Abstract
Purpose
This study aimed to investigate the clinical outcomes of morula stage transfer derived from post-thawed cleavage embryos undergoing overnight culture in frozen embryo transfer (FET) cycles.
Methods
We performed a retrospective study that included 392 FET cycles with 784 thawed embryos undergoing overnight culture between January 2014 and December 2018. Embryos were divided into three groups in terms of their status: 8–16 cells without morula (group I), one morula (group II), and two morulae (group III). The clinical outcomes of these cycles were then compared between the three groups. Logistic regression analysis was performed to control for confounders.
Results
Group III was associated with a significantly higher clinical pregnancy rate (odds ratio [OR] 2.35; 95% confidence interval [CI] 1.29–4.27; P = 0.005), implantation rate (OR 3.00; CI 1.75–5.16; P < 0.001), multiple pregnancy rate (OR 4.91; CI 2.11–11.40; P < 0.001), and live birth rate (OR 1.96; CI 1.10–3.49; P = 0.022) than group I. Group II had a higher live birth rate than group I after adjustment (OR 1.70; CI 1.04–2.79; P = 0.035). There was no difference in the rate of premature delivery when compared across the three groups after adjustment.
Conclusion
The transfer of morula stage embryos following the overnight culture of post-thawed cleavage embryos led to an improvement in the clinical outcomes of FET cycles. It is important to reduce the number of morula embryos transferred in order to achieve a singleton pregnancy.
Keywords: Morula, Overnight culture, Frozen embryo transfer, Live birth, Multiple pregnancy
Introduction
Recent improvements in the techniques used to cryopreserve embryos have led to the wide-scale application of frozen embryo transfer (FET) in assisted reproductive technology (ART), and an increased proportion of in vitro fertilization-embryo transfer (IVF-ET) cycles [1]. FET reduces the risk of ovarian hyper-stimulation syndrome (OHSS), maximizes the utilization of embryos following oocyte retrieval per cycle, and also provides an option for patients who cannot proceed with fresh transfer [2, 3]. Some recent studies have also indicated that the use of FET could result in better clinical pregnancy rates than fresh embryo transfer [4–6], particularly in patients with polycystic ovary syndrome (PCOS) [7].
There are two methods for routine embryo transfer in FET cycles: transfer after a short post-thaw culture period (2 h) and transfer following overnight culture. Several studies have suggested that the overnight culture in FET cycles would result in a better clinical outcome for FET cycles than short culture periods [8, 9]. In our center, we mostly select the post-thaw overnight culture approach for FET cycles, as we believe that this technique provides optimal outcomes. By investigating the overnight culture protocol during FET cycles, we will be able to acquire more information relating to the development and potency of thawed embryos, thus, providing us with a better understanding of the relationship between embryos and clinical outcomes. We observed that a proportion of thawed embryos undergoing overnight culture could develop further into the morula stage. However, literature has neglected to consider the role of morula embryo in terms of embryo transfer (ET). Consequently, little is known of the morula stage with regard to ART, even though this stage forms an essential step for the transition of cleavage stage embryos into blastocysts [10].
The European Society of Human Reproduction and Embryology (ESHRE) Atlas of Human Embryology defines the morula embryo as an indistinguishable mass of cells on day 4 [10]. The lack of well-established evaluation criteria for the morula stage, which has been overlooked for a long time, creates a major limitation for the clinical application of this embryonic stage in the ART process. The first report of morula stage embryo transfer was published in 1994 and indicated higher rates of implantation than day 3 transfers; a subsequent publication indicated that morulae could be used for ET following day 3 blastomere biopsy [11, 12]. Tao et al. went on to suggest that day 4 ET could lead to superior implantation and clinical pregnancy rates when compared with the transfer of day 3 embryos [13]; these results were supported by several subsequent studies [14, 15]. Furthermore, the selective transfer of a single morula was associated with similar clinical outcomes when compared with blastocyst transfer in IVF-ET cycles [16]. In another study, Fabozzi et al. highlighted the fact that the morphology of morula stage embryos played a more decisive role in predicting the quality and formation of blastocysts than our conventional embryo scoring systems [17]. While these previous studies have shown an encouraging outcome, there have been no specific investigations of the transfer of morula stage embryos in FET cycles.
This study was a retrospective study that aimed to investigate the impact of morulae derived from post-thawed cleavage embryos undergoing overnight culture on the clinical outcomes of FET cycles. We also evaluated the value of morula embryo transfer and its application in FET cycles.
