Increased blastomere number in D3 embryos is associated with higher live birth rates in vitrified– thawed single blastocyst transfer cycles

doi:10.21203/rs.3.rs-3865314/v1

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Increased blastomere number in D3 embryos is associated with higher live birth rates in vitrified– thawed single blastocyst transfer cycles

https://doi.org/10.21203/rs.3.rs-3865314/v1

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Background

The number of D3 embryo blastomeres affects pregnancy outcomes in patients undergoing cleavage-stage embryo transfer. However, the association between blastomere number in D3 embryos and pregnancy outcomes after vitrified–thawed single blastocyst transfer remains unknown.

Methods

This retrospective cohort follow-up study included 2,274 cases of vitrified–thawed single blastocyst transfer,all patients were divided into six groups according to blastomere number of D3 embryos: ≤5, 6, 7, 8, 9, and ≥ 10 cells. The primary outcome was the live birth rate (LBR). The secondary outcomes were the clinical pregnancy rate, miscarriage rate, and neonatal outcomes. Statistical analyses were performed using a multivariate logistic regression model to explore the association between blastomere number in D3 embryos and LBR.

Results

The LBR significantly increased with the number of blastomeres in D3 embryos (28.4%, 36.4%, 42.5%, 46.1%, 45.2%, and 58.1%; p < 0.001). Furthermore, the results of the high- and low-quality blastocyst subgroup analyses showed significant differences in the LBR among the groups (p < 0.01). As the number of blastomeres in D3 embryos increased, the miscarriage rate significantly decreased (23.3%, 18.6%, 14.0%, 15.9%, 13.6%, and 8.9%; p < 0.05). However, the number of blastomeres did not affect perinatal outcomes. Multivariate logistic regression analysis after adjusting for confounding factors revealed significantly decreased LBR in the ≤ 5-cell group (adjusted odds ratio [aOR]: 0.627, 95% confidence interval [CI]: 0.442–0.891; p < 0.01) and significantly increased LBR in the ≥ 10-cell group (aOR: 1.612, 95% CI: 1.230–2.112; p < 0.01) compared with that in the 8-cell group.

Conclusions

The number of blastomeres in D3 embryos may be an important factor in selecting blastocysts during vitrified–thawed single blastocyst transfer cycles. The transfer of a single blastocyst arising from ≥ 10-blastomere D3 embryo may reduce the miscarriage rate and improve LBR.

day 3 blastomere number

blastocyst quality

frozen embryo transfer

single blastocyst transfer

live birth rate

The primary goal of assisted reproductive technology (ART) is to select the best embryo with high development potential for achieving a single healthy live birth. With the development of ART, selective single embryo transfer has been widely promoted to reduce adverse health outcomes in mothers and neonates. Although the transfer of multiple embryos improves the pregnancy rate and live birth rate (LBR), it results in multiple pregnancies, which may increase maternal and neonatal complications such as pregnancy-induced hypertension, diabetes mellitus, hemorrhage, preterm delivery, and low birth weight [1, 2]. Therefore, it is crucial to select the highest developmental competitive embryo for the transfer.

The Istanbul consensus recommends that the optimal developmental speed for a 3-day-old (D3) embryo is the achievement of the cleavage stage with eight blastomeres [3]. Some studies have shown that embryos with either lower or higher blastomere numbers have significantly reduced developmental potential, which can negatively influence the pregnancy rate[4–6].

Recently, advancements in embryo culture media and the improved utilization of vitrification have shifted the preference of using cleavage-stage embryo transfer to using blastocyst-stage embryo transfer in ART[7]. Single blastocyst-stage embryo transfer is generally accepted as a more effective method than multiple cleavage-stage embryo transfer to reduce the rate of multiple pregnancies without reducing the clinical pregnancy rate [8]. The blastocyst grading system proposed by Gardner DK et al.[9] is commonly used to select blastocysts for embryo transfer. Blastocysts with high inner cell mass (ICM) and trophectoderm cell (TE) grading are associated with higher euploidy and implantation rates than blastocysts with low morphology scores [10].

The effects of the transfer of single blastocysts arising from cleavage-stage embryos with different cell numbers are controversial. A small number of blastomeres in D3 embryos is associated with reduced clinical pregnancy rates and LBR[11]. However, information regarding clinical pregnancy rates and LBRs in the transfer of blastocysts arising from fast-developing embryos is limited.Kroener et al.[12]suggested that blastocysts derived from fast-developing embryos are associated with significantly increased aneuploidy rates compared with those derived from embryos with 6–9 cells. Previous studies have reported that fast-developing D3 embryos have euploidy rates similar to those of 8-cell embryos[13, 14]and the LBR of blastocyst arising from fast-developing embryos is similar in > 8-cell and 8-cell groups [11, 15]. Therefore, it is important to identify whether fast-developing D3 embryos are superior to 8-cell embryos in predicting pregnancy outcomes in blastocysts with similar morphological quality. This study aimed to explore the impact of D3 blastomere number on pregnancy outcomes in patients undergoing vitrified–thawed single blastocyst transfers.

