Comparison of pregnancy outcomes between 4th day morula and 5th day blastocyst after embryo transfer:a retrospective cohort study

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Methods From September 2017 to September 2020, 1963 fresh transfer cycles that underwent early follicular phase extra-long protocol for assisted conception in our fertility center were divided into D4 (324 cases) and D5 (1639 cases) groups based on the number of days of in vitro culture after fertilization, and the general basic information, clinical and embryonic conditions of patients in both groups were compared. To compare the differences in pregnancy outcomes, the D4 and D5 groups were further divided into groups A and B based on single and double embryo transfer. Results In single embryo transfer, there was no significant difference between groups D4A and D5A ( P > 0.05). In double embryo transfer, group D4B had a lower newborn birthweight and a larger proportion of low birthweight infants ( P < 0.05). The preterm delivery rate, twin delivery rate, cesarean delivery rate, and percentage of low birthweight infants were lower in the D5A group than in the D5B group ( P < 0.05). Conclusion When factors such as working life and hospital holidays are being considered, D4 morula transfer may be a good alternative to D5 blastocyst transfer. Given the in vitro fertilization/intracytoplasmic sperm injection(IVF/ICSI) success rate and risk of twin pregnancy, D4 morula transfer requires an adapted decision between single and double embryo transfer, although a single blastocyst transfer is recommended for the D5 transfer in order to decrease the twin pregnancy rate. Early follicular phase prolonged protocol Morula Blastocyst Assisted reproduction IVF/ICSI outcome Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The advancement of Assisted Reproductive Technology (ART), including the improvement of embryo culture media and the optimization of culture techniques, has enabled the development of embryos into blastocysts in vitro[ 1 ]. Numerous studies have demonstrated that compared to cleavage stage embryo, blastocysts have greater implantation and developmental potential[ 2 – 4 ]. As a result, many centers have shifted their transfer strategy from cleavage embryo transfers on day 3 to blastocyst transfers on day 5. Due to the lack of typical morphological markers[ 5 ], morulae on day 4 have long been understudied in humans, and the corresponding value of morula transfers has frequently been overlooked. However, it has recently been demonstrated that the densification process at the morula stage involves multiple self-correcting mechanisms that may be critical in determining embryo quality[ 6 ], as well as being critical for blastocyst formation, establishment of the first cell lineage, and the entire developmental process[ 7 , 8 ], which means that morula transfer may also be a feasible transfer strategy. In addition, studies have shown that the use of ART techniques, particularly dual embryo transfer, significantly increases the rate of twin pregnancies[ 9 ], which can increase the risk of pregnancy and seriously endanger the mother's and child's life and health[ 10 ]. To reduce the number of twin pregnancies while successfully assisting pregnancy, our center prefers to use a single blastocyst transfer strategy. However, many patients choose D4 morula transfer and dual embryos transfer due to factors such as time constraints and the desire for a high success rate. In this study, we compared the pregnancy outcomes of D4 morula and D5 blastocyst, single embryo and double embryos using the early follicular stage extra-long protocol, and investigated the benefits and drawbacks of various transfer strategies in order to provide a foundation for individualized transfer protocols. Materials and Methods Research Subject Retrospective analysis of 1963 cycles undergoing early follicular phase ultra-long protocol assisted conception at our fertility center from September 2017 to September 2020, divided into D4 (324 cases) and D5 (1639 cases) groups based on the number of days of in vitro culture after fertilization, and further divided D4 and D5 into groups A and B based on single and double embryo transfer to compare their outcomes, as show in Fig. 1 . To compare D4A with D4B, D5A with D5B, D4A with D5A, D4B with D5B, and to clarify the differences in pregnancy outcomes between D4 morula and D5 blastocyst, as well as single embryos and double embryos for transfer. Inclusion criteria: 1. patient age between 20 and 42 years; 2. early follicular phase ultra-long protocol was used to assist pregnancy; 3. number of day3(D3) high-quality embryos ≥ 3. Exclusion criteria:1. uterine pathologies such as adenomyosis, submucous myomas, severe uterine adhesions, uterine malformations; 2. history of adverse pregnancy such as recurrent miscarriage, stillbirth and multiple inductions; 3. chromosomal abnormalities. Assisted reproductive process To limit the impact of diverse IVF regimens on pregnancy outcomes, all cycles in this study were completed using the early follicular phase extra-long regimen, which was routinely utilized to facilitate pregnancy in our center. On days 2–5 of menstruation, 3.75 mg of gonadotropin releasing hormone agonist (GnRH-a) was administered intramuscularly to suppress pituitary function. After 28–38 days, an ultrasound and endocrine examination would be conducted. When endometrial thickness was less than 5mm,follicle stimulating hormone(FSH) was less than 5mIU/ml, luteinizing hormone (LH) was less than 5mIU/ml, and estradiol(E2) was less than 50pg/ml, the pituitary gland was considered completely downregulated. At this time, gonadotropin (Gn) would be used to initiate ovulation, and the type and dosage of Gn would be adjusted continuously based on ultrasound and serum sex hormone level. An intramuscular injection of 4000–10,000 IU of chorionic gonadotropin was given when at least one follicle measures at least 20 mm in diameter or when two follicles measure at least 18 mm in diameter. Oocytes were carried out under vaginal ultrasonography guidance 36 hours later, and embryos were cultivated after in vitro fertilization of the oocytes, embryo transfer was performed under abdominal ultrasound guidance. Serum human chorionic gonadotropin(hCG) levels were examined two weeks after transfer to see if there was a pregnancy. If the hCG level was positive, an ultrasound two weeks later revealed an intrauterine gestational sac, indicating a clinical pregnancy. Embryo culture and classification The quality of embryos was closely related to clinical outcomes[ 11 , 12 ], and we had strict requirements for embryo quality in order to control the interference of different embryo quality on study outcomes. In this study, cycles with at least three high-quality embryos on D3 were chosen for grouping. All embryos were cultured in order, with high-quality D3 embryos being transferred to blastocyst culture. The culture was continued until the fourth or fifth days, after which the best 1–2 embryos were chosen for transplantation on D4 and D5, as shown in Fig. 2 . According to the Istanbul consensus[ 13 ], the embryo quality was graded according to the size, uniformity and amount of nucleus-free fragments of the D3 oocyte cleavage spheres. A high-quality embryo should have 6–10 cells, uniform blastomere size, few fragments, and no multinucleation on D3 after fertilization. The agreement on Day 4 was that an optimum embryo at this stage would be compacted or compacting, the compaction should include virtually all the embryo volume and would have entered a fourth round of cleavage.[ 14 ]. Blastocyst scoring used the Gardner scale[ 15 , 16 ], which scored the degree of blastocyst expansion, inner cell mass, and trophectoderm cell development, respectively. Blastocysts with tightly arranged cells, almost free of debris and not yet hatched were usually considered as the most suitable blastocysts for transfer. Observation indicators Each group was examined for demographic characteristics, clinical and laboratory outcomes, pregnancy outcomes and other indicators. The primary outcome indicators were the clinical pregnancy rate (number of clinical pregnancy cycles/number of transplantation cycles×100%), miscarriage rate (number of miscarriage cycles/number of clinical pregnancy cycles×100%), and live birth rate (number of live births/number of transplantation cycles×100%)[ 17 ]. Secondary outcome indicators were the preterm birth rate, normal birth rate, cesarean delivery rate, neonatal weight, and other observations. As a major complication of ovulation promotion, we also compared the incidence of ovarian hyperstimulation syndrome (OHSS) in each group. Statistics Analysis For statistical analysis, SPSS 25.0 software (IBM Corp, Armonk, NY, USA) was utilized. The data was examined for normality. The average value of normally distributed measures was expressed as the mean ± standard deviation, and the T-Test for two independent samples was used to compare groups. While non-normally distributed measures were expressed as M (P25, P75), and the Wilcoxon rank sum test was used to compare groups. Count data was reported as percentages, and group comparisons were performed using the Chi-Square Test or Fisher-Yates-Test. P values < 0.05 were considered significant. Results General information Among 1963 cycles, we transferred morulae in 324 cycles and blastocysts in 1639 cycles. Table 1 summarizes the demographic characteristics of the D4 and D5 groups. There were no significant differences between these two groups in terms of age, Body mass index(BMI), years of infertility, Anti-Müllerian Hormone(AMH), basal FSH, or etiology of infertility. As shown in Table 2 , with respect to clinical and laboratory outcomes, there were no differences in LH, E2, Progesterone(P) after downregulation, and LH, P, endometrial thickness at hCG triggering, normal fertilization rate and quality embryo rate. However, the E2 at hCG triggering, and the number of retrieved oocytes, fertilized oocytes, D3 embryos, and quality D3 embryos were lower in the D4 group than in the D5 group, while the percentage of single embryo transfer was higher in the D5 group than in the D4 group. Because single and double embryo transfer rates differed between D4 and D5, we further separated D4 and D5 into D4A, D4B, D5A, D5B according to the number of embryos transferred. Table 1 Comparison of demographic characteristics in two groups Variables D4 group(n = 324) D5 group(n = 1639) P value Age (years) 31.36 ± 4.42 31.18 ± 4.27 0.325 BMI (kg/m2) 21.79 ± 3.19 21.96 ± 3.12 0.406 Duration of infertility (years) 3.34 ± 2.43 3.34 ± 2.39 0.911 AMH (ng/ml) 3.68 ± 3.10 4.26 ± 3.38 0.670 Basic FSH (mIU/mL) 7.99 ± 2.98 7.93 ± 4.89 0.215 Etiology of infertility (%) Tubal 47.53(154/324) 46.31(759/1639) 0.687 PCOS 10.80(35/324) 14.15(232/1639) 0.108 Endometriosis 2.47(8/324) 1.65(27/1639) 0.307 Ovarian 5.86(19/324) 4.09(67/1639) 0.153 Combined 11.73(38/324) 11.65(191/1639) 0.969 Other 21.60(70/324) 22.15(363/1639) 0.830 * BMI = Body mass index, AMH = Anti-Müllerian Hormone, FSH = Follicle stimulating hormone, PCOS = Polycystic ovary syndrome Table 2 Comparison of clinical and laboratory outcomes between the two groups Variables D4 group(n = 324) D5 group(n = 1639) P value LH after deregulation (IU/L) 0.44 ± 0.39 0.55 ± 2.23 0.289 E2 after deregulation (pg/ml) 24.48 ± 12.82 27.83 ± 121.13 0.449 P after deregulation (nmol/L) 0.51 ± 0.42 0.56 ± 2.11 0.474 LH on trigger day (IU/L) 0.73 ± 0.56 0.72 ± 0.64 0.826 E2 on trigger day (pg/ml) 1833.07 ± 1023.23 2400.88 ± 1168.62 0.004 P on trigger day (nmol/L) 0.63 ± 0.32 1.44 ± 26.18 0.401 Endometrial thickness on trigger day (mm) 10.70 ± 2.18 11.17 ± 2.25 0.122 NO. of retrieved oocytes 9.5(7,14) 13(10,16) 0.001 NO. of fertilized oocytes 7(5,10) 9(7,12) 0.001 NO. of D3 embryos 7(5,10.75) 9(7,12) 0.001 NO. of quality D3 embryos 5(4,8) 7(5,10) 0.001 Normal fertilization (%) 75.5(64.29,85.71) 75(62.5,85.71) 0.223 Quality embryo (%) 83.33(71.43,1) 85(68.75,1) 0.292 Single embryo transfer (%) 33.64(109/324) 87.37(1432/1639) 0.001 Double embryo transfer (%) 66.36(215/324) 12.63(207/1639) 0.001 * LH = Luteinizing hormone, E2 = serum estradiol, P = serum progesterone Comparison of pregnancy outcomes D4A and D5A groups at single embryo transfer In single embryo transfer, we have performed 109 cycles of morula transfers and 1432 cycles of blastocyst transfers. The clinical pregnancy rate, miscarriage rate, live birth rate, preterm birth rate, overdue birth rate, twin