Materials and methods
Patient population
We conducted a retrospective cohort study relating to the transfer of morula stage embryos in FET cycles between January 2014 and December 2018 at the Reproductive Medicine Center, Shanxi Provincial People’s Hospital. A total of 392 cycles, with 784 cleavage stage embryos that were thawed and cultured overnight (17–19 h), were ultimately recruited for analysis. Our inclusion criteria were as follows: (1) two post-thawed embryos transferred with at least one blastomere ≥ 8 cells, (2) maternal age ≤ 38 years, (3) no more than three transfer cycles, and (4) patient undergoing hormone replacement therapy (HRT) for endometrium preparation. Exclusion criteria were as follows: (1) thawed embryos with damaged blastomeres, (2) the transfer of one or three embryos, (3) patients with natural cycles undergoing endometrium preparation, and (4) patients experiencing repeated implantation failure. A flow diagram describing the protocol for this study is shown in Fig. 1.
Fig. 1.
Flow diagram showing the distribution of the study populations
IVF/ICSI procedures
Ovarian stimulation was performed on the basis of a patient’s individual characteristics by using one of the three following protocols: a gonadotropin-releasing hormone (GnRH) agonist long protocol, a GnRH antagonist protocol, or a micro-dose GnRH agonist flare-up. The final maturation of oocytes was induced by injecting 6000–10,000 IU of hCG when at least two follicles reached 18 mm in diameter. Oocyte collection was performed 36–38 h after hCG stimulation and under the guideline of transvaginal ultrasound aspiration. The insemination of retrieved oocytes was accomplished by conventional IVF or intracytoplasmic sperm injection (ICSI). The assessment of fertilization and embryonic development was carried out at set checkpoints, in accordance with Istanbul’s Consensus [18]. Standard day 3 frozen embryos were defined as follows: number of blastomeres ≥ 4 cells of equal/unequal size, ≤ 20% fragmentation, the presence of an integrated zona pellucida, and no vacuoles and no multiple nuclei.
Freezing and thawing of day 3 embryos
Day 3 embryos were frozen using a Vitrification Freezing Kit (Kitazato, Japan). Selected day 3 embryos were removed from cleavage medium to equilibrium solution (ES) for 4 min and then transferred into vitrification solution (VS) for 30 s. Embryos, covered by only a minimal volume of vitrification solution, were then placed onto a Cryotip (Kitazato, Japan) and immediately plunged into liquid nitrogen.
For thawing, the Cryotip holding the embryo was removed from the liquid nitrogen and placed into 1.0 ml of thawing solution (TS) for 1 min at 37 °C. Then, the embryos were transferred to 0.5 ml of dilution solution (DS) for 4 min, WS1 solution for 5 min, and WS2 solution for 5 min. The warming process was carried out in accordance with the Thawing Kit (Kitazato, Japan). After warming, each embryo was individually transferred into a 20-μl drop of cleavage medium (1026, Quinn’s, American). Embryos were then checked for blastomere survival under an inverted microscope and cultured overnight for 17–19 h. The next morning, we observed and evaluated the cultured embryos. Morulae were evaluated used the scoring system proposed by Tao et al [13]. The zona pellucida underwent laser thinning prior to transfer with a ZILOS-tkTM laser system (Hamilton Thorn Bioscience Inc., Beverly, MA, USA).
Endometrium preparation and embryo transfer
Hormone replacement therapy (HRT) was used to prepare the endometrium of patients undergoing FET cycles. Oral estrogen (Progynova, Bayer, Germany) was commenced (2 mg twice daily) on the 2nd or 3rd day of the menstrual cycle, and subsequently increased by a step-up protocol to 8 mg/day. An ultrasound endometrial assessment was performed after 11 days to check whether the endometrial thickness is ≥ 7 mm. If the endometrial thickness had not reached 7 mm, then oral estrogen was provided for three further days and the endometrial thickness rechecked. When the endometrial thickness reached 7 mm, we injected 60 mg/day of progesterone prior to FET; this dosage was maintained after FET until 10 weeks of gestation. If the endometrial thickness failed to reach 7 mm, the cycle was canceled.