Study design and patients

In this study, data of all patients (n = 2,274 cycles) who underwent vitrified–thawed single blastocyst transfer on day 5 from January 2016 to December 2021 at the Reproductive Medicine Center of Guangdong Provincial Fertility Hospital were retrospectively analyzed. The patients were classified into six groups based on the number of blastomeres in D3 embryos: (1) ≤ 5 cells, (2) 6 cells, (3) 7 cells, (4) 8 cells, (5) 9 cells, and (6) ≥ 10 cells. The LBR, clinical pregnancy rate, miscarriage rate, and neonatal outcomes were compared among the six groups. The exclusion criteria were as follows: patients who had undergone embryo transfer and those with factors that can adversely affect implantation, including hydrosalpinx, endometrial polyps, intrauterine adhesion, adenomyosis, myomas (≥ 4cm in diameter or the submucous type), and uterine abnormalities. Preimplantation genetic testing is not performed at the center; therefore, none of the patients included in this study used preimplantation genetic testing.

Embryo culture and assessment

Controlled ovarian stimulation was performed according to the standard long and short antagonist protocols in the center. Ovulation was induced by administering 250-µg recombinant human chorionic gonadotropin (hCG; Ovidrel, Merck Serono, Switzerland) when at least two or three dominant follicles had a diameter of ≥ 17 mm. Oocytes were retrieved 34–36 h after hCG injection under vaginal ultrasound guidance. Insemination/injection was performed 39–40 h after hCG injection. Embryos were individually cultured in 30 µL of G1 medium droplets (G1 PLUS, Vitrolife Sweden AB, Sweden) under mineral oil (Vitrolife Sweden AB, Sweden) and incubated at 37°C in a 6% CO₂ and 5% O₂ environment. Fertilization was assessed 16–18 h after insemination/injection, and embryo quality was assessed on D3 (67–69 h), D5, and D6. After patients’ 1–2 high-quality embryos were frozen on D3, all supernumerary embryos were individually cultured in 30 µL of G2 medium droplet (G2 PLUS; Vitrolife Sweden AB, Sweden) until D5 or D6.

D3 cleavage-stage embryos were graded according to blastomere symmetry, fragmentation percentage, and blastomere number. The assessment criteria were based on the Istanbul consensus[3]. The blastocyst stage was assessed using the Gardner scoring system[9]. High-quality blastocysts were defined as blastocysts with stage 3 or higher blastocyst expansion and ICM and TE scores higher than grade B, whereas those graded lower than grade 3BB were defined as low-quality blastocysts. Blastocysts graded with CC were excluded and destroyed in this study. Blastocysts with better grades were given priority for transfer, irrespective of the D3 cell number.

Blastocyst vitrification and thawing

Blastocoels were artificially shrunken using a laser pulse (RI Saturn Active Laser System, England) before vitrification. Vitrification and warming were performed according to the instructions provided in Vit Kits (Kitazato Corporation, Japan). On the day of blastocyst transfer, blastocysts with the highest score were selected for thawing and transfer.

Endometrial preparation

Natural cycles were used for patients with regular menstrual cycles, whereas artificial hormone replacement therapy or stimulated cycles were used for patients with irregular menstruation or those with a history of thin endometrium.

Pregnancy and perinatal outcomes

The primary outcome of this study was the LBR, which was defined as delivery of a living baby after 28 weeks of gestation. Secondary endpoints included the clinical pregnancy and miscarriage rates. Neonatal outcomes among singletons were also evaluated, which included gestational age, birth weight, very preterm birth (VPTM; <32 weeks’ gestation), preterm birth (PTM; <37 weeks’ gestation), very low birth weight (VLBW; ≤1,500 g at birth), low birth weight (LBW, ≤ 2,500 g at birth), and high birth weight (HBW; ≥4,000 g at birth). Furthermore, congenital malformations were observed. Clinical pregnancy was identified by detecting an intrauterine gestational sac with fetal heartbeat on day 35 after frozen embryo transfer. Miscarriage was defined as clinical pregnancy loss before the 28th gestational week.

