With the development of blastocyst culture technology, elective blastocyst culture and single blastocyst transfer have become an important way of improving the pregnancy rate and reducing the multiple pregnancy rate. However, there is no consensus regarding the inclusion criteria for blastocyst culture, especially about the effects of the insemination method on the outcome of elective blastocyst culture.

Intracytoplasmic sperm injection (ICSI), which is used mostly for severe male factor infertility and after fertilization failure in previous conventional in vitro fertilization (IVF) treatment, has been widely used. However, the ICSI procedure lacks the natural selection of sperm and bypasses the process in which spermatozoa penetrate the cumulus oophores and zona pellucida. This is unlike conventional IVF. Therefore, some concerns regarding embryo quality and pregnancy outcomes have been raised. Some research has demonstrated that the quality of embryos deriving from ICSI is inferior to the quality of those deriving from conventional IVF [1,2,3]. However, other studies have found that embryo quality was comparable between these insemination methods [4,5], or even superior in ICSI [6]. Therefore, it remains unclear whether ICSI affects embryo quality. It is well-known that the outcome of blastocyst culture is closely related to the quality of the embryos at the cleavage stage. Therefore, it is unclear whether with the same inclusion criteria for blastocyst culture, the culture results would be different between ICSI and conventional IVF. For this reason, the present study assessed the effects of the insemination method on the outcomes of elective blastocyst culture by retrospectively analyzing elective blastocyst culture data from January 2011 to December 2014.

A total of 2,003 conventional IVF and 336 ICSI cycles were included in this study. Table 1 summarizes the outcomes of our study. The mean age of female patients, the basal FSH level, the basal luteinizing hormone level, and average body mass index were similar between these groups.

In the conventional IVF group, 25,652 of the 35,521 oocytes were fertilized. The mean fertilization rate among retrieved oocytes was 72.22%. In the ICSI group, 4,770 of the 5,845 oocytes were metaphase II and were injected with sperm; of these, 4,164 oocytes were fertilized. On the basis of the number of oocytes, there was no significant difference in the fertilization rate between the conventional IVF and ICSI groups (72.22% vs. 71.24%, p>0.05). Overall, 25,652 embryos in the conventional IVF group and 4,164 embryos in the ICSI group progressed to blastocysts, and the good-embryo rate on day 3 was similar (IVF, 59.57%; ICSI, 58.36%, p>0.05), but the blastocyst formation rate was significantly different. In the conventional IVF group, 14,032 blastocysts were formed, including 13,106 usable blastocysts; in contrast, there were 2,121 blastocysts, including 1,984 usable blastocysts, in the ICSI group. Therefore, the blastocyst formation rate among fertilized oocytes in the conventional IVF group was significantly higher than in the ICSI group (54.70% vs. 50.94%, p<0.05) and the embryo utilization rate was also better in the conventional IVF group (51.09% vs. 47.65%, p<0.05).

Regarding clinical parameters, no differences were found in the clinical pregnancy rate (IVF, 50.93%; ICSI, 55.10%), the miscarriage rate (IVF, 12.57%; ICSI, 16.29%), the ectopic pregnancy rate (IVF, 4.19%; ICSI, 3.70%), or the live birth rate (IVF, 42.12%; ICSI, 44.08%) (p>0.05) (Table 2).

As is known, the ultimate goal of assisted reproductive technology is for the patient to take one term healthy baby home per cycle [8]. Therefore, single-embryo transfer was proposed. Furthermore, elective blastocyst culture and single blastocyst transfer have been demonstrated as the best way to achieve this goal [9,10,11]. However, blastocyst culture is not suitable for all patients, so choosing patients appropriately is critical. Nonetheless, there are no uniform inclusion criteria for blastocyst culture. The parameters of the inclusion criteria have included the number of oocytes retrieved, the number of good embryos on day 3, and the patients' age [12]. No direct analysis has been conducted on the effect of the insemination method.