birth rate, normal birth rate, cesarean delivery rate, neonatal birthweight, proportion of fetal macrosomia, proportion of low birthweight infants, and incidence of OHSS were all significantly the same between the D4A group and D5A group, as shown in Table 3 . Table 3 Comparison of pregnancy outcomes between the D4A and D5A groups at single embryo transfer Variables D4A group(n = 109) D5A group(n = 1432) P value Clinical pregnancy (%) 59.63(65/109) 64.73(927/1432) 0.284 Miscarriage (%) 16.92(11/65) 13.05(121/927) 0.374 Live birth (%) 49.54(54/109) 56.28(806/1432) 0.346 Preterm birth (%) 1.83(2/109) 4.12(59/1432) 0.355 Overdue birth (%) 3.67(4/109) 3.63(52/1432) 1.000 Twin birth (%) 0(0/65) 1.83(17/927) 0.620 Normal birth (%) 42.59(23/54) 47.89(386/806) 0.450 Cesarean (%) 57.41(31/54) 52.11(420/806) 0.450 Newborn birthweight(g) 3209.56 ± 429.69 3260.10 ± 546.69 0.506 Fetal Macrosomia (%) 3.70(2/54) 5.98(49/820) 0.696 Low birthweight infant (%) 5.56(3/54) 7.44(61/820) 0.806 OHSS incidence (%) 0.92(1/109) 1.33(19/1432) 1.000 * Unknown birthweight of D5A babies for 3 cycles D4B group and the D5B group at double embryo transfer In double embryo transfer, we have performed 215 cycles of morula transfers and 207 cycles of blastocyst transfers. There were no significant differences in clinical pregnancy rate, miscarriage rate, live birth rate, preterm birth rate, overdue birth rate, twin birth rate, normal birth rate, cesarean section rate, proportion of fetal macrosomia and OHSS incidence between the D4B and D5B group populations, but the birthweight of newborns in the D4B group was lower than that in the D5B group and the proportion of low birthweight infants was higher than that in the D5B group, as shown in Table 4 . Table 4 Comparison of pregnancy outcomes between the D4B and D5B groups at double embryo transfer Variables D4B group(n = 215) D5B group(n = 207) P value Clinical pregnancy (%) 69.30(149/215) 65.22(135/207) 0.371 Miscarriage (%) 11.41(17/149) 8.15(11/135) 0.357 Live birth (%) 61.40(132/215) 59.90(124/207) 0.754 Preterm birth (%) 14.88(32/215) 13.04(27/207) 0.586 Overdue birth (%) 2.33(5/215) 2.90(6/207) 0.712 Twin birth (%) 36.24(54/149) 34.81(47/135) 0.802 Normal birth (%) 31.06(41/132) 27.42(34/124) 0.522 Cesarean (%) 68.94(91/132) 72.58(90/124) 0.522 Newborn birthweight(g) 2777.33 ± 652.96 2915.59 ± 602.46 0.039 Fetal Macrosomia (%) 3.23(6/186) 2.34(4/171) 0.823 Low birthweight infant (%) 34.95(65/186) 21.05(36/171) 0.004 OHSS incidence (%) 1.86(4/215) 1.45(3/207) 1.000 Single and double embryo transfer in group D4 Among 324 D4 morula transfer cycles, we transferred one embryo in 109 cycles and two embryos in 215 cycles. There were no significant differences in clinical pregnancy rate, miscarriage rate, overdue delivery rate, normal delivery rate, cesarean delivery rate, proportion of fetal macrosomia and OHSS incidence between patients in D4A and D4B groups, but the live birth rate, preterm delivery rate, twin birth rate and proportion of low birthweight infants were lower in D4A group than in D4B group; neonatal birthweight was higher than in D4B group. as shown in Table 5 and Fig. 3 . Table 5 Comparison of single and double embryo transfer assisted pregnancy outcomes in the D4 group Variables D4A group(n = 109) D4B group(n = 215) P value Clinical pregnancy (%) 59.63(65/109) 69.30(149/215) 0.082 Miscarriage (%) 16.92(11/65) 11.41(17/149) 0.271 Live birth (%) 49.54(54/109) 61.40(132/215) 0.037 Preterm birth (%) 1.83(2/109) 14.88(32/215) 0.001 Overdue birth (%) 3.67(4/109) 2.33(5/215) 0.735 Twin birth (%) 0(0/65) 36.24(54/149) 0.001 Normal birth (%) 42.59(23/54) 31.06(41/132) 0.133 Cesarean (%) 57.41(31/54) 68.94(91/132) 0.133 Newborn birthweight(g) 3209.56 ± 429.69 2777.33 ± 652.96 0.001 Fetal Macrosomia (%) 3.70(2/54) 3.23(6/186) 1.000 Low birthweight infant (%) 5.56(3/54) 34.95(65/186) 0.001 OHSS incidence (%) 0.92(1/109) 1.86(4/215) 0.667 Single and double embryo transfer in group D5 Among 1639 D5 blastocyst transfer cycles, we transferred one embryo in 1432 cycles and two embryos in 207 cycles. There were no significant differences in clinical pregnancy rate, miscarriage rate, live birth rate, overdue birth rate, proportion of fetal macrosomia, or incidence of OHSS between patients in the D5A and D5B groups. However, the preterm birth rate, twin birth rate, cesarean section rate, and proportion of low birthweight infants were lower in the D5A group; the normal birth rate and neonatal birthweight were higher in the D5A group, as shown in Table 6 and Fig. 4 . Table 6 Comparison of single and double embryo transfer assisted pregnancy outcomes in the D5 group Variables D5A group(n = 1432) D5B group(n = 207) P value Clinical pregnancy (%) 64.73(927/1432) 65.22(135/207) 0.892 Miscarriage (%) 13.05(121/927) 8.15(11/135) 0.107 Live birth (%) 56.28(806/1432) 59.90(124/207) 0.326 Preterm birth (%) 4.12(59/1432) 13.04(27/207) 0.001 Overdue birth (%) 3.63(52/1432) 2.90(6/207) 0.594 Twin birth (%) 1.83(17/927) 34.81(47/135) 0.001 Normal birth (%) 47.89(386/806) 27.42(34/124) 0.001 Cesarean (%) 52.11(420/806) 72.58(90/124) 0.001 Newborn birthweight(g) 3260.10 ± 546.69 2915.59 ± 602.46 0.007 Fetal Macrosomia (%) 5.98(49/820) 2.34(4/171) 0.055 Low birthweight infant (%) 7.44(61/820) 21.05(36/171) 0.001 OHSS incidence (%) 1.33(19/1432) 1.45(3/207) 1.000 * Unknown birthweight of D5A babies for 3 cycles Discussion The selection of embryos with optimal implantation potential for transfer is a critical step in optimizing pregnancy outcomes in vitro fertilization-embryo transfer(IVF-ET) treatment[ 18 ]. Studies have shown that embryo culture is a meritocratic process and that a large number of embryonic genes are activated and expressed on day 4 compared to day 3 transfer, indicating that D4 morula development is an essential screen for future embryo survival[ 8 , 19 , 20 ]. Sang et al. examined morula and maternal endometrium transcriptome datasets to identify the expression of receptor genes expressed by morula and corresponding ligand genes in endometrium, and to further clarify the details of communication between embryo and mother[ 21 ].Human embryos enter the uterine cavity on day 4 after fertilization, when the uterus provides a different nutritional environment than the oviduct, uterine contractility is reduced, and synchronization of embryos with the endometrium is improved[ 22 ], giving D4 morula and D5 blastocyst transfers an advantage over D3 cleavage embryo. Two main morphogenetic phases build the basic architecture of the pre-implantation embryo: the formation of the morula and of the blastocyst[ 23 ]. Embryo culture up to day 5 may aid in the rejection of unsuitable embryos, but it is unknown whether D5 blastocyst is superior to D4 morula in this regard. However, compared to D5 blastocyst, transfer of D4 morula reduces the exposure time of embryos in vitro, reduces the impact of environmental factors on embryos in vitro[ 24 , 25 ], reduces the risk of developmental arrest and embryo quality decline[ 26 ], and makes better use of embryos. Patients undergoing blastocyst culture may have fewer total embryos available for transfer, or no embryos available for transfer and transfer cycles cancelled. Combining the advantages and risks, the outcome of transferring D4 morula versus D5 blastocyst has not been clearly established. The results of this study show that there are no significant differences in general baseline conditions such as age and BMI between the D4 and D5 groups, but the E2 at hCG triggering, of retrieved oocytes, number of fertilized oocytes, number of D3 embryos, number of quality D3 embryos, and percentage of single embryo transfer are lower in the D4 group than in the D5 group, and the percentage of double embryo transfer is higher than in the D5 group. This could be related to our transfer strategy, in which we choose single blastocyst transfer as the primary strategy when the overall circumstances are favorable and the blastocyst-raising success rate is high. In contrast, we preferentially transfer one or two D4 morulae when there are few embryos, an anticipated delay in embryo growth on day 4, a risk of cycle cancellation, or a conflict in schedule for day 5 transfer. Therefore, the clinical and embryonic profile of the D5 group may be slightly better than that of the D4 group, while the single embryo transfer rate is significantly higher than that of the D4 group. This study performed additional stratified analysis of single and double embryo transfer in order to rule out the influence of embryo transfer quantity on pregnancy outcome. Kang et al. performed a retrospective analysis of 271 cycles and discovered no significant changes in clinical pregnancy, live birth, or miscarriage rates between D4 and D5 single embryo transfers, despite the fact that the miscarriage rate was somewhat higher in D4. They suggested that the results may have been affected by sample size constraints and that additional samples are required for validation[ 27 ]. In a retrospective study of 427 GnRH antagonist protocol cycles, Li et al. found no significant difference in clinical pregnancy rate, live birth rate, or miscarriage rate between D4 and D5, however the full-term birth rate was greater in the D4 group than in the D5 group. They also presented an embryo quality assessment system for D4 to help with embryo selection[ 28 ]. Simopoulo et al. reviewed 6 prospective studies and 9 retrospective cohort studies, and found that the rates of clinical pregnancy, sustained pregnancy/live birth, cancelled pregnancy, and miscarriage of D4 and D5 transplantation had no statistical difference. Additionally, statistically speaking, D4 had a much lower preterm birth rate than D5[ 18 ]. In our research, we retrospectively analyzed 1963 cycles using the early follicular phase extra-long protocol and further stratified D4 and D5 based on the number of embryo transfers, finding that there was no significant difference in D4 transfer and D5 transfer outcomes for single embryo transfer, and the results were generally consistent with the previous studies. Moreover, there was no significant difference in the primary pregnancy outcome between D4 and D5 double embryo transfers. Although, the birthweight of babies in the D4B group was lower than that of babies in the D5B group, and the number of babies with low birthweight was higher in the D4B group than in the D5B group. Additionally, we found a higher rate of cesarean delivery in our study for both D4 and D5. The reasons for this are diverse. On the one hand, the increased rate of multiple births in assisted reproduction increases the probability of difficult deliveries[ 20 , 29 ]. On the other hand, the relatively advanced maternal age[ 30 ], the mental stress of late pregnancy, and the difficulty in obtaining a fetus result in the request for cesarean delivery by the mother and her family. In response, the indications for cesarean delivery were relaxed clinically, which led to an increase in the rate of cesarean delivery. In order to find the optimal transfer strategy, we compared the outcomes of single and double embryo transfer on days D4 and D5, respectively, and found that the live birth rate, preterm birth rate, twin birth rate, and low birthweight infant percentage were lower in the D4A group than in the D4B group at D4 transfer, while the newborn birthweight was higher than in the D4B group. In addition, the clinical pregnancy rate in the D4A group was lower than that in the D4B group, and the normal birth rate was higher than that in the D4B group. Although the differences were not statistically significant, they still showed a corresponding trend, which may be related to the small sample size of D4 transplants in this study, and further increasing the sample size may make the above differences significant. The results of our study suggest that the outcome of double embryo transfer in D4 will be better than single embryo transfer in terms of success rate. According to Duffy et al, twin pregnancies lead to the occurrence of outcomes such as preterm delivery, cesarean delivery, and low birthweight infants[ 10 ], thus, we primarily blamed the difference in the twin pregnancy rate for the difference in secondary pregnancy outcomes between the D4A and D4B groups. In our study, double embryo transfer in D4 row significantly increased the twin rate and increased the risk of pregnancy such as preterm delivery and low birthweight. Several studies have shown that reducing the number of embryos transferred helps to reduce the occurrence