Based on the data from TLI of PA, the duration of pronucleus (PN) formation and oocytes cleavage rate were analyzed. Oct4 and Cdx2 of cleavage embryos were detected by immunofluorescence staining. Oocytes were collected from five patients with a mean age of 36.0 ± 4.0 years, and IVM was performed on 50 oocytes. 15 oocytes were assigned to the early group and 13 to the late group. The rate of the oocytes which released fPB was 56.0%. The duration of PN formation was significantly longer in the early group (60.2 ± 13.0 hours) than in the late group (23.9 ± 8.4 hours) (p=0.045). The over all cleavage rate was 39.3%, and the results was summarized in table 1. The rate of cleavage to 8 or more cells were significantly higher in the late group than in the early group. The overall rate of cleavage to morula was 7.1%. Revers cleavage was observed in 54.5% (early group 80.0%, late group 15.4%) of all cleaved embryos ,and these did not all cleaved to morula. Oct4 was detected in the HPB in late group.Tabled 1Early group (n=15)Late group (n=13)p value≥ 2cell33.3% (n=5)46.2% (n=6)p=0.488≥ 8cell0%30.8% (n=4)p=0.020≥ morula0%15.4% (n=2)p=0.206 Open table in a new tab The timing of release of fPB within IVM may related to the results of PA. This finding is an important point in creating an efficient pHESC, which we have clarified for the first time. It is probable that the time difference between nuclear maturation and cytoplasmic maturation is related, though the impact of the duration of PN formation is unkown at this time, and should be investigated in the future.