Statistical analysis

All statistical analyses were performed using Statistical Package for the Social Sciences (version 23.0; IBM Corp, Armonk, NY, USA). The patients were divided into six groups according to the number of D3 embryo blastomeres: ≤5, 6, 7, 8, 9, and ≥ 10 cells. Continuous variables are presented as means ± standard deviations and were compared using one-way analysis of variance. Categorical variables are expressed as numbers and percentages and were compared using Pearson’s chi-square test or Fisher’s exact test. Because five comparisons were made, p-values < 0.01 (calculated as 0.05/5) were used to denote statistical significance in the analysis according to Bonferroni adjustment for multiple comparisons. Logistic regression analysis was performed to investigate the independent effects of the number of D3 blastomeres on clinical outcomes after controlling for potential confounders. The number of D3 blastomeres, female’s age, infertility duration, infertility type (primary or secondary), scarred uterus, body mass index (BMI), stimulation protocol, fertilization protocol, number of oocytes retrieved, frozen embryo transfer preparation, endometrial thickness, blastomere symmetry, D3 embryo fragmentation, and D5/D6 blastocyst and individual blastocyst grades (expansion stage, ICM, and TE) were used as independent variables, and the clinical pregnancy rate and LBR were used as dependent variables. Using the 8-cell embryos group as reference, adjusted odd ratios (aORs) of clinical pregnancy and live birth were calculated. The results are reported as aORs with 95% confidence intervals (CIs). Two-tailed p-values of < 0.05 were used to denote statistical significance.

Comparison of general characteristics

A total of 2,274 patients were included in this study. According to the number of D3 blastomeres, the patients were divided into six groups: ≤5 (n = 225), 6 (n = 236), 7 (n = 431), 8 (n = 814), 9 (n = 217), and ≥ 10 (n = 351) cells. The baseline demographics of the patient cohort are presented in Table 1. The results showed no significant differences in age, infertility duration, BMI, basal follicle-stimulating hormone level, type of infertility, causes of infertility, scarred uterus, induced ovulation protocol in in vitro fertilization (IVF)/intracytoplasmic sperm injection cycles, fertilization methods, frozen embryo transfer preparation protocol, and endometrial thickness on the day of embryo transfer among the six groups (all p ≥ 0.05). However, the number of oocytes retrieved per cycle, blastomere symmetry, and fragmentation percentage of D3 embryos were significantly different among the six groups (p < 0.05). The number of oocytes retrieved was significantly lower in the ≤ 5-cell group (12.0 ± 4.78) than in the 8-cell group (14.33 ± 4.98). The fragmentation percentage (< 10%) was significantly lower in the ≤ 5-cell (28.9%), 6-cell (31.4%), and 7-cell (27.4%) groups than in the 8-cell (41.0%) group; however, the fragmentation percentage < 10% was higher in the 10-cell (63.8%) group than in the 8-cell group (p < 0.01). In the fragmentation percentage > 25% groups, the results were exactly the opposite.