of multiple pregnancies as well as maternal and infant adverse pregnancy outcomes[ 9 , 31 ], and we also tend to control the number of embryos transferred in our transfer strategy and promote single embryo transfer to reduce the risk of twin pregnancies. In the D5 transfer, there was no significant difference in the main pregnancy outcomes between single and twin embryo transfer, and the preterm delivery rate, twin birth rate, cesarean delivery rate, and low birthweight infant ratio in the D5A group were lower than those in the D5B group, while the normal delivery rate and neonatal birthweight were higher than those in the D5B group, indicating that single embryo transfer would be more advantageous in the D5 transfer. As an important complication of IVF, OHSS can cause increased vascular permeability, ascites, and even life-threatening respiratory failure, and the increased number of embryos obtained can increase the risk of OHSS[ 32 ]. Xin et al. found that when patients at high risk for OHSS underwent embryo transfer, the incidence of OHSS was lower in the D5 transfer group than in the D3 group. They hypothesized that this may be because the delayed culture gave doctors more time to monitor and treat patients' symptoms as well as the lower rate of multiple births with D5 transfer[ 33 ].However, no studies have found a link between D4 or D5 transfer and OHSS. In our study, no significant differences were seen in the incidence of OHSS under different transplantation strategies, which may be due to the limitations of the retrospective study model we adopted and the inadequate sample size, and further studies are needed to explore the association in the future. Conclusion Our findings suggest that D4 morula embryo transfer does not reduce IVF success in the early follicular phase extra-long protocol. The D4 embryo transfer can be a good alternative to D5 blastocyst transfer in case of holiday or work schedule conflicts. For D4 morula embryo transfer, single and double embryo transfer should be selected individually considering the success rate and the risk of twin pregnancy, while single blastocyst transfer is more recommended for D5 blastocyst transfer to reduce the rate of double pregnancy and optimize pregnancy outcome. The sample size of D4 in this study was relatively small, and the study didn’t analyze the quality and grading of embryos on D4 and D5 days, and didn’t compare pregnancy complications and subsequent neonatal mental and physical development. Further prospective randomized controlled studies are needed to verify the benefits of the above transfer strategies. Abbreviations Day 4 D4 Day 5 D5 In vitro fertilization/intracytoplasmic sperm injection IVF/ICSI Assisted Reproductive Technology ART Day 3 D3 Gonadotropin releasing hormone agonist GnRH-a Follicle stimulating hormone FSH Luteinizing hormone LH Estradiol E2 Gonadotropin Gn Human chorionic gonadotropin hCG Ovarian hyperstimulation syndrome OHSS Body mass index BMI in vitro fertilization-embryo transfer IVF-ET Polycystic ovary syndrome PCOS Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University ( 2021-K-381-01),and registered on December 31, 2021, retroactive registration.As a retrospective study,this study passed the informed consent exemption application by the Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University. Consent for publication Not applicable. Availability of data and materials All authors had full access to the data and materials. Data is provided in the supplementary information file. If anyone wants to obtain data from this study, they can contact the corresponding author. Competing interests All authors declare that they have no competing interests. Funding The National Key Research and Development Program (grant no. 2018YFC1003103) Authors’ contributions Junzhao Zhao conceived the idea and wrote the proposal for the study. Yiqun Sun collected the data, analyzed the data, interpreted the data and drafted the manuscript. Qi Shen and Haitao Xi analyzed and interpreted the data. Liucai Sui and Yanghua Fu processed tables and figures. All authors read and approved the final manuscript. Acknowledgements We would like to thank Reproductive Center of the Second Affiliated Hospital of Wenzhou Medical University for support. References Gardner, D.K. and M. Lane, Culture and selection of viable blastocysts: a feasible proposition for human IVF? Human reproduction update, 1997. 3 (4): p. 367-82. De Croo, I., P. De Sutter, and K. Tilleman, A stepwise approach to move from a cleavage-stage to a blastocyst-stage transfer policy for all patients in the IVF clinic. Hum Reprod Open, 2020. 2020 (3): p. hoaa034. Glujovsky, D., et al., Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology. Cochrane Database Syst Rev, 2016(6): p. CD002118. Long, X., et al., Pregnancy Outcomes of Single/Double Blastocysts and Cleavage Embryo Transfers: a Retrospective Cohort Study of 24,422 Frozen-Thawed Cycles. Reprod Sci, 2020. 27 (12): p. 2271-2278. Coticchio, G., et al., The enigmatic morula: mechanisms of development, cell fate determination, self-correction and implications for ART. Hum Reprod Update, 2019. 25 (4): p. 422-438. Lagalla, C., et al., Embryos with morphokinetic abnormalities may develop into euploid blastocysts. Reprod Biomed Online, 2017. 34 (2): p. 137-146. Brison, D.R., R.G. Sturmey, and H.J. Leese, Metabolic heterogeneity during preimplantation development: the missing link? Hum Reprod Update, 2014. 20 (5): p. 632-40. Vassena, R., et al., Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development. Development, 2011. 138 (17): p. 3699-709. Sunderam, S., et al., Assisted Reproductive Technology Surveillance - United States, 2017. Morbidity and mortality weekly report. Surveillance summaries (Washington, D.C. : 2002), 2020. 69 (9): p. 1-20. Duffy, C.R., Multifetal Gestations and Associated Perinatal Risks. NeoReviews, 2021. 22 (11): p. e734-e746. Zhao, Y.Y., Y. Yu, and X.W. Zhang, Overall Blastocyst Quality, Trophectoderm Grade, and Inner Cell Mass Grade Predict Pregnancy Outcome in Euploid Blastocyst Transfer Cycles. Chin Med J (Engl), 2018. 131 (11): p. 1261-1267. Wang, L., et al., IVF embryo choices and pregnancy outcomes. Prenat Diagn, 2021. 41 (13): p. 1709-1717. Medicine, A.S.i.R. and E.S.I.G.o. Embryology, The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Human reproduction (Oxford, England), 2011(6): p. 1270-83. Tao, J., et al., The neglected morula/compact stage embryo transfer. Hum Reprod, 2002. 17 (6): p. 1513-8. Gardner, D.K. and W.B. Schoolcraft, In vitro culture of human blastocyst. Towards Reproductive Certainty Infertility & Genetics Beyond, 1999. Gardner, D.K. and W.B. Schoolcraft, Culture and transfer of human blastocysts. Curr Opin Obstet Gynecol, 1999. 11 (3): p. 307-11. Zhang, H.N., et al., Comparison of Pregnancy Outcomes Between Single-Morula Embryo Transfer and Single-Blastocyst Transfer in Fresh IVF/ICSI Cycles. Med Sci Monit, 2021. 27 : p. e928737. Simopoulou, M., et al., Should the flexibility enabled by performing a day-4 embryo transfer remain as a valid option in the IVF laboratory? A systematic review and network meta-analysis. J Assist Reprod Genet, 2019. 36 (6): p. 1049-1061. Strelchenko, N., et al., Morula-derived human embryonic stem cells. Reproductive biomedicine online, 2004. 9 (6): p. 623-9. Hirate, Y., et al., Polarity-dependent distribution of angiomotin localizes Hippo signaling in preimplantation embryos. Current biology : CB, 2013. 23 (13): p. 1181-94. Sang, L., et al., Atlas of receptor genes expressed by the bovine morula and corresponding ligand-related genes expressed by uterine endometrium. Mol Reprod Dev, 2021. 88 (10): p. 694-704. Lesny, P., et al., Uterine junctional zone contractions during assisted reproduction cycles. Human reproduction update, 1998. 4 (4): p. 440-5. Maitre, J.L., Mechanics of blastocyst morphogenesis. Biol Cell, 2017. 109 (9): p. 323-338. Morbeck, D.E., Air quality in the assisted reproduction laboratory: a mini-review. Journal of Assisted Reproduction and Genetics, 2015. 32 (7): p. 1019-1024. Braga, D.P., et al., The importance of the cleavage stage morphology evaluation for blastocyst transfer in patients with good prognosis. J Assist Reprod Genet, 2014. 31 (8): p. 1105-10. Simon J. Phillips, 2 Nicola L. Dean,1 William M. Buckett,1 and Seang Lin Tan, Consecutive transfer of day 3 embryos and of day 5-6 blastocysts increases overall pregnancy rates associated with blastocyst culture. Assisted Reproduction and Genetics, 2003. Kang, S.M., 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): p. 423-8. Li, R.S., et al., Day 4 good morula embryo transfer provided compatible live birth rate with day 5 blastocyst embryo in fresh IVF/ET cycles. Taiwan J Obstet Gynecol, 2018. 57 (1): p. 52-57. Luke, B., Pregnancy and birth outcomes in couples with infertility with and without assisted reproductive technology: with an emphasis on US population-based studies. Am J Obstet Gynecol, 2017. 217 (3): p. 270-281. Luke, B., Pregnancy and birth outcomes in couples with infertility with and without assisted reproductive technology: with an emphasis on US population-based studies. American journal of obstetrics and gynecology, 2017. 217 (3): p. 270-281. Hui, D., et al., Morula transfer achieves better clinical outcomes than post-thawed cleavage embryos after overnight culture in frozen embryo transfer (FET) cycles. J Assist Reprod Genet, 2020. 37 (4): p. 945-952. Blumenfeld, Z., The Ovarian Hyperstimulation Syndrome. Vitam Horm, 2018. 107 : p. 423-451. Xin, Z.M., et al., Pregnancy outcomes of day 5 embryo transfer in patients at high risk of developing ovarian hyperstimulation syndrome and analysis of factors affecting blastocyst formation. J Int Med Res, 2013. 41 (4): p. 1127-34. Additional Declarations No competing interests reported. Supplementary Files researchdata.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 22 Mar, 2024 Reviews received at journal 22 Mar, 2024 Reviewers agreed at journal 11 Mar, 2024 Reviews received at journal 28 Jan, 2024 Reviewers agreed at journal 28 Jan, 2024 Reviewers invited by journal 18 Jan, 2024 Editor assigned by journal 18 Jan, 2024 Editor invited by journal 12 Jan, 2024 Submission checks completed at journal 12 Jan, 2024 First submitted to journal 19 Dec, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3775383","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266696373,"identity":"c82cd8fb-ff8b-4d11-b652-de7568bc08ba","order_by":0,"name":"Yiqun Sun","email":"","orcid":"","institution":"Second Affiliated Hospital \u0026 Yuying Children's Hospital of Wenzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yiqun","middleName":"","lastName":"Sun","suffix":""},{"id":266696374,"identity":"57e2150e-50aa-4a03-b0fc-66364e3de902","order_by":1,"name":"Qi Shen","email":"","orcid":"","institution":"Second Affiliated Hospital \u0026 Yuying Children's Hospital of Wenzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Shen","suffix":""},{"id":266696375,"identity":"05c80619-19a5-4916-84da-730a3a960a62","order_by":2,"name":"Haitao Xi","email":"","orcid":"","institution":"Second Affiliated Hospital \u0026 Yuying Children's Hospital of Wenzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haitao","middleName":"","lastName":"Xi","suffix":""},{"id":266696376,"identity":"c0367fcd-ec50-46fb-bfa3-7a118117babc","order_by":3,"name":"Liucai Sui","email":"","orcid":"","institution":"Second Affiliated Hospital \u0026 Yuying Children's Hospital of Wenzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liucai","middleName":"","lastName":"Sui","suffix":""},{"id":266696377,"identity":"dd89ca3c-692a-4218-9b16-573a1d6b2bb9","order_by":4,"name":"Yanghua Fu","email":"","orcid":"","institution":"Second Affiliated Hospital \u0026 Yuying Children's Hospital of Wenzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yanghua","middleName":"","lastName":"Fu","suffix":""},{"id":266696378,"identity":"b8097ccc-8c01-4451-ae78-d5d5df3c93b9","order_by":5,"name":"Junzhao Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYHCCBBDBw8/e2PjwAyla5CR7DjcbS5BilbHBjfQ2AR5ilMo3MDx8zFNjkzhz5sM2BgkGOzndBgJaGBsYko15jqUl9ksntj0oAHLMDhDQwszAkCbN23A4cebsxHYDCYYDidsIaWGDaPmfuOHmwTYJHmK08EC0HAB6n5FILRLMDMmGc44lAwM5ERjIBkT4Rb69J/HBmxo7YFQef/jwQ4WdHEEtDMw8CUyI6DAgpBwM2A8w/iBK4SgYBaNgFIxYAAD3hT5ATyH20QAAAABJRU5ErkJggg==","orcid":"","institution":"Second Affiliated Hospital \u0026 Yuying Children's Hospital of Wenzhou Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Junzhao","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2023-12-19 