Elective single morula transfer achieved better clinical outcomes than two cleavage embryos transfer in fresh IVF-ET cycles.

A total of 577 cycles from women who met the inclusion criteria were included in the study. These were further subdivided into two matched groups (Day 3 double embryo transfer group and Day 4 single morula embryo transfer group) based on the PSM method. This was done in order to achieve unbiased baseline characteristics (Fig.  2 ). In the entire cohort ( n  = 577), after adjusting for maternal age, cause of infertility, baseline AMH, endometrial thickness, estradiol on trigger day, and number of oocytes, multivariate adjusted Poisson regression analyses similarly demonstrated that the difference in clinical pregnancy rates between the two groups was not statistically significant. The statistical analysis revealed that there was no significant difference in the pregnancy rates between the Day3 double embryo transfer and the Day4 single morula embryo transfer (OR = 0.77; 95% CI = 0.49–1.19; P  = 0.237). The relationship remained stable when the propensity score was adjusted in a single step (OR = 0.78; 95% CI = 0.51–1.20, P  = 0.257). Furthermore, IPTW was weighed in univariate modified Poisson regression analyses, yielding comparable ORs. The OR remained consistent for the weighted SMRW, PA, and OW models (Fig.  2 ). Fig. 2 Forest plot shows CRPs in eSMT group using a variety of models. Notes: Each group adjusted for these factors: maternal age, maternal BMI, fertility method, male age, number of retrieved oocytes Forest plot shows CRPs in eSMT group using a variety of models. Notes: Each group adjusted for these factors: maternal age, maternal BMI, fertility method, male age, number of retrieved oocytes Before propensity score matching (PSM), the baseline characteristics of the two groups were unevenly distributed (Table  1 ). The eSMT group had higher baseline AMH levels and higher antral follicle counts compared to the DCT group (AMH:3.45 versus 3.16, P  < 0.01; antral follicle counts: 12.64 ± 5.78 vs. 11.09 ± 5.18, P  < 0.01). On the trigger day, the eSMT group also had higher estradiol levels and more oocytes retrieved compared to the DCT group (estradiol on trigger day: 10,497.21 vs. 8,838.42, P  < 0.001; retrieved oocytes: 11.00 vs. 8.00, P   0.05). Table 1 Women’s demographic and treatment cycle characteristics in different groups Characteristic Before PSM ( N  = 577) After PSM ( N  = 312) DCT group ( N = 380) eSMT group ( N  = 197) P DCT group ( N  = 156) eSMT group ( N  = 156) P Maternal age(yr) a 29.81 ± 3.33 29.83 ± 3.09 0.924 29.70 ± 3.31 29.73 ± 2.99 0.929 Paternal age (yr) a 31.55 ± 4.18 31.24 ± 3.60 0.379 31.63 ± 4.59 30.99 ± 3.27 0.152 Fertilization method n (%) b 0.711 0.142 IVF 306 (80.53) 163 (82.74) 128 (82.05) 128 (82.05) ICSI 41 (10.79) 17 (8.63) 22 (14.10) 15 (9.62) IVF + ICSI 33 (8.68) 17 (8.63) 6 (3.85) 13 (8.33) Maternal BMI (kg/m 2 ) a 22.32 ± 3.28 21.92 ± 3.21 0.160 22.65 ± 3.44 21.93 ± 3.25 0.059 Infertility type n (%) b 0.281 0.733 Primary 178 (46.84) 83 (42.13) 70 (44.87) 73 (46.79) Secondary 202 (53.16) 114 (57.87) 86 (55.13) 83 (53.21) Infertility cause n (%) b 0.629 0.974 Male 37 (9.74) 26 (13.2) 19 (12.18) 19 (12.18) Ovulation dysfunction 56 (14.74) 25 (12.69) 22 (14.1) 20 (12.82) Tubal factor 182 (47.89) 98 (49.75) 80 (51.28) 77 (49.36) Mixed 21 (5.53) 7 (3.55) 6 (3.85) 7 (4.49) Unexplained 61 (16.05) 32 (16.24) 23 (14.74) 24 (15.38) Endometriosis 23 (6.05) 9 (4.57) 6 (3.85) 9 (5.77) Infertility duration (yr) c 3.00 (2.00, 4.00) 3.00 (2.00, 4.00) 0.333 3.00 (2.00, 4.00) 3.00 (2.00, 4.00) 0.407 Basal FSH(U/L) c 6.71 (5.60, 7.76) 6.42 (5.67, 7.40) 0.112 6.68 (5.43, 7.74) 6.38 (5.54, 7.36) 0.244 Basal LH (mIU/ml) c 4.58 (3.35, 6.36) 4.86 (3.47, 6.76) 0.087 4.75 (3.50, 6.61) 5.07 (3.44, 6.83) 