Early rescue ICSI is effective for TFF and near-TFF. It can provide more transplantable embryos and get a normal neonatal outcome for the TFF cycles(2, 3, 6, 15, 16). However, the early rescue ICSI must be combined with early cumulus cells removal. And previous studies only compared the MPN rate and other outcomes of the early cumulus cells removal (4–6 h post-insemination) with those of the late cumulus cells removal (18–20 h post-insemination)(1, 11, 15). They did not compare the MPN rate among the different timings of the early cumulus cells removal, such as the timings of 4, 5 and 6 h post-insemination. Therefore, when all the removal timings belong to the early cumulus cells removal, it is still unknown whether the early removal timing has a significant effect on the MPN. Another study showed that the different timings of early rescue ICSI (< 6 h, 6–8 h, and > 8 h post-insemination) have significant effects on the outcomes of rescue ICSI (3). Although the different timings of early rescue ICSI usually be combined with the different timings of early cumulus cells removal, the previous study still did not analyzed whether the timing of early cumulus cells removal have a significant effect on the MPN. Therefore, our study tried to answer this question.
In our study, after adjustment for the other variables, we found that the timing of early cumulus cells removal was associated with the MPN. And time ≤ 4 group had the highest rate of the cycles with MPN > 0%, 5.5 < time ≤ 6 group had the lowest rate of the cycles with MPN > 0% (Fig. 1). In cohort study, MPN rate also had a significant difference among the six timing groups, the highest and lowest mean of MPN rate were also the time ≤ 4 group and 5.5 < time ≤ 6 group, respectively (Table IV). These results were consist with the two previous studies (1, 11) and could explain the conflicting results of the two studies. Even if both the two studies were early cumulus cells removal, the timing of early cumulus cells removal still had a significant effect on MPN. In the Hong paper(1), the authors had a speculation about that why the studies of Zhang Wei and Sun Ying-pu (cumulus cells were removed at 2–4 h of insemination) had a higher MPN rate than their study (cumulus cells were removed at 6 h of insemination). They speculated that it is due to the different timings of early cumulus cells removal. And our results confirmed the speculation.
Interestingly, in our study, the 2PN rate was not significantly different among the different timings of early cumulus cells removal. And previous studies(1, 11) showed that the 2PN rate was also not significantly different between the early and late cumulus cells removal groups. It seems that the timing of cumulus cells removal does not have a significant effect on the 2PN. In addition, compared with the two previous studies, our study had a further analysis on the 0PN with cleavage rate and 0PN without cleavage rate, and found that 0PN without cleavage rate had a significant difference among the six timing groups. The 0PN without cleavage oocytes are usually the fertilization failure oocytes.
According to the result of 2PN rate, we speculated that the oocytes which can be normally fertilized did not affected by the timing of cumulus cells removal. The affected oocytes were the poor quality ones. These poor quality oocytes manifested MPN when the cumulus cells were removed at time ≤ 4 h post-insemination and manifested fertilization failure when the cumulus cells were removed at 5.5 < time ≤ 6 h post-insemination.
In the Kotil study(17), the authors evaluated fine structural morphology and cytoskeletal features of 3PN oocytes after ICSI(17). They found that the 3PN oocytes have the features of poor oocyte quality, such as lipofuscin granules, lack of cytoplasmic halo, multilamellar body formation, diminished cytoskeletal fine filaments, disrupted γ-tubulin accumulation and degenerated mitochondria(17–19). These results confirmed our speculation that most of the MPN oocytes in the time ≤ 4 group are the poor quality oocytes. And only the poor quality oocytes are easy to be affected by the early cumulus cells removal.
There are three major reasons for the formation of MPN. 1) Polyspermy. 2) Failure of the second polar body (PBII) extrusion. 3) Abnormal pronucleus formation(17, 20). In the Hong study(1), the authors speculated that the high MPN rate is due to the damage on oocytes, when the cumulus cells are completely removed at 2 ~ 4 h post-insemination. They speculated that the early cumulus cells removal maybe damages the zona penucida, and makes the zona penucida lost the ability to prevent polyspermy. However, in our study, the oocytes were washed and transferred into a new medium without sperm immediately after early cumulus cells removal. Therefore, even though the zona penucida was damaged, no a new sperm could enter into the oocytes. Thus, the main cause of the highest MPN rate in time ≤ 4 groups is not the polyspermy.