06:59:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3775383/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3775383/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49646711,"identity":"e1c05906-3fa4-4741-ab64-9f670d10254f","added_by":"auto","created_at":"2024-01-15 21:09:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2842933,"visible":true,"origin":"","legend":"\u003cp\u003eGrouping of morulae on day 4 and blastocysts on day 5 after fertilization using an early follicular phase prolonged protocol\u003c/p\u003e\n\u003cp\u003eD4, the fourth day after fertilization; D5, the fifth day after fertilization; D4A, single morula was transplanted; D4B, double morulae were transplanted; D5A, single blastocyst was transplanted; D5B, double blastocysts were transplanted\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3775383/v1/c2f1fe3c26916f57b4aa4d59.png"},{"id":49646130,"identity":"c70e5d10-048a-465e-8770-38d2f0bd94df","added_by":"auto","created_at":"2024-01-15 21:01:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1951431,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of different grades of D3 cleavage embryo, D4 morula, and D5 blastocyst\u003c/p\u003e\n\u003cp\u003e(a) Inferior D3 cleavage embryo. The embryo had uneven surfaces, irregular shapes, and was filled with more fragments and visible cell granules. (b) Inferior D4 morula. Only some blastomeres underwent compaction, that was partial compaction, and other cells remained in a separated status, then a smaller compacted multicellular mass would form. (c) Inferior D5 blastocyst. The blastocyst cavity was not significantly dilated, the number of ICM cells was low, and the TE cells were sparse. (d) High quality D3 cleavage embryo. The embryo was of uniform size, regular shape, full hyaline band, uniform cell quality, no granularity and few fragments. (e) High quality D4 morula. The embryo was nearly fully compacted with all blastomeres undergoing the compact process, and a large multicellular mass was expected. (f) High quality D5 blastocyst. The blastocyst cavity was significantly enlarged in volume, the zona pellucid was thinned, the number of ICM cells was high and tightly arranged, and the number of TE cells was high, forming a tight epithelium. Original magnification: x200.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3775383/v1/d70ce3c0345604c7e550e329.png"},{"id":49646128,"identity":"0753dd3c-052e-4645-90db-ce45cfdfa2c9","added_by":"auto","created_at":"2024-01-15 21:01:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":424241,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of single and double embryo transfer assisted pregnancy outcomes in the D4 group\u003c/p\u003e\n\u003cp\u003eD4A, single morula was transplanted; D4B, double morulae were transplanted; CPR, Clinical pregnancy rate; LBR, Live birth rate; PBR, Preterm birth rate; TBR, Twin birth rate; LBIR, Low birthweight infant rate; np＞ 0.05, *p ＜ 0.05, **p ＜ 0.01\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3775383/v1/7b3ef666d19ee2a8a6ac07f3.png"},{"id":49646129,"identity":"f0d2a3e3-8b22-4917-a871-5130020974fc","added_by":"auto","created_at":"2024-01-15 21:01:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":98988,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of single and double embryo transfer assisted pregnancy outcomes in the D5 group\u003c/p\u003e\n\u003cp\u003eD5A, single blastocyst was transplanted; D5B, double blastocysts were transplanted. PBR, Preterm birth rate; TBR, Twin birth rate; NBR, Normal birth rate; CR, Cesarean rate; NB, Newborn birthweight; LBIR, Low birthweight infant rate; **p ＜ 0.01\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3775383/v1/0ab5d6ac9237da3b81a6800e.png"},{"id":49646903,"identity":"327856d4-b7f5-4f73-b1d5-466169f9fab2","added_by":"auto","created_at":"2024-01-15 21:17:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2664756,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3775383/v1/d0ed8894-4892-4400-9d19-cd46c646cef7.pdf"},{"id":49646132,"identity":"d70e5563-82fd-4bff-8267-18eac3903b15","added_by":"auto","created_at":"2024-01-15 21:01:42","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":570458,"visible":true,"origin":"","legend":"","description":"","filename":"researchdata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3775383/v1/d65f46e49f68837706f9bcc0.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparison of pregnancy outcomes between 4th day morula and 5th day blastocyst after embryo transfer:a retrospective cohort study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe advancement of Assisted Reproductive Technology (ART), including the improvement of embryo culture media and the optimization of culture techniques, has enabled the development of embryos into blastocysts in vitro[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Numerous studies have demonstrated that compared to cleavage stage embryo, blastocysts have greater implantation and developmental potential[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As a result, many centers have shifted their transfer strategy from cleavage embryo transfers on day 3 to blastocyst transfers on day 5. Due to the lack of typical morphological markers[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], morulae on day 4 have long been understudied in humans, and the corresponding value of morula transfers has frequently been overlooked. However, it has recently been demonstrated that the densification process at the morula stage involves multiple self-correcting mechanisms that may be critical in determining embryo quality[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], as well as being critical for blastocyst formation, establishment of the first cell lineage, and the entire developmental process[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], which means that morula transfer may also be a feasible transfer strategy. In addition, studies have shown that the use of ART techniques, particularly dual embryo transfer, significantly increases the rate of twin pregnancies[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], which can increase the risk of pregnancy and seriously endanger the mother's and child's life and health[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. To reduce the number of twin pregnancies while successfully assisting pregnancy, our center prefers to use a single blastocyst transfer strategy. However, many patients choose D4 morula transfer and dual embryos transfer due to factors such as time constraints and the desire for a high success rate. In this study, we compared the pregnancy outcomes of D4 morula and D5 blastocyst, single embryo and double embryos using the early follicular stage extra-long protocol, and investigated the benefits and drawbacks of various transfer strategies in order to provide a foundation for individualized transfer protocols.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eResearch Subject\u003c/h2\u003e \u003cp\u003eRetrospective analysis of 1963 cycles undergoing early follicular phase ultra-long protocol assisted conception at our fertility center from September 2017 to September 2020, divided into D4 (324 cases) and D5 (1639 cases) groups based on the number of days of in vitro culture after fertilization, and further divided D4 and D5 into groups A and B based on single and double embryo transfer to compare their outcomes, as show in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. To compare D4A with D4B, D5A with D5B, D4A with D5A, D4B with D5B, and to clarify the differences in pregnancy outcomes between D4 morula and D5 blastocyst, as well as single embryos and double embryos for transfer. Inclusion criteria: 1. patient age between 20 and 42 years; 2. early follicular phase ultra-long protocol was used to assist pregnancy; 3. number of day3(D3) high-quality embryos\u0026thinsp;\u0026ge;\u0026thinsp;3. Exclusion criteria:1. uterine pathologies such as adenomyosis, submucous myomas, severe uterine adhesions, uterine malformations; 2. history of adverse pregnancy such as recurrent miscarriage, stillbirth and multiple inductions; 3. chromosomal abnormalities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAssisted reproductive process\u003c/h2\u003e \u003cp\u003eTo limit the impact of diverse IVF regimens on pregnancy outcomes, all cycles in this study were completed using the early follicular phase extra-long regimen, which was routinely utilized to facilitate pregnancy in our center. On days 2\u0026ndash;5 of menstruation, 3.75 mg of gonadotropin releasing hormone agonist (GnRH-a) was administered intramuscularly to suppress pituitary function. After 28\u0026ndash;38 days, an ultrasound and endocrine examination would be conducted. When endometrial thickness was less than 5mm,follicle stimulating hormone(FSH) was less than 5mIU/ml, luteinizing hormone\u003c/p\u003e \u003cp\u003e(LH) was less than 5mIU/ml, and estradiol(E2) was less than 50pg/ml, the pituitary gland was considered completely downregulated. At this time, gonadotropin (Gn) would be used to initiate ovulation, and the type and dosage of Gn would be adjusted continuously based on ultrasound and serum sex hormone level. An intramuscular injection of 4000\u0026ndash;10,000 IU of chorionic gonadotropin was given when at least one follicle measures at least 20 mm in diameter or when two follicles measure at least 18 mm in diameter. Oocytes were carried out under vaginal ultrasonography guidance 36 hours later, and embryos were cultivated after in vitro fertilization of the oocytes, embryo transfer was performed under abdominal ultrasound guidance. Serum human chorionic gonadotropin(hCG) levels were examined two weeks after transfer to see if there was a pregnancy. If the hCG level was positive, an ultrasound two weeks later revealed an intrauterine gestational sac, indicating a clinical pregnancy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEmbryo culture and classification\u003c/h2\u003e \u003cp\u003eThe quality of embryos was closely related to clinical outcomes[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and we had strict requirements for embryo quality in order to control the interference of different embryo quality on study outcomes. In this study, cycles with at least three high-quality embryos on D3 were chosen for grouping. All embryos were cultured in order, with high-quality D3 embryos being transferred to blastocyst culture. The culture was continued until the fourth or fifth days, after which the best 1\u0026ndash;2 embryos were chosen for transplantation on D4 and D5, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. According to the Istanbul consensus[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], the embryo quality was graded according to the size, uniformity and amount of nucleus-free fragments of the D3 oocyte cleavage spheres. A high-quality embryo should have 6\u0026ndash;10 cells, uniform blastomere size, few fragments, and no multinucleation on D3 after fertilization. The agreement on Day 4 was that an optimum embryo at this stage would be compacted or compacting, the compaction should include virtually all the embryo volume and would have entered a fourth round of cleavage.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Blastocyst scoring used the Gardner scale[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], which scored the degree of blastocyst expansion, inner cell mass, and trophectoderm cell development, respectively. Blastocysts with tightly arranged cells, almost free of debris and not yet hatched were usually considered as the most suitable blastocysts for transfer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eObservation indicators\u003c/h2\u003e \u003cp\u003eEach group was examined for demographic characteristics, clinical and laboratory outcomes, pregnancy outcomes and other indicators. The primary outcome indicators were the clinical pregnancy rate (number of clinical pregnancy cycles/number of transplantation cycles\u0026times;100%), miscarriage rate (number of miscarriage cycles/number of clinical pregnancy cycles\u0026times;100%), and live birth rate (number of live births/number of transplantation cycles\u0026times;100%)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Secondary outcome indicators were the preterm birth rate, normal birth rate, cesarean delivery rate, neonatal weight, and other observations. As a major complication of ovulation promotion, we also compared the incidence of ovarian hyperstimulation syndrome (OHSS) in each group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistics Analysis\u003c/h2\u003e \u003cp\u003eFor statistical analysis, SPSS 25.0 software (IBM Corp, Armonk, NY, USA) was utilized. The data was examined for normality. The average value of normally distributed measures was expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, and the T-Test for two independent samples was used to compare groups. While non-normally distributed measures were expressed as M (P25, P75), and the Wilcoxon rank sum test was used to compare groups. Count data was reported as percentages, and group comparisons were performed using the Chi-Square Test or Fisher-Yates-Test. \u003cb\u003eP\u003c/b\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGeneral information\u003c/h2\u003e \u003cp\u003eAmong 1963 cycles, we transferred morulae in 324 cycles and blastocysts in 1639 cycles. