The outcomes of sequential embryo transfer in patients undergoing in vitro fertilization with frozen-thawed embryos: A retrospective study

Aim To explore whether sequential embryo transfer benefits patients with repeated implantation failure (RIF) undergoing frozen–thawed embryo transfer (FET) cycles.

We included 311 patients with a history of RIF in this retrospective study. We did sequential transfers with a cleavage embryo on day 3 and a blastocyst on day 5 in 77 patients; blastocyst transfers with two blastocysts on day 5 in 80 patients; and cleavage embryo transfers with two cleavage embryos on day 3 in 154 patients. We compared clinical outcomes between the three groups.

The clinical pregnancy rate was comparable between the blastocyst transfer group (48.8%), the sequential transfer group(48.1%) and the cleavage embryo transfer group (48.1%). There was no statistically significant difference found (p > 0.05). The ongoing pregnancy and multipregnancy rates were also comparable between the three groups (p > 0.05). The early miscarriage rate was significantly higher in the sequential transfer group (32.4%) compared with the blastocyst group (12.8%) and the cleavage embryo group (12.2%) (p < 0.05). However, after adjusting for confounders, there was no significant difference in early miscarriage rates in the sequential transfer group compared with the blastocyst group (odds ratio [OR], 2.97; 95% confidence interval [CI], 0.85–9.24; p = 0.07) and the cleavage embryo group (OR, 3.03; 95% CI, 0.94–8.06; p = 0.08).

Sequential embryo transfer failed to improve clinical outcomes for patients with RIF.

blastocyst, cleavage embryo, frozen–thawed embryo transfer, sequential embryo transfer

Successful implantation requires high‐quality embryos, a receptive endometrium, and good synchrony between the embryo and endometrium. 1 Human embryo implantation is a relatively inefficient process compared with other species. 2 Repeated implantation failure (RIF) is a frustrating and challenging issue for both patients and reproductive endocrinologists. Although controversial, RIF is commonly defined as three or more embryo transfer failures after using 3 to 10 good‐quality embryos. 3 , 4 , 5 , 6 During the treatment of infertility, couples experience severe stress and a decline in quality of life. 7 Every single failure aggravates their anxiety and depression. 8 Nearly 50% to 60% of couples discontinue IVF‐ET (in vitro fertilization‐embryo transfer) treatment after repeated failed attempts because it is a time‐consuming and costly treatment. 9 Other patients desperately seek explanations for their failures and amelioration strategies to increase their pregnancy rate. Clinicians tend to propose interventions such as sequential embryo transfer as a response to these requests. Sequential embryo transfer was first carried out by Abramovici et al. 10 Embryo freezing was not available in his center, so to avoid wasting embryos and improve chances of finding the implantation window, he performed double embryo transfers (the first transfer performed on day 2 after oocyte retrieval and the second on day 3) on 16 patients who had more than four embryos. They yielded a clinical pregnancy rate of 25%, whereas the pregnancy rate among those with only one transfer was 6.2%. Goto et al. 11 performed two‐step transfers (a cleavage embryo and a blastocyst or morula transfer on days 2 and 5, respectively) that had significantly higher pregnancy and implantation rates than conventional day 2 transfers (33.3% vs. 18.9%). Inspired by these satisfying reports, specialists started applying sequential transfer to patients with RIF. One of the most critical causes of RIF should be the suboptimal endometrial receptivity. 12 The sequential embryo transfer in the same cycle may improve the endometrium receptivity by maximizing the “window of implantation.” 10 The first embryo transferred into the uterine cavity may initiate crosstalk with the endometrium, thus facilitating the implantation of the second embryo. 13 Co‐culture studies predicted that developmentally competent embryos could induce the endometrium to be receptive to implantation by upregulating implantation‐promoting factors including the heparin‐binding epidermal growth‐like growth factor (HB‐EGF), leukemia inhibitory factor (LIF), or interleukin‐1 beta (IL‐1b). 14 Culture media from the developmentally competent embryos could also improve the uterine environment by inducing peri‐implantation factors such as COX‐2, Prss28, and Prss29 that trigger Ca2+ oscillations. 15 Additionally, the use of a transfer catheter in the first embryo transfer may mildly stimulate the endometrium making it conducive to embryo implantation. 16 , 17 , 18 Based on these mechanisms mentioned above, several scholars used the strategy of sequential transfer in RIF patients and found that it improved pregnancy rates compared with cleavage embryo transfers. 13 , 18 , 19 However, there is still inadequate evidence comparing sequential embryo transfers with blastocyst transfers. Previous studies only focused on fresh embryo transfers. Our study evaluates the clinical outcomes of sequential embryo, blastocyst, and cleavage embryo transfers among women with a history of RIF undergoing frozen–thawed embryo transfer (FET) cycles.

