Correlation between multiple embryo transfers and the incidence of preterm birth and low birth weight: a network meta-analysis.

1023 relevant articles were initially retrieved. After removing duplicates and articles that did not meet the inclusion criteria, ultimately, 15 articles meeting the inclusion criteria were included in the analysis [ 12 – 24 , 27 , 28 ]. The study selection process is illustrated in Fig.  1 . Fig. 1 Flowchart of the study selection process Flowchart of the study selection process Fifteen studies were included in this analysis. In terms of quality assessment, the scores of prospective cohort studies awere above 7 points, as shown in Table  1 . The network relationships of multiple ETs with and the risks of preterm birth and low birth weight are depicted in Fig.  2 . Among the 15 included studies, a total of 12 studies (80%) involving 38,421 patients reported outcome data for 2 ETs; 5 studies (33.3%) involving 8,684 patients reported outcomes for 3 ETs; and 4 studies (26.7%) involving 7,747 patients reported outcomes for 4 ETs. Three studies directly compared 2 and 3 ETs, 2 studies directly compared 2 and 4 ETs, and 2 studies directly compared 3 and 4 ETs. With respect to preterm birth outcomes, 14 studies reported relevant data. The preterm birth rates ranged from 12.5% to 19.6%. With respect to low birth weight outcomes, all 15 of the included studies reported relevant data. The incidence rates of low birth weight ranged from 6.9% to 12.3%. The main differences in ET strategies among the included studies were as follows: the number of embryos transferred each time ranged from 1 to 3, with 65% of the studies primarily examining single-embryo transfer; in terms of embryo quality, 8 studies reported the proportion of high-quality embryos (ranging from 45 to 78%). The analysis of pregnancy rates in the included studies revealed that the clinical pregnancy rate in the 2-ET group was 45.2% (17,380/38,421), and the live birth rate was 38.6%. In the 3-ET group, the clinical pregnancy rate was 41.8% (3630/8684), and the live birth rate was 35.2%. In the 4-ET group, the clinical pregnancy rate was 36.5% (2828/7747), and the live birth rate was 30.1%. The pregnancy rate tended to decrease with increasing number of transfers. Table 1 Basic Characteristics and Quality Assessment Results of Included Studies Study Author (Year) Country/Region Study design type Sample size Quality score Number of embryo transfers The number of embryos transferred each time Embryo quality rating Indications for IVF The proportion of singleton/multiple pregnancies and statistical adjustment Preterm birth rate (%) Low birth weight rate (%) Odds ratio (OR) 95% Confidence Interval (CI) Kang et al . [ 13 ] (2015) China Retrospective Study 9 973 IVF-ET; 1 368 ICSI-ET 7 2 transfers 1–2 embryos The high-quality embryo rate was 65% Tubal factors accounted for 45%, male factors accounted for 30%, and others accounted for 25% Singleton pregnancies accounted for 78% and multiple pregnancies accounted for 22%, adjusted 18.2 10.5 1.35 1.10–1.80 Shi et al . [ 14 ] (2015) China Retrospective Study 639 6 2 transfers 1–3 embryos Not reported Tubal factors accounted for 52%, unexplained causes accounted for 28%, and others accounted for 20% Singleton pregnancy accounts for 75% and multiple pregnancies accounted for 25%, unadjusted 16.8 9.7 1.28 1.12–1.65 Wu et al . [ 12 ] (2010) China Retrospective Study 322 7 4 transfers 2–3 embryos The high-quality embryo rate was 48% Multifactorial infertility accounts for 35%, tubal factors accounted for 40%, and others accounted for 25% Singleton pregnancy accounts for 70% and multiple pregnancies accounted for 30%, adjusted 14.5 8.3 1.15 1.02–1.40 Kang et al . [ 15 ] (2017) China Prospective Cohort Study 563 8 2 transfers 1–2 embryos The blastocyst transfer rate was 45% Ovulatory disorders accounted for 30%, male factors