Effect of day 3 embryo cell number on the pregnancy and neonatal outcomes of day 4 single embryo transfer from fresh cycles

doi:10.21203/rs.3.rs-4887460/v1

Background

The aim of this retrospective cohort study was to assess the impact of day 3(D3) embryo cell number on the clinical pregnancy and neonatal outcomes of day 4(D4) single embryo transfer in fresh cycles.

Methods

The study included 431 day 4 single embryo transfer in fresh cycles conducted between December 2018 and June 2023. These cycles were divided into three groups according to the day 3 embryo cell number: 248 cycles in the 7 ~ 9-cell group, 149 cycles in the 10 ~ 13-cell group and 34 cycles in the >13-cell group, and clinical pregnancy outcomes and neonatal outcomes were compared among the three groups.

Results

The clinical outcomes with 10 ~ 13-cell were significantly higher than those of the 7 ~ 9-cell group, regardless of whether the female age was < 30 or ≥ 30 years. The same result could be found when the insemination pattern was IVF, and when the transferred embryos were the grade of complete fusion. There were no differences in neonatal outcomes between different groups.After adjusting for confounding factors, the 7 ~ 9-cell group was associated with lower clinical pregnancy rates and live birth rates compared with the 10 ~ 13-cell group (clinical pregnancy rates: aOR 0.521, 95% CI -1.198~-0.105, P = 0.019; LBR: aOR 0.539, 95% CI -1.122~-0.114, P = 0.016)

Conclusion

The cell number of D3 embryos can be an important reference indicator for D4 embryo selection. When performing day 4 single embryo transfer in fresh cycles, embryos with 10 ~ 13-cell on D3 can be preferentially selected for transplantation to enhance clinical outcomes, especially when the insemination pattern is IVF,and when the transferred embryos are the grade of compaction stage

Currently, with the continuous optimization of various technologies in assisted reproductive technology (ART) ,more fertility centers tend to culture embryos to blastocysts and then perform single blastocyst transfers(SBT).Compared with cleavage-stage embryos, blastocysts have better synchronization with the endometrium and the embryonic genome has been activated, enabling selection of embryos with higher aneuploidy rates and relatively higher vitality [1–2].At the same time, the selection of higher quality blastocysts for SBT can help reduce the rate of multiple pregnancies and avoid adverse obstetric outcomes.

Morula embryos on the fourth day after fertilization have the same advantage of embryonic genome activation as blastocysts[3].And as the embryo enters the uterine cavity from the fallopian tube on the fourth day under physiological conditions, D4 embryo transfer is closer to the physiological process[4]. Meanwhile, uterine peristalsis decreases on the fourth day, which favors embryo implantation. Compared with day 5(D5) transfer, D4 embryo transfer shortens the in vitro culture time and reduces the risk of abnormalities in acquired modifications of genes due to the stress of continuous exposure of embryos to in vitro culture conditions. Therefore, D4 embryo transfer is able to increase the number of transferable embryos than D5[5] and reduce the probability of having no embryos to transfer. And studies have shown that D4 embryo transfer is able to achieve pregnancy outcomes similar to blastocysts in both fresh and thawed cycles [6–9].In addition, several studies have associated fresh blastocyst transfer with a high risk of placental and perinatal complications [10–12].

Embryonic fusion is essential for the formation of blastocyst trophoblast and inner cell mass [13].On the fourth day of development, the embryo will have 16–32 blastomeres, with signs of fusion between the blastomeres and the cell membrane boundaries between cells gradually disappear. On the fourth day after fertilization, the embryos will have different states, including different blastomeres fusion ratios, fragmentation ratios, and other states. In faster developing embryos, the blastomeres fuse completely or even form early blastocysts. The 2011 Istanbul Consensus [14] proposed criteria for the evaluation of embryos at the D4 morula stage, which provided some reference for the scoring of D4 embryos. This consensus considers embryos that develop to full fusion or more on the fourth day to be high-quality embryos. For cycles with a larger number of oocytes, multiple embryos that have developed beyond full fusion may be obtained on D4.Therefore, how to select embryos with high implantation potential for single-embryo transfer is the key to increasing pregnancy rates and decreasing multiple birth rates.

The embryo cell number on the third day is one of the most important predictors of embryonic developmental potential. Based on national data from the Society for Assisted Reproductive Technology Clinical Outcomes Reporting System (SART CORS),when the cell numbers was less than or equal to 8, the live birth rate (LBR) increased with the number of cells, but the live birth rate was diminished in embryos > 8 cells[15].Similarly, in the Istanbul Consensus of the European Society of Human Reproduction and Embryology (ESHRE)-Alpha scientists, the best-quality day 3 embryos have eight identical-sized blastomeres, whereas slower- or faster-developing embryos may have anomalies that can lead to decreased implantation rates[16].However, recent studies have reported that rapidly developing embryos at day 3 have similar or even significantly higher rates of blastocyst formation compared to 8-cell embryos [17–22].Pons et al [21] further demonstrated comparable ploidy between > 11-cell and 8-cell embryo embryos. But there is still no consensus on the developmental potential of rapidly developing embryos. And, the effect of day 3 embryo cell number on the clinical outcomes of day 4 embryo transfer has not been reported. The aim of this paper is to investigate the effect of day 3 embryo cell number on the pregnancy and neonatal outcomes of day 4 single embryo transfer in fresh cycles, and to provide a basis for the selection of suitable embryos.