0.458 Basal E 2 (pg/ml) c 149.43 (111.09, 194.35) 153.55 (116.00, 207.40) 0.272 139.70 (108.20, 182.64) 154.40 (116.90, 204.72) 0.051 Basal P(ng/ml) c 0.39 (0.27, 0.60) 0.37 (0.23, 0.58) 0.203 0.37 (0.23, 0.60) 0.31 (0.18, 0.55) 0.046 Endometrial thickness (mm) a 11.28 ± 1.64 11.06 ± 1.69 0.152 11.03 ± 1.64 11.12 ± 1.72 0.614 Basal AMH (ng/ml) c 3.16 (2.24, 4.68) 3.45 (2.58, 5.21) 0.014 3.96 (2.53, 5.50) 3.48 (2.58, 5.34) 0.749 Antral follicle counts 11.09 ± 5.18 12.64 ± 5.78 0.001 11.92 ± 5.21 12.94 ± 6.00 0.110 Estradiol on the day of trigger (pg/ml) c 8838.42 (6360.25, 11,010.00) 10,497.21 (7703.03, 11,010.00)  < 0.001 10,122.4 (7590.7, 11,010.0) 9924.6 (7422.1, 11,010.0) 0.387 Progesterone on the day of trigger (ng/ml) c 0.75 (0.51, 1.02) 0.79 (0.57, 1.02) 0.638 0.83 (0.58, 1.10) 0.78 (0.57, 0.98) 0.072 Number of retrieved oocytes 8.00 (6.00, 11.00) 11.00 (8.00, 14.00)  < 0.001 10.00 (8.00, 13.00) 11.00 (8.00, 13.00) 0.608 DCT group- Day 3 double cleavage-stage embryo transfer group;eSMT group-Day 4 single morula transfer group PSM Propensity score matching, BMI Body mass index, IVF In vitro fertilization, ICSI Intracytoplasmic sperm injection, FSH Follicle-stimulating hormone, LH Luteinizing hormone, P Progesterone, E 2 Estradiol, AMH Anti-Mullerian hormone Data are presented as a: Mean ± SD and b: Number (%) c: Median (IQR) a: Analysis using T-test b: Analysis using Chi-square test c: Analysis using Kruskal–Wallis test Women’s demographic and treatment cycle characteristics in different groups DCT group- Day 3 double cleavage-stage embryo transfer group;eSMT group-Day 4 single morula transfer group PSM Propensity score matching, BMI Body mass index, IVF In vitro fertilization, ICSI Intracytoplasmic sperm injection, FSH Follicle-stimulating hormone, LH Luteinizing hormone, P Progesterone, E 2 Estradiol, AMH Anti-Mullerian hormone Data are presented as a: Mean ± SD and b: Number (%) c: Median (IQR) a: Analysis using T-test b: Analysis using Chi-square test c: Analysis using Kruskal–Wallis test Table 2 compares clinical and neonatal outcomes between groups. Before propensity score matching, the eSMT group demonstrated significantly reduced rates of twin pregnancy, preterm birth, and cesarean section versus the DCT group ( P < 0.01), alongside a higher single live birth rate ( P < 0.05). After matching, eSMT maintained significantly lower twin pregnancy and preterm birth rates ( P < 0.001), with a concurrently higher single live birth rate ( P 0.05). Neither before nor after matching were significant differences observed in clinical pregnancy rate, abortion rate, or live birth rates ( P >0.05). Table 2 Clinical outcomes and Birth outcomes of each group Outcomes n(%) Before PSM(N = 577) After PSM( N  = 312) DCT group ( N  = 380) eSMT group( N  = 197) P DCT group ( N  = 156) eSMT group ( N  = 156) P Clinical pregnancy rate 279 (73.42) 139 (70.56) 0.466 117 (75.00) 108 (69.23) 0.256 Clinical pregnancy rate of twins 102 (36.56) 1 (0.72)  < 0.001 38 (32.48) 0 (0)  < 0.001 Abortion rate 35 (12.54) 22 (15.83) 0.357 19 (16.24) 16 (14.81) 0.768 Early abortion rate 20 (7.17) 17 (12.23) 0.086 12 (10.26) 13 (12.04) 0.671 Late abortion rate 14 (5.02) 5 (3.6) 0.511 6 (5.13) 3 (2.78) 0.502 Preterm birth rate 43 (18.14) 11 (9.4) 0.031 20 (21.51) 9 (9.78) 0.028 Live birth rate 237 (62.37) 117 (59.39) 0.486 93 (59.62) 92 (58.97) 0.908 Single live birth rate 167 (43.95) 116 (58.88)  < 0.001 68 (43.59) 92 (58.97) 0.007 Cesarean section rate 141 (59.49) 55 (47.01) 0.026 48 (51.61) 38 (41.3) 0.16 DCT group- Day 3 double cleavage-stage embryo transfer group; eSMT group-Day 4 single morula transfer group Data are presented as Number (%); PSM: propensity score Matching; DCT group- Day 3 double cleavage-stage embryo transfer group; eSMT group-Day 4 single morula transfer