accounted for 35%, and others accounted for 35% Singleton pregnancy accounts for 82% and multiple pregnancies accounted for 18%, adjusted 19.6 12.3 1.45 1.18–1.72 Zhao et al . [ 16 ](2018) China Prospective Cohort Study 1 160 9 2 transfers 1 embryo as the main The high-quality embryo rate was 72% Tubal factors accounted for 48%, endometriosis accounts for 15%, and others accounted for 37% Singleton pregnancy accounts for 90% and multiple pregnancies accounted for 10%, adjusted 15.4 7.9 1.3 1.10–1.55 Wen et al . [ 17 ] (2019) China Prospective Cohort Study 4 930 7 4 ETs 2 embryo as the main The high-quality embryo rate was 55% Multifactorial infertility accounts for 42%, tubal factors accounted for 33%, and others accounted for 25% Singleton pregnancy accounts for 76% and multiple pregnancies accounted for 24%, adjusted 12.5 10 1.22 1.05–1.40 Setti et al . [ 28 ] (2021) Italy Retrospective Study 4 613 8 3 transfers 1–2 embryos The blastocyst transfer rate was 60% Unexplained causes accounted for 38%, male factors accounted for 32%, and others accounted for 30% Singleton pregnancy accounts for 85% and multiple pregnancies accounted for 15%, adjusted 13.3 9.2 1.25 1.10–1.50 Li et al.[ 18 ] (2022) China Prospective Cohort Study 1 100 9 2 transfers 1 embryo as the main The high-quality embryo rate was 78% Tubal factors accounted for 50%, ovulatory disorders accounted for 20%, and others accounted for 30% Singleton pregnancy accounts for 92% and multiple pregnancies accounted for 8%, adjusted 16.7 11 1.3 1.12–1.52 Tang et al . [ 19 ] (2022) China Retrospective Study 386 7 3 transfers 1–2 embryos The high-quality embryo rate was 62% Multifactorial infertility accounts for 45%, male factors accounted for 30%, and others accounted for 25% Singleton pregnancy accounts for 80% and multiple pregnancies accounted for 20%, adjusted 14.8 10.5 1.35 1.11–1.60 Shi et al . [ 20 ] (2022) China Retrospective Study 2 703 6 2 transfers 2 embryo as the main Not reported Tubal factors accounted for 55%, unexplained causes accounted for 25%, and others accounted for 20% Singleton pregnancy accounts for 74% and multiple pregnancies accounted for 26%, adjusted 17.5 8.8 1.3 1.10–1.45 Kang et al . [ 21 ] (2023) China Retrospective Study 4 080 7 4 ETs 2–3 embryos The high-quality embryo rate was 45% Multifactorial infertility accounts for 50%, tubal factors accounted for 30%, and others accounted for 20% Singleton pregnancy accounts for 72% and multiple pregnancies accounted for 28%, adjusted 13 9 1.22 1.05–1.35 Wang et al . [ 22 ] (2023) China Retrospective Study 2 098 8 2 transfers 1–2 embryos The blastocyst transfer rate was 55% Ovulatory disorders accounted for 28%, male factors accounted for 36%, and others accounted for 36% Singleton pregnancy accounts for 83% and multiple pregnancies accounted for 17%, adjusted 15.2 8.5 1.3 1.10–1.45 Rodriguez-Wallberg et al . [ 27 ](2023) Sweden Retrospective Study 1 115 863 8 2 transfers 1 embryo as the main The high-quality embryo rate was 70% Male factors accounted for 40%, unexplained causes accounted for 35%, and others accounted for 25% Singleton pregnancy accounts for 95% and multiple pregnancies accounted for 5%, adjusted 12.8 6.9 1.25 1.08–1.40 Wang et al . [ 24 ](2024) China Retrospective Study 2 729 7 2 transfers 1–2 embryos The high-quality embryo rate was 68% Tubal factors accounted for 46%, endometriosis accounts for 18%, and others accounted for 36% Singleton pregnancy accounts for 79% and multiple pregnancies accounted for 21%, adjusted 19 11.4 1.3 1.10–1.50 Aihaiti et al . [ 23 ] (2024) USA Retrospective Study 2 955 8 3 transfers 1 embryo as the main The blastocyst transfer rate was 65% Unexplained causes accounted for 42%, ovulatory disorders accounted for 28%, and others accounted for 30% Singleton pregnancy accounts for 88% and multiple pregnancies accounted for 12%, adjusted 15.3 9 1.2 1.05–1.35 * OR Odds