Reproductive and Developmental Medicine

: 2018  |  Volume : 2  |  Issue : 2  |  Page : 95--99

Clinical Outcomes of Transfer Vitrified-Thawed Day 4, 8-Cell Embryos into Endometrium Prepared for Day 3 Embryos

Ji-Qiang Si, Hannah Ya-Ning Chang, Yan-Ping Kuang, Qi-Feng Lyu 
 Department of Assisted Reproduction, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200001, People's Republic of China

Correspondence Address:
Qi-Feng Lyu
Department of Assisted Reproduction, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, 639 Zhizaoju Road, Shanghai 200001
People's Republic of China


Objective: To determine the clinical outcomes of transfer of day 3, 8-cell embryos into endometrium prepared for day 3 embryos. Methods: This study performed a retrospective analysis of 1,190 retarded embryos. These embryos underwent extended culture in vitro to select and vitrify day 4, 8-cell embryos, followed by 176 frozen embryo transfer cycles (study group), matching 660 transfer cycles with single frozen day 3, 8-cell embryos as the control (control group). Results: The study group achieved successful implantation rates, clinical pregnancy rate, and live-birth rates (20.45%, 15.91%, 14.20%, respectively), although these were lower than that in the control group (30.45%, 27.42%, and 20.91%; P = 0.009, 0.002, and 0.046, respectively). The miscarriage rate was similar to that in the control group (4.55% vs. 3.33%, P > 0.05), and the mean birth weight of the study group was higher than that in the control group (3,556 ± 381 g vs. 3,311 ± 570 g, P = 0.012). Conclusions: Transfer of frozen day 4, 8-cell embryos into endometrial prepared for day 3 embryos can be a new and safe alternative for patients with delayed embryos.

How to cite this article:
Si JQ, Chang HY, Kuang YP, Lyu QF. Clinical Outcomes of Transfer Vitrified-Thawed Day 4, 8-Cell Embryos into Endometrium Prepared for Day 3 Embryos.Reprod Dev Med 2018;2:95-99

How to cite this URL:
Si JQ, Chang HY, Kuang YP, Lyu QF. Clinical Outcomes of Transfer Vitrified-Thawed Day 4, 8-Cell Embryos into Endometrium Prepared for Day 3 Embryos. Reprod Dev Med [serial online] 2018 [cited 2022 Jun 30 ];2:95-99
Available from:

Full Text


Synchronization between competent embryos and endometrial secretory patterns is vital to achieve successful implantation in assisted reproductive technologies. Three prerequisites for successful implantation include an embryo with implantation competency, an endometrium in a receptive state, and actual synchronization between them.[1],[2] Asynchronous cleavage resulted in day 3 embryos with 3, 5, 6, or 7 cells, while others cleaved synchronously to 8 cells.[3] Embryos that were less than 6-cell stage on day 3 or 12-cell stage on day 4 were defined as retarded embryos.[4]

How to effectively utilize day 3 retarded embryos has been a dilemma in the field of assisted reproduction technology. Fresh transfer of day 3 or day 4 retarded embryos resulted in lower implantation rates, and less slow embryos could develop in vitro to usable blastocysts compared with day 3, 8-cell embryos.[5],[6],[7] This phenomenon did not improve even with a refined accessing system that combined cleavage rate and pattern with conventional morphology and degree of fragment.[3],[8],[9] This implied that extended culture time was essential to induce delayed embryos to reach preimplantation status.[10] However, little has been published about clinical outcomes of transfer day 4, 8-cell embryos, which are derived from day 3, 4/5-cell embryos, into endometrium prepared for day 3 embryos.

The objective of this study was to determine whether transfer of frozen day 4, 8-cell embryos, derived from day 3 retarded embryos, into endometrium prepared for day 3 embryos could achieve successful implantation and increased cumulative pregnancy rates.