Main outcome measures
Clinical pregnancy was defined as the detection of gestational sacs at 5 weeks by ultrasound after FET transfer. The clinical pregnancy rate was defined as the number of clinical pregnancy cycles divided by the number of FET cycles. Implantation rate was defined as the number of observed gestational sacs divided by the number of thawed embryo transferred. Miscarriage rate was defined as a clinical pregnancy lost before 12 weeks of gestation per clinical pregnancy cycle. Multiple pregnancy rate was defined as the number of multiple pregnancy cycles (two observed intrauterine gestational sacs with heartbeats) divided by the clinical pregnancy cycles. Live birth rate was defined as the number of live deliveries after 24 weeks of pregnancy per FET cycles. The rate of premature delivery was defined as the number of deliveries before 37 weeks divided by the number of live births.
Statistical analysis
All data analyses were performed using SPSS software version 20.0 (IBM, New York, USA). Continuous data are shown as the mean ± standard deviation (SD) and were subjected to one-way analysis of variance (ANOVA). Non-normally distributed data were compared by the Kruskal-Wallis test. Categorical data were described as percentages and analyzed by the chi-square test or Fisher’s exact test. Logistic regression analysis was used to adjust for confounders, including demographic variables and the further development of thawing embryos. A P < 0.05 was considered as statistically significant.
Results
A total of 784 frozen-thawed embryos, in 392 hormonal replacement transfer cycles, were included in this study. The flow diagram for this study is shown in Fig. 1. According to the development of post-thawed embryos after overnight culture, embryos were assigned to a non-morula stage group (8–16 cells group, group I, n = 153), a one morula stage group (group II, n = 145), and a two morulae stage group (group III, n = 94). The demographic and clinical characteristics of the three groups are shown in Table 1. The maternal age, duration of infertility, primary infertility, and factors responsible for infertility were comparable across the three groups (P > 0.05). However, body mass index (BMI) and basal FSH level showed significant differences between group II and group I (P = 0.001; P = 0.028, respectively); BMI and basal FSH were similar when compared between group III and group I (P > 0.05). The total gonadotropin dose used showed significant differences among the three groups (P < 0.001 and P = 0.006, respectively).
Table 1.
Demographic and clinical characteristics of the three groups
| Group I | Group II | Group III | P valuea | P valueb | |
|---|---|---|---|---|---|
| No. of cycles | 153 | 145 | 94 | ||
| Maternal age (years) | 29.4 ± 3.5 | 29.6 ± 3.6 | 29.5 ± 3.7 | 0.618 | 0.908 |
| BMI (kg/m2) | 22.4 ± 3.3 | 23.7 ± 3.5 | 23.2 ± 3.6 | 0.001 | 0.059 |
| Duration of infertility (years) | 3.3 ± 2.1 | 3.4 ± 2.5 | 3.6 ± 2.4 | 0.841 | 0.336 |
| Primary infertility (%) | 111 (72.5) | 108 (74.5) | 68 (72.3) | 0.705 | 0.972 |
| Factors of infertility (%) | 0.677 | 0.436 | |||
| Female | 37 (24.2) | 31 (21.4) | 23 (24.5) | ||
| Male | 34 (22.2) | 32 (22.1) | 13 (13.8) | ||
| Both | 76 (49.7) | 80 (55.2) | 53 (56.4) | ||
| Unexplained | 4 (2.6) | 1 (0.7) | 2 (2.1) | ||
| Basal FSH (IU/L) | 7.4 ± 2.4 | 6.8 ± 2.1 | 7.3 ± 2.6 | 0.028 | 0.898 |
| Total gonadotropin dose (IU) | 2370.7 ± 791.4 | 2033.0 ± 736.7 | 2086.3 ± 872.7 | < 0.001 | 0.006 |
Values are reported as means ± standard deviations or numbers (percentages); BMI, body mass index; FSH, follicle-stimulating hormone
aGroup II vs. group I
bGroup III vs. group I
The laboratory characteristics of post-thawed embryos following overnight culture in FET cycles is shown in Table 2. In total, 784 embryos were observed: 306 embryos in group I, 290 embryos in group II, and 188 embryos in group III. In total, 333/784 (42.5%) embryos had reached the morula stage after overnight culture; of these, 60.7% were derived from 8 cells, 11.1% from less than 8 cells, and 28.2% from more than 8 cells. The number of oocytes retrieved in group III was slightly higher than group I (P = 0.018), but no significant difference was shown between group II and group I (P > 0.05). The proportion of IVF cycles was not significantly different when compared across the three groups (P > 0.05). However, the further development potency of post-thawed embryos was significantly different when compared across the three groups (P < 0.001).