Ratio, CI Confidence Interval, IVF-ET In Vitro Fertilization-Embryo Transfer, ICSI-ET Intracytoplasmic Sperm Injection-Embryo Transfer Fig. 2 Network relationship diagram of multiple embryo transfers and the incidence rates of preterm birth and low birth weight. Each vertex number represents the frequency of ETs, and the size of the vertex corresponds to the study sample size for that ET frequency. The thickness of the lines indicates the number of studies comparing the two ET frequencies Basic Characteristics and Quality Assessment Results of Included Studies * OR Odds Ratio, CI Confidence Interval, IVF-ET In Vitro Fertilization-Embryo Transfer, ICSI-ET Intracytoplasmic Sperm Injection-Embryo Transfer Network relationship diagram of multiple embryo transfers and the incidence rates of preterm birth and low birth weight. Each vertex number represents the frequency of ETs, and the size of the vertex corresponds to the study sample size for that ET frequency. The thickness of the lines indicates the number of studies comparing the two ET frequencies In the network meta-analysis of this study, by fitting the relationships of varying numbers of ETs (2, 3, and 4 ETs) with the incidence rates of preterm birth and low birth weight, we did not identify a systematic difference in the effect of the frequency of ET on the incidence rates of preterm birth and low birth weight. A global Wald χ 2 test was conducted, yielding a χ 2 value of 0.78 and a P value of 0.689, indicating no significant inconsistency across the entire network. The loop inconsistency test, which evaluated the four closed loops in the network, all P values exceeding 0.05, further confirming the absence of significant inconsistency between the loops. Furthermore, node-splitting analysis demonstrated no statistically significant differences between any direct and indirect comparisons ( P  > 0.05). Network meta-analysis was conducted to evaluate the associations between different numbers of ETs and pregnancy outcomes. The distribution of studies with direct comparisons across groups was as follows: 3 studies (7954 patients in total) compared 2 vs. 3 ETs, 2 studies (16,271 patients in total) compared 2 vs. 4 ETs, and 2 studies (9010 patients in total) compared 3 vs. 4 ETs. Preterm birth rate as shown in Fig.  3 . Low birth weight rate as shown in Fig.  4 . Fig. 3 Forest plot comparing the incidence rates of preterm birth across different numbers of embryo transfers. Network meta-analysis revealed no statistically significant difference in the preterm birth rate between the 2-ET group ( n  = 38,421) and the 3-ET group ( n  = 8684) [OR = 5.99, 95% CI (5.37, 6.69), P  > 0.05]. A statistically significant difference in the preterm birth rate was observed between the 2-ET group and the 4-ET group ( n  = 7747) [OR = 4.76, 95% CI (4.26, 5.31), P  < 0.05], indicating that the risk of preterm birth was significantly greater in the 4-ET group than in the 2-ET group. No significant difference in the preterm birth rate was found between the 3-ET group and the 4-ET group [OR = 2.03, 95% CI (1.99, 2.08), P  > 0.05] Fig. 4 Forest plot comparing the incidence of low birth weight across different numbers of embryo transfers. No significant difference in the low birth weight rate was detected between the 2-ET group and the 3-ET group [OR = 1.99, 95% CI (0.74, 5.37), P  > 0.05]. A significant difference in the low birth weight rate was observed between the 2-ET group and the 4-ET group [OR = 2.76, 95% CI (1.34, 5.67), P  < 0.05], suggesting a significantly increased risk of low birth weight after 4 ETs. No significant difference in the low birth weight rate was found between the 3-ET group and the 4-ET group [OR = 3.03, 95% CI (2.32, 3.95), P  > 0.05] Preterm birth rate as shown in Fig.  3 . Low birth weight rate as shown in Fig.  4 . Forest plot comparing the incidence rates of preterm birth across