Study material

This retrospective study was conducted at the Xingtai Infertility Specialist Hospital between December 2018 and June 2023.The inclusion criteria were as follows: maternal age ≤ 35 years, day 4 single embryo transfer in fresh cycles, and IVF or ICSI insemination pattern. The exclusion criteria included acquired or congenital uterine abnormalities (such as congenital uterine malformations, intrauterine adhesions, endometrial polyps and submucosal fibroids, and severe adenomyosis) diagnosed using 3D ultrasound, a lack of core data, and lost cases. The study included 431 day 4 single embryo transfer in fresh cycles. These cycles were divided into three groups according to the day 3 embryo cell number: 248 cycles in the 7 ~ 9-cell group, 149 cycles in the 10 ~ 13-cell group and 34 cycles in the >13-cell group, and compared the clinical pregnancy outcomes and neonatal outcomes among the three groups. Since there were only 13 cycles in the <7-cell group, they were not discussed in this study(Fig. 1). This study was approved by the Ethics Committee of Xingtai Infertility Specialist Hospital (Approval number: 2023-LX-09). According to the Ethics Committee of Xingtai Reproduction and Genetics Specialist Hospital, the requirement for informed consent was waived owing to the retrospective nature of the study, and data from all patients were used anonymously.

Ovarian stimulation

Ovulation promotion protocols were based on routine protocols established by the department. Agonist and antagonist regimens were selected to stimulate ovulation based on the patient's ovarian reserve function, homogeneity of follicle size in the basal antral follicle, and the receptivity of the endometrium. 6000–10,000 IU of human chorionic gonadotropin (Zhuhai Lizon Pharmaceutical) were injected when at least two leading follicles measured ≥ 18 mm. After 36 ~ 37 h, under intravenous anesthesia and using ultrasound guidance, oocytes were taken out through a vaginal puncture.

Embryo culture

All oocytes were inseminated using in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) methods according to semen parameters. Embryos were cultured using the G5 sequential medium (Vitrolife, Göteborg, Sweden) at 37°C under conditions of 6% CO2, 5% O2 and 89% N2

. The 2011 ESHRE Istanbul Consensus and Ryh-sheng Li's approach [17] were used in our center to score embryos.The number of blastomeres, the level of fragmentation, and the existence of multinucleated cells were used to evaluate D3 embryos. D4 embryos, which lose all of the boundaries of the blastomere(compaction stage) or early blastocyst(Including stage 1, 2 and 3 blastocysts) were called high-quality embryos [17]. In this study, the embryos transferred in all cycles were of high quality.This is because in our center, if no high-quality embryos are formed on the fourth day after fertilization, the patients will be transferred with 2 embryos, which has been excluded in this study.

Definition of clinical outcomes

Twelve to fourteen days post-transplant, serum β-hCG was tested to ascertain whether a biochemical pregnancy had occurred. Transvaginal ultrasound was used to view the heart canal and the beat of the gestational sac approximately four weeks after the transplant. The quantity of gestational sacs was noted, and the gestational sac was verified to be a clinical pregnancy characteristic. Up until 12 weeks of gestation, luteal support was given, and then there was postpartum follow-up.

Statistical analysis

We performed statistical analyses using SPSS 22.0. Categorical variables were expressed as frequencies and percentages, while continuous variables that did not conform to the normal distribution were expressed as the median (25th, 75th percentile)M(Q1, Q3). Continuous variables with a normal distribution were expressed as mean ± standard deviation. and the median comparison was performed using the Kruskal–Wallis test. Rates (%) in enumeration data were compared using the adjusted χ² test or Fisher’s exact test. The Bonferroni correction was used for data analysis, and P < 0.017(0.05/3) was considered statistically significant. A logistic regression model was used to analyze the effect of D3 cell number on clinical pregnancy rate(CPR) and LBR after adjusting for confounding factors. The odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Results with P < 0.05 were considered statistically significant.

Maternal and cycle characteristics

As shown in Table 1, no significant differences were observed in female BMI, duration of infertility, cycle number, pattern of infertility,hormone value,infertility factors,Gn used dosage,Gn used duration, pattern of insemination,Fragmentation of day 3 embryo, number of oocytes retrieved or endometrial thickness among the three groups. However, the female age decreased significantly as the day 3 embryo cell number increased(P<0.001).The percentage of IVF insemination method was significantly higher in 10 ~ 13-cell group than in 7 ~ 9-cell group(P = 0.011).Additionally, The evenness of the blastomeres of the day 3 embryo and grade of transferred morula transferred on 7 ~ 9-cell, 10 ~ 13-cell, and >13-cell group were different(P<0.001).