Participants Between January 2017 and July 2021, 571 patients with at least five embryos (including three or more embryos of top‐quality) but had not conceived after three or more embryo transfer cycles were screened and defined as RIF. Of these patients, 311 met the inclusion criteria and were enrolled in this retrospective study. The inclusion criteria was as follows: 40 years old or younger, normal hysteroscopy, normal karyotype, and two frozen embryos transferred during the study period. Patients with a thin endometrium (thickness less than 6 mm) on the transfer day, repeated pregnancy loss, severe endometriosis (stage IV according to the revised American Fertility Society classification system) or any autoimmune diseases were excluded. Based on the developmental stage of their embryos, the participants were divided into the following three groups: the sequential transfer group (n = 77), the blastocyst transfer group (n = 80), and the cleavage embryo transfer group (n = 154). Informed consents stating that data during treatment could be used in research anonymously were obtained from all the participants before IVF treatment. The study was approved by the ethics committee of the Zhejiang Provincial People's Hospital (grant number: 2021QT425). FET cycles All patients underwent FET cycles. The endometrium preparation protocol included both hormone replacement (HRT) and natural cycles. For HRT cycles, oral estradiol valerate (Delpharm Lille S.A.S) was administered at a dose of 6 mg daily starting from day 3 of the menstrual cycle. After 7 days, patients whose endometrium was thinner than 7 mm were given transdermal estradiol gels (Besins Manufacturing) at a dose of 5 g daily. After at least 12 days of preparation, when the endometrial thickness no longer increased, progesterone (Zhejiang Xianju Pharmaceutical Co. Ltd) was injected at a dose of 40 mg per day. For the natural cycles, progesterone treatment started from the ovulation day or 2 days after the luteinizing hormone (LH) surge. The progesterone priming day was defined as day 0. For patients in the sequential transfer group, a cleavage embryo was transferred on day 3 and a blastocyst on day 5. Blastocysts were transferred on day 5 in the blastocyst transfer group and cleavage embryos were transferred on day 3 in the cleavage embryo group. The cleavage‐stage embryos were morphologically assessed by the number, size, distribution of blastomeres, and the cytoplasmic fragmentation percentage. 20 Embryos graded as 1 and 2 with 7–10 cells on day 3 were defined as top‐quality embryos. The Gardner grading system was used for evaluating the blastocysts. 21 Blastocysts graded higher than 3BB on day 5 or 4BB on day 6 were deemed as top‐quality blastocysts. Pregnancy outcomes assessment The blood human chorionic gonadotropin (hCG) levels were tested 12 days after FET and a transvaginal ultrasound was performed 30–35 days after FET. If a gestational sac was detected, clinical pregnancy was confirmed. Spontaneous miscarriage in the first trimester was deemed as an early miscarriage. Pregnancy beyond 20 weeks was defined as an ongoing pregnancy. Statistical analysis Statistical analysis was performed by SPSS (version 21; Chicago, IL, USA). All measured data were expressed as mean ± SD. The one‐way analysis of variance (ANOVA) test was adopted for continuous variables with normal distribution. The Bons, SNK, and Dunnett methods were used to compare the differences between the two groups. The Kruskal–Wallis test was performed for data with uneven variance. The Pearson chi‐square test was applied to perform proportion comparisons among groups. A p value of less than 0.05 was defined as statistically significant. The generalized estimate equation (GEE) analysis model was used to control confounding factors and evaluate the differences in clinical end points. The female age, female body mass index (BMI), primary infertility, infertility duration, previous failed cycles, the number of top‐quality embryo transferred, and the endometrial thickness on transfer day were all included in the GEE model. In all the GEE models, binomial distribution and logit connection function were used except for the embryo implantation, for which a Poisson distribution model was adopted.