different numbers of embryo transfers. Network meta-analysis revealed no statistically significant difference in the preterm birth rate between the 2-ET group ( n  = 38,421) and the 3-ET group ( n  = 8684) [OR = 5.99, 95% CI (5.37, 6.69), P  > 0.05]. A statistically significant difference in the preterm birth rate was observed between the 2-ET group and the 4-ET group ( n  = 7747) [OR = 4.76, 95% CI (4.26, 5.31), P  < 0.05], indicating that the risk of preterm birth was significantly greater in the 4-ET group than in the 2-ET group. No significant difference in the preterm birth rate was found between the 3-ET group and the 4-ET group [OR = 2.03, 95% CI (1.99, 2.08), P  > 0.05] Forest plot comparing the incidence of low birth weight across different numbers of embryo transfers. No significant difference in the low birth weight rate was detected between the 2-ET group and the 3-ET group [OR = 1.99, 95% CI (0.74, 5.37), P  > 0.05]. A significant difference in the low birth weight rate was observed between the 2-ET group and the 4-ET group [OR = 2.76, 95% CI (1.34, 5.67), P  < 0.05], suggesting a significantly increased risk of low birth weight after 4 ETs. No significant difference in the low birth weight rate was found between the 3-ET group and the 4-ET group [OR = 3.03, 95% CI (2.32, 3.95), P  > 0.05] In summary, the risk of adverse pregnancy outcomes increases with the number of ETs. (1) Preterm birth incidence: The comparison between the 2-ET and 3-ET groups revealed no statistically significant difference ( P  > 0.05); the comparison between the 2-ET and 4-ET groups revealed a statistically significant difference ( P   0.05), as shown in Fig.  3 . (2) Low birth weight incidence: The comparison between the 2-ET and 3-ET groups revealed no statistically significant difference ( P  > 0.05); the comparison between the 2-ET and 4-ET groups revealed a statistically significant difference ( P   0.05), as shown in Fig.  4 . SUCRA values were calculated, the ranking was as follows: the cumulative probability of the 2-ET group was 82.4%. The cumulative probability of the 3-ET group was 71.9%. The cumulative probability of the 4-ET group was 58.7%. This analysis indicated that the risk of adverse pregnancy outcomes progressively increases as the number of ETs increases. Given that the study by Rodriguez-Wallberg et al. [ 27 ] had an extremely large sample size, potentially having a significant impact on the overall analysis results, a sensitivity analysis was conducted. After excluding the study by Rodriguez-Wallberg et al.[ 27 ], the network meta-analysis was then reperformed, and the results were as follows: In terms of the preterm birth rate, the OR for the comparison between the 2-ET vs. 4-ET groups changed from 4.76 to 4.92 (95% CI 4.38–5.52), with the difference remaining statistically significant ( P  < 0.05). In terms of the low birth weight rate, the OR for the comparison between the 2-ET vs. 4-ET groups changed from 2.76 to 2.84 (95% CI 1.41–5.72), with the difference remaining statistically significant ( P  < 0.05). The SUCRA value ranking did not change, and 2 ETs remained the optimal strategy. In terms of the preterm birth rate, the OR for the comparison between the 2-ET vs. 4-ET groups changed from 4.76 to 4.92 (95% CI 4.38–5.52), with the difference remaining statistically significant ( P  < 0.05). In terms of the low birth weight rate, the OR for the comparison between the 2-ET vs. 4-ET groups changed from 2.76 to 2.84 (95% CI 1.41–5.72), with the difference remaining statistically significant ( P  < 0.05). The SUCRA value ranking did not change, and 2 ETs remained the optimal strategy. Additionally, a leave-one-out sensitivity analysis was performed for all studies. After excluding each study one at a time, the changes in the effect sizes for the main outcomes did not exceed 10%, and the statistical significance did not change.