Table 1

Maternal and cycle characteristics among the diferent groups
Characteristics	7 ~ 9-cell	10 ~ 13-cell	>13-cell	χ²		P-value
Number of transfer cycles(n)	248	149	34
Female age [years,M(Q1,Q3)]	31(28,33)^a	29(26, 32)^b	27(25, 31)^b	14.472		<0.001
Female BMI [kg/m2 ,,M(Q1,Q3)]	24.4(21.9,26.9)	24.7(22.2,26.8)	23.8(21.8,26.9)	1.252		0.535
Duration of infertility [years,,M(Q1,Q3)]	3.5(2,6)	3(2,5)	4(3,6)	1.418		0.492
Cycle number(%)				1.550		0.817
1	91.13(226/248)	91.28(136/149)	88.24(30/34)
2	7.26(18/248)	5.22(13/249)	8.82(3/34)
3	1.61(4/248)	0.67(1/149)	2.94(1/34)
Pattern of infertility(%)				0.993		0.609
Primary	34.68(86/248)	39.60(59/149)	35.29(12/34)
Secondary	65.73(163/248)	60.40(90/149)	64.71(22/34)
Basal FSH[U/L,,M(Q1,Q3)]	6.46(5.41,7.49)	6.01(5.3,7.21)	5.80(4.86,7.09)	2.879		0.237
Basal LH[U/L,,M(Q1,Q3)]	4.16(3.01,5.98)	3.79(2.78,5.43)	3.75(2.17,4.96)	4.385		0.112
AMH[µg/L,,M(Q1,Q3)]	3.72(2.7,5.29)	3.92(2.77,6.09)	4.15(1.98,5.43)	1.979		0.372
Gn used dosage[IU,,M(Q1,Q3)]	2450(2013,3000)	2400(2000,3150)	2300(2125,3069)	0.012		0.994
Gn used duration[d,,M(Q1,Q3)]	11(10,13)	11,(10,12)	11(10,12)	4.397		0.111
Hormone value on hCG injection day[,M(Q1,Q3)]
E₂(pmol/L)	2698(1831,3833)	2628(1898,3887)	2069(1692,3137)	3.8837		0.143
LH(U/L)	0.95(0.73,1.25)	0.86(0.665,1.13)	0.89(0.75,1.24)	3.6291		0.163
P(nmol/L)	0.74(0.58,0.90)	0.72(0.57,0.88)	0.66(0.57,0.80)	2.1911		0.334
Proportion of infertility factors(%)				6.4		0.78
Fallopian tube factor	70.97(176/248)	70.47(105/149)	63.65(23/34)
Ovulation disorder	12.90(32/248)	11.41(17/149)	14.71(5/34)
Endometriosis	3.23(8/248)	1.34(2/149)	2.94(1/34)
Male factor	8.47(21/248)	7.38(11/149)	8.82(3/34)
Unknown cause	2.02(5/248)	4.03(6/149)	2.94(1/34)
Others	2.02(5/248)	5.37(8/149)	2.94(1/34)
Pattern of insemination(%)				6.56		0.038
IVF	79.44(197/248)^a	89.26(133/149)^b	85.29(29/34)^a
ICSI	20.56(51/248)^a	10.74(16/149)^b	14.71(5/34)^a
Evenness of the blastomeres of day 3 embryo(%)				30.8		<0.001
0	31.45(78/248)^a	9.40(14/149)^b	23.53(8/34)^a
1	65.32(162/248)^a	89.93(134/149)^b	76.47(26/34)^a
2	3.23(8/248)	0.67(1/149)	0(0/34)
Fragmentation of day 3 embryo(%)					7.09	0.131
< 10%	52.82(131/248)	52.35(78/149)	52.94(18/34)
10–25%	42.74(106/248)	47.65(71/249)	44.12(15/34)
> 25%	4.44(11/248)	0(0/149)	2.94(1/34)
Grade of transferred Morula(%)				41.4		<0.001
Compaction stage	32.66(81/248)^a	10.74(16/149)^b	2.94(1/34)^b
Stage I blastocyst	52.01(129/248)	63.76(95/149)	55.88(19/34)
Stage2 blastocyst	13.31(33/248)^a	21.48(32/149)^a	38.24(13/34)^b
Stage3 blastocyst	2.02(5/248)	4.03(6/149)	2.94(1/34)
Number of oocytes retrieved, [,M(Q1,Q3)]	13.5(9,18)	14(11,18)	13(10,16)	2.161		0.339
Endometrial thickness[mm,M(Q1,Q3)]	11.5(10,13)	11.5(10,13.3)	12(10.1,13)	0.076		0.963
Note: 1. P value indicates differences among the three groups. P < 0.05 indicates statistically significant difference in results
2. a, b: different superscript letters in the same row indicate significant differences between groups. According to Bonferroni correction for multiple comparisons,
the difference was statistically significant at P < 0.017 (0.05/3)

Clinical outcomes and neonatal outcomes

The clinical outcomes and neonatal outcomes of different D3 cell number groups are listed in Table 2. The clinical pregnancy rate,implantation rate and live birth rate of the 10 ~ 13-cell group were significantly higher than that in the the 7 ~ 9-cell group(all P < 0.017).However, although it was lower in the > 13-cell group than in the 10 ~ 13-cell group, it was not significantly different.It is apparent from this table that there were no differences in neonatal outcomes among the three groups.