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the demographic characteristics of the D4 and D5 groups. There were no significant differences between these two groups in terms of age, Body mass index(BMI), years of infertility, Anti-M\u0026uuml;llerian Hormone(AMH), basal FSH, or etiology of infertility. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, with respect to clinical and laboratory outcomes, there were no differences in LH, E2, Progesterone(P) after downregulation, and LH, P, endometrial thickness at hCG triggering, normal fertilization rate and quality embryo rate. However, the E2 at hCG triggering, and the number of retrieved oocytes, fertilized oocytes, D3 embryos, and quality D3 embryos were lower in the D4 group than in the D5 group, while the percentage of single embryo transfer was higher in the D5 group than in the D4 group. Because single and double embryo transfer rates differed between D4 and D5, we further separated D4 and D5 into D4A, D4B, D5A, D5B according to the number of embryos transferred.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of demographic characteristics in two groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD4 group(n\u0026thinsp;=\u0026thinsp;324)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD5 group(n\u0026thinsp;=\u0026thinsp;1639)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.36\u0026thinsp;\u0026plusmn;\u0026thinsp;4.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e31.18\u0026thinsp;\u0026plusmn;\u0026thinsp;4.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.325\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI (kg/m2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.79\u0026thinsp;\u0026plusmn;\u0026thinsp;3.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e21.96\u0026thinsp;\u0026plusmn;\u0026thinsp;3.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.406\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of infertility (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.34\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e3.34\u0026thinsp;\u0026plusmn;\u0026thinsp;2.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.911\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMH (ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.68\u0026thinsp;\u0026plusmn;\u0026thinsp;3.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e4.26\u0026thinsp;\u0026plusmn;\u0026thinsp;3.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.670\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasic FSH (mIU/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.99\u0026thinsp;\u0026plusmn;\u0026thinsp;2.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e7.93\u0026thinsp;\u0026plusmn;\u0026thinsp;4.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.215\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEtiology of infertility (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.53(154/324)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e46.31(759/1639)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.687\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCOS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.80(35/324)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e14.15(232/1639)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.108\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndometriosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.47(8/324)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e1.65(27/1639)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.307\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOvarian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.86(19/324)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e4.09(67/1639)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.153\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCombined\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.73(38/324)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e11.65(191/1639)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.969\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.60(70/324)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e22.15(363/1639)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.830\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e* BMI\u0026thinsp;=\u0026thinsp;Body mass index, AMH\u0026thinsp;=\u0026thinsp;Anti-M\u0026uuml;llerian Hormone, FSH\u0026thinsp;=\u0026thinsp;Follicle stimulating hormone, PCOS\u0026thinsp;=\u0026thinsp;Polycystic ovary syndrome\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of clinical and laboratory outcomes between the two groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD4 group(n\u0026thinsp;=\u0026thinsp;324)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD5 group(n\u0026thinsp;=\u0026thinsp;1639)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLH after deregulation (IU/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.289\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE2 after deregulation (pg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.48\u0026thinsp;\u0026plusmn;\u0026thinsp;12.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.83\u0026thinsp;\u0026plusmn;\u0026thinsp;121.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.449\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP after deregulation (nmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.474\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLH on trigger day (IU/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.826\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE2 on trigger day (pg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1833.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1023.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2400.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1168.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP on trigger day (nmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;26.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndometrial thickness on trigger day (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO. of retrieved oocytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.5(7,14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13(10,16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO. of fertilized oocytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7(5,10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9(7,12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO. of D3 embryos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7(5,10.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9(7,12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO. of quality D3 embryos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5(4,8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7(5,10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal fertilization (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.5(64.29,85.71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75(62.5,85.71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuality embryo (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83.33(71.43,1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85(68.75,1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.292\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSingle embryo transfer (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.64(109/324)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87.37(1432/1639)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDouble embryo transfer (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.36(215/324)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.63(207/1639)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e* LH\u0026thinsp;=\u0026thinsp;Luteinizing hormone, E2\u0026thinsp;=\u0026thinsp;serum estradiol, P\u0026thinsp;=\u0026thinsp;serum progesterone\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eComparison of pregnancy outcomes\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003eD4A and D5A groups at single embryo transfer\u003c/h2\u003e \u003cp\u003eIn single embryo transfer, we have performed 109 cycles of morula transfers and 1432 cycles of blastocyst transfers. The clinical pregnancy rate, miscarriage rate, live birth rate, preterm birth rate, overdue birth rate, twin birth rate, normal birth rate, cesarean delivery rate, neonatal birthweight, proportion of fetal macrosomia, proportion of low birthweight infants, and incidence of OHSS were all significantly the same between the D4A group and D5A group, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of pregnancy outcomes between the D4A and D5A groups at single embryo transfer\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD4A group(n\u0026thinsp;=\u0026thinsp;109)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD5A group(n\u0026thinsp;=\u0026thinsp;1432)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical pregnancy (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59.63(65/109)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.73(927/1432)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.284\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMiscarriage (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.92(11/65)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.05(121/927)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.374\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLive birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.54(54/109)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56.28(806/1432)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.346\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePreterm birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.83(2/109)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.12(59/1432)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.355\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOverdue birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.67(4/109)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.63(52/1432)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTwin birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0(0/65)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.83(17/927)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.620\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.59(23/54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.89(386/806)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.450\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCesarean (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.41(31/54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52.11(420/806)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.450\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNewborn birthweight(g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3209.56\u0026thinsp;\u0026plusmn;\u0026thinsp;429.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3260.10\u0026thinsp;\u0026plusmn;\u0026thinsp;546.