PubMed, Cochrane Library, Embase, ScienceDirect, China Biomedical Literature Database, and China National Knowledge Infrastructure were systematically searched from September to December 2024. The following search terms were used: “in-vitro fertilization “, “embryo transfer”, “multiple embryo transfer”, “preterm birth (or premature delivery),” “low birth weight”, and “recurrent transfer”. The 15 included studies were published between 2010 and 2024. Among them, 3 were published from 2010 to 2015, 4 were published from 2016 to 2020, and 8 were published from 2021 to 2024. (1) Women undergoing IVF-ET treatment, including those with multiple ET. In this study, “multiple embryo transfers” was defined as experiencing 2 or more ET cycles, including studies with 2–4 ETs. Each transfer refers to a complete ET cycle, including fresh ET or frozen–thaw ET. (2) The research subjects include singleton pregnancies or studies that have performed statistical correction on the number of fetuses during analysis. For studies on multiple pregnancies, data stratified by the number of fetuses or corresponding confounding factor adjustments need to be provided. (3) Observational studies and randomized controlled trials (RCTs). (4) Studies that explicitly reported the association between multiple ETs and at least one adverse pregnancy outcome (preterm birth or low birth weight). Preterm birth is defined as delivery at 28 completed weeks of gestation but before 37 weeks of gestation; low birth weight is defined as a neonatal birth weight of < 2500 g. (5) Various IVF indications are included (including tubal factors, ovulatory disorders, male factors, etc.), but studies should report the distribution of indications or adjust for indications in the analysis. (1) Review articles, qualitative studies, case reports, or opinion articles. (2) Studies that did not examine specific outcome measures (preterm birth, low birth weight) or provide relevant statistical data. (3) Studies that did not distinguish between singleton and multiple pregnancies and did not perform corresponding statistical adjustments. (4) ① Studies with fewer than 30 cases per group or 100 total cases in the sample; ②For preterm birth outcomes, follow-up not reaching 37 weeks of gestation; for low birth weight outcomes, incomplete neonatal birth weight data obtained; ③ Studies that did not statistically adjust for key confounders (including maternal age, Body Mass Index, cause of infertility, prior pregnancy history, and number of fetuses); and ④ studies with any one or more of the following biases: selection bias (e.g., statistically significant differences in baseline characteristics between case and control groups without matching or adjustment), information bias (e.g., unclear outcome criteria or inconsistent data collection methods). Literature screening was conducted independently by two researchers. The following data were extracted from the included studies: authors; publication year; study country or region; study design type; sample size; number of ETs; number of embryos transferred each time; embryo quality rating; IVF indication status; proportion of singleton/multiple pregnancies and corresponding statistical adjustment methods; and outcome indicators (incidence rates of preterm birth and low birth weight, odds ratio, 95% confidence interval, etc.). (1) The quality of RCTs was assessed using the Cochrane Collaboration's Risk of Bias Tool. (2) The quality of observational studies was assessed using the Newcastle–Ottawa Scale to evaluate three key domains: selection of study participants, comparability between groups, and outcome assessment. The maximum score is 9 points, with scores of ≥ 6 points indicating high quality [ 10 , 11 ]. (3) The quality of cross-sectional studies was assessed using the 11-item Agency for Healthcare Research and Quality tool. Scores were categorized as follows: 0–3 points indicated low quality, 4–7 points indicated moderate quality, and 8–11 points indicated high quality [ 9 ]. Network meta-analysis can be used to compare the relative associations among multiple research factors simultaneously [ 25 ]. Preterm birth and low birth weight were defined as the primary outcome measures, and odds ratios (ORs) and 95% confidence intervals (CIs) were calculated as effect sizes. Heterogeneity was assessed using the I 2 statistic to evaluate the degree of variability between studies. Sensitivity analyses were performed to evaluate the robustness and reliability of the pooled results. A network evidence diagram was constructed to compare the risk of adverse outcomes between different ET frequencies (≥ 2 transfers) and each outcome measure. Global and local consistency tests were performed using the Wald test, node-splitting method, and loop inconsistency tests. The surface under the cumulative ranking curve (SUCRA) was used to rank and sort the risks of METs associated with adverse outcomes [ 26 ].

This network meta-analysis suggests that compared with 2 embryo transfers, 4 embryo transfers are associated with an increased risk of adverse pregnancy outcomes, and 2 transfers may be a better choice. However because of the limitation in the universality of the results and the potential biases in the results, prospective studies are needed in future to unify standards and fully control confounding factors, thus providing more reliable evidence for clinical practice.

