Since its advent almost 40 years ago, in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) has helped many infertile couples to become parents. Controlled ovarian stimulation (COS) is an essential step in IVF/ICSI treatment, with a subset of patients found to be sensitive to COS. These patients are known as high responders and are characterized by a total follicle count of 15 or more and/or an estradiol (E2) concentration $>$3000 pg/mL on the day of ovulation induction [1].

Many studies have shown that frozen embryo transfer (FET) is preferable for high responders since it has a higher live birth rate (LBR) and a lower incidence of ovarian hyperstimulation syndrome (OHSS) compared to fresh embryo transfer (ET) [2, 3]. However, other studies have reported that FET had no advantage over fresh ET in terms of reducing OHSS in high responders [4]. Moreover, for infertile women with polycystic ovary syndrome (PCOS), it was reported that FET was associated with a higher risk of preeclampsia after the first transfer than fresh ET [2]. Furthermore, fresh ET can result in shorter times to pregnancy and live birth than FET, thus making it more cost-effective [5]. In addition, the use of gonadotropin-releasing hormone (GnRH) antagonists is associated with a reduced incidence of OHSS and does not seem to compromise ongoing pregnancy rates [6]. Therefore, fresh ET is worth considering for some high responders undergoing GnRH antagonist cycles.

To obtain an optimal clinical pregnancy rate (CPR), two cleavage-stage embryos are usually transferred. However, this can have adverse consequences related to multiple pregnancy in women and their new-borns [7]. Evidence shows that single blastocyst transfer (SBT) may be an effective way to lower the risk of multiple pregnancy without affecting the CPR [8, 9, 10]. Nevertheless, several retrospective studies on high responders in IVF/ICSI cycles have shown that fresh SBT was inferior to fresh day-3 ET (two embryo transfer) in terms of the CPR [3, 11]. Thus, clinicians face a dilemma between the use of day-3 ET or day-5 SBT during fresh cycles, especially when multiple high-quality embryos are available for high responders. Further studies are therefore needed to provide more reliable evidence when faced with this dilemma.

To the best of our knowledge, clinical outcomes with day-3 ET or day-5 SBT for high responders stimulated with GnRH antagonists in fresh cycles have not been reported to date. In the present study, we compared the CPR, LBR, OHSS and other IVF/ICSI-associated complications between fresh day-5 SBT and fresh day-3 ET using high-quality embryos in high responders. The European Society of Human Reproduction and Embryology (ESHRE) guidelines recommend the GnRH antagonist protocol for high responders due to its better safety and equivalent efficacy [12]. This comparative analysis therefore included only patients who were stimulated with GnRH antagonists.

As shown in Table 1, no significant differences in age, body mass index (BMI), duration of infertility, primary cause of infertility, AMH, and baseline LH or E2 level were detected between the day-5 SBT and day-3 ET groups (p $>$ 0.05). The SBT group had significantly lower baseline FSH levels than the day-3 ET group (p = 0.002).

As shown in Table 2, no significant differences were observed between the two groups in terms of the FSH, LH, E2 and P levels on the hCG trigger day, the trigger rate for GnRH agonist plus hCG, the number of oocytes, or the incidence of moderate/severe OHSS (p $>$ 0.05).

Table 3 compares the outcomes between the day-5 SBT and day-3 ET groups. No significant differences were found with regard to the incidence of CPR, LBR, early pregnancy loss, ectopic pregnancy, prematurity, macrosomia, congenital anomalies, or neonatal gender. The day-5 SBT group had a significantly higher IR than the day-3 ET group, but lower incidence of multiple pregnancy and LBW.

Table 4 shows the comparison of outcomes between day-5 SBT and day-3 ET groups according to patient age. For both age groups, no significant differences were observed between day-5 SBT and day-3 ET groups with regard to CPR, LBR, early pregnancy loss, ectopic pregnancy, prematurity or macrosomia, congenital anomalies or neonatal gender. For patients aged $\le$30 years, the day-5 SBT group had a significantly lower incidence of multiple pregnancy and LBW than the day-3 ET group. For patients aged $>$30 years, the day-5 SBT group had a significantly higher IR, but lower incidence of multiple pregnancy and LBW than the day-3 ET group.

This study found that day-5 SBT and day-3 ET showed similar CPR and LBR following fresh transfer of high-quality embryos in high responders stimulated with a GnRH antagonist protocol. Moreover, day-5 SBT showed significantly higher IR and lower incidence of multiple pregnancy and LBW than day-3 ET.