The baseline data in the three groups including the average male and female age, anti‐Mullerian hormone (AMH) levels, BMI, duration of infertility, previous failed cycles, and causes of infertility were all comparable (Table 1). TABLE 1. | Variables | Sequential embryo transfer (n = 77) | Blastocyst transfer (n = 80) | Cleavage embryo transfer (n = 154) | p‐Value | |---|---|---|---|---| | Female age (years) | 33.39 ± 3.89 | 32.50 ± 3.79 | 33.63 ± 3.97 | 0.10 | | Male age (years) | 34.90 ± 4.70 | 33.98 ± 4.66 | 35.42 ± 5.30 | 0.11 | | BMI | 21.38 ± 2.72 | 21.16 ± 2.65 | 21.53 ± 2.61 | 0.60 | | Infertility duration (years) | 4.09 ± 3.19 | 3.53 ± 2.60 | 3.84 ± 2.83 | 0.47 | | Previous failed cycles | 3.66 ± 1.28 | 3.43 ± 0.90 | 3.64 ± 0.89 | 0.24 | | Primary infertility ratio (n, %) | 32/60 (41.6) | 30/80 (37.5) | 71/201 (35.3) | 0.63 | | AMH (ng/mL) | 3.71 ± 2.92 | 3.49 ± 2.29 | 3.43 ± 2.95 | 0.91 | | Cause of infertility (n,%) | |||| | Tubal factor | 32/77 (41.6) | 40/80 (50.0) | 65/154 (42.2) | 0.40 | | Male factor | 18/77 (23.4) | 16/80 (20.0) | 26/154 (16.2) | | | Ovulation disorder | 10/77 (13.0) | 7/80 (8.8) | 15/154 (9.7) | | | Endometriosis | 4/77 (5.2) | 8/80 (10.0) | 23/154 (14.9) | | | Unexplained | 13/77 (16.9) | 9/80 (11.3) | 25/154 (16.2) | Abbreviations: AMH, anti‐Müllerian hormone; BMI, body mass index. Continuous variables are presented as mean ± SD, categorical variables were presented as frequencies (percentages). The endometrium preparation protocols for the three groups were similar and the HRT protocol accounted for nearly three‐fourths of the protocols. The thickness of the participants' endometrium on the transfer day was comparable among the three groups. The average number of top‐quality embryos transferred was significantly higher in the cleavage embryo group (1.90 ± 0.33) compared with the sequential transfer group (1.53 ± 0.50) and the blastocyst group (1.10 ± 0.76) (p = 0.000). No statistical significance was found in the implantation rate and the clinical rate in the blastocyst group (33.8%, 48.8%), the sequential transfer group (31.8%, 48.1%) and the cleavage embryo group (31.5%, 48.1%, p > 0.05). The early miscarriage rate in the sequential embryo transfer group was as high as 32.4%, which was significantly higher than those of the blastocyst group and the cleavage embryo group, 12.8% and 12.2%, respectively (p 0.05) and multipregnancy rate (32.4%, 38.5%, 29.7%; p > 0.05) in the sequential group, the blastocyst group and the cleavage embryo group (Table 2). TABLE 2. | Variables | Sequential transfer (n = 77) | Blastocyst transfer (n = 80) | Cleavage embryo transfer (n = 154) | p‐Value | |---|---|---|---|---| | Thickness of endometrium (mm) | 9.91 ± 1.71 | 9.88 ± 2.29 | 9.54 ± 1.84 | 0.26 | | Top‐quality embryo | 1.53 ± 0.50 | 1.10 ± 0.76 | 1.90 ± 0.33 | 0.000 | | Top‐quality embryo ratio on transfer day (n, %) | 118/154 (76.6) | 88/160 (55.0) | 292/308 (94.8) | 0.00 | | FET protocols (n, %) | |||| | HRT | 65/77 (84.4) | 61/80 (76.3) | 121/154 (78.6) | 0.42 | | NC | 12/77 (15.6) | 19/80 (23.8) | 33/154 (21.4) | | | Implantation rate (n, %) | 49/154 (31.8) | 54/160 (33.8) | 97/308 (31.5) | 0.88 | | Clinical pregnancy