Table 2.
The laboratory characteristics of thawed-embryos following overnight culture
| Group I (n = 153) | Group II (n = 145) | Group III (n = 94) | P valuea | P valueb | |
|---|---|---|---|---|---|
| No. of post-thawed embryos | 306 | 290 | 188 | ||
| No. of oocytes retrieved | 11.2 ± 5.4 | 11.7 ± 5.8 | 13.0 ± 6.2 | 0.402 | 0.018 |
| Conventional IVF (%) | 113 (73.9) | 108 (74.5) | 73 (77.7) | 0.786 | 0.501 |
| Proliferation cycles | < 0.001 | < 0.001 | |||
| Without further development cycles | 22 (14.4) | 1 (0.7) | 0 | ||
| One for further development cycles | 67 (43.8) | 43 (29.7) | 10 (10.6) | ||
| Two for further development cycles | 64 (41.8) | 101 (69.7) | 84 (89.4) |
Values are reported as means ± standard deviations, or as numbers (percentages). The Kruskal-Wallis test was used to investigate proliferation capacity between the three groups
aGroup II vs. group I
bGroup III vs. group I
Clinical outcomes of the three groups are shown in Table 3. The thickness of the endometrium prior to transfer was not significantly different when compared across the three groups (P > 0.05). The clinical pregnancy rate (CPR), multiple pregnancy rate (MPR), and the rate of premature delivery were similar between group II and group I (P > 0.05). While the implantation rate (IR) and live birth rate (LBR) were significantly different when compared between group II and group I (P = 0.035; P = 0.022, respectively). The CPR, IR, MPR, and LBR showed significant differences between group III and group I (P < 0.01). The rate of premature delivery in group III was higher than in group I (P = 0.022), although there was no significant difference between group II and group I (P > 0.05). The miscarriage rate in group III (16.7%) was slightly higher than group I and group II, but this was not statistically significant.
Table 3.
Clinical outcomes of the three groups
| Group I (n = 153) | Group II (n = 145) | Group III (n = 94) | P valuea | P valueb | |
|---|---|---|---|---|---|
| Endometrial thickness (mm) | 9.1 ± 1.1 | 9.1 ± 1.3 | 9.2 ± 1.2 | 0.900 | 0.711 |
| Clinical pregnancy rate (%) | 74/153 (48.4) | 84/145 (57.9) | 66/94 (70.2) | 0.098 | 0.001 |
| Implantation rate (%) | 92/306 (30.1) | 111/290 (38.3) | 97/188 (51.6) | 0.035 | < 0.001 |
| Multiple pregnancy rate (%) | 13/74 (17.6) | 22/84 (26.2) | 26/66 (39.4) | 0.193 | 0.004 |
| Miscarriage rate (%) | 11/74 (14.9) | 5/84 (6.0) | 11/66 (16.7) | 0.064 | 0.770 |
| Live birth rate (%) | 60/153 (39.2) | 75/145 (51.7) | 52/94 (55.3) | 0.030 | 0.014 |
| Premature delivery (%) | 7/60 (11.7) | 14/75 (18.7) | 15/52 (28.8) | 0.265 | 0.022 |
The chi-square test, or Fisher’s exact test, was used to compare the three groups
aGroup II vs. group I
bGroup III vs. group I
Logistic regression analysis is shown in Table 4. Several variables, including BMI, oocytes retrieved, and proliferation cycles, were employed in the logistic regression analysis to eliminate the effects on clinical outcomes. After controlling for confounders, group III was shown to be associated with higher CPR (odds ratio (OR) 2.35; 95% confidence interval (CI)1.29–4.27; P = 0.005), IR (OR 3.00; CI 1.75–5.16; P < 0.001), and LBR (OR 1.96; CI 1.10–3.49; P = 0.022) than group I. Moreover, group III also had a higher MPR than group I (OR 4.91; CI 2.11–11.40; P < 0.001). The LBR in group II was higher than group I after adjusting for confounders (OR 1.70; CI 1.04–2.79; P = 0.035), but the other clinical outcomes, including CPR, IR, and MPR, were similar between groups. The rate of premature delivery was similar across the three groups after adjustment.
Table 4.