group Clinical outcomes and Birth outcomes of each group DCT group- Day 3 double cleavage-stage embryo transfer group; eSMT group-Day 4 single morula transfer group Data are presented as Number (%); PSM: propensity score Matching; DCT group- Day 3 double cleavage-stage embryo transfer group; eSMT group-Day 4 single morula transfer group Table 3 presents the results of multivariable regression analysis. After adjusting for covariates including maternal age, infertility etiology, baseline AMH (bAMH), endometrial thickness, estradiol levels on trigger day, and the number of oocytes retrieved, the results showed that the eSMT group had a significantly lower preterm birth rate compared to the DCT group ( P  = 0.05), and a significantly higher singleton live rate ( P   0.05). Table 3 Multivariable logistic regression analyses of SMT and clinical outcomes Outcomes n (%) Total ( n  = 312) DCT group ( n  = 156) eSMT group ( n  = 156) Crude Model Adjusted Model OR (95% CI) P OR (95% CI) P Clinical pregnancy rate 225(72.11) 117 (75.00) 108 (69.20) 0.75 (0.46 ~ 1.23) 0.257 0.78 (0.47 ~ 1.30) 0.334 Abortion rate 35(11.21) 19 (12.17) 16 (10.30) 0.9 (0.44 ~ 1.85) 0.768 0.9 (0.41 ~ 2.01) 0.804 Preterm birth rate 29(9.29) 20 (12.82) 9 (5.77) 0.4 (0.17 ~ 0.92) 0.032 0.39 (0.16 ~ 1.00) 0.05 Live birth rate 185(59.29) 93 (59.61) 92 (58.98) 0.97 (0.62 ~ 1.53) 0.908 1.00(0.62 ~ 1.60) 0.993 Single live birth rate 160(51.28) 68 (43.59) 92 (58.98) 1.86 (1.19 ~ 2.92) 0.007 1.91 (1.19 ~ 3.06) 0.007 Cesarean section rate 86(46.48) 48 (51.61) 38 (41.30) 0.66 (0.37 ~ 1.18) 0.161 0.53 (0.28 ~ 1.01) 0.054 Crude model: no other covariates were adjusted Adjusted model: we adjusted maternal age, infertility cause, basic AMH, endometrial thickness, estradiol on the day of trigger, number of retrieved oocytes Multivariable logistic regression analyses of SMT and clinical outcomes Crude model: no other covariates were adjusted Adjusted model: we adjusted maternal age, infertility cause, basic AMH, endometrial thickness, estradiol on the day of trigger, number of retrieved oocytes The results of the subgroup analysis are presented in Figs.  3 and 4 , showing clinical outcomes for different subgroups. The analysis revealed no significant differences between the two embryo transfer strategies in terms of clinical pregnancy rate and live birth rate ( P  > 0.05). Moreover, the results remained stable across different subgroups, indicating a high degree of stability in the analysis. Fig. 3 Forest plot shows CRPs of each group in subgroup analyses after PSM. Notes: Each group adjusted for these factors: maternal age, maternal BMI, fertility method, male age, number of retrieved oocytes Fig. 4 Forest plot shows LBRs of each group in subgroup analyses after PSM. Notes: Each group adjusted for these factors: maternal age, maternal BMI, fertility method, male age, number of retrieved oocytes Forest plot shows CRPs of each group in subgroup analyses after PSM. Notes: Each group adjusted for these factors: maternal age, maternal BMI, fertility method, male age, number of retrieved oocytes Forest plot shows LBRs of each group in subgroup analyses after PSM. Notes: Each group adjusted for these factors: maternal age, maternal BMI, fertility method, male age, number of retrieved oocytes