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.506\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFetal Macrosomia (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.70(2/54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.98(49/820)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.696\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow birthweight infant (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.56(3/54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.44(61/820)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.806\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOHSS incidence (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.92(1/109)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.33(19/1432)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e* Unknown birthweight of D5A babies for 3 cycles\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eD4B group and the D5B group at double embryo transfer\u003c/h2\u003e \u003cp\u003eIn double embryo transfer, we have performed 215 cycles of morula transfers and 207 cycles of blastocyst transfers. There were no significant differences in clinical pregnancy rate, miscarriage rate, live birth rate, preterm birth rate, overdue birth rate, twin birth rate, normal birth rate, cesarean section rate, proportion of fetal macrosomia and OHSS incidence between the D4B and D5B group populations, but the birthweight of newborns in the D4B group was lower than that in the D5B group and the proportion of low birthweight infants was higher than that in the D5B group, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of pregnancy outcomes between the D4B and D5B groups at double embryo transfer\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD4B group(n\u0026thinsp;=\u0026thinsp;215)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD5B group(n\u0026thinsp;=\u0026thinsp;207)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical pregnancy (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69.30(149/215)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.22(135/207)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.371\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMiscarriage (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.41(17/149)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.15(11/135)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.357\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLive birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61.40(132/215)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59.90(124/207)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.754\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePreterm birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.88(32/215)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.04(27/207)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.586\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOverdue birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.33(5/215)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.90(6/207)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.712\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTwin birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.24(54/149)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.81(47/135)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.802\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.06(41/132)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.42(34/124)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.522\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCesarean (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.94(91/132)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72.58(90/124)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.522\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNewborn birthweight(g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2777.33\u0026thinsp;\u0026plusmn;\u0026thinsp;652.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2915.59\u0026thinsp;\u0026plusmn;\u0026thinsp;602.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.039\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFetal Macrosomia (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.23(6/186)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.34(4/171)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.823\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow birthweight infant (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.95(65/186)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.05(36/171)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOHSS incidence (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.86(4/215)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.45(3/207)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSingle and double embryo transfer in group D4\u003c/h2\u003e \u003cp\u003eAmong 324 D4 morula transfer cycles, we transferred one embryo in 109 cycles and two embryos in 215 cycles. There were no significant differences in clinical pregnancy rate, miscarriage rate, overdue delivery rate, normal delivery rate, cesarean delivery rate, proportion of fetal macrosomia and OHSS incidence between patients in D4A and D4B groups, but the live birth rate, preterm delivery rate, twin birth rate and proportion of low birthweight infants were lower in D4A group than in D4B group; neonatal birthweight was higher than in D4B group. as shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of single and double embryo transfer assisted pregnancy outcomes in the D4 group\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD4A group(n\u0026thinsp;=\u0026thinsp;109)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD4B group(n\u0026thinsp;=\u0026thinsp;215)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical pregnancy (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59.63(65/109)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.30(149/215)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.082\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMiscarriage (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.92(11/65)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.41(17/149)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.271\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLive birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.54(54/109)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.40(132/215)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.037\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePreterm birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.83(2/109)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.88(32/215)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOverdue birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.67(4/109)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.33(5/215)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.735\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTwin birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0(0/65)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.24(54/149)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.59(23/54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.06(41/132)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.133\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCesarean (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.41(31/54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68.94(91/132)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.133\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNewborn birthweight(g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3209.56\u0026thinsp;\u0026plusmn;\u0026thinsp;429.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2777.33\u0026thinsp;\u0026plusmn;\u0026thinsp;652.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFetal Macrosomia (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.70(2/54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.23(6/186)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow birthweight infant (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.56(3/54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.95(65/186)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOHSS incidence (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.92(1/109)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.86(4/215)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.667\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSingle and double embryo transfer in group D5\u003c/h2\u003e \u003cp\u003eAmong 1639 D5 blastocyst transfer cycles, we transferred one embryo in 1432 cycles and two embryos in 207 cycles. There were no significant differences in clinical pregnancy rate, miscarriage rate, live birth rate, overdue birth rate, proportion of fetal macrosomia, or incidence of OHSS between patients in the D5A and D5B groups. However, the preterm birth rate, twin birth rate, cesarean section rate, and proportion of low birthweight infants were lower in the D5A group; the normal birth rate and neonatal birthweight were higher in the D5A group, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of single and double embryo transfer assisted pregnancy outcomes in the D5 group\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD5A group(n\u0026thinsp;=\u0026thinsp;1432)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD5B group(n\u0026thinsp;=\u0026thinsp;207)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical pregnancy (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64.73(927/1432)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.22(135/207)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.892\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMiscarriage (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.05(121/927)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.15(11/135)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.107\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLive birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.28(806/1432)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59.90(124/207)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.326\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePreterm birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.12(59/1432)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.04(27/207)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOverdue birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.63(52/1432)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.90(6/207)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.594\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTwin birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.83(17/927)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.81(47/135)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal birth (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.89(386/806)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.42(34/124)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCesarean (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.11(420/806)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72.58(90/124)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNewborn birthweight(g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3260.10\u0026thinsp;\u0026plusmn;\u0026thinsp;546.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2915.59\u0026thinsp;\u0026plusmn;\u0026thinsp;602.