It is known that once the sperm enters the oocyte, it immediately triggers calcium oscillation, further inducing cortical granular exocytosis. Then the released cortical granule proteins induce zona pellucida reaction, blocking both the entry of other spermatozoa , and the bi-directional communication between the oocyte and the cumulus cells. In this large retrospective cohort study, we showed that, early cumulus cell removal 4 h after insemination had no detrimental effects on pregnancy and neonatal outcomes in patients undergoing IVF treatment as compared with conventional cumulus cell removal 20 h after insemination. Thus, our findings suggested that once the mature oocytes are fertilized, the surrounding cumulus cells may not be essential for the subsequent embryonic development.
In our study, no significant differences were observed in the rates of biochemical pregnancy, clinical pregnancy, ongoing pregnancy, live birth, miscarriage, ectopic pregnancy, multiple pregnancy and twin delivery between the 4 h and 20 h groups. Similarly, a small prospective randomized sibling-oocyte study involving 61 patients showed that 4 h group had no adverse influence on the biochemical pregnancy and clinical pregnancy rates when compared with 20 h group . Furthermore, another large sample retrospective study also indicated that early cumulus cell removal had no detrimental effects on clinical pregnancy, miscarriage and live birth rates when compared with conventional cumulus cell removal, in patients with high-risk of fertilization failure . Taken together, the data indicate that early cumulus cell removal may have no detrimental effects on pregnancy outcomes in fresh embryo transfer cycles.
There are always concerns about the safety, when any type of modification is introduced into ART. Therefore, focusing on neonatal outcomes is well justified. The early cumulus cell removal is an important variable in conventional ART , and its potential effects need to be considered. Increasing evidence suggests that the birthweight is related to the risk of perinatal and infant morbidity and mortality as well as future adult chronic diseases [24, 25]. Gestational age-specific birth weight is a commonly assessed perinatal outcome. Furthermore, fetal weight estimation using the customized birth weight percentiles has led to more accurate predictions of adverse perinatal outcomes . So far, studies on the neonatal outcomes of early cumulus cell removal are limited. We found that both the singleton and twin pregnancies between 4 h and 20 h groups had similar neonatal outcomes such as birth weight, gestational age at delivery and preterm birth rate. In contrast, Guo et al  showed that early cumulus cell removal had higher rates of low birth weight compared with conventional cumulus cell removal protocol. However, those results may be debated, because no distinction was made between singletons and twins, and the number of live births was small (n = 54). In the present study, we analyzed the neonatal outcomes in singleton and twin pregnancies separately, because twin pregnancies were associated with increased risk of adverse pregnancy and neonatal outcomes. We also adjusted for newborn gender and gestational age in singleton pregnancies, and Z scores in two groups were also comparable. Thus, the data suggested that the early cumulus cell removal 4 h after insemination had no detrimental effects on neonatal outcomes in fresh embryo transfer cycles.
During the conventional IVF procedure, oocytes and cumulus cells were co-incubated for 19–20 h, and then the cumulus cells were removed to observe the fertilization status . It was known that cumulus cells provide oocytes with a series of factors, which play important roles in nuclear and cytoplasmic maturation of oocytes, fertilization and development . Our data showed comparable rates of normal fertilization, high-quality embryo, blastocyst formation rate between the 4 h and the 20 h group, suggesting that normal fertilization and embryonic development are not affected by early cumulus cell removal. These results are consistent with the previous reports [11, 22].
Consistent with the previous reports [11, 27], our results showed a significantly higher polyspermy rate in 4 h group as compared to the 20 h group. The oocytes may be more vulnerable due to having active spindles and microtubules shortly after insemination. It is possible that repeated aspirations for an earlier cumulus cell removal may have some effects on the integrity of oocyte structure which reduces its defense against polyspermy . In addition, the potential temperature and pH fluctuations during the process of early cumulus cell removal and observation may also play a role. In contrast, several studies have shown that early cumulus cell removal does not increase the polyspermy rate [15, 28]. This discrepancy may be due to the different degree or time of cumulus cell removal in different studies. Nevertheless, in our study, the increased polyspermy rate with early cumulus removal did not affect the major ART outcomes. Therefore, the clinical significance of this finding may be limited.
In China, the indication and proportion of ICSI cycles per center is strictly regulated by the government . Therefore, early cumulus cell removal was applied for patients with a higher risk of fertilization failure to avoid the excessive use of ICSI technique in clinical practice. Previous studies suggested that primary infertility and longer infertility duration were important risk factors for total fertilization failure [29, 30]. The incidence of total fertilization failure was also higher in patients with unexplained infertility [31, 32]. Patients with these causes of infertility were included in the 4 h group, and early rescue ICSI was performed if necessary, in our center. This can explain that the proportion of primary infertility, duration of infertility, type of infertility and causes of infertility were significantly different between 4 h and 20 h groups in this study.
The major strength of the current study is that we showed evidence for the safety of early cumulus cell removal with a large sample size. In addition, the laboratory practices did not substantially change during the study period, which should minimize the possible confounders associated with pregnancy and neonatal outcomes. There are some limitations to this study. As a retrospective design, our data were collected from medical records, which could not provide all information on personal covariates. We minimized the limitations of this retrospective study by adjusting for the known factors related to the IVF outcomes in the multivariable analysis as independent variables. The information on congenital malformations was obtained by parental report after delivery and the data in patients with miscarriage were not available. The rate of congenital malformations was calculated using live newborns, and therefore the data did not represent all birth defects. Another limitation is the selection of patient population and different sample size between the two study groups. Therefore, prospective multicenter trials in general population are needed to eliminate the effect of confounders.