This study included 843 patients who underwent 916 controlled ovarian hyperstimulation (COH) cycles with 1190 day 3, 4/5-cell embryos (retarded embryos) between January 2015 and May 2017. The protocols for COH included use of a GnRH agonist or a GnRH antagonist protocol or a progestin-primed ovarian hyperstimulation protocol.[11] Delayed embryos on day 3 underwent extended culture to day 4 to select more viable embryos. Embryos that reached 8-cell stage were vitrified on day 4 for frozen embryo transfer (FET), while others were continuously cultured for blastocysts. The extended culture was used for freezing in all FET cycles. We studied the clinical outcomes of 176 transfers with single frozen day 4, 8-cell embryos into endometrium prepared for day 3 embryos (study group) and matched 660 thawing cycles with single day 3, 8-cell embryos as the control (control group). In this study, not all embryos on day 4 were vitrified for subsequent FET, because most embryos that had not reached 8-cell stage on day 4 would be arrested, other than for a few vitrified blastocysts. FET with possible arrested embryos was not used for infertile couples who desired pregnancy. In this study, parameters for analysis included age of women, body mass index (BMI), factors of infertility, number of follicles aspirated, number of retrieved oocytes, method of fertilization, number of transferred embryos, implantation rate (implanted sacs per 100 transferred embryos), clinical pregnancy rate (clinical pregnancies per 100 FET), ongoing pregnancy rate (ongoing pregnancies per 100 FET), live-birth rate, and mean birth weight. This study was approved by the Ethics Committee (Institutional Review Board) of the Ninth People's Hospital of Shanghai (No. 2014-94).

Frozen embryo transfer

Cryopreservation of embryos using a Cryotop (Kitazato, Japan) was performed using established methods.[12] For warming of frozen embryos, the Cryotop was removed from liquid nitrogen and immediately placed in warming solution 1 at 37°C for 1 min. Embryos were then placed in warming solution 2 at room temperature for 3 min. Finally, embryos were washed at room temperature for 5 min with warming solution 3 and 4, respectively. The embryos were cultured at 37°C for at least 2 h before ET. All transfers were performed with a Wallace catheter under ultrasound guidance. Endometrial preparation and FET were performed in natural cycles, stimulation cycles, or hormone therapy cycles.[13],[14]


All statistical analyses were performed with SPSS 13.0 software (IBM SPSS Statistics Inc., Chicago, IL, USA). Dichotomous variables were evaluated with Chi-squared test and t-test or one-way analysis of variance was used for comparison of quantitative variables. P < 0.05 was considered statistically significant.


Results after extended culture of retarded embryos

In this study, 160 embryos on day 3 afternoon, 362 embryos with 8-cell on day 4, and 68 that had developed into blastocysts were cryopreserved, while 1,190 retarded embryos on day 3 underwent extended culture. The general utilization rate was 49.58%. The characteristics of patients and related results after extended culture of 1,190 retarded embryos are shown in [Table 1]. For the basic characteristics of patients, there were not statistically differences in Group A (160 embryos with 8-cell on day 3 afternoon) and Group B (362 embryos with 8-cell on day 4), which are shown in [Table 2].{Table 1}{Table 2}

The characteristics of frozen embryo transfer patients

The characteristics of patients who underwent single frozen embryos transfer, including age, BMI, duration of infertility, and causes of infertility, are shown in [Table 3].{Table 3}

Clinical outcomes of frozen embryo transfer patients

A total of 176 transfer cycles with single frozen day 4, 8-cell embryos achieved successful implantation rates, clinical pregnancy rate, and live-birth rate (20.45%, 15.91%, and 14.20%, respectively), although these were lower than that of day 3, 8-cell embryos (30.45%, 27.42%, and 20.91%, respectively). There was a similar miscarriage rate between the two groups. Among 23 women who delivered singletons in the study group, the mean birth weight was 3,556 ± 381 g (23) which was higher than 3,311 ± 570 g in the control group (134). No adverse outcome was observed in singletons newborns. Related results are shown in [Table 4].{Table 4}


In this study, transfer of day 4, 8-cell embryos into endometrium prepared for day 3 embryos achieved successfully the implantation rates, fetal heart pulse rates, and live-birth rates, which were lower than that in day 3, 8-cell embryos. After successful implantation, the clinical outcomes following transfer of day 4, 8-cell embryos were comparable to day 3, 8-cell embryos. The variance of implantation rates between day 4 8-cell embryos and day 3 8-cell embryos in this study might due to less potential of delayed embryos, because FET enabled more appropriate synchronization between embryonic and endometrial development than fresh transfer.[15],[16] The implantation rate of day 4, 8-cell embryos in this study was higher than the previously reported implantation rate of 2.2% (3/134) after fresh transfer of premorula on day 4 into endometrium prepared for day 4 embryos.[4] The increase could be ascribed to better synchronization between day 4, 8-cell embryos and day 3 endometrium in FET cycles. Our results found that 8-cell embryos on day 4 could not predict the same implantation potential as day 3, 8-cell embryos, although 8-cell embryo is a critical stage during which complete embryonic genome was activated. However, cryopreserved blastocysts had similar implantation potential regardless of whether they developed by days 5, 6, or 7, which could be explained by asynchrony with endometrial receptivity, instead of poorer embryo quality.[10] These suggest that the implantation potential of embryos can be assessed better after an extended culture period, especially to blastocysts. Delayed embryos should be cultured to blastocysts and transfer them in FET cycles, while transfer of frozen day 4, 8-cell embryos into endometrium prepared for day 3 embryos can be an alternative, owing to its predictive precision, and accuracy for potential is less than that for the blastocyst stage.