Extended culture to the blastocyst stage is considered a useful tool for embryo selection, since it allows only high-quality embryos to develop into blastocysts [18]. A meta-analysis of 5 randomized clinical trials (RCTs) found that SBT was associated with a higher rate of ongoing pregnancy and LBR than single cleavage-stage transfer [19]. A recent publication involving 15 studies and 2219 women also found the LBR was higher in blastocyst transfer patients than in cleavage-stage ET patients (odds ratio (OR) 1.27, 95% confidence interval (CI) 1.05 to 1.51) following fresh ET [10]. However, in the present study with high-quality embryos we observed a trend (p = 0.057) for day-3 ET to have higher LBR than day-5 SBT. This trend was observed in both the younger and older patient groups. Moreover, the CPR with day-3 ET was reported to be significantly higher than SBT in high responders with the GnRH agonist protocol and fresh cycles [3]. A previous study reported no significant difference in the LBR between SBT and two cleavage-stage ET [20]. Because blastocysts have good developmental potential, blastocyst transfer with high-quality embryos should result in better clinical outcomes than cleavage-stage ET. However, in the present study blastocyst transfer did not result in significantly better LBR compared to cleavage-stage ET (41.55% vs. 52.86%, p = 0.057). This may be due to the fact that two embryos were transferred in the cleavage-stage ET group, and a high serum E2 level ($>$3000 pg/mL) on the hCG trigger day is thought to reduce uterine receptivity [21]. Joo et al. [22] also found the serum E2 level negatively affects the CPR and IR in a concentration-dependent manner after it exceeds a certain level during COS. COS has consistently been found to cause histological endometrial maturation and changes in endometrial gene expression, which can adversely affect embryo implantation [23]. In the present study cohort, all participants were high responders with very high levels of serum E2. Therefore, excessive serum E2 levels during COS may advance the window for implantation and thus decrease endometrial receptivity, thereby affecting the CPR, LBR and IR in fresh cycles. Blastocysts are cultured for two days longer than day-3 embryos and this may have a greater effect on endometrial receptivity for SBT following fresh transfer than for day-3 ET.

The day-5 SBT group was observed here to have a higher IR than the day-3 ET group, indicating the rate of successful implantation between the two groups was different. The IR was not significantly different when the patients were aged $\le$30 years, although this may be related to the small sample size. The high-responder subjects in the present study who underwent ET on day-3 received two high-quality embryos. However, not all of the cleavage-stage embryos transferred on day-3 would have developed into blastocysts. Blastocysts have a higher potential for implantation than cleavage-stage embryos and are likely to be more representative of the totipotency of embryo development [3, 10]. Therefore, considering the efficacy of ET, SBT is recommended for high responders in fresh ET cycles.

It has been proposed that GnRH antagonists have the advantage of reducing the risk of OHSS in women with PCOS [6]. Moreover, a GnRH agonist for oocyte triggering resulted in a lower incidence of OHSS in normal patients [24, 25]. In the present study, the number of oocytes induced by GnRH agonist plus hCG was almost identical in the day-5 SBT and day-3 ET groups. Since all patients in the current study were stimulated with the GnRH antagonist protocol, the incidence of OHSS was already rather low in high responders due to the beneficial effect of this protocol.

Numerous studies have recommended that SBT be considered first in routine practice when high-quality blastocysts are available, as this reduces the risk of multiple pregnancy and has no effect on the LBR [7, 26]. In accordance with these studies, our analysis also found the multiple pregnancy rate with day-5 SBT was markedly lower than with day-3 ET. Multiple pregnancy is considered an important risk factor for LBW and is associated with increased medical, psychological, economic and social costs [27, 28]. As expected, the incidence of LBW in the current study was found to be markedly higher in the day-3 ET group than in the day-5 SBT group. Advanced age is an important risk factor for female infertility, pregnancy loss, fetal anomalies, stillbirth, and obstetric complications [29]. Patients who have a good response to COS and undergo fresh ET are usually young [1]. The median age of patients in the day-5 SBT and day-3 ET groups in the current study was very similar at around 30 years. There was no evidence of any differences between the two groups in terms of the incidence of early pregnancy loss, ectopic pregnancy, prematurity, macrosomia, congenital anomalies, or in terms of neonatal gender. These results concur with those of previous studies [30].

This study was limited by its retrospective design and the use of data from a single-center. The inclusion and exclusion criteria were strictly obeyed in order to minimize the risk of bias in the selection of patients. In view of its limitations, this was a preliminary study to examine the outcome of two fresh ET strategies, day-5 SBT and day-3 ET, for high responders in GnRH antagonist cycles.

In conclusion, when considering the CPR, IR, LBR and various complications such as LBW and multiple pregnancy, day-5 SBT appears to be preferable over day-3 ET for high responders undergoing IVF/ICSI cycles with GnRH antagonist in fresh ET cycles with high-quality embryos. However, further RCTs are needed to determine more accurately which fresh ET strategy is best for high responders with high-quality embryos.

The raw data generated in this study are available upon reasonable request from the corresponding author.

Conceptualization: TX, YZ, JO, WX. Data curation: FY, XL, WX. Formal analysis and investigation: TX, FY, JO. Methodology: WX, FY, XL. Resources: FY, XL. Supervision: JO. Writing – original draft: WX, JO. Writing – review & editing: WX, JO, TX. All authors contributed to editorial changes in the manuscript. 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.

This study was approved by the Institutional Review Board of the Third Affiliated Hospital of Sun Yat-sen University ([2021]02-143-01). All patients in this study signed informed consent forms to undergo IVF/ICSI treatments in our center, and the anonymity of the patients has been strictly protected.

Not applicable.

This project was supported by the Medical Scientific Research Foundation of Guangdong Province of China (Grant No.: A2020575).

The authors declare no conflict of interest.