Table 2

Clinical outcomes among the diferent groups
	7 ~ 9-cell	10 ~ 13-cell	>13-cell	χ²	P
Number of transfer cycles	248	149	34
Clinical Pregnancy rate(%)	54.03(134/248)^a	71.14(106/149)^b	55.88(19/34)^ab	11.6	0.003
Miscarriage rate(%)	21.64(29/134)^a	15.09(16/106)^ab	0(0/19)^b	6.079	0.048
Implantation rate(%)	54.44(135/248)^a	71.14(106/149)^b	52.94(18/34)^ab	11.619	0.003
Live birth rate(%)	41.94(104/248)^a	59.73(89/149)^b	55.88(19/34)^ab	12.456	0.002
Ectopic pregnancy rate(%)	0.75(1/134)	0(0/106)	0(0/19)	0.740	0.691
Multiple pregnancy rate(%)	0.75(1/134)	0(0/106)	0(0/19)	0.740	0.691
Monozygotic twins rate(%)	0.75(1/134)	0.94(1/106)	0(0/19)	0.317	0.854
Premature delivery rate(%)	5.97(8/134)	5.66(6/106)	0(0/19)	1.153	0.562
Stillbirth rate(%)	0.75(1/134)	0.94(1/106)	0(0/19)	0.317	0.854
Cesarean section rate(%)	50.75(68/134)	48.11(51/106)	63.16(12/19)	2.459	0.293
Gestational week of delivery[weeks, ±s]	38.37 ± 1.72	38.47 ± 1.74	36.68 ± 8.92	0.317	0.853
Male/female ratio	47/58	50/44	11/9	1.695	0.429
Live birth weight [g, ±s]	3198.65 ± 495.22	3138.99 ± 755.72	3430.56 ± 387.35	4.408	0.110
Note: 1. P value indicates differences among the three groups. P < 0.05 indicates statistically significant difference in results
2. a, b: different superscript letters in the same row indicate significant differences between groups. According to Bonferroni correction for multiple comparisons,
the difference was statistically significant at P < 0.017 (0.05/3)

Subgroup analysis

Due to the age, the insemination method and the morula grade of the three groups being different among three groups, we stratified patients by age,insemination method and morula grade in Tables 3 and 4, and 5. Interestingly, the clinical pregnancy rates,implantation rates and live birth rates of the 10 ~ 13-cell group were also significantly higher than that in the the 7 ~ 9-cell group in ≥ 30 years women(all P < 0.017). In<30 years women, the 10 ~ 13-cell group had a greater live birth rate and a lower miscarriage rate than the 7 ~ 9-cell group (all P < 0.017). Although there were no statistically significant differences in clinical pregnancy rate and implantation rate of the <30 years group patients, a similar trend was presented; When the insemination method was IVF and with transferred compaction stage embryos, the clinical pregnancy rate,implantation rate and live birth rate of the 10 ~ 13-cell group were also significantly higher than that in the the 7 ~ 9-cell group(all P < 0.017). When the insemination method was ICSI and with transferred stage I,stage 2 and stage 3 blastocysts, there were no statistically significant differences in clinical pregnancy rates, implantation rates and live birth rates, but a similar trends was observed.From this data, we also could see that there were no significant differences in neonatal outcomes in any of the subgroups of this study.

Table 3

The effect of ages on the clinical pregnancy rates and neonatal outcomes
	7 ~ 9-cell	10 ~ 13-cell	>13-cell	χ²	P
≥ 30 years
Number of transfer cycles	152	71	12
Clinical Pregnancy rate(%)	52.63(80/152)^a	70.42(50/71)^b	58.33(7/12)^ab	6.301	0.043
Miscarriage rate(%)	18.75(15/80)	16(8/50)	0(0/7)	1.655	0.437
Implantation rate(%)	52.63(80/152)^a	70.42(50/71)^b	58.33(7/12)^ab	6.301	0.043
Live birth rate(%)	42.11(64/152)^a	59.15(42/71)^b	58.33(7/12)^ab	6.167	0.046
Cesarean section rate(%)	57.50(46/80)	44(22/50)	57.14(4/7)	2.311	0.315
Gestational week of delivery[weeks, ±s]	37.54 ± 5.14	38.52 ± 1.71	33.43 ± 14.76	1.214	0.545
Male/female ratio	29/36	25/18	5/2	3.103	0.212
Live birth weight [g, ±s]	3137.89 ± 657.74	3183.14 ± 845.73	3107.14 ± 1453.28	1.163	0.559
<30 years
Number of transfer cycles	96	78	22
Clinical Pregnancy rate(%)	56.25(54/96)	71.79(56/78)	54.55(12/22)	5.050	0.080
Miscarriage rate(%)	25.93(14/54)^a	14.29(8/56)^b	0(0/12)^ab	8.496	0.014
Implantation rate(%)	57.29(55/96)	71.79(56/78)	54.55(12/22)	4.587	0.101
Live birth rate(%)	41.67(40/96)^a	60.26(47/78)^b	54.55(12/22)^ab	6.111	0.047
Cesarean section rate(%)	40.74(22/54)	51.79(29/56)	66.67(8/12)	30129	0.209
Gestational week of delivery[weeks, ±s]	38.75 ± 0.98	38.42 ± 1.78	38.58 ± 0.79	0.412	0.813
Male/female ratio	18/22	25/26	6/7	0.151	0.927
Live birth weight [g, ±s]	3218.25 ± 439.82	3101.77 ± 677.03	3296.15 ± 291.16	1.172	0.557
Note: 1. P value indicates differences among the three groups. P < 0.05 indicates statistically significant difference in results
2. a, b: different superscript letters in the same row indicate significant differences between groups. According to Bonferroni correction for multiple comparisons,
the difference was statistically significant at P < 0.017 (0.05/3)