rate (n, %) | 37/77 (48.1) | 39/80 (48.8) | 74/154 (48.1) | 0.99 | | Ongoing pregnancy rate (n, %) | 25/77 (32.5) | 34/80 (42.5) | 62/154 (40.3) | 0.39 | | Early miscarriage rate (n, %) | 12/37 (32.4) | 5/39 (12.8) | 9/74 (12.2) | 0.03 | | Multipregnancy rate (n, %) | 12/37 (32.4) | 15/39 (38.5) | 22/74 (29.7) | 0.64 | Abbreviations: HRT, hormone replacement therapy; NC, natural cycles. Continuous variables are presented as mean ± SD, categorical variables were presented as percentages (frequencies). We used the GEE models to adjust for confounding factors such as women's age, BMI, primary infertility, infertility duration, previous failed cycles, endometrial thickness on transfer day, and the number of top‐quality embryos transferred. Results before adjusting was shown in Table 3. The odds ratio (OR) of the early pregnancy rate in the sequential group against the cleavage embryo group and the blastocyst group was 3.08 (95% confidence interval [CI] 1.13–9.78) and 3.12 (95% CI 1.04–8.56) respectively, which was statistically significant (p < 0.05). However, after adjusting, there was no significant difference in early miscarriage rates in the sequential group compared with the blastocyst group (OR, 2.97; 95% CI, 0.85–9.24; p = 0.07) and the cleavage group (OR, 3.03; 95% CI, 0.94–8.06; p = 0.08) (Table 4). TABLE 3. | Parameters | Sequential transfer versus cleavage embryo transfer | Sequential transfer versus blastocyst transfer | Blastocyst transfer versus cleavage embryo transfer | ||| |---|---|---|---|---|---|---| | Crude OR (95% CI) | p‐Value | Crude OR (95% CI) | p‐Value | Crude OR (95% CI) | p‐Value | | | Implantation rate | 1.09 (0.91–1.31) | 0.33 | 0.91 (0.78–2.01) | 0.33 | 1.23 (0.71–1.84) | 0.25 | | Clinical pregnancy rate | 1.02 (0.61–1.72) | 0.92 | 0.97 (0.49–1.91) | 0.74 | 1.06 (0.61–1.73) | 0.82 | | Ongoing pregnancy rate | 0.83 (0.37–4.56) | 0.54 | 0.62 (0.31–1.24) | 0.17 | 1.15 (0.72–3.03) | 0.66 | | Early miscarriage rate | 3.08 (1.13–9.78) | 0.03 | 3.12 (1.04–8.56) | 0.03 | 1.22 (0.29–4.40) | 0.77 | | Multipregnancy rate | 1.17 (0.50–2.74) | 0.73 | 0.96 (0.68–1.37) | 0.32 | 1.69 (0.76–3.76) | 0.20 | TABLE 4. | Parameters | Sequential transfer versus cleavage embryo transfer | Sequential transfer versus blastocyte transfer | Blastocyst transfer versus cleavage embryo transfer | ||| |---|---|---|---|---|---|---| | Adjusted OR (95% CI) | p‐Value | Adjusted OR (95% CI) | p‐Value | Adjusted OR (95% CI) | p‐Value | | | Implantation rate | 1.03 (0.72 ~ 2.78) | 0.25 | 0.89 (0.61 ~ 1.78) | 0.13 | 1.04 (0.91–1.19) | 0.29 | | Clinical pregnancy rate | 1.07 (0.68 ~ 2.43) | 0.46 | 0.91 (0.31–2.97) | 0.46 | 1.43 (0.91–3.19) | 0.25 | | Ongoing pregnancy rate | 0.72 (0.66 ~ 1.29) | 0.14 | 0.57 (0.14 ~ 1.39) | 0.34 | 1.18 (0.88–3.35) | 0.11 | | Early miscarriage rate | 3.03 (0.94 ~ 8.06) | 0.08 | 2.97 (0.85 ~ 9.24) | 0.07 | 1.38 (0.13–1.14) | 0.48 | | Multipregnancy rate | 1.20 (0.75 ~ 2.65) | 0.54 | 0.87 (0.55 ~ 1.93) | 0.44 | 1.57 (0.43 ~ 2.87) | 0.32 | Adjusted by female age, female BMI, primary infertility, infertility duration, previous failed cycles, the number of top‐quality embryo transferred, endometrial thickness on transfer day.