This retrospective study analyzed clinical data from patients undergoing fresh Day 3 or Day 4 embryo transfers following IVF/ICSI-ET at the Department of Reproductive Medicine Department of the Affiliated Ganzhou Hospital of Nanchang University between January 2021 and December 2022.Cycles deviating from the protocol were excluded, including those using mild stimulation or progesterone primed ovarian stimulation (PPOS), transfers involving non-2PN-derived embryos, and cases with abnormal ovarian reserve, uterine pathology, or impaired endometrial receptivity (see flowchart). After propensity score Matching, 156 Day 3 double cleavage-stage embryo transfer cycles and 156 Day 4 single morula transfer cycles were included. All patients provided written informed consent for their respective protocols. The scoring system for morula was based on the Istanbul Consensus Update. The study complied with STROBE guidelines and received ethical approval from the hospital’s ethics committee (Reference No. TY-ZKY2023-051–01). This retrospective study compared clinical outcomes between two embryo transfer strategies in sub fertile women aged ≤ 35 years undergoing IVF-ET.We excluded cycles that deviated from the study protocol, including those using mild stimulation or Progestin Primed Ovarian Stimulation (PPOS) protocols, cycles involving the transfer of embryos derived from non-2PN origins, and cases with abnormal ovarian reserve or uterine conditions (see flowchart for details Fig.  1 ). After propensity score Matching, a total of 156 Day 3 double cleavage-stage embryo transfer cycles (DCT group) and 156 Day 4 single morula transfer cycles (eSMT group) were included. All patients provided written informed consent for either single morula transfer or double cleavage-stage embryo transfer. The scoring system for morula was based on the criteria outlined by Tao et al. The research adhered to the guidelines outlined in the STROBE Statement. This study was conducted at the Ganzhou Hospital, affiliated with Southern Hospital of Jiangxi Province, from January 2021 to December 2022, and received ethical approval from the hospital’s ethics committee (Reference No. TY-ZKY2023-051–01). Fig. 1 Flow-chart of the study population, enrollment, allocation and analysis Flow-chart of the study population, enrollment, allocation and analysis In this study, all participants underwent controlled ovarian hyperstimulation (COH) using a long-acting gonadotropin-releasing hormone agonist (GnRH agonist) protocol in the early follicular phase. Transvaginal ultrasound was performed on days 2–4 of the menstrual cycle to exclude ovarian functional cysts, and pituitary downregulation was initiated with a 3.75 mg intramuscular injection of GnRH-a (leuprorelin acetate; Livzon Group, China). Confirmation of pituitary suppression required the fulfillment of all the following criteria: follicle-stimulating hormone (FSH) < 5 IU/L, luteinizing hormone (LH) < 5 IU/L, serum estradiol  5 mm in diameter bilaterally, and endometrial thickness < 5 mm. After confirming downregulation, ovarian stimulation was initiated with daily doses of 75–300 IU of gonadotropins (Gn; Gonal-f®, Merck, Germany or Elisheba®, Livzon Pharmaceutical, China), with dose adjustments individualized based on patient age, body mass index (BMI), ovarian reserve markers, follicular growth kinetics, and serial hormone levels. When ultrasound revealed ≥ 3 follicles with a diameter ≥ 18 mm, ovulation was triggered with 250 μg of recombinant human chorionic gonadotropin (Ovidrel®; Merck, Switzerland). The peak serum estradiol level was recorded on the day of hCG injection, and oocyte retrieval was performed under transvaginal ultrasound guidance 36 h after hCG administration. Oocyte retrieval was performed 36–38 h after hCG injection, followed by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). For IVF, oocytes were fertilized 3–4 h after retrieval; for ICSI, cumulus cells were removed 2 h post-retrieval, and fertilization followed the standard protocol. Fertilization was confirmed by the presence of two pronuclei (2PN) 16–18 h later. On Day 3 of post-retrieval, physicians provided patients with a brief update on embryo development. Day 4 single morula transfer was recommended to patients with more than two high-quality cleavage-stage embryos or