Day 4, 8-cell embryos had the same safe clinical outcome as day 3, 8-cell embryos after successful implantation. Miscarriage rates of the study embryos were similar to those of the control group, which correlates with chromosome abnormalities.[17] The mean birth weight after transfer of frozen day 4, 8-cell embryo was higher than that of day 3, 8-cell embryos, which may be related to the extended culture period and the lower rate of preterm birth than day 3, 8-cell embryos group.[18] This result indicated that retarded embryos that had developed to the blastocyst stage could yield outcomes comparable to those using normally developed embryos.[7]

Embryos must be at a certain cleavage stage instead of a particular day of development to achieve optimal synchronization, leading us to transfer day 4, 8-cell embryos into endometrium prepared for day 3 embryos. Transfers of frozen embryos on day 3 after rescue intracytoplasmic sperm injection (ICSI) resulted in higher implantation rates than transfers of fresh embryos on day 2 after rescue ICSI (13.33% vs. 4.5%).[1] Similarly, transfer day 6 or 7 blastocysts could achieve better implantation rate in FET cycles than fresh cycles.[2],[10] Slow embryos only underwent extended culture in vitro to day 4 in this study based on the following. First, this provided a retarded embryo with an additional day to cleave and activate the complete embryonic genome during 4-cell to 8-cell period.[19] Second, potential-arrested embryos were identified and almost had the same number of blastomeres at 2 sequential times from day 3 to day 4 (24 h).[20] Third, transfers of day 4, 8-cell embryos could expose the study embryos to the uterine environment for the maximum period before implantation, where some unknown substance essential to embryo development may be present, while not available in an in vitro culture medium.

Factors related to cleavage rate included the culture media, exposure time of embryos outside the incubator, and inadequate maturation of oocytes or sperm-derived oocyte-activating substances.[21],[22],[23] Moreover, nonearly cleavage embryos were prone to result in 4/5-cell and even arrested embryos on day 3.[24] A prospective study with a larger sample and further investigation is needed to illuminate specific mechanisms of embryo growth retardation.