Successful implantation of the embryo involves complex interplay between the embryo and the endometrium. 22 , 23 Sequential embryo transfer was applied in patients with a history of RIF with the intention to improve endometrial receptivity. However, the effect of sequential embryo transfer is controversial. Machtinger et al. 18 found that the implantation rate and clinical pregnancy rate were significantly higher in the sequential transfer group than in the cleavage embryo transfer group among patients who underwent three or more IVF failures but had an adequate ovarian response. Fang et al. 13 performed sequential day 2 and 3 embryo transfers and found that the clinical pregnancy rate was significantly higher than that of day 3 embryo transfer. However, the clinical pregnancy rate of sequential day 3 and 5 transfers was comparable with that of day 5 blastocyst transfer. They attributed the negative result to the small sample size. This result urged clinicians to find out whether the advantage of sequential transfers was related to blastocyst transfers rather than double transfers. In 2013, Yazbeck et al. 24 divided cleavage embryo transfer patients into two subgroups depending on whether blastocysts were cryopreserved with extended culture. It turned out that the clinical pregnancy rate of the sequential transfer group was similar to that of the blastocysts subgroup but significantly higher than those without blastocysts. This suggests that it was the embryonic development potential, not the sequential transfer, that determined the clinical outcome. In a randomized controlled trial, Tehraninejad et al. 25 did not find any advantage of sequential embryo transfer over blastocyst embryo transfer. A meta‐analysis carried out in 2021 found that sequential transfer has a positive impact on clinical pregnancy compared with cleavage embryo transfer, but not with blastocyst transfer. 26 These results suggest that blastocysts, which possess a higher implantation potential, contribute to the encouraging results of sequential transfers. Our study found the implantation rates (31.8%, 33.8% vs. 31.5%), clinical pregnancy rates (48.1%, 48.8% vs. 48.1%) and ongoing pregnancy rates (32.5%, 42.5% vs. 40.3%) for the two groups versus the sequential transfer group were comparable. The outcomes of the sequential transfer group were not superior to those of either the cleavage or the blastocyst group. A possible reason is that the embryos transferred in previous studies were all top‐quality, but in our study, the top‐quality embryo rate was highest in the cleavage embryo group (94.8%) and lowest in the blastocyst group (55.0%). This is because on day 3, the top‐quality embryos in our center are transferred or frozen, leaving the suboptimal quality embryos for extended culture. The good quality of the cleavage embryos may compensate for their relatively early developmental stage as evidenced by the Karacan M's study, which found that cleavage embryos with optimal quality were as efficient as blastocyst transfer in pregnancy outcomes. 27 Suboptimal quality blastocysts could yield similar live birth rates to top‐quality ones if frozen on day 5. 28 We performed sequential embryos transfer in FET cycles, whereas previous studies mainly focused on fresh cycles. It is reported that the “window of implantation” for fresh cycles is narrower due to premature progesterone and supraphysiologic estrogen levels. 29 , 30 The advantage of more chances for the embryo to identify the “window of implantation” in sequential embryo transfers may not be so effective in FET cycles. Unexpectedly, we found a significantly higher early miscarriage rate (32.4%) in the sequential transfer group than in the other two groups (12.8%, 12.2%, p < 0.05). However, the adjusted OR showed no significant difference in early miscarriage rates in the sequential transfer group compared with the blastocyst group (OR, 2.97; 95% CI, 0.85–9.24; p = 0.07) or the cleavage embryo group (OR, 3.03; 95% CI, 0.94–8.06; p = 0.08). In previous studies, only Yazbeck et al. 24 and Fang et al. 13 mentioned the miscarriage rate. Both studies reported a lower or similar miscarriage rate in the sequential transfer group compared with the control groups. The meta‐analysis carried out by Zhang et al. 26 did not find any significant difference between the sequential transfer and blastocyst transfer groups (OR, 0.99; 95% CI, 0.68–1.44; p = 0.95). Our adjusted results were consistent with the above study. Regrettably, only 8 out of 12 cases with