contraindications to multiple pregnancies. Patients were given the option to choose between Day 4 single morula transfer and Day 3 double cleavage-stage embryo transfer based on their personal preference. High-quality cleavage-stage embryos were defined as 2PN-derived embryos observed 72 h post-retrieval, with 7–9 cells of uniform size, less than 10% fragmentation, and no abnormalities such as vacuoles or multinucleation. According to the Istanbul Consensus Update, the morula stage denotes a day 4 post-fertilization embryo defined by cohesive cellular aggregation resulting from complete (fully compacted morula, FCM) or partial (partially compacted morula, PCM) blastomere compaction [ 8 ]. All embryo cultures followed the user guidelines for the G-series culture media (Vitrolife,Sweden) provided by Relieve Company, Sweden. Following transfer, progesterone oil (60 mg) was administered via intramuscular injection daily until Day 12, with serum hCG levels monitored (produced by Zhe Jiang Xian Ju Pharmaceutical Co., Ltd.). Pregnancy was confirmed by transvaginal ultrasound one week after hCG elevation. Clinical pregnancy was defined by the detection of a gestational sac 14 days post-transfer via ultrasound. Singleton and twin pregnancies were defined by the presence of one or two gestational sacs, respectively. Abortions were categorized as early (before 12 weeks) or late (after 12 weeks). Gestational age was calculated by adding 19 days to the embryo transfer date. Preterm birth was defined as delivery before 37 weeks of gestation. Live birth rate was defined as the proportion of Live births occurring after 24 weeks of gestation per embryo transfer (ET) cycle. The R statistical software ( http://www.R-project.org , The R Foundation) and Free Statistics software version 2.0 were used to conduct analyses. Due to the non-randomized study design, propensity score matching (PSM) analysis was performed to evaluate covariates. Multivariate logistic regression was used to estimate the PSM. Maternal age, cause of infertility, baseline AMH, endometrial thickness, estradiol on trigger day and number of oocytes retrieved were considered as potential confounders. This study used a 1:1 ratio scheme for PS matching without replacement (greedy-matching algorithm) with a caliper width of 0.2 standard deviations. Balance of covariates was judged by standardized differences. Here, balance is considered satisfactory when the standardized difference is less than 10%. Patient characteristics, as well as clinical and neonatal outcomes, were analyzed as continuous variables using one-way analysis of variance (ANOVA) for statistical comparisons. Depending on the incidence of various outcomes, chi-square tests, Yates’ continuity correction, or Fisher’s exact test were applied as appropriate.

This study demonstrates that, in infertile women under 35 years old, elective single morula transfer (eSMT) on Day 4 achieves clinical pregnancy and live birth rates comparable to those of double cleavage-stage embryo transfer (eDCT) on Day 3, while significantly reducing the risks of twin pregnancy and preterm birth. While these preliminary findings suggest clinical value, we emphasize that the level of evidence remains constrained compared to RCT-derived data. Furthermore, eSMT provides an alternative treatment strategy for patients concerned about blastocyst culture failure. The reduced multiple gestation risk must be weighed against center-specific embryology capabilities and patient preferences in shared decision-making. Thus, within the parameters of this study, eSMT emerges as a potentially safer and more effective technique that may offer personalized embryo transfer options for younger patients, while generating preliminary insights that could inform future ART optimization strategies.

