With the development of assisted reproductive technologies (ART), particularly in the domains of embryo culture and cryopreservation, the implantation rate (IR) is increasing along with the multiple pregnancy rate (MPR). Multiple pregnancies were commonly regarded as the most consequential adverse outcomes correlated with ART, as they are associated with elevated risks of maternal and neonatal morbidity. Although, the reduction in the number of transferred embryos has been posited as a critical strategy for mitigating multiple pregnancies, this strategy has not been widely used in clinical practice owing to apprehensions regarding diminished pregnancy rates [1]. Contrary to cleavage-stage embryos, extending embryo culture to the blastocyst stage allowed for better evaluation of the implantation potential, yielding a higher IR [2, 3, 4]. Previous studies have posited that elective single blastocyst transfer could yield comparable clinical pregnancy rates (CPR) for patients with a good prognosis [1, 5, 6]. Although live birth rate (LBR) equivalence was not demonstrated, it was thought the additional complications associated with multiple gestations outweighed the potentially higher LBR [5]. Additionally, frozen-thawed single blastocyst transfers have been found to result in enhanced CPR relative to fresh single blastocyst transfers in ovulatory women with a good prognosis [6]. In the context of advanced maternal age ($\ge$40 years), both single and double blastocyst transfers yielded similar CPR and LBR, while MPR was lower for single blastocyst transfers [7]. Despite burgeoning research in the domain of different embryo-stage and fresh vs frozen embryo transfers [8, 9], a comprehensive analysis examining the impact of blastocyst number, quality and maternal age on pregnancy and birth outcomes remains lacking. The present study aims to fill this gap by analyzing these variables to formulate a more effective blastocyst transfer strategy for decreasing MPR while achieving desirable pregnancy and birth outcomes.

Pertaining to the total age group under study, group G exhibited the highest IR, the lowest MPR, and analogous CPR, AR, and LBR when compared to other cohorts employing good-quality blastocysts. Likewise, group P had a higher IR, lower MPR, and similar CPR, AR and LBR in relation to group PP, which is in consonance with previous studies [11, 12, 13, 14]. In regard to double blastocyst transfer, the MPR of groups GG, GP and PP were 56.8%, 36.3% and 29.6%, respectively. Importantly, these trends across diverse age subgroups were consistent with those observed in the total age group. Consequently, the obtained results indicated that single blastocyst transfer appeared to be an efficacious strategy for minimizing MPR while achieving favorable LBR. The logistic regression analysis further accentuated that maternal age and transferred blastocysts were factors significantly correlated with LBR. Specifically, within the age group of 35, the MPRs for groups GG, GP, and PP were 67.4%, 41.0% and 34.2%, respectively. Hence, our findings implicitly advocate for the utilization of single blastocyst transfer strategies over their double blastocyst counterparts, particularly for younger patients. Notably, our analyses also revealed that increasing the number of transferred blastocysts of the same grade did not engender a substantial elevation in CPR and LBR but did significantly increase MPR. The results were similar to those of previous studies [12, 15, 16]. One limitation that merits acknowledgment is that our study classified blastocyst quality only dichotomously, potentially introducing variance within quality levels that could have nuanced implications for pregnancy outcomes.