Therefore, the main purpose of this study was to compare the effect of different fertilization methods on the outcomes of elective blastocyst culture. No significant differences were found between conventional IVF and ICSI cycles in the mean age, basal levels of FSH or luteinizing hormone, body mass index, the number of oocytes, or the fertilization rate. All included cycles progressed to the blastocyst stage, and the good-embryo rate on day 3 was similar. Therefore, the main influencing factor in this study was the insemination method. Our data demonstrated that blastocyst formation in vitro was affected by the insemination method. The blastocyst formation rate in the ICSI group was significantly lower than in the conventional IVF group. This finding also confirmed the results that have been reported by others [1,3,13]. Griffiths et al. [3] used sibling oocytes with the same semen sample for conventional IVF or ICSI, and demonstrated that the ICSI procedure contributed to a reduced capacity for blastocyst formation. Other studies using surplus embryos [1,13,14], which were not able to control for potential confounders such as surplus embryo quality, also showed that blastocyst formation was compromised by ICSI. However, some other studies did not support the conclusion that ICSI decreased the blastocyst formation rate [5,15]. This study, to our knowledge, was the first to only include cycles in which elective blastocyst culture was performed.

The different blastocyst formation rate in conventional IVF and ICSI is mainly related to the different binding mechanism of the egg and sperm. During conventional IVF fertilization, natural selection of sperm takes place, just like in natural fertilization, in which the sperm must penetrate the oocyte-corona-cumulus complex, zona pellucida, and plasmalemma, and only then can the sperm and oocyte be combined. However, in the ICSI procedure, the operation bypasses such selection, and so-called ‘ forced fertilization’ can be achieved using spermatozoa that might never have attained natural fertilization or with genetic defects. In theory, embryonic development is influenced by the quality of the sperm and egg. The first three cleavage divisions are mainly regulated by maternal genes, and then at the 4-to 8-cell stage of development, the embryonic genome is activated and the paternal genome starts to play a role [16]. Many studies have demonstrated that spermatozoa from patients with severe male infertility had an adverse effect on blastocyst formation [17]. This may be related to the fact that spermatozoa from infertile men have higher rates of defects such as DNA damage and aneuploidy. Fluorescent in situ hybridization results have shown that chromosomal aneuploidy was related to the presence of sperm with abnormal characteristics, such as round-headed sperm and large-headed sperm [18]. Therefore, the embryo quality on day 3 may be associated with oocyte-related factors, while genetically abnormal sperm may show a negative influence on blastulation. Our study also demonstrated that the good-embryo rates on day 3 in the conventional IVF and ICSI groups were similar, while the blastocyst formation rate in the ICSI group was significantly worse. This may have occurred because the condition of the female patients included in these groups was similar, but the semen parameters in the ICSI group were poorer. Inconsistent conclusions have been made about the effect of sperm on early embryo development. Some researchers have shown that severe teratozoospermia had no effect on blastocyst formation [19,20].

In addition, the ICSI operation itself may be detrimental to the oocyte, and furthermore to the development of the embryo. A study by Dumoulin et al. [21] indicated that the volume of aspirated cytoplasm, type of membrane breakage, and skill in performing ICSI had an effect on blastocyst formation. Meanwhile, manipulation of the degranulated cell and the injection of polyvinylpyrrolidone and culture medium into the oocyte may also influence the subsequent development of the embryo. Furthermore, the injection needle may injure the spindle, which may cause errors in the first cleavage division, then lead to aneuploidy. A paper showed that ICSI affected embryonic development and gene expression in cattle [22]. However, another animal model reported that ICSI did not perturb embryonic development [23].

Our study compared the clinical outcomes between conventional IVF and ICSI groups, and showed that there were no differences in the clinical pregnancy/implantation rate, miscarriage rate, or live birth rate. These findings suggest that the subsequent developmental potential of blastocysts derived from ICSI did not appear to be affected. These results are also supported by those of other studies [14,24]. It may be beneficial to delay culture to day 5 or 6, such that some chromosomally abnormal embryos fail to undergo blastulation in culture.

In conclusion, blastocyst formation ability was significantly affected by ICSI. Therefore, in order to avoid cases of cycle cancellation due to the formation of no blastocysts, it can be suggested that more care should be taken in choosing blastocyst culture for ICSI patients.