Elective single morula transfer (eSMT) is becoming a clinical trend in assisted reproductive technology (ART) owing to its enhanced implantation potential and developmental competence. Despite this, patient reluctance persists due to concerns regarding cycle cancellation if blastulation fails, or perceptions that dual cleavage-stage embryo transfer improves pregnancy success. Our findings demonstrate comparable clinical pregnancy rates (CPR), abortion rates (AR), and live birth rates (LBR) between eSMT and double cleavage-stage transfer (DCT) in women  0.05). This indicates that transferring a single high-potential morula maintains pregnancy rates while substantially mitigating multiple pregnancy risks. Previous studies have demonstrated comparable clinical pregnancy and live birth rates between elective single morula transfer (eSMT) and elective single blastocyst transfer (eSBT). A retrospective cohort study reported clinical pregnancy rates of 59.63% for single morula transfer (D4 A group) and 64.73% for single blastocyst transfer (D5 A group), with no statistically significant difference ( P  = 0.284) [ 9 ]. Importantly, Rijnders et al. [ 10 ] found that approximately 59.2% of high-quality cleavage-stage embryos developed to the morula stage by Day 4, while 47% reached the blastocyst stage. Notably, the cycle cancellation rate for morula-stage transfer is significantly lower than for blastocyst transfer, highlighting the developmental advantage of morula-stage embryos. Additionally, morula-stage embryos tend to exhibit better symmetric division and a lower incidence of chromosomal abnormalities providing an important physiological basis for the application of eSMT [ 11 ]. The morula stage marks a crucial transition for embryos as they move from the fallopian tube to the uterine cavity, carrying significant physiological implications for embryonic development. Research indicates that morula-stage embryo transfer serves as a natural selection point, favoring embryos with higher developmental potential. Moreover, recent studies utilizing time-lapse imaging have identified abnormal compaction patterns at this stage as being potentially linked to chromosomal abnormalities, further establishing this stage as a key physiological checkpoint in embryonic development [ 12 ]. From the perspective of natural conception, embryos typically pass through the morula stage during their transit to the uterine cavity, highlighting the relevance of this developmental phase for successful embryonic development. In our study, propensity score matching (PSM) and multivariable regression analyses revealed that the clinical twin pregnancy rate (TCPR) and preterm birth rate in the eSMT group were significantly lower than those in the DCT group ( P  < 0.05). Crucially, the significantly lower preterm birth rate observed in our eSMT group is primarily attributable to the drastic reduction in twin pregnancies resulting from the single embryo transfer strategy inherent to eSMT, compared to DCT. In a meta-analysis [ 13 ] pooled data from 45 studies show a 95% reduction in multiple pregnancy rate (MPR) for SET (OR = 0.05, 95% CI:0.04–0.06, P  < 0.001), while 13 studies demonstrate a 75% lower preterm birth rate in SET (OR = 0.25, 0.21–0.30, P  < 0.001). Sensitivity analyses using adjusted ORs (0.06, 0.02–0.16, P  < 0.001 for MPR) and propensity score-matched models further validate these findings, aligning with the conclusion that SET minimizes twin pregnancies and preterm deliveries. The potential mechanisms underlying the advantages of eSMT over DCT May be attributed to the following aspects. First, Day 4 morula aligns with the natural endometrial receptivity window, as human embryos normally enter the uterus on Day 4 post-fertilization, thereby reducing uterine contractility and optimizing implantation conditions. Second, compaction from Day 3 cleavage -stage embryo to Day 4 morula provides natural screening by eliminating developmentally abnormal or aneuploid embryos. This is supported by higher implantation rates in fully compacted morula(46.4%) vs. Day 3 embryos (21.4%) [ 13 , 14 ]. Based on those underlying mechanisms, eSMT may provide comparable or even better clinical outcomes than those of DCT. Through subgroup stratification by patient characteristics (age, infertility type, endometrial thickness, baseline anti-Müllerian hormone (AMH) levels, and oocyte yield), comparable clinical pregnancy and live birth rates were observed between eSMT and DCT groups. Notably, eSMT was associated with a significant reduction in twin pregnancy and preterm birth risks. These findings indicate that eSMT represents not only an appropriate strategy for younger patients but also an individualized, safe, and effective transfer option across diverse reproductive profiles. This aligns with reports that individual factors—including age, ovarian reserve, and uterine receptivity—significantly influence ART outcomes [ 15 – 18 ]. For instance, younger patients typically demonstrate superior embryonic developmental competence, and eSMT serves as a versatile transfer approach that optimizes treatment based on specific patient parameters. Furthermore, while eSMT may also be a viable option for older patients or those with a poor ovarian response, further research is needed to validate its efficacy in these populations. Single morula transfer (SMT) strategy has been increasingly applied due to its unique advantages, and its safety has been evidenced by many clinical studies and animal models. On the one hand, our research findings demonstrate that elective SMT (eSMT) maintains a comparable abortion rate to double cleavage-stage transfer (DCT) (10.30% vs. 