In the Coticchio study(21), the authors showed that the timing of PBII extrusion is 3.3 ± 1.1 h post-insemination. In the Kotil study(17), their results indicated that MPN oocytes are the poor quality oocytes with diminished cytoskeletal fine filaments and disrupted γ-tubulin accumulation. According to these studies, we speculated that one main cause of the highest MPN rate in time ≤ 4 group is the failure of PBII extrusion. Because the timing of cumulus cells removal was exactly the timing of PBII extrusion in time ≤ 4 groups, and the mechanical operation of cumulus cells removal would damage the cytoskeleton organization of the poor quality oocytes. These would result in the failure of PBII extrusion. Note that the damage generally only happened to the poor quality oocytes, it was the cause that why the time ≤ 4 group had a normal 2PN rate.
In addition, the damage of the cytoskeleton organization also affects the pronucleus formation. And assembly error of pronucleus can result in MPN too(20). Therefore, abnormal pronucleus formation is another main cause of the highest MPN rate in the time ≤ 4 group. The Mutia study showed that 33.3% of 3PN embryos had no chromosomal abnormalities(22). The formation cause of these 3PN embryos may be the abnormal pronucleus formation.
Coticchio et al(21) also showed that the female and male PN appearance are 6.2 ± 1.4 and 6.3 ± 1.4 h post-insemination, respectively. Therefore, in 5.5 < time ≤ 6 group, the mechanical operation of cumulus cells removal would interfere the pronucleus formation. It might be the reason why this group had the highest 0PN without cleavage rate. As well as the time ≤ 4 group, most of the affected oocytes were the poor quality oocytes, because this gourp also had a normal 2PN rate.
In binary logistic regression, after adjustment for the other variables, number of oocytes retrieval, gonadotrophins per oocyte, and MII rate also had significant effects on the MPN. Our results showed that the more oocytes are retrieved in a cycle, the more chance of MPN > 0% in the cycle. It is due to the number of poor quality oocytes often increases with the number of retrievable oocytes. And the poor quality oocytes are easy to be fertilization failure or fertilization abnormality. The previous research had a similar result(23), it showed that the fertilization rate decreases with the number of oocytes retrieval(23). Although our study showed that the MPN > 0% group had a higher MII rate than the MPN = 0% group, it was unclear whether the MII oocytes had a mature cytoplasmic in the MPN > 0% group. Only when the cytoplasmic maturation is concomitant with nuclear maturation can the oocyte achieve its full fertilization and developmental potential(24, 25). And the cytoplasmic maturation is regulated by microenvironmental factors, such as gonadotrophin and growth factors, in the follicles(26). However, in our study, gonadotrophin dose per oocyte was lower in the MPN > 0% group. The lower gonadotrophin dose per oocyte might not be enough to support the cytoplasmic maturation of all oocytes. Therefore, some of these MII oocytes did not complete the cytoplasmic maturation and were easy to be MPN in the MPN > 0% groups. In a word, these results showed that if there are too many retrievable oocytes in a cycle, the gonadotrophin dose per oocyte will be low. Even though the MII rate is high in the cycle, some of these MII oocytes do not complete the cytoplasmic maturation and are poor quality. These oocytes can be fertilized, but they often are MPN. These results confirmed our speculation that the affected oocytes by the timing of cumulus cells removal are the poor quality oocytes. In addition, a high dose of gonadotrophins can affect the oocyte quality too(27). However, there was no significant difference in the total dose of gonadotrophins between MPN = 0% and MPN > 0% group (33.25 ± 13.41 vs 33.53 ± 10.74, ampoule, Mann-Whitney Test, P = 0.713).
The highest grade 1–2 embryo rate at day 3 in time ≤ 4 group was due to the shortest co-incubation of gametes. A high concentration of sperm and the corresponding metabolic products have a negative effect on embryo quality(28, 29). Our result was in consistent with the previous studies(28, 29).
There were some important limitations in this study due to it was a retrospective study. First, some confounding factors might be missed due to the data could not be collected. Second, we decided the removal timing according to workloads and the number of embryologists on duty. And the cycles could not be randomly assigned to the six removal timing groups. It might lead to a population selection bias. Therefore, a randomized controlled trial should be done to confirm our results. Third, we speculated that one main cause of the highest MPN rate in time ≤ 4 group is the failure of PBII extrusion. However, we could not collect the data of PBII extrusion to verify the speculation. It was due to the data were not recorded in the clinical work. In addition, some good quality embryos were selected to transfer or freeze at day 3 in some cycles, but in the other cycles, all embryos were cultured to blastocyst stage. Therefore, the result of blastocyst formation rate also had a bias. It needed to be confirmed in a targeted research.