Logistic regression analysis: the relationship between groups and clinical outcomes
| Group II vs. group I | Group III vs. group I | |||
|---|---|---|---|---|
| OR (95%CI) | P value | OR (95%CI) | P value | |
| Clinical pregnancy ratea | ||||
| Unadjusted | 1.47 (0.93–2.32) | 0.099 | 2.52 (1.46–4.34) | 0.001 |
| Adjusted | 1.38 (0.85–2.26) | 0.196 | 2.35 (1.29–4.27) | 0.005 |
| Implantation ratea | ||||
| Unadjusted | 1.51 (0.98–2.32) | 0.062 | 2.88 (1.77–4.70) | < 0.001 |
| Adjusted | 1.48 (0.93–2.36) | 0.096 | 3.00 (1.75–5.16) | < 0.001 |
| Multiple pregnancy ratea | ||||
| Unadjusted | 1.93 (0.93–3.99) | 0.077 | 4.12 (1.99–8.51) | < 0.001 |
| Adjusted | 2.23 (1.04–5.01) | 0.041 | 4.91 (2.11–11.40) | < 0.001 |
| Live birth ratea | ||||
| Unadjusted | 1.67 (1.05–2.63) | 0.031 | 1.92 (1.14–3.23) | 0.014 |
| Adjusted | 1.70 (1.04–2.79) | 0.035 | 1.96 (1.10–3.49) | 0.022 |
| Premature rateb | ||||
| Unadjusted | 2.18 (0.83–5.72) | 0.114 | 3.53 (1.32–9.47) | 0.012 |
| Adjusted | 2.37 (0.71–7.89) | 0.160 | 2.96 (0.77–11.39) | 0.115 |
aAfter adjusting for BMI, oocytes retrieved, and proliferation cycles
bAfter adjusting for BMI, proliferation cycles, and multiple pregnancy
OR, odds ratio; 95%CI, 95% confidence interval
Discussion
The present study was designed to investigate the impact of transfer of morula embryos on reproductive outcomes following FET cycles. Our data suggested that the transfer of morulae, derived from post-thawed cleavage-stage embryos after overnight culture, yielded better clinical outcomes than non-morula stages in FET cycles, including a higher clinical pregnancy rate (CPR), implantation rate (IR), and live birth rate (LBR). However, as two morulae stage embryos were transferred in FET, this resulted in a much higher CPR, IR, and LBR than the other groups, but at the expense of significantly increased multiple pregnancy rates. Although the rate of multiple pregnancy was higher in group III, the rate of premature delivery was similar across the three groups after controlling for confounders. Our results indicate that the appearance of the morula stage is an efficient predictive indicator of embryo development potential and may represent an alternative strategy for achieving singleton delivery in FET cycles.
Due to recent advancements in culture system design, the extension of embryo culture in vitro has become a quicker, more straightforward, and a more cost-effective method with which to select a viable embryo than non-invasion time-lapse microscopy (TLM) or invasive pre-implantation genetic testing (PGT) [19]. Over recent years, the elective transfer of a single blastocyst transfer has become a tendency for IVF/ICSI procedures, irrespective of whether the cycle involves ET or FET; this practice has led to a remarkable increase in the ongoing pregnancy rate and LBR [20, 21]. However, the failure to form blastocysts in patients with few day 3 cleavage stage embryos, or low ovarian responders, would lead to an increase in cycle cancelation, leading to frustration on the part of clinicians, embryologists, and patients [22]. Therefore, a cleavage transfer policy is still adopted in most centers, as this practice can be used for a wider range of infertility patients. Our study showed that the proportion of morula developed from post-thawed embryos was 42.5% (333/784); of these, 60.7% were derived from 8 cells; these results were in accordance with a previous study [13]. However, there was a reduction in the blastulation rate of day 3 good quality embryos by day 5 [23]. Therefore, we believe that clinicians and embryologists should investigate the value of the morula stage embryo with regard to clinical applications in ART.
Morulae, referred to as day 4 embryos, are characterized by cell compaction beginning at the 8- to 10-cell stages in early embryonic development, and are described as “an indistinguishable mass of cells” [10]. Morulae are crucial for the embryo’s transition from cleavage stage to the blastocyst stage; however, the transfer of morulae has been largely neglected. The first morula stage embryo transfer occurred in 1994 [11]. Subsequently, a few studies demonstrated that day 4 morula embryos led to higher implantation and clinical pregnancy rates when compared with day 3 embryos in ET cycles [12–15]. Kang et al. suggested that a single day 4 embryo transfer could achieve a comparable CPR, IPR, and LBR, when compared with day 5 blastocyst (51.5% vs. 51.8%; 52.3% vs. 52.5%; 39.2% vs. 44.7%, respectively) [16]. Although these results are encouraging, little is known of how the transfer of morula stage embryos might influence the outcomes of FET cycles.