12.17%, P  = 0.768), confirming no increased risk of pregnancy loss. Furthermore, eSMT significantly improves the single live birth rate (58.98% vs. 43.59%, P  = 0.007) and trends toward lower cesarean section rates (41.30% vs. 51.61%, P  = 0.054), suggesting potential maternal safety benefits. These results highlight that eSMT not only preserves pregnancy viability but also enhances reproductive outcomes by reducing multifetal pregnancy risks. On the other hand, supporting evidence from Ryh-Sheng Li’s study further validates the safety of day 4 morula transfer, showing no significant differences in implantation (36.3% vs. 39.6%, P  = 0.500), clinical pregnancy (49.5% vs. 51.9%, P  = 0.737), or live birth rates (42.1% vs. 45.6%, P  = 0.574) compared to day 5 blastocyst transfer. Notably, the term birth rate was even higher in the morula group (95.7% vs. 79.5%, P  = 0.006), reinforcing its safety and efficacy [ 19 ]. Beyond clinical studies, the biological feasibility of morula transfer is underscored by its extensive and successful use in animal models, particularly in livestock breeding. Decades of safe implementation in this field provide empirical support for the stability and reliability of morula-stage embryo manipulation [ 20 ]. The parallels between animal models and clinical applications suggest that SMT is not only a scientifically sound approach but also one with a proven safety record across species. Propensity score matching was employed to balance baseline characteristics between the single embryo transfer group and the double cleavage-stage embryo transfer group. However, medical advice-driven selection of eSMT for patients with multiple pregnancy contraindications may have introduced systematic differences in clinical profiles and outcomes, potentially affecting the comparative assessment of both strategies. Although matching controlled for key confounders, results interpretation merits caution.Furthermore, our follow-up concluded at live birth, precluding assessment of neonatal outcomes such as congenital anomalies (e.g., chromosomal abnormalities, structural malformations) or developmental milestones (e.g.,Apgar scores, neurodevelopmental indices at one year of age). This limitation mirrors the Majority of ART studies conducted prior to 2022, as standardized protocols for longitudinal child follow-up in reproductive research remain logistically challenging to implement.Additionally, with the advancement of embryo culture technologies—such as time-lapse imaging and non-invasive embryo assessment tools—methods for evaluating morula development potential and selecting the optimal embryo for transfer may further enhance the safety and efficacy of eSMT [ 21 , 22 ]. Notably, future studies could explore long-term comparative outcomes between eSMT and eSBT across different patient populations, including maternal and neonatal health outcomes and intergenerational effects. Moreover, integrating artificial intelligence (AI)-assisted embryo assessment systems may further refine embryo selection accuracy, thereby optimizing the clinical application of eSMT [ 23 , 24 ].

Assisted reproductive technology (ART) remains the most effective intervention for infertility; However, multiple pregnancies present significant clinical challenge in reproductive medicine. These pregnancies are associated with elevated maternal risks, such as gestational hypertension, and adverse neonatal outcomes including low birth weight and preterm birth [ 1 – 3 ]. The dual objectives of achieving high pregnancy rates while minimizing the incidence of multiple pregnancies continue to challenge healthcare providers and patients alike. To address this challenge, the selective single embryo transfer (SET) strategy has garnered increasing adoption, particularly through Day 5 elective single blastocyst transfer (eSBT), which enhances embryo selection precision and implantation rates [ 4 ]. Extended culture to Day 5 facilitates more accurate assessment of embryonic developmental potential and chromosomal integrity, consequently improving clinical outcomes relative to Day 3 cleavage-stage embryo transfer [ 5 ]. Nevertheless, challenges persist, including the risk of culture failure and the additional resources required for prolonged culture [ 6 ]. In this context, Day 4 elective single morula transfer (eSMT) represents as a viable alternative. The morula stage represents a more advanced developmental phase than the cleavage-stage characterized by embryonic genome activation and substantial morphological remodeling. This advancement suggests potential advantages in embryo viability, as morula have consistently demonstrated higher survival rates post-cryopreservation compared to their cleavage-stage and blastocyst counterparts [ 7 ]. Furthermore, Day 4 morula transfer offers distinct practical utility in clinical settings where Day 3 or Day 5 transfers are precluded, including during non-routine laboratory hours or scheduling limitations. Despite the promising prospects of eSMT, there remains a paucity of real-world data directly comparing the clinical outcomes of Day 4 single morula transfer with those of Day 3 cleavage-stage embryo transfer. Addressing this gap is crucial, as previous studies suggest that eSMT outcomes May be comparable to those of Day 5 SBT, yet further investigation is warranted to substantiate these findings. Therefore, our center undertook a retrospective study aimed at evaluating the clinical pregnancy outcomes of Day 4 selective single morula transfer relative to Day 3 cleavage-stage embryo transfer, thereby providing valuable insights for clinical practice.

Supplementary Material 1. Supplementary Material 1.