Pregnancy is often conceptualized as a nuanced interplay, mediated by localized secretion of key factors, between a developmentally competent embryo and a receptive endometrium. Existing research posits that decidualized human endometrial stromal cells possess the ability to selectively identify developmentally impaired embryos and respond by inhibiting the secretion of key implantation mediators such as Interleukin-1$\beta$ (IL-1$\beta$) and heparin-binding EGF-like growth factor (HB-EGF), as well as immunomodulators including IL-5, -6, -10, -11, -17, and eotaxin [17]. Impaired embryos elicited an endoplasmic stress response in human decidual cells. Conversely, signals originating from developmentally competent embryos activated a focused gene network enriched in metabolic enzymes and implantation factors [18]. Moreover, high-quality embryos have been observed to incite transient, oscillatory Ca21 fluxes, whereas low-quality embryos induce an elevated and prolonged Ca21 response [18]. These dichotomous mechanisms may contribute to diminished IR among poor-quality blastocysts. This inhibitory phenomenon might be cumulative, thereby further depressing the IR when two low-quality blastocysts were transferred simultaneously. A solitary good-quality blastocyst has been noted to yield a higher IR compared to the transfer of two such blastocysts, potentially due to insufficient secretion of critical implantation factors in the latter scenario. Our observations with regard to IRs in age groups 35 and 35–39, as well as the broader patient demographic, echo existing literature, indicated that a reduced IR for GP (one good-quality and one poor-quality blastocyst) might be attributed to biochemical reactions that obstructed the implantation of the lower-quality blastocyst, consequently affecting the implantation potential of its higher-quality blastocyst [12, 14]. Moreover, our data showed credence to the hypothesis that competitive interactions might exist between co-transferred blastocysts, manifesting as compromised IR for double blastocyst transfers. Previous findings had demonstrated that IRs were high in the following order: single good embryo, double good embryos, one good embryo with a poor embryo, single bad embryo and double bad embryos [14]. Blastocyst score and proportion of top-scoring blastocyst affected implantation, and the degree of blastocoele re-expansion have been affirmed as potent predictors of live birth outcomes in warmed single blastocyst transfer cycles [10, 19], corroborating the findings of our study.

Multiple pregnancy is considered the most significant adverse event associated with ART and linked to an increased risk of maternal and neonatal morbidity. The results indicated that single blastocyst transfer had a lower MPR (2.6% vs 39.3%, p/OR/95% CI 0.01/0.04/0.03–0.05), AR (16.8% vs 19.1%, p/OR/95% CI = 0.07/0.85/0.72–1.01), cesarean section rate (60.6% vs 76.0%, p/OR/95% CI 0.01/0.49/0.41–0.57), preterm labor rate (8.4% vs 25.7%, p/OR/95% CI 0.01/0.26/0.22–0.32) and ectopic pregnancy rate (0.38% vs 1.81%, p/OR/95% CI 0.01/0.21/0.10–0.44) than two blastocyst transfer, while the singleton group had a higher average gestational age and birthweight as well as a lower birthweight rate. Importantly, LBR were observed to be statistically analogous between single and double blastocyst transfers, irrespective of blastocyst quality categorizations. Consequently, the implementation of single blastocyst transfer strategies is advocated as a potent measure for mitigating the associated morbidity risks intrinsic to multiple pregnancies. Intriguingly, our study delineated distinct health markers between monozygotic and dizygotic twins. The former had a lower gestational age and birthweight in conjunction with a higher preterm birth rate and low birthweight rate. Therefore, monozygotic twins deserved more attention in the context of ART treatments.

In vitro culture has been shown to induce precocious X-chromosome inactivation, and intracytoplasmic sperm injection (ICSI) is implicated in reducing the number of trophectoderm cells in female blastocysts [20]. Sex ratio was significantly higher toward males in the transfer of blastocyst compared to transfer of cleavage stage embryo [20, 21, 22, 23]. Our data further substantiate a significant positive correlation between sex ratio (male/female) and the proportion of good-quality blastocysts transferred. As, the practice of blastocyst culture and selective single good-quality blastocyst transfer gains scholarly endorsement [6, 12, 24], the potential for such strategies to engender imbalances in neonatal sex ratios remains an emergent area requiring further research.

Our empirical analyses confirm the efficacy of single blastocyst transfer as an optimal strategy for significantly attenuating MPR while ensuring favorable pregnancy and birth outcomes. Nonetheless, it should be noted that this strategy may engender a skewed sex ratio among the neonates. Furthermore, the distinct health metrics of monozygotic twins deserve more attention in the context of ART treatments.

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

The study was designed by YL and BM. LZ, PY, NL and YL were involved in planning and managing the data collection. YL and BM were involved in the statistical analysis and wrote the manuscript with support. Critical review was from all authors. 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.

Ethical approval was granted by the Ethics Committee of Haikou Mary Hospital on the March 8, 2020 (NO. 02/MLYY/2020). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

The authors would like to thank all participants and staff at IVF Center, Mali Hospital, Haikou, China and the Hospital of Shenzhen Immigration Inspection, Shenzhen, China.

Hainan Medical and Health Research Project (21A200331).

The authors declare no conflict of interest.