Our study found that morula embryos, developed from post-thawed cleavage embryos, resulted in a higher CPR, IR, and LBR than non-morula embryos (8–16 cells group) in FET cycles. After a logistic regression analysis was used to adjust for BMI, oocytes retrieved, and proliferation cycle, the CPR, IR, and LBR remained statistically significant when compared between group III and group I. These results were consistent with previous studies that day 4 embryo transfer showed better clinical outcomes than day 3 embryos in ET cycles [13–15]. The higher IR and CPR shown in the morula embryo group might be associated with two possible mechanisms. First, embryonic genome activation (EGA) occurred during the morula stage; this process involves the self-correction mechanism and the exclusion of aneuploid cells from the compacting embryo. Secondly, the morula stage was close to physiological condition, thus providing a better embryo-endometrium synchronization [10, 24]. However, as the number of morula stage embryos transferred increased, the LBR in group III was similar with group II. Notably, the rate of miscarriage for the one morula transfer group (4.6%) was lower than for the two morula group (15.7%). Moreover, the CPR and IR in the two morula transfer group were slightly higher than in the one morula group, although this was not statistically significant. We concluded that the morula stage embryo had a good developmental and implantation potency when cultured overnight during FET cycles. Although this finding was enlightening, this should be confirmed further by performing a randomized, controlled pilot trial in the future.
The higher multiple pregnancy rate in the two morula group (39.4%) caught our attention and suggested that morula stage embryos were associated with better embryo viability than non-morula stages. Since this study used two embryos for transfer, the multiple pregnancy rate in the non-morula group (18.6%) were still higher than the guidelines reported by the Human Fertility and Embryology Authority (< 10%) [25]. Therefore, we speculated that the multiple pregnancy rate was associated with the number of embryos transferred in the same cycle, even if a poorer quality embryo was included. Previous studies have demonstrated that day 3 embryos, with low scores, might still result in a viable pregnancy if they are transferred into the uterus at the appropriate time [26]. Our hypothesis was supported by a previous research study, which evaluated the impact of a poor quality embryo, compared wih a top-quality embryo, during double embryo transfer (DET), on IVF outcomes. The authors of this previous study found that the number of multiple births was significantly increased when DET was conducted when compared with single embryo transfer (SET), regardless of the quality of the transferred embryo [27]. We therefore suggest that the transfer of a single morula embryo can achieve a comparable clinical outcome and reduce the risk of multiple gestations.
Multiple pregnancies would lead to a higher risk of preterm birth. Therefore, we compared the rate of premature delivery across the three groups. Before controlling for confounders, group III showed a higher rate of preterm births, which might be due to the higher number of multiple pregnancies. However, the differences in the rate of premature delivery between the three groups were eliminated after adjustment. Thus, we speculated that morula stage embryo transfer might not affect preterm births. This hypothesis needs to be further confirmed in future research.
Our analysis was limited by its small sample size, observational design, and a risk of bias, including epidemiological factors and the numbers of embryos used for transfer. Double embryo transfer would create bias when evaluating the value of morula stage embryos. However, we tried to control the quality and developmental rate of the embryos used in our study, so that the three groups were comparable. We also adjusted for confounders in our statistical analysis. Our results implied that transfer of one or two morulae in FET led to a higher LBR,but having no effect on the premature delivery. A randomized controlled trial is strongly recommended to confirm that the morula stage will be suitable for transfer and valuable in clinical application.
In conclusion, our study showed that the transfer of morula embryos from post-thawed cleavage embryos will be a viable option for clinical application in FET cycles, and also represents an effective approach with which to reduce multiple pregnancies by reducing the number of embryos transferred.
Compliance with ethical standards
The study was approved by the Ethics Review Board of Shanxi Provincial People’s Hospital ([2019] Provincial Medical Opinions No. 75).