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFetal Macrosomia (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.98(49/820)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.34(4/171)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.055\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow birthweight infant (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.44(61/820)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.05(36/171)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOHSS incidence (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.33(19/1432)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.45(3/207)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e* Unknown birthweight of D5A babies for 3 cycles\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe selection of embryos with optimal implantation potential for transfer is a critical step in optimizing pregnancy outcomes in vitro fertilization-embryo transfer(IVF-ET) treatment[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Studies have shown that embryo culture is a meritocratic process and that a large number of embryonic genes are activated and expressed on day 4 compared to day 3 transfer, indicating that D4 morula development is an essential screen for future embryo survival[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Sang et al. examined morula and maternal endometrium transcriptome datasets to identify the expression of receptor genes expressed by morula and corresponding ligand genes in endometrium, and to further clarify the details of communication between embryo and mother[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].Human embryos enter the uterine cavity on day 4 after fertilization, when the uterus provides a different nutritional environment than the oviduct, uterine contractility is reduced, and synchronization of embryos with the endometrium is improved[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], giving D4 morula and D5 blastocyst transfers an advantage over D3 cleavage embryo. Two main morphogenetic phases build the basic architecture of the pre-implantation embryo: the formation of the morula and of the blastocyst[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Embryo culture up to day 5 may aid in the rejection of unsuitable embryos, but it is unknown whether D5 blastocyst is superior to D4 morula in this regard. However, compared to D5 blastocyst, transfer of D4 morula reduces the exposure time of embryos in vitro, reduces the impact of environmental factors on embryos in vitro[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], reduces the risk of developmental arrest and embryo quality decline[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and makes better use of embryos. Patients undergoing blastocyst culture may have fewer total embryos available for transfer, or no embryos available for transfer and transfer cycles cancelled. Combining the advantages and risks, the outcome of transferring D4 morula versus D5 blastocyst has not been clearly established.\u003c/p\u003e \u003cp\u003eThe results of this study show that there are no significant differences in general baseline conditions such as age and BMI between the D4 and D5 groups, but the E2 at hCG triggering, of retrieved oocytes, number of fertilized oocytes, number of D3 embryos, number of quality D3 embryos, and percentage of single embryo transfer are lower in the D4 group than in the D5 group, and the percentage of double embryo transfer is higher than in the D5 group. This could be related to our transfer strategy, in which we choose single blastocyst transfer as the primary strategy when the overall circumstances are favorable and the blastocyst-raising success rate is high. In contrast, we preferentially transfer one or two D4 morulae when there are few embryos, an anticipated delay in embryo growth on day 4, a risk of cycle cancellation, or a conflict in schedule for day 5 transfer. Therefore, the clinical and embryonic profile of the D5 group may be slightly better than that of the D4 group, while the single embryo transfer rate is significantly higher than that of the D4 group. This study performed additional stratified analysis of single and double embryo transfer in order to rule out the influence of embryo transfer quantity on pregnancy outcome.\u003c/p\u003e \u003cp\u003eKang et al. performed a retrospective analysis of 271 cycles and discovered no significant changes in clinical pregnancy, live birth, or miscarriage rates between D4 and D5 single embryo transfers, despite the fact that the miscarriage rate was somewhat higher in D4. They suggested that the results may have been affected by sample size constraints and that additional samples are required for validation[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In a retrospective study of 427 GnRH antagonist protocol cycles, Li et al. found no significant difference in clinical pregnancy rate, live birth rate, or miscarriage rate between D4 and D5, however the full-term birth rate was greater in the D4 group than in the D5 group. They also presented an embryo quality assessment system for D4 to help with embryo selection[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Simopoulo et al. reviewed 6 prospective studies and 9 retrospective cohort studies, and found that the rates of clinical pregnancy, sustained pregnancy/live birth, cancelled pregnancy, and miscarriage of D4 and D5 transplantation had no statistical difference. Additionally, statistically speaking, D4 had a much lower preterm birth rate than D5[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In our research, we retrospectively analyzed 1963 cycles using the early follicular phase extra-long protocol and further stratified D4 and D5 based on the number of embryo transfers, finding that there was no significant difference in D4 transfer and D5 transfer outcomes for single embryo transfer, and the results were generally consistent with the previous studies. Moreover, there was no significant difference in the primary pregnancy outcome between D4 and D5 double embryo transfers. Although, the birthweight of babies in the D4B group was lower than that of babies in the D5B group, and the number of babies with low birthweight was higher in the D4B group than in the D5B group. Additionally, we found a higher rate of cesarean delivery in our study for both D4 and D5. The reasons for this are diverse. On the one hand, the increased rate of multiple births in assisted reproduction increases the probability of difficult deliveries[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. On the other hand, the relatively advanced maternal age[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], the mental stress of late pregnancy, and the difficulty in obtaining a fetus result in the request for cesarean delivery by the mother and her family. In response, the indications for cesarean delivery were relaxed clinically, which led to an increase in the rate of cesarean delivery.\u003c/p\u003e \u003cp\u003eIn order to find the optimal transfer strategy, we compared the outcomes of single and double embryo transfer on days D4 and D5, respectively, and found that the live birth rate, preterm birth rate, twin birth rate, and low birthweight infant percentage were lower in the D4A group than in the D4B group at D4 transfer, while the newborn birthweight was higher than in the D4B group. In addition, the clinical pregnancy rate in the D4A group was lower than that in the D4B group, and the normal birth rate was higher than that in the D4B group. Although the differences were not statistically significant, they still showed a corresponding trend, which may be related to the small sample size of D4 transplants in this study, and further increasing the sample size may make the above differences significant. The results of our study suggest that the outcome of double embryo transfer in D4 will be better than single embryo transfer in terms of success rate. According to Duffy et al, twin pregnancies lead to the occurrence of outcomes such as preterm delivery, cesarean delivery, and low birthweight infants[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], thus, we primarily blamed the difference in the twin pregnancy rate for the difference in secondary pregnancy outcomes between the D4A and D4B groups. In our study, double embryo transfer in D4 row significantly increased the twin rate and increased the risk of pregnancy such as preterm delivery and low birthweight. Several studies have shown that reducing the number of embryos transferred helps to reduce the occurrence of multiple pregnancies as well as maternal and infant adverse pregnancy outcomes[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and we also tend to control the number of embryos transferred in our transfer strategy and promote single embryo transfer to reduce the risk of twin pregnancies. In the D5 transfer, there was no significant difference in the main pregnancy outcomes between single and twin embryo transfer, and the preterm delivery rate, twin birth rate, cesarean delivery rate, and low birthweight infant ratio in the D5A group were lower than those in the D5B group, while the normal delivery rate and neonatal birthweight were higher than those in the D5B group, indicating that single embryo transfer would be more advantageous in the D5 transfer.\u003c/p\u003e \u003cp\u003eAs an important complication of IVF, OHSS can cause increased vascular permeability, ascites, and even life-threatening respiratory failure, and the increased number of embryos obtained can increase the risk of OHSS[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Xin et al. found that when patients at high risk for OHSS underwent embryo transfer, the incidence of OHSS was lower in the D5 transfer group than in the D3 group. They hypothesized that this may be because the delayed culture gave doctors more time to monitor and treat patients' symptoms as well as the lower rate of multiple births with D5 transfer[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].However, no studies have found a link between D4 or D5 transfer and OHSS. In our study, no significant differences were seen in the incidence of OHSS under different transplantation strategies, which may be due to the limitations of the retrospective study model we adopted and the inadequate sample size, and further studies are needed to explore the association in the future.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings suggest that D4 morula embryo transfer does not reduce IVF success in the early follicular phase extra-long protocol. The D4 embryo transfer can be a good alternative to D5 blastocyst transfer in case of holiday or work schedule conflicts. For D4 morula embryo transfer, single and double embryo transfer should be selected individually considering the success rate and the risk of twin pregnancy, while single blastocyst transfer is more recommended for D5 blastocyst transfer to reduce the rate of double pregnancy and optimize pregnancy outcome. The sample size of D4 in this study was relatively small, and the study didn\u0026rsquo;t analyze the quality and grading of embryos on D4 and D5 days, and didn\u0026rsquo;t compare pregnancy complications and subsequent neonatal mental and physical development. Further prospective randomized controlled studies are needed to verify the benefits of the above transfer strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDay 4 D4 \u003c/p\u003e\n\u003cp\u003eDay 5 D5\u003c/p\u003e\n\u003cp\u003eIn vitro fertilization/intracytoplasmic sperm injection IVF/ICSI\u003c/p\u003e\n\u003cp\u003eAssisted Reproductive Technology ART\u003c/p\u003e\n\u003cp\u003eDay 3 D3\u003c/p\u003e\n\u003cp\u003eGonadotropin releasing hormone agonist GnRH-a\u003c/p\u003e\n\u003cp\u003eFollicle stimulating hormone FSH\u003c/p\u003e\n\u003cp\u003eLuteinizing hormone LH\u003c/p\u003e\n\u003cp\u003eEstradiol E2\u003c/p\u003e\n\u003cp\u003eGonadotropin Gn\u003c/p\u003e\n\u003cp\u003eHuman chorionic gonadotropin hCG \u003c/p\u003e\n\u003cp\u003eOvarian hyperstimulation syndrome OHSS\u003c/p\u003e\n\u003cp\u003eBody mass index BMI\u003c/p\u003e\n\u003cp\u003ein vitro fertilization-embryo transfer IVF-ET\u003c/p\u003e\n\u003cp\u003ePolycystic ovary syndrome PCOS\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of the Second Affiliated Hospital of Wenzhou\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMedical University (\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e2021-K-381-01),and registered on December 31, 2021, retroactive registration.As a retrospective study,this study passed the informed consent exemption application by the Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors had full access to the data and materials. Data is provided in the supplementary information file. If anyone wants to obtain data from this study, they can contact the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe National Key Research and Development Program\u0026nbsp;(grant no. 2018YFC1003103)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJunzhao Zhao conceived the idea and wrote the proposal for the study. Yiqun Sun collected \u0026nbsp;\u003c/p\u003e\n\u003cp\u003ethe data, analyzed the data, interpreted the data and drafted the manuscript. Qi Shen and Haitao Xi analyzed and interpreted the data. Liucai Sui and Yanghua Fu processed tables and figures. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Reproductive Center of the Second Affiliated Hospital of Wenzhou\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMedical University for support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGardner, D.K. and M. Lane, \u003cem\u003eCulture and selection of viable blastocysts: a feasible proposition for human IVF?\u003c/em\u003e Human reproduction update, 1997. \u003cstrong\u003e3\u003c/strong\u003e(4): p. 367-82.\u003c/li\u003e\n\u003cli\u003eDe Croo, I., P. De Sutter, and K. Tilleman, \u003cem\u003eA stepwise approach to move from a cleavage-stage to a blastocyst-stage transfer policy for all patients in the IVF clinic.