Table 4

The effect of insemination pattern on the clinical pregnancy rates and neonatal outcomes
	7 ~ 9-cell	10 ~ 13-cell	>13-cell	χ²	P
IVF
Number of transfer cycles	197	133	29
Clinical Pregnancy rate(%)	53.30(105/197)^a	70.68(94/133)^b	58.62(17/29)^ab	10.035	0.007
Miscarriage rate(%)	20.95(22/105)	13.83(13/94)	0(0/17)	5.421	0.066
Implantation rate(%)	53.30(105/197)^a	70.68(94/133)^b	58.62(17/29)	10.35	0.007
Live birth rate(%)	42.13(83/197)^a	60.15(80/133)^b	58.62(17/29)	11.219	0.004
Cesarean section rate(%)	56.19(59/105)	47.87(45/94)	70.59(12/17)	3.496	0.174
Gestational week of delivery[weeks, ±s]	38.42 ± 1.71	38.46 ± 1.71	36.41 ± 9.42	0.160	0.923
Male/female ratio	37/46	45/39	10/8	1.621	0.445
Live birth weight [g, ±s]	3218.19 ± 491.82	3169.52 ± 767.13	3202.78 ± 897.95	0.948	0.622
ICSI
Number of transfer cycles	51	16	5
Clinical Pregnancy rate(%)	56.86(29/51)	75(12/16)	40(2/5)	2.534	0.282
Miscarriage rate(%)	24.14(7/29)	25(3/12)	0(0/2)	0.639	0.726
Implantation rate(%)	58.82(30/51)	75(12/16)	40(2/5)	2.349	0.309
Live birth rate(%)	41.18(21/51)	56.25(9/16)	40(2/5)	1.462	0.481
Cesarean section rate(%)	31.03(9/29)	50(6/12)	0(0/2)	2.468	0.291
Gestational week of delivery[weeks, ±s]	36.41 ± 8.32	38.56 ± 2.13	39.00 ± 0.00	1.462	0.481
Male/female ratio	10/12	5/5	1/1	0.064	0.968
Live birth weight [g, ±s]	2979.55 ± 832.90	2882.50 ± 626.50	3475.00 ± 106.07	2.240	0.326
Note: 1. P value indicates differences among the three groups. P < 0.05 indicates statistically significant difference in results
2. a, b: different superscript letters in the same row indicate significant differences between groups. According to Bonferroni correction for multiple comparisons,
the difference was statistically significant at P < 0.017 (0.05/3)

Table 5

The effect of transferred morula grade on the clinical pregnancy rates and neonatal outcomes
	7 ~ 9-cell	10 ~ 13-cell	>13-cell	χ²	P
Compaction stage
Number of transfer cycles	81	16	1
Clinical Pregnancy rate(%)	48.15(39/81)^a	87.5(14/16)^b	100(1/1)^a	9.186	0.010
Miscarriage rate(%)	17.95(7/39)	21.43(3/14)	0(0/1)	0.314	0.855
Implantation rate(%)	49.38(40/81)^a	87.5(14/16)^b	100(1/1)^a	8.673	0.013
Live birth rate(%)	38.27(31/81)^a	68.75(11/16)^b	100(1/1)^a	6.332	0.042
Cesarean section rate(%)	58.97(23/39)	42.86(6/14)	0(0/1)	2.258	0.323
Gestational week of delivery[weeks, ±s]	37.25 ± 6.97	38.00 ± 1.61	39.00	0.853	0.653
Male/female ratio	13/19	7/4	1/0	2.858	0.240
Live birth weight [g, ±s]	2948.44 ± 701.21	2863.64 ± 1007.99	3400.00	1.504	0.471
Stage I blastocyst
Number of transfer cycles	129	95	19
Clinical Pregnancy rate(%)	52.71(68/129)	65.26(62/95)	57.89(11/19)	3.54	0.171
Miscarriage rate(%)	20.59(14/68)	12.90(8/62)	0(0/11)	3.660	0.160
Implantation rate(%)	52.71(68/129)	65.26(62/95)	57.89(11/19)	3.538	0.171
Live birth rate(%)	41.86(54/129)	55.79(53/95)	57.89(11/19)	4.968	0.083
Cesarean section rate(%)	48.53(33/68)	48.39(30/62)	90.91(10/11)ab	7.319	0.026
Gestational week of delivery[weeks, ±s]	38.32 ± 1.88	38.54 ± 1.79	38.55 ± 0.82	0.843	0.656
Male/female ratio	25/29	28/27	6/6	0.240	0.887
Live birth weight [g, ±s]	3312.78 ± 506.75	3149.36 ± 643.92	3445.83 ± 443.36	3.119	0.210
Stage2 blastocyst
Number of transfer cycles	33	32	13
Clinical Pregnancy rate(%)	75.76(25/33)	81.25(26/32)	46.15(6/13)	5.996	0.050
Miscarriage rate(%)	32(8/25)	19.23(5/26)	0(0/6)	3.162	0.206
Implantation rate(%)	75.76(25/33)	81.25(26/32)	46.15(6/13)b0.019	5.996	0.050
Live birth rate(%)	51.52(17/33)	65.63(21/32)	46.15(6/13)	1.983	0.371
Cesarean section rate(%)	44(11/25)	50(13/26)	33.33(2/6)	0.593	0.744
Gestational week of delivery[weeks, ±s]	38.29 ± 1.61	38.38 ± 1.83	32.50 ± 15.95	0.045	0.978
Male/female ratio	7/10	12/12	3/3	0.339	0.844
Live birth weight [g, ±s]	3051.18 ± 373.86	3202.08 ± 913.60	2808.33 ± 1412.95	0.510	0.775
Stage3 blastocyst
Number of transfer cycles	5	6	1
Clinical Pregnancy rate(%)	40(2/5)	66.66(4/6)	100(1/1)	1.577	0.454
Miscarriage rate(%)	0	0	0	-	-
Implantation rate(%)	40(2/5)	66.66(4/6)	100(1/1)	1.577	0.454
Live birth rate(%)	40(2/5)	66.66(4/6)	100(1/1)	1.577	0.454
Cesarean section rate(%)	50(1/2)	50(2/4)	0(0/1)	0.875	0.646
Gestational week of delivery[weeks, ±s]	39 ± 1.41	39.25 ± 0.96	39	0.150	0.928
Male/female ratio	2/0	3/1	1/0	0.875	0.646
Live birth weight [g, ±s]	3775.00 ± 813.17	3375.00 ± 263.00	3000.00 ± NaN	1.814	0.404
Note: 1. P value indicates differences among the three groups. P < 0.05 indicates statistically significant difference in results
2. a, b: different superscript letters in the same row indicate significant differences between groups. According to Bonferroni correction for multiple comparisons,
the difference was statistically significant at P < 0.017 (0.05/3)