In conclusion, delayed embryos on day 3 should be cultured to blastocysts and transfer of them in FET cycles, while transfer of frozen day 4, 8-cell embryos into endometrium prepared for day 3 embryos in our study can be an alternative protocol, which provides guidance when cleavage embryos were delayed.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Ming L, Liu P, Qiao J, Lian Y, Zheng X, Ren X, et al. Synchronization between embryo development and endometrium is a contributing factor for rescue ICSI outcome. Reprod Biomed Online 2012;24:527-31. doi: 10.1016/j.rbmo.2012.02.001.
2Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Ross R. Contrasting patterns in in vitro fertilization pregnancy rates among fresh autologous, fresh oocyte donor, and cryopreserved cycles with the use of day 5 or day 6 blastocysts may reflect differences in embryo-endometrium synchrony. Fertil Steril 2008;89:20-6. doi: 10.1016/j.fertnstert.2006.08.092.
3Sela R, Samuelov L, Almog B, Schwartz T, Cohen T, Amit A, et al. An embryo cleavage pattern based on the relative blastomere size as a function of cell number for predicting implantation outcome. Fertil Steril 2012;98:650-6.e4. doi: 10.1016/j.fertnstert.2012.05.041.
4Huisman GJ, Fauser BC, Eijkemans MJ, Pieters MH. Implantation rates after in vitro fertilization and transfer of a maximum of two embryos that have undergone three to five days of culture. Fertil Steril 2000;73:117-22.
5Weitzman VN, Schnee-Riesz J, Benadiva C, Nulsen J, Siano L, Maier D, et al. Predictive value of embryo grading for embryos with known outcomes. Fertil Steril 2010;93:658-62. doi: 10.1016/j.fertnstert.2009.032.
6Shapiro BS, Harris DC, Richter KS. Predictive value of 72-hour blastomere cell number on blastocyst development and success of subsequent transfer based on the degree of blastocyst development. Fertil Steril 2000;73:582-6.
7Zhao P, Li M, Lian Y, Zheng X, Liu P, Qiao J, et al. The clinical outcomes of day 3 4-cell embryos after extended in vitro culture. J Assist Reprod Genet 2015;32:55-60. doi: 10.1007/10815-014-0361-6.
8Mizobe Y, Oya N, Iwakiri R, Yoshida N, Sato Y, Miyoshi K, et al. Effects of early cleavage patterns of human embryos on subsequent in vitro development and implantation. Fertil Steril 2016;106:348-53.e2. doi: 10.1016/j.fertnstert.2016.04.020.
9Yang ST, Shi JX, Gong F, Zhang SP, Lu CF, Tan K, et al. Cleavage pattern predicts developmental potential of day 3 human embryos produced by IVF. Reprod Biomed Online 2015;30:625-34. doi: 10.1016/j.rbmo.2015.02.008.
10Hiraoka K, Hiraoka K, Miyazaki M, Fukunaga E, Horiuchi T, Kusuda T, et al. Perinatal outcomes following transfer of human blastocysts vitrified at day 5, 6 and 7. J Exp Clin Assist Reprod 2009;6:4.
11Kuang Y, Chen Q, Fu Y, Wang Y, Hong Q, Lyu Q, et al. Medroxyprogesterone acetate is an effective oral alternative for preventing premature luteinizing hormone surges in women undergoing controlled ovarian hyperstimulation for in vitro fertilization. Fertil Steril 2015;104:62-70.e3. doi: 10.1016/j.fertnstert.2015.03.022.
12Cobo A, de los Santos MJ, Castellò D, Gámiz P, Campos P, Remohí J, et al. Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: Evaluation of 3,150 warming cycles. Fertil Steril 2012;98:1138-460. doi: 10.1016/j.fertnstert.2012.07.1107.
13Du T, Chen H, Fu R, Chen Q, Wang Y, Mol BW, et al. Comparison of ectopic pregnancy risk among transfers of embryos vitrified on day 3, day 5, and day 6. Fertil Steril 2017;108:108-160. doi: 10.1016/j.fertnstert.2017.05.027.
14Mao X, Zhang J, Chen Q, Kuang Y, Zhang S. Short-term copper intrauterine device placement improves the implantation and pregnancy rates in women with repeated implantation failure. Fertil Steril 2017;108:55-610. doi: 10.1016/j.fertnstert.2017.05.014.
15Richter KS, Shipley SK, McVearry I, Tucker MJ, Widra EA. Cryopreserved embryo transfers suggest that endometrial receptivity may contribute to reduced success rates of later developing embryos. Fertil Steril 2006;86:862-6. doi: 10.1016/j.fertnstert.2006.02.114.
16Gomaa H, Baydoun R, Sachak S, Lapana I, Soliman S. Elective single embryo transfer: Is frozen better than fresh? JBRA Assist Reprod 2016;20:3-7. doi: 10.5935/1518-0557.20160002.
17Dekel-Naftali M, Aviram-Goldring A, Litmanovitch T, Shamash J, Yonath H, Hourvitz A, et al. Chromosomal integrity of human preimplantation embryos at different days post fertilization. J Assist Reprod Genet 2013;30:633-48. doi: 10.1007/s10815-013-9988-y.
18Zhu J, Lin S, Li M, Chen L, Lian Y, Liu P, et al. Effect of in vitro culture period on birthweight of singleton newborns. Hum Reprod 2014;29:448-54. doi: 10.1093/humrep/det460.
19Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 1988;332:459-61. doi: 10.1038/332459a0.
20Wu HY, Lin YC, Lan KC. Twin live births following transfer using eight-cell cleavage stage embryos on day 4 with developmental arrest. Taiwan J Obstet Gynecol 2011;50:381-4. doi: 10.1016/j.tjog.2010.06.001.
21Magli MC, Gianaroli L, Fiorentino A, Ferraretti AP, Fortini D, Panzella S, et al. Improved cleavage rate of human embryos cultured in antibiotic-free medium. Hum Reprod 1996;11:1520-4.
22Zhang JQ, Li XL, Peng Y, Guo X, Heng BC, Tong GQ, et al. Reduction in exposure of human embryos outside the incubator enhances embryo quality and blastulation rate. Reprod Biomed Online 2010;20:510-5. doi: 10.1016/j.rbmo.2009.12.27.
23Tesarik J, Mendoza C, Greco E. Paternal effects acting during the first cell cycle of human preimplantation development after ICSI. Hum Reprod 2002;17:184-9.
24Lee MJ, Lee RK, Lin MH, Hwu YM. Cleavage speed and implantation potential of early-cleavage embryos in IVF or ICSI cycles. J Assist Reprod Genet 2012;29:745-50. doi: 10.1007/s10815-012-9777-z.