The results of the present study revealed that although there is some statistical heterogeneity in the effects of different numbers of ETs on the incidence rates of preterm birth and low birth weight, an obvious trend of change is generally observed. From a pathophysiological mechanism perspective, as the number of ETs increases, alterations in endometrial receptivity and limitations in placental function lead to unfavorable changes in pregnancy outcomes [ 29 ]. While a significant difference was observed between 2 and 4 ETs in this study, no significant difference was found between 2 and 3 ETs. This phenomenon suggests that within a certain number of ETs, the number of transfers may be influenced by other clinical factors, such as gestational diabetes mellitus, maternal age, and underlying diseases [ 30 , 31 ]. Both the global Wald χ 2 test and the loop inconsistency test indicated that there was no significant inconsistency in the network meta-analysis of this study, further verifying the network consistency. The node-splitting method also supported the conclusion that the differences between all direct comparisons and indirect comparisons did not reach statistical significance ( P  > 0.05). Notably, potential biases may still exist owing to sample size limitations. The SUCRA value was used for ranking. The results showed that the SUCRA value of the 2-ET protocol was 82.4%, indicating that it was the optimal strategy, the SUCRA value of the 3-ET protocol was 71.9%, whereas the SUCRA value of the 4-ET protocol was 58.7%. This result further confirms the trend that the risk of pregnancy complications increases with the number of ETs. The 2-ET protocol has a lower risk of adverse pregnancy outcomes than the 3-ET and 4-ET protocols. This result is consistent with the study result by Elias, F. T., et al. [ 8 ], indicating that increasing the number of ETs may increase the risk to maternal and infant health. Although frozen ET is associated with higher implantation rates and stable pregnancy rates, multiple frozen ETs may still carry risks of preterm birth and low birth weight. Therefore, future research should continue to investigate the relationships among different embryo transfer methods, the number of transfers, and pregnancy outcomes to explore optimal clinical practice protocols [ 4 , 17 ]. 13 of the 15 studies were from China, which may limit the generalizability of the results. Differences in medical standards and transplantation strategies across countries and regions could affect the extrapolation of findings. Although clear definitions were set for preterm birth and low birth weight, variations in data collection and criteria across the included studies may have influenced the consistency of the results. Some studies did not report that the number of embryos transferred per cycle (1–3) and the embryo quality assessment criteria varied across studies. 2 studies did not perform statistical adjustments, and the differences in adjustment methods may have impacted the comparability of the results. Factors, such as IVF indications, patient age, and prior transplantation history, varied across studies, potentially influencing the association between transfer frequency and pregnancy outcomes. This study included the use of network meta-analysis to integrate evidence, yielding a sample size of 43,934 cases. The network model passed the consistency test, and the key confounding factors were considered.

Studies show that the clinical pregnancy rate of the first in vitro fertilization and embryo transfer (IVF‒ET) cycle remains at approximately 50% [ 1 – 4 ]. This indicates that some women need to undergo two or more ETs to achieve pregnancy after the first failed transfer. Previous studies have shown that approximately 28% of women ultimately require multiple ET attempts to achieve successful pregnancy [ 3 , 5 ]. However, undergoing multiple ETs also carries a risk of adverse outcomes, including preterm birth and low birth weight. And both of these outcomes have long-term effects on a child’s health, growth, and development [ 6 , 7 ]. Moreover, some studies have explored the relationship between undergoing multiple ETs and the risks of these outcomes [ 1 ]; however, the findings have been inconsistent, thus highlighting the need for further research. The analysis by Rodriguez-Wallberg et al. [ 27 ] has shown that the incidence rates of preterm birth and low birth weight are higher in women who have undergone two or more ETs than in those who underwent a single ET. A multicenter clinical cohort study in China also indicated that as the number of transfers increases, the preterm birth rate can reach 19% in some populations, while the incidence rate of low birth weight approaches 12% [ 13 , 15 , 24 ]. These risks are influenced not only by the number of transfers but also by factors, such as the number of embryos, embryo quality, maternal baseline status, and IVF indications. Differences in terms of research backgrounds, proportions of complicated cases, transfer techniques, and embryo processing standards across countries and regions have limited the comparability and consistency of study conclusions. Currently, there are few systematic studies on this issue; these studies are unable to comprehensively compare the risks of adverse pregnancy outcomes associated with different numbers of ETs. Therefore, there is an urgent need for large-sample, multicenter systematic studies to reveal the true associations between different numbers of ET and core outcomes (e.g., preterm birth and low birth weight). Therefore, the current network meta-analysis will not only help integrate existing heterogeneous evidence and optimize pregnancy management decisions but also provide a theoretical basis for mechanistic and interventional studies.