A logistic regression analysis

The effect of D3 cell number on CPR and LBR after adjusting for confounding factors

As shown in Table 6, after adjusting for confounding factors,the 7 ~ 9-cell group was associated with lower clinical pregnancy rate and live birth rate compared with the 10 ~ 13-cell group (CPR: aOR 0.521, 95% CI -1.198~-0.105, P = 0.019; LBR: aOR 0.539, 95% CI -1.122~-0.114, P = 0.016).Moreover, the grade of transferred morula,E2 on hCG day and number of oocytes retrieved also significantly affected the clinical pregnancy.

Table 6

Results of the logistic regression analysis of clinical pregnancy rates and live birth rates after adjustments for confounding factors according to the maternal and cycle characteristics
	Clinical pregnancy rate			live birth rate
	OR	95%CI	p值	OR	95%CI	p值
Day 3 cell number :
10 ~ 13-cell	1 (Reference)
7 ~ 9-cell	0.52	-1.19~-0.11	0.02	0.54	-1.12~-0.114	0.02
>13-cell	0.40	-1.82 ~ 0.01	0.05	0.88	-1.00 ~ 0.744	0.77
Grade of transferred Morula
Stage2 blastocyst	1 (Reference)
Compaction stage	0.58	-1.35 ~ 0.26	0.18	1.09	-0.47 ~ 0.646	0.76
Stage I blastocyst	0.46	-1.45~-0.10	0.02	1.10	-0.51 ~ 0.692	0.76
Stage3 blastocyst	0.43	-2.40 ~ 0.73	0.30	1.34	-1.20 ~ 1.796	0.70
Female age	0.98	-0.10 ~ 0.05	0.53	0.99	-0.08 ~ 0.057	0.72
Duration of infertility	1.00	-0.10 ~ 0.09	0.94	1.01	-0.09 ~ 0.104	0.84
Female BMI	0.98	-0.10 ~ 0.07	0.65	0.99	-0.10 ~ 0.165	0.72
Proportion of infertility factors
Primary	1 (Reference)
Secondary	1.06	-0.50 ~ 0.61	0.85	0.96	-0.57 ~ 0.491	0.88
Basal FSH	0.97	-0.17 ~ 0.11	0.70	0.99	-0.15 ~ 0.122	0.86
Basal LH	0.96	-0.10 ~ 0.03	0.25	0.96	-0.10 ~ 0.022	0.21
AMH	1.03	-0.10 ~ 0.17	0.64	1.05	-0.07 ~ 0.165	0.45
Proportion of infertility factors
Others	1 (Reference)
Unknown cause	0.34	-3.01 ~ 0.85	0.27	1.78	-1.17 ~ 2.325	0.52
Endometriosis	0.14	-4.22 ~ 0.27	0.09	0.17	-4.25 ~ 0.698	0.16
Ovulation disorder	0.83	-1.75 ~ 1.38	0.82	1.58	-0.88 ~ 1.788	0.50
Male factor	0.25	-3.15 ~ 0.37	0.12	1.56	-1.11 ~ 2.009	0.58
Fallopian tube factor	0.49	-2.13 ~ 0.71	0.33	1.28	-0.95 ~ 1.435	0.69
Gn used dosage	1	-3.31E − 4-6.48E-04	0.53	1	-0.00 ~ 0.00	0.76
Gn used duration	0.87	-0.32 ~ 0.05	0.14	0.98	-0.19 ~ 0.15	0.84
E2	1	0.00 ~ 0.00	0.04	1	0 ~ 0	0.81
LH	0.89	-0.40 ~ 0.16	0.40	0.92	-0.35 ~ 0.18	0.53
P	0.76	-1.23 ~ 0.70	0.59	0.60	-1.44 ~ 0.42	0.28
Endometrial thickness	1.07	-0.04 ~ 0.17	0.21	1.04	-0.06 ~ 0.14	0.43
Number of oocytes retrieved	0.94	-0.11~-0.01	0.03	0.98	-0.07 ~ 0.03	0.39
Fragmentation of day 3 embryo
10–25%	1 (Reference)
<10%	0.95	-0.55 ~ 0.45	0.84	1.02	-0.45 ~ 0.49	0.95
>25%	1.63	-0.86 ~ 1.84	0.48	1.41	-0.93 ~ 1.62	0.60
Evenness of the blastomeres of day 3 embryo
0	1 (Reference)
1	1.40	-0.22 ~ 0.90	0.24	1.17	-0.39 ~ 0.70	0.57
2	1.88	-0.95 ~ 2.21	0.435	1.62	-0.98 ~ 1.94	0.52
Pattern of insemination
ICSI	1 (Reference)
IVF	0.61	-1.36 ~ 0.37	0.26	1.34	-0.49 ~ 1.08	0.47
Cycle number
1	1 (Reference)
2	1.77	-0.43 ~ 1.57	0.26	1.41	-0.53 ~ 1.21	0.44
3	5.26E + 06	-1370.51-1401.46	0.98	3.12	-0.92 ~ 3.20	0.28