early miscarriage in the sequential transfer group examined the chromosome of embryos, and 6 cases were found to be with aneuploidy. Immunological factors such as antiphospholipid syndrome (APS) and undifferentiated connective tissue disease (UCTD) may also lead to early pregnancy loss. 31 Nevertheless, these diseases were not routinely screened in the patients included in this study. Consequently, it is difficult to deduce the cause of such a high early miscarriage rate and whether it is relevant to the procedure of sequential transfer needs to be further explored. Multiple pregnancies have always been a safety concern with IVF. 32 Based on previous sequential transfer studies, 13 , 18 , 24 , 25 only Machtinger et al. 18 found that it resulted in significantly higher multiple pregnancy rates. In our study, the multipregnancy rate was highest in the blastocyst group (38.5%), followed by the sequential transfer group (32.4%) and the cleavage embryo group (29.7%). The statistical difference was insignificant, which is consistent with the result of a 2021 meta‐analysis. 26 Since all the three groups carried out double embryo transfers, it seems what determines the multipregnancy rate is the number of embryos transferred, not the two‐step transfer process. This can be further validated by the findings showing that the multipregnancy rate was significantly higher in patients who received two embryos (a good quality embryo and a poor quality embryo) regardless of whether they were cleavage embryos or blastocysts. 33 After RIF, most patients wished to transfer two embryos to maximize the clinical pregnancy rate. Nevertheless, the above findings lead us to reflect on the benefits of double embryo transfers in patients with RIF. Patients did not report lower abdominal discomfort or vaginal bleeding before the second transfer and no infection occurred after the double transfers. However, patients in the sequential transfer group needed to pay twice as much as patients in the other two groups. Our findings suggest that sequential transfers are a harmless but ineffective and sometimes costly procedure for patients with RIF. In conclusion, our study investigated the value of sequential embryo transfers in FET cycles. Unfortunately, we did not find any improved clinical outcomes favoring sequential embryo transfers. Possible reasons may be as follows: the small sample size, the uneven distribution of top‐quality embryos, FET cycles, and the limitations of retrospective studies. It is noteworthy that the sequential transfers did not result in higher multipregnancy and early miscarriage rates. For patients who have had repeated IVF failures with different developmental stages of embryos, a sequential embryo transfer is a plausible choice. Further prospective studies are needed to investigate its value in RIF patients. Author contributions All authors contributed to the study conception and design. Study conception and design, patients recruitment, data collection, analysis and manuscript writing were performed by Mengxia Ji. Ling Zhang contributed significantly to data analysis; Xiaohua Fu contributed to study design; Wenjie Xie performed data collection. Xiangli Wu contributed to data collection, manuscript editing and revision; Jing Shu helped manuscript revision. Both of Mengxia Ji and Jing Shu obtained fund and supported this research. All authors have read and approved the final manuscript. Conflict of interest All authors do not have any financial and personal relationships with other people or organizations that could inappropriately influence (bias) this work. There are no potential conflicts of interest include employment, consultancies, stock ownership, honoraria, paid expert testimony and patent applications/registrations. Acknowledgments This work was supported by basic public welfare research program of Zhejiang Province of China (Grant Number: LGF20H040012), medical and health clinical research project of Zhejiang Province of China (Grant Number: 2021KY525) and adjunct talent fund of Zhejiang Provincial People's Hospital. Furthermore, the authors thank TopEdit (www.topeditsci.com) for its linguistic assistance during the preparation of this manuscript. Contributor Information Xiangli Wu, Email: [email protected]. Jing Shu, Email: [email protected]. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request.

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[DOI] [PMC free article] [PubMed] [Google Scholar] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request.