\u003c/em\u003e Hum Reprod Open, 2020. \u003cstrong\u003e2020\u003c/strong\u003e(3): p. hoaa034.\u003c/li\u003e\n\u003cli\u003eGlujovsky, D., et al., \u003cem\u003eCleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology.\u003c/em\u003e Cochrane Database Syst Rev, 2016(6): p. CD002118.\u003c/li\u003e\n\u003cli\u003eLong, X., et al., \u003cem\u003ePregnancy Outcomes of Single/Double Blastocysts and Cleavage Embryo Transfers: a Retrospective Cohort Study of 24,422 Frozen-Thawed Cycles.\u003c/em\u003e Reprod Sci, 2020. \u003cstrong\u003e27\u003c/strong\u003e(12): p. 2271-2278.\u003c/li\u003e\n\u003cli\u003eCoticchio, G., et al., \u003cem\u003eThe enigmatic morula: mechanisms of development, cell fate determination, self-correction and implications for ART.\u003c/em\u003e Hum Reprod Update, 2019. \u003cstrong\u003e25\u003c/strong\u003e(4): p. 422-438.\u003c/li\u003e\n\u003cli\u003eLagalla, C., et al., \u003cem\u003eEmbryos with morphokinetic abnormalities may develop into euploid blastocysts.\u003c/em\u003e Reprod Biomed Online, 2017. \u003cstrong\u003e34\u003c/strong\u003e(2): p. 137-146.\u003c/li\u003e\n\u003cli\u003eBrison, D.R., R.G. Sturmey, and H.J. Leese, \u003cem\u003eMetabolic heterogeneity during preimplantation development: the missing link?\u003c/em\u003e Hum Reprod Update, 2014. \u003cstrong\u003e20\u003c/strong\u003e(5): p. 632-40.\u003c/li\u003e\n\u003cli\u003eVassena, R., et al., \u003cem\u003eWaves of early transcriptional activation and pluripotency program initiation during human preimplantation development.\u003c/em\u003e Development, 2011. \u003cstrong\u003e138\u003c/strong\u003e(17): p. 3699-709.\u003c/li\u003e\n\u003cli\u003eSunderam, S., et al., \u003cem\u003eAssisted Reproductive Technology Surveillance - United States, 2017.\u003c/em\u003e Morbidity and mortality weekly report. Surveillance summaries (Washington, D.C. : 2002), 2020. \u003cstrong\u003e69\u003c/strong\u003e(9): p. 1-20.\u003c/li\u003e\n\u003cli\u003eDuffy, C.R., \u003cem\u003eMultifetal Gestations and Associated Perinatal Risks.\u003c/em\u003e NeoReviews, 2021. \u003cstrong\u003e22\u003c/strong\u003e(11): p. e734-e746.\u003c/li\u003e\n\u003cli\u003eZhao, Y.Y., Y. Yu, and X.W. Zhang, \u003cem\u003eOverall Blastocyst Quality, Trophectoderm Grade, and Inner Cell Mass Grade Predict Pregnancy Outcome in Euploid Blastocyst Transfer Cycles.\u003c/em\u003e Chin Med J (Engl), 2018. \u003cstrong\u003e131\u003c/strong\u003e(11): p. 1261-1267.\u003c/li\u003e\n\u003cli\u003eWang, L., et al., \u003cem\u003eIVF embryo choices and pregnancy outcomes.\u003c/em\u003e Prenat Diagn, 2021. \u003cstrong\u003e41\u003c/strong\u003e(13): p. 1709-1717.\u003c/li\u003e\n\u003cli\u003eMedicine, A.S.i.R. and E.S.I.G.o. Embryology, \u003cem\u003eThe Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting.\u003c/em\u003e Human reproduction (Oxford, England), 2011(6): p. 1270-83.\u003c/li\u003e\n\u003cli\u003eTao, J., et al., \u003cem\u003eThe neglected morula/compact stage embryo transfer.\u003c/em\u003e Hum Reprod, 2002. \u003cstrong\u003e17\u003c/strong\u003e(6): p. 1513-8.\u003c/li\u003e\n\u003cli\u003eGardner, D.K. and W.B. Schoolcraft, \u003cem\u003eIn vitro culture of human blastocyst.\u003c/em\u003e Towards Reproductive Certainty Infertility \u0026amp; Genetics Beyond, 1999.\u003c/li\u003e\n\u003cli\u003eGardner, D.K. and W.B. Schoolcraft, \u003cem\u003eCulture and transfer of human blastocysts.\u003c/em\u003e Curr Opin Obstet Gynecol, 1999. \u003cstrong\u003e11\u003c/strong\u003e(3): p. 307-11.\u003c/li\u003e\n\u003cli\u003eZhang, H.N., et al., \u003cem\u003eComparison of Pregnancy Outcomes Between Single-Morula Embryo Transfer and Single-Blastocyst Transfer in Fresh IVF/ICSI Cycles.\u003c/em\u003e Med Sci Monit, 2021. \u003cstrong\u003e27\u003c/strong\u003e: p. e928737.\u003c/li\u003e\n\u003cli\u003eSimopoulou, M., et al., \u003cem\u003eShould the flexibility enabled by performing a day-4 embryo transfer remain as a valid option in the IVF laboratory? A systematic review and network meta-analysis.\u003c/em\u003e J Assist Reprod Genet, 2019. \u003cstrong\u003e36\u003c/strong\u003e(6): p. 1049-1061.\u003c/li\u003e\n\u003cli\u003eStrelchenko, N., et al., \u003cem\u003eMorula-derived human embryonic stem cells.\u003c/em\u003e Reproductive biomedicine online, 2004. \u003cstrong\u003e9\u003c/strong\u003e(6): p. 623-9.\u003c/li\u003e\n\u003cli\u003eHirate, Y., et al., \u003cem\u003ePolarity-dependent distribution of angiomotin localizes Hippo signaling in preimplantation embryos.\u003c/em\u003e Current biology : CB, 2013. \u003cstrong\u003e23\u003c/strong\u003e(13): p. 1181-94.\u003c/li\u003e\n\u003cli\u003eSang, L., et al., \u003cem\u003eAtlas of receptor genes expressed by the bovine morula and corresponding ligand-related genes expressed by uterine endometrium.\u003c/em\u003e Mol Reprod Dev, 2021. \u003cstrong\u003e88\u003c/strong\u003e(10): p. 694-704.\u003c/li\u003e\n\u003cli\u003eLesny, P., et al., \u003cem\u003eUterine junctional zone contractions during assisted reproduction cycles.\u003c/em\u003e Human reproduction update, 1998. \u003cstrong\u003e4\u003c/strong\u003e(4): p. 440-5.\u003c/li\u003e\n\u003cli\u003eMaitre, J.L., \u003cem\u003eMechanics of blastocyst morphogenesis.\u003c/em\u003e Biol Cell, 2017. \u003cstrong\u003e109\u003c/strong\u003e(9): p. 323-338.\u003c/li\u003e\n\u003cli\u003eMorbeck, D.E., \u003cem\u003eAir quality in the assisted reproduction laboratory: a mini-review.\u003c/em\u003e Journal of Assisted Reproduction and Genetics, 2015. \u003cstrong\u003e32\u003c/strong\u003e(7): p. 1019-1024.\u003c/li\u003e\n\u003cli\u003eBraga, D.P., et al., \u003cem\u003eThe importance of the cleavage stage morphology evaluation for blastocyst transfer in patients with good prognosis.\u003c/em\u003e J Assist Reprod Genet, 2014. \u003cstrong\u003e31\u003c/strong\u003e(8): p. 1105-10.\u003c/li\u003e\n\u003cli\u003eSimon J. Phillips, 2 Nicola L. Dean,1 William M. Buckett,1 and Seang Lin Tan, \u003cem\u003eConsecutive transfer of day 3 embryos and of day 5-6 blastocysts increases overall pregnancy rates associated with blastocyst culture.\u003c/em\u003e Assisted Reproduction and Genetics, 2003.\u003c/li\u003e\n\u003cli\u003eKang, S.M., et al., \u003cem\u003eClinical outcomes of elective single morula embryo transfer versus elective single blastocyst embryo transfer in IVF-ET.\u003c/em\u003e J Assist Reprod Genet, 2012. \u003cstrong\u003e29\u003c/strong\u003e(5): p. 423-8.\u003c/li\u003e\n\u003cli\u003eLi, R.S., et al., \u003cem\u003eDay 4 good morula embryo transfer provided compatible live birth rate with day 5 blastocyst embryo in fresh IVF/ET cycles.\u003c/em\u003e Taiwan J Obstet Gynecol, 2018. \u003cstrong\u003e57\u003c/strong\u003e(1): p. 52-57.\u003c/li\u003e\n\u003cli\u003eLuke, B., \u003cem\u003ePregnancy and birth outcomes in couples with infertility with and without assisted reproductive technology: with an emphasis on US population-based studies.\u003c/em\u003e Am J Obstet Gynecol, 2017. \u003cstrong\u003e217\u003c/strong\u003e(3): p. 270-281.\u003c/li\u003e\n\u003cli\u003eLuke, B., \u003cem\u003ePregnancy and birth outcomes in couples with infertility with and without assisted reproductive technology: with an emphasis on US population-based studies.\u003c/em\u003e American journal of obstetrics and gynecology, 2017. \u003cstrong\u003e217\u003c/strong\u003e(3): p. 270-281.\u003c/li\u003e\n\u003cli\u003eHui, D., et al., \u003cem\u003eMorula transfer achieves better clinical outcomes than post-thawed cleavage embryos after overnight culture in frozen embryo transfer (FET) cycles.\u003c/em\u003e J Assist Reprod Genet, 2020. \u003cstrong\u003e37\u003c/strong\u003e(4): p. 945-952.\u003c/li\u003e\n\u003cli\u003eBlumenfeld, Z., \u003cem\u003eThe Ovarian Hyperstimulation Syndrome.\u003c/em\u003e Vitam Horm, 2018. \u003cstrong\u003e107\u003c/strong\u003e: p. 423-451.\u003c/li\u003e\n\u003cli\u003eXin, Z.M., et al., \u003cem\u003ePregnancy outcomes of day 5 embryo transfer in patients at high risk of developing ovarian hyperstimulation syndrome and analysis of factors affecting blastocyst formation.\u003c/em\u003e J Int Med Res, 2013. \u003cstrong\u003e41\u003c/strong\u003e(4): p. 1127-34.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pregnancy-and-childbirth","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prch","sideBox":"Learn more about [BMC Pregnancy and Childbirth](http://bmcpregnancychildbirth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/prch/default.aspx","title":"BMC Pregnancy and Childbirth","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Early follicular phase prolonged protocol, Morula, Blastocyst, Assisted reproduction, IVF/ICSI outcome","lastPublishedDoi":"10.21203/rs.3.rs-3775383/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3775383/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThis study was designed to evaluate pregnancy outcomes between morulae transferred on day 4 (D4) and blastocysts transferred on day 5 (D5).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFrom September 2017 to September 2020, 1963 fresh transfer cycles that underwent early follicular phase extra-long protocol for assisted conception in our fertility center were divided into D4 (324 cases) and D5 (1639 cases) groups based on the number of days of in vitro culture after fertilization, and the general basic information, clinical and embryonic conditions of patients in both groups were compared. To compare the differences in pregnancy outcomes, the D4 and D5 groups were further divided into groups A and B based on single and double embryo transfer.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn single embryo transfer, there was no significant difference between groups D4A and D5A (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In double embryo transfer, group D4B had a lower newborn birthweight and a larger proportion of low birthweight infants (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The preterm delivery rate, twin delivery rate, cesarean delivery rate, and percentage of low birthweight infants were lower in the D5A group than in the D5B group (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eWhen factors such as working life and hospital holidays are being considered, D4 morula transfer may be a good alternative to D5 blastocyst transfer. Given the in vitro fertilization/intracytoplasmic sperm injection(IVF/ICSI) success rate and risk of twin pregnancy, D4 morula transfer requires an adapted decision between single and double embryo transfer, although a single blastocyst transfer is recommended for the D5 transfer in order to decrease the twin pregnancy rate.\u003c/p\u003e","manuscriptTitle":"Comparison of pregnancy outcomes between 4th day morula and 5th day blastocyst after embryo transfer:a retrospective cohort study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-15 21:01:37","doi":"10.21203/rs.3.rs-3775383/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-22T08:14:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-22T04:30:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75319c6c-c60c-468a-8082-b11d1b3e99a7","date":"2024-03-11T22:30:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-01-28T15:58:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0a0f0fe4-2491-4c3d-89ef-1cff01e52b4a_SNPRID","date":"2024-01-28T14:07:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-18T09:19:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-18T09:15:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-01-12T13:36:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-12T13:32:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pregnancy and Childbirth","date":"2023-12-19T06:53:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pregnancy-and-childbirth","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prch","sideBox":"Learn more about [BMC Pregnancy and Childbirth](http://bmcpregnancychildbirth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/prch/default.aspx","title":"BMC Pregnancy and Childbirth","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2b641eed-94b8-4bf6-8357-595832c4f016","owner":[],"postedDate":"January 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-20T13:00:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-15 21:01:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3775383","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3775383","identity":"rs-3775383","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}