Selecting embryos with the highest developmental potential for single embryo transfer on D4 of a fresh cycle is the key to improving IVF clinical outcomes and reducing the multiple birth rate.The present study suggested that when performing day 4 single embryo transfer in fresh cycles,the clinical outcomes of embryos with 10 ~ 13-cell were significantly higher than those of the 7 ~ 9-cell group, regardless of whether the female age was < 30 or ≥ 30 years,so embryos with 10 ~ 13 cells on day 3 can be prioritized to enhance clinical outcome. The same result could be found when the insemination pattern was IVF,and when the transferred embryos were the compaction stage. Whereas there were no differences in neonatal outcomes between different groups,indicating that different cell numbers on D3 had no difference in pregnancy outcomes after single embryo transfer on D4.

Following the fourth day after fertilization, the embryo enters the fourth round of cleavage, which is observed morphologically as a gradual fusion of the blastomeres and even the appearance of a blastocyst cavity. Compared with other stages of preimplantation embryo development, the morula embryo on D4 and its associated cell fusion process have received little attention.Day 4 embryos are in the process of transitioning from the cleavage stage to the fusion stage [24] and have usually completed the fusion process.It is optimal for an embryo to reach a specific stage of development at a specific time; either too fast or too slow development predicts a lower embryonic developmental potential.Several studies have analyzed the process of cell fusion in human embryos in vitro,the initiation of fusion before 8 cells is associated with cell dynamics failure and abnormal embryonic development [24].At the same time,delayed and/or incompletely fused morula embryos have been reported to be less likely to develop into high-quality blastocysts [25].In a retrospective study analyzing the development of 2059 embryos,developmental delay at post-fusion stages was more pronounced in the group showing partial fusion,which affects blastocyst formation, implantation, and live birth [26].Embryos cultured in a time-lapse incubator were found that develop into fully fused embryos at 94.9 hours and form regular blastocysts at 113.9 hours after ICSI fertilization show high pregnancy rates [27]. In addition, another study showed that the embryos had a high implantation potential by completing the first division within 25.90 h, the second division within 37.88 h, and complete fusion within 79.3 hours after culture before reaching the blastocyst stage [28].However,the exact time point for embryonic development is currently unknown.

In this study, since there were only 13 cycles in the <7-cell group and had a low pregnancy rate, they were not discussed in this study.After transferred single high-quality embryo on D4 of the fresh cycle,the clinical outcomes was significantly higher in the 10 ~ 13-cell group than in the 7 ~ 9-cell groups.Due to differences in some basic information between groups, the same results were obtained after stratification according to age and the logistic regression analysis after adjustments for confounding factors according to the maternal and cycle characteristics.Although there were no significant differences when the insemination pattern is ICSI,and when the transferred embryos are the grades of grade 1, grade 2 and grade 3 blastocysts, the trends were the same. The reason for the statistical insignificance may be related to the small amount of data.This finding is consistent with the general consensus that slower developing day 3 embryos have reduced developmental potential[15–16].Possible causes include prolonged cell cycle, fragmentation resulting in fewer surviving blastomeres, and developmental arrest or unexplained developmental delay [29].Slow-developing embryos have been reported to have a higher rate of aneuploidy and a reduced likelihood of forming euploidy blastocysts [21–22].Thus, chromosomal abnormalities may be responsible for the failure of implantation after embryo transfer.

There were fewer studies on the effect of increased number of D3 cells on pregnancy outcome. Racowsky et al [15] found that embryos with a higher number of blastomeres had significantly lower LBR than 8-cell embryos. However, the data may have some bias because confounding factors such as female age and infertility were not excluded, and double embryo transfer made it difficult to differentiate the morphological characteristics of independent embryos.On the contrary, Kong et al [29] found that after excluding embryos with abnormal divisions, LBR tended to increase with increasing cell number on day 3.Zhao et al [30] reported no significant difference in LBR (60.0% vs. 59.90%) in patients with > 10-cell embryo transfers compared to the 8-cell transfer group, but the miscarriage rate of > 10-cell embryos (4.3% vs. 13.5%; P = 0.04) was significantly lower.The study by Tian et al [31] was based on 2237 fresh day 3 single embryo transfer cycles and showed that LBR of ≥ 10-cell embryos was significantly higher than that of 8-cell embryos, and there was no significant difference in the miscarriage rate.Our results showed that D4 embryos with fast development (10 ~ 13 cells) at D3 had a higher clinical outcome after transfer than the 7 ~ 9 cell group.The exact reason for the increased number of blastomeres leading to improved pregnancy outcomes is not known.It may be related to the higher rate of blastocyst formation and quality of blastocysts in faster developing embryos.While some studies have shown that blastocyst formation rates in faster developing embryos of D3 are comparable to those of 8-cell embryos [17, 18, 20, 22], other researchers have reported statistically significant elevations [21, 29].A retrospective study by Luna et al [19] showed that faster developing embryos (≥ 10 cells) were more likely to grow into high quality blastocysts of 4AA or 5AA than those with intermediate developmental rates.Thus, an increase in the cell number on day 3 may predict higher developmental potential, leading to higher clinical outcomes.

