In vitro fertilization (IVF) is widely used to treat infertile couples. Compared with intracytoplasmic sperm injection (ICSI), the combination of oocytes and sperm in IVF more closely mimics the natural human physiological state [1, 2]. However, even with normal semen parameters, approximately 20% of IVF cycles result in a low fertilization rate (25%), and 5% to 15% of cycles experience complete fertilization failure [3, 4]. Such failures place considerable pressure on both doctors and patients. To avoid fertilization failure, some centers perform ICSI on all or part of the oocytes, which leads to the over-application of ICSI [5]. Compared with conventional IVF, the use of ICSI does not improve the transplantation or live birth rates in non-male factor infertility couples [1]. Given the risks of oocyte damage and the potential transmission of genetic defects in male infertility to the next generation, IVF remains the first option for treating non-severe male infertility. A viable option for addressing fertilization failure after IVF is early rescue ICSI [6]. It was provided to those oocytes with unclear release of the second polar body 6 h after initial insemination. The short co-incubation of gametes combined with early rescue ICSI can alleviate the stress of fertilization failure and effectively limit the overuse of ICSI [7]. However, whether short co-incubation of gametes should be used in all IVF cycles remains unclear.

There is still no consensus on the optimal duration for oocyte-sperm co-incubation during IVF. Some studies suggest that short co-incubation times (1–6 hours) improve IVF outcomes compared to longer co-incubation times (16–24 hours) [8, 9]. Conversely, other studies have found no such advantage [10, 11]. A previous study indicated that prolonged exposure of oocytes to sperm might harm early embryonic development [12]. Barraud-Lange et al. [13] found that while short co-incubation of gametes reduced the fertilization rate compared to the standard overnight method, embryo quality remained comparable. Fan et al. [9] meta-analysis revealed that reduced gamete co-incubation time benefits clinical pregnancy and implantation rates compared to overnight IVF, with no significant differences in fertilization rates or embryo quality. Our study aimed to investigate these findings further and provide a reference for clinical practice.

Conventional IVF is a widely used treatment for female infertility factors, unexplained infertility, and some male infertility factors. Total fertilization failure (TFF) remains challenging for clinicians and embryologists [17]. Short-term fertilization is a preferable method to avoid TFF, leading to the widespread use of short co-incubation of gametes combined with early rescue ICSI. It has been suggested that early removal of granulosa cells is beneficial for embryo quality [18], but some studies have shown conflicting results [10, 11]. Our study found a trend toward a higher polyspermy rate in the S group (8.71%) compared to the L group (7.96%), although this difference was not statistically significant. The top cleavage embryo rate was significantly higher in the S group, which can be attributed to the younger mean age and better ovarian function compared to the L group. In partial short fertilization, there was no discernible difference in the top cleavage embryo rate between the two groups. The rates of top-quality blastocyst, clinical pregnancy and implantation rates showed no significant differences between the two groups (p $>$ 0.05). These findings are similar to other reports [11, 19].

Gianaroli et al. [12] indicated that sperm enter cumulus cells within 15 minutes, interact with them within 1 hour, and reach the oocyte cortex 4 hours later. These findings suggest that prolonged co-incubation of oocytes and sperm may not be necessary. Additionally, some researchers have found that prolonged co-incubation time may lead to high levels of reactive oxygen species (ROS) [5], which can negatively affect gamete interactions and embryo quality. However, Kong et al. [20] suggested that removing the cumulus-oocyte complex (COC) too early may interfere with the crucial communication between oocytes and the COC. While short co-incubation can decrease ROS levels, it may not provide sufficient time for oocyte maturation compared to longer incubation [20, 21]. We found that the normal fertilization rate (2PN) was slightly lower in the S group compared to the L group, with statistical significance (p 0.01). Additionally, a self-matched control analysis of 79 partial short-duration patients showed no difference in 2PN rates between embryos in the short and long gamete co-incubation segments (p $>$ 0.05). The analysis results are inconsistent. The populations of S Group and L Group are different, which affects inferences when analyzing the results of these 2 populations. The number of oocytes retrieved is considered a key factor in ovarian responsiveness to exogenous gonadotropin, and the number of oocytes retrieved for IVF treatment is related to clinical outcomes [17, 19]. According to the 2017 Vienna Consensus and the “Expert Consensus on Quality Control of Key Indicators of the Embryo Laboratory” the ideal normal fertilization rate of 2PN is 75% [22]. We stratified the number of oocytes obtained and conducted a regression analysis to further evaluate the association between co-incubation time and 2PN. The analysis demonstrated that co-incubation time did not affect normal insemination rate after stratifying by the number of oocytes obtained controlling for factors such as sterility type, female age, AMH levels, MⅡ oocytes, and stimulation protocol.

There are some limitations to our study. Due to the nature of retrospective study, it is possible that important confounder was not taken into account in the present study. It is a single-center study with a relatively small sample size. Cycles with three or fewer eggs, overnight fertilization was used to avoid, and fresh embryo transfer is not performed in these cycles in our center, which may potentially introduce bias. The live birth rates were not included in the results. Our plan is to accumulate more cases to further verify the conclusions.

Short-term insemination does not affect the fertilization rate and embryo development [3]. Furthermore, short co-incubation may require embryologists to work at night, resulting in longer working hours. Therefore, it is unnecessary to choose short fertilization in all IVF cycles. Clinicians should identify patients at risk for low or complete fertilization failure, and the fertilization method should be based on the history of infertility, the experience of clinicians and embryology laboratory personnel, and the patient’s medical history. The inclusion of additional procedures during fertilization evaluation may pose a potential risk of adverse effects on the fertilized egg [23]. Therefore, the method of selecting only some fertilized eggs, rather than all fertilized eggs for polar body observation helps to reduce the risk. Partial short insemination may be an appropriate method to reduce IVF fertilization failure or low fertilization rates.

Short and long oocyte-sperm co-incubation times have similar in vitro developmental outcomes. It is safe to use short-term fertilization for patients who may be at risk of fertilization failure.

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

FC: investigation; formal analysis; writing—original draft; data curation. LL: investigation; formal analysis; writing—original draft. XD: formal analysis; writing—original draft. XX: formal analysis; writing—original draft. LC: conceptualization; methodology; writing—review and editing; funding acquisition. YW: conceptualization; methodology; writing—review and editing; funding acquisition; project administration. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Changzhou Maternal and Child Health Care Hospital (approval number: 2022071).

We gratefully acknowledge the assistance and instruction from Dr. Zhu of the Department of Reproductive Medicine Center.

This research was funded by the China Reproductive Public Welfare Fund “Pilotage Plan” (grant SZ202412) and the Key Project of Changzhou Clinical Medical Center (grant CMCM202203).

The authors declare no conflict of interest.