Footnotes
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Contributor Information
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References
- 1.Adamson GD, de Mouzon J, Chambers GM, Zegers-Hochschild F, Mansour R, Ishihara O, Banker M, Dyer S. International Committee for Monitoring Assisted Reproductive Technology: world report on assisted reproductive technology, 2011. Fertil Steril. 2018;110(6):1067–1080. doi: 10.1016/j.fertnstert.2018.06.039. [DOI] [PubMed] [Google Scholar]
- 2.Roque M, Haahr T, Geber S, Esteves SC, Humaidan P. Fresh versus elective frozen embryo transfer in IVF/ICSI cycles: a systematic review and meta-analysis of reproductive outcomes. Hum Reprod Update. 2019;25(1):2–14. doi: 10.1093/humupd/dmy033. [DOI] [PubMed] [Google Scholar]
- 3.Balaban B, Urman B, Ata B, Isiklar A, Larman MG, Hamilton R, Gardner DK. A randomized controlled study of human day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Hum Reprod. 2008;23(9):1976–1982. doi: 10.1093/humrep/den222. [DOI] [PubMed] [Google Scholar]
- 4.Shapiro BS, Daneshmand ST, Restrepo H, Garner FC, Aguirre M, Hudson C. Matched-cohort comparison of single-embryo transfers in fresh and frozen-thawed embryo transfer cycles. Fertil Steril. 2013;99(2):389–392. doi: 10.1016/j.fertnstert.2012.09.044. [DOI] [PubMed] [Google Scholar]
- 5.Toner JP, Coddington CC, Doody K, Van Voorhis B, Seifer DB, Ball GD, et al. Society for assisted reproductive technology and assisted reproductive technology in the United States: a 2016 update. Fertil Steril. 2016;106(3):541–546. doi: 10.1016/j.fertnstert.2016.05.026. [DOI] [PubMed] [Google Scholar]
- 6.Belva F, Bonduelle M, Roelants M, Verheyen G, van Landuyt L. Neonatal health including congenital malformation risk of 1072children born after vitrified embryo transfer. Hum Reprod. 2016;31(7):1610–1620. doi: 10.1093/humrep/dew103. [DOI] [PubMed] [Google Scholar]
- 7.Chen ZJ, Shi Y, Sun Y, Zhang B, Liang X, Cao Y, Yang J, Liu J, Wei D, Weng N, Tian L, Hao C, Yang D, Zhou F, Shi J, Xu Y, Li J, Yan J, Qin Y, Zhao H, Zhang H, Legro RS. Fresh versus frozen embryos for infertility in the polycystic ovary syndrome. N Engl J Med. 2016;375(6):523–533. doi: 10.1056/NEJMoa1513873. [DOI] [PubMed] [Google Scholar]
- 8.Guo L, Luo C, Quan S, Chen LN, Li H, Guo YC, Han Z, Ou X. The outcome of different post-thawed culture period in frozen-thawed embryo transfer cycle. J Assist Reprod Genet. 2013;30(12):1589–1594. doi: 10.1007/s10815-013-0120-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Haas J, Meriano J, Bassil R, Barzilay E, Robert F. Prolonged culture of blastocysts after thawing as a tool for improving prediction of success. J Assist Reprod Genet. 2018;35(12):2195–2199. doi: 10.1007/s10815-018-1308-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Coticchio G, Lagalla C, Sturmey R, Pennetta F, Borini A. The enigmatic morula: mechanisms of development, cell fate determination, self-correction and implications for ART. Hum Reprod Update. 2019;25(4):422–438. doi: 10.1093/humupd/dmz008. [DOI] [PubMed] [Google Scholar]
- 11.Huisman GJ, Alberda AT, Leerentveld RA, Verhoeff A, Zeilmaker GH. A comparison of in vitro fertilization results after embryo transfer after 2, 3 and 4 days of embryo culture. Fertil Steril. 1994;61(5):970–971. doi: 10.1016/S0015-0282(16)56715-6. [DOI] [PubMed] [Google Scholar]
- 12.Grifo JA, Giatras K, Tang YX, Krey LC. Successful outcome with day 4 embryo transfer after preimplantation diagnosis for genetically transmitted diseases. Hum Reprod. 1998;13(6):1656–1659. doi: 10.1093/humrep/13.6.1656. [DOI] [PubMed] [Google Scholar]
- 13.Tao J, Tamis R, Fink K, Williams B, Nelson-White T, Craig R. The neglected morula/compact stage embryo transfer. Hum Reprod. 2002;17(6):1513–1518. doi: 10.1093/humrep/17.6.1513. [DOI] [PubMed] [Google Scholar]