Faster developing embryos are often considered to have a higher probability of chromosomal abnormalities.Among the supporting evidence [21, 32, 33], one of the most recent studies was a 2015 embryo biopsy showing that > 9-cell embryos at day 3 were significantly associated with increased aneuploidy rates [21].However, Moayeri et al.'s [34] study concluded that embryo fragmentation, rather than cell number, can be a sensitive predictor of chromosomal normalcy or not.In another retrospective study, Zhao et al [30] also found that embryos of > 10-cell origin had a similar aneuploidy rate as those of 8-cell origin (55.6% vs. 55.9%).Pons et al [22] analyzed PGT-A data from a total of 4028 embryos and further confirmed that the ploidy of blastocysts from > 11-cell is comparable to that of 8-cell.Thus, the implantation potential of faster developing embryos may not be affected by their chromosomal status.All of the above studies classified > 10 cells as a group without further detailed grouping, while the present study further detailed > 10 cells into 10 ~ 13 cells and ≥ 14 cells groups. The results showed that D4 embryos with 10 ~ 13-cell at D3 had the highest pregnancy and live birth rate after transfer, while those with too fast embryo development (≥ 14 cells) tended to have lower pregnancy rates.The reduced implantation potential of too faster developing embryos may be associated with an increased abnormal cleavage behavior of direct division in too faster developing embryos,the exact reason for which is not known.Embryos with higher cell numbers have smaller blastomeres, and larger fragments can easily be mistaken for blastomeres, so some fragmented embryos are at risk of being mistaken for faster developing embryos. This may also be one of the reasons why there is a tendency for clinical outcomes to decline in faster developing embryos.However, different culture media and culture environments in reproduction centers may affect the embryos cleavage and influence the metabolic activity of embryos, leading to inconsistent results in different studies.For example, embryos cultured under 5% O₂ conditions develop faster than 20% O₂[35]. In addition, culture conditions and male factors affect the duration of the S phase and cytoplasmic maturation[36, 37].

There are some limitations in this study, firstly, this study is a retrospective cohort study and the number of cycles in each group is not uniformly distributed, but this situation is reasonable because D4 high-quality embryo formation rate in ≥ 14-cell embryos on D3 is inherently low. Additionally, the amount of data in this study was limited, and further prospective studies with larger sample sizes are needed to obtain stronger evidence.

In conclusion, when performing day 4 single embryo transfer in fresh cycles ,the clinical outcomes of embryos with 10 ~ 13-cell on day 3 were significantly higher than those of the 7 ~ 9-cell group, regardless of whether the female age was < 30 or ≥ 30 years. The same result can be found when the insemination pattern is IVF,and when the transferred embryos are the grades of complete fusion. There were no differences in neonatal outcomes between different groups.Therefore, when transplantation is performed on D4 of the fresh cycle,embryos with 10 ~ 13 cells on day 3 can be preferentially selected for transplantation to enhance clinical outcomes.The results of this study provide evidence for improving the reference indicator for embryo selection on the D4 of a fresh cycle.

IVF:In vitro fertilization;ICSI:Intracytoplasmic sperm injection;CPR:clinical pregnancy rates,LBR :live birth rates ;Gn: Gonadotropin; D3:Day 3;D4:Day 4;D5:Day 5;BMI:Body mass index;

CI:Confidence interval;FSH :Follicle-stimulating hormone ;OR: Odds ratio ;PGT:Preimplantation genetic testing

Ethics approval and consent to participate

The study was approved by the research ethics board at Xingtai Infertility Specialist Hospital (approval number：2023-LV-09). The requirement for informed consent was waived owing to the retrospective nature of the study, and data from all patients were used anonymously.The research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki.

Consent for publication

Not applicable.

Data Availability

The datasets during the current study are available from the corresponding author on reasonable reque

Competing interests

The authors declare no competing interests.

Funding

Hebei Medical Science Research Project (20240234).

Authors' contributions

Fangfang Dai and Shusong Wang contributed to the conception and design of the study. Linlin Tao

and Bo zheng contributed to data interpretation and drafted the manuscript. Guozhen Li and Yasong Geng contributed to data analysis. Yuying Guo and Haoyang Dai contributed to data acquisition. The authors read and approved the final manuscript.

Acknowledgements

Not applicable.

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No competing interests reported.

Effect of day 3 embryo cell number on the pregnancy and neonatal outcomes of day 4 single embryo transfer from fresh cycles

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Background

Materials and methods

Study material

Ovarian stimulation

Embryo culture

Definition of clinical outcomes

Statistical analysis

Results

Maternal and cycle characteristics

Clinical outcomes and neonatal outcomes

Subgroup analysis

A logistic regression analysis

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1