- 14.Skorupski JC, Stein DE, Acholonu U, Field H, Keltz M. Successful pregnancy rates achieved with day 4 embryo transfers. Fertil Steril. 2007;87(4):788–791. doi: 10.1016/j.fertnstert.2006.08.093. [DOI] [PubMed] [Google Scholar]
- 15.Pantos K, Makrakis E, Chronopoulou M, Biba M, Perdikaris A, Dafereras A. Day 4 versus day 3 embryo transfer: a prospective study of clinical outcomes. Fertil Steril. 2008;89(3):573–577. doi: 10.1016/j.fertnstert.2007.03.056. [DOI] [PubMed] [Google Scholar]
- 16.Kang SM, Lee SW, Jeong HJ, Yoon S-H, Koh MW, Lim J-H, et al. Clinical outcomes of elective single morula embryo transfer versus elective single blastocyst embryo transfer in IVF-ET. J Assist Reprod Genet. 2012;29(5):423–428. doi: 10.1007/s10815-012-9736-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Fabozzi G, Alteri A, Rega E, Starita MF, Piscitelli C, Giannini P, Colicchia A. Morphological assessment on day 4 and its prognostic power in selecting viable embryos for transfer. Zygote. 2016;24(4):477–484. doi: 10.1017/S0967199415000404. [DOI] [PubMed] [Google Scholar]
- 18.ALPHA Scientists In Reproductive Medicine. ESHRE Special Interest Group Embryology Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Reprod Biomed Online. 2011;22(6):632–646. doi: 10.1016/j.rbmo.2011.02.001. [DOI] [PubMed] [Google Scholar]
- 19.Sanchez T, Seidler EA, Gardner DK, Needleman D, Sakkas D. Will noninvasive methods surpass invasive for assessing gametes and embryos? Fertil Steril. 2017;108(5):730–737. doi: 10.1016/j.fertnstert.2017.10.004. [DOI] [PubMed] [Google Scholar]
- 20.Eftekhar M, Aflatoonian A, Mohammadian F, Tabibnejad N. Transfer of blastocysts derived from frozen thawed cleavage stage embryos improved ongoing pregnancy. Arch Gynecol Obstet. 2012;286(2):511–516. doi: 10.1007/s00404-012-2303-9. [DOI] [PubMed] [Google Scholar]
- 21.Wei DM, Liu JY, Sun Y, Shi YH, Zhang B, Chen ZJ, et al. Frozen versus fresh single blastocyst transfer in ovulatory women: a multicentre, randomized controlled trial. Lancet. 2019;393(10178):1310–1318. doi: 10.1016/S0140-6736(18)32843-5. [DOI] [PubMed] [Google Scholar]
- 22.Glujovsky D, Farquhar C. Cleavage-stage or blastocyst transfer: what are the benefits and harms? Fertil Steril. 2016;106(2):244–250. doi: 10.1016/j.fertnstert.2016.06.029. [DOI] [PubMed] [Google Scholar]
- 23.Rijnders PM, Jansen CAM. The predictive value of day 3 embryo morphology regarding blastocyst formation, pregnancy and implantation rate after day 5 transfer following in-vitro fertilization or intracytoplasmic sperm injection. Hum Reprod. 1998;13(10):2869–2873. doi: 10.1093/humrep/13.10.2869. [DOI] [PubMed] [Google Scholar]
- 24.Tannus S, Cohen Y, Henderson S, Ma’mari Al N, Shavit T, Son W-Y, Dahan MH. Fresh transfer of day 5 slow-growing embryos versus deferred transfer of vitrified, fully expanded day 6 blastocysts: which is the optimal approach? Hum Reprod. 2019;34(1):44–51. doi: 10.1093/humrep/dey351. [DOI] [PubMed] [Google Scholar]
- 25.Human Fertility and Embryology Authority (HFEA). Our campaign to reduce multiple births. Available at: https://www.hfea.gov.uk/about-us/our-campaign-to-reduce-multiple-births/. Accessed February 5, 2018.
- 26.Gleicher N, Kushnir VA, Barad DH. Is it time for a paradigm shift in understanding embryo selection? Reprod Biol Endocrinol. 2015;13:3. doi: 10.1186/1477-7827-13-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Dobson Samuel JA, Lao MT, Essam M, Varghese AC, Kannamannadiar J. Effect of transfer of a poor quality embryo along with a top quality embryo on the outcome during fresh and frozen in vitro fertilization cycles. Fertil Steril. 2018;110(4):655–659. doi: 10.1016/j.fertnstert.2018.05.010. [DOI] [PubMed] [Google Scholar]