Table 1

Demographic and treatment characteristics of patients with single blastocyst transfers.
Variable	≤ 5 cells (n = 225)	6 cells (n = 236)	7 cells (n = 431)	8 cells (n = 814)	9 cells (n = 217)	≥ 10 cells (n = 351)	P value
Maternal age at transfer(years)	32.63 ± 4.71	32.29 ± 4.63	32.62 ± 4.13	31.96 ± 4.23	32.11 ± 4.18	32.05 ± 4.31	0.093
Maternal age at oocyte retrieval(years)	31. 67 ± 4.71	31.30 ± 4.64	31.57 ± 4.13	31. 00 ± 4.23	31.23 ± 4.20	31.23 ± 4.32	0.197
Infertility duration (years)	5.06 ± 3.55	4.78 ± 3.30	5.01 ± 2.94	4.69 ± 2.93	4.74 ± 2.76	4.92 ± 2.99	0.393
Maternal BMI(kg/m2 )	21.46 ± 2.68	21.32 ± 3.04	21.25 ± 2.76	21.34 ± 2.93	21.40 ± 2.61	21.39 ± 2.96	0.966
Basal FSH((IU/L))	6.39 ± 1.73	6.26 ± 1.71	6.19 ± 1.53	6.29 ± 1.57	6.21 ± 1.62	6.23 ± 1.54	0.697
Infertility type n (%)
Primary infertility	150(66.7)	147(62.3)	253(58.7)	502(61.7)	134(61.8)	214(61.0)	0.538
Secondary infertility	75(33.3)	89(37.7)	178(41.3)	312(38.3)	83(38.2)	137(39.0)	-
Causes of infertility n (%)
Female	59(26.2)	77(32.6)	135(31.3)	276(33.9)	66(30.4)	100 (28.5)	0.234
Male	122(54.2)	128(54.2)	221 (51.3)	425(52.2)	110(50.7)	201(57.3)	0.601
Mixed	34(15.1)	24(10.2)	65(15.1)	90 (11.1)	33(15.2)	39(11.1)	0.117
Unexplained	10(4.4)	7(3.0)	10(2.3)	23(2.8)	8(3.7 )	11(3.1)	0.743
Scarred uterus, n (%)
Yes	30(13.3)	34(14.4)	76(17.6)	133(16.3)	37(17.1)	40 (11.4)	0.162
No	196(86.7)	202(85.6)	355(82.4)	681(83.7)	180(82.9)	311(88.6)	-
Stimulation protocol, n (%)
Agonist	157(69.8)	174(73.7)	310(71.9)	570(70.0)	148(68.2)	247(70.4)	0.808
Antagonist	68(30.2)	62(26.3)	121(28.1)	244(30.0)	69(31.8)	104(29.6)	-
Fertilization Methods, n (%)
IVF	182(80.9)	191(80.9)	360(83.5)	702(86.2)	184(86.4)	284(80.9)	0.078
ICSI	43(19.1)	45(19.1)	71(16.5)	112(13.8)	29(13.6)	67(19.1)	-
Number of oocytes retrieved	12.0 ± 4.78*	12.72 ± 4.80	14.14 ± 5.12	14.33 ± 4.98	13.84 ± 4.90	13.85 ± 4.89	0.000
FET preparation, n (%)
Natural cycle	33(14.7)	42(17.8)	71(16.5)	137(16.8)	31(14.3)	52(14.8)	0.825
Hormonal replacement cycle	175(77.8)	179(75.8)	333(77.3)	635(78.0)	177(81.6)	285(81.2)	0.511
Mild Stimulation cycle	17(7.6)	15(6.4)	27(6.3)	42(5.2)	9(4.1)	14(4.0)	0.396
Endometrium thickness on the day of embryo transfer (mm)	10.66 ± 2.26	11.04 ± 2.26	10.77 ± 2.25	10.83 ± 2.28	10.93 ± 2.20	10.87 ± 2.18	0.535
D3 embryo parameters
Blastomere symmetry, n (%)
Even	80(35.6)	75(31.8)	141(32.7)	325(39.9)	68(31.3)	115(32.8)	0.027
Uneven	145(64.4)	161(68.2)	290(67.3)	489(60.1)	149(68.7)	236(67.2)	-
Fragmentation percentage, n (%)
< 10%	65(28.9)*	74(31.4)*	118(27.4)*	334(41.0)	98(45.2)	224(63.8)*	0.000
10–25%	97(43.1)	83(35.2)*	216(50.1)	397(48.8)	94(43.3)	108(30.8)*	0.000
> 25%	63(28.0)*	79(33.5)*	97(22.5)*	83(10.2)	25(11.5)	19(5.4)*	0.000
Data are presented as mean ± standard deviation or number (percentage).*P < 0.01 represent signifcant diferences in the analysis according to Bonferroni adjustment (using the 8-cell embryo group as reference) .

Developmental potential of D3 embryos with different numbers of blastomeres in extended culture

The blastocyst formation, available blastocyst, and high-quality blastocyst rates were compared among the six groups to explore the development potential of embryos with different blastomere numbers. Overall, 2,274 cycles of 14,370 embryos were compared. The results revealed that the blastocyst formation and available blastocyst rates increased in groups with cell numbers of up to eight cells (p < 0.001) (Fig. 1) and decreased in the 9- and 10-cell groups compared with those in the 8-cell group (p < 0.001). The high-quality blastocyst rate also increased as the cell number increased; however, the ≥ 10-cell group had a higher high-quality blastocyst rate than the 8-cell group (31.47% vs. 27.44%; p < 0.01; Fig. 1).

Quality of transferred blastocysts developed from D3 embryos with different numbers of blastomeres

The high- and low-quality blastocyst rates, speed of blastocyst development (D5 or D6), and individual blastocyst grades (ICM, TE, and expansion stage) were compared among the six groups to explore the development potential of embryos with different blastomere numbers. The results revealed significant differences in all these comparison parameters among the groups(p < 0.001, Table 2). The proportions of high-quality blastocysts and D5 blastocysts in the ≤ 5-cell, 6-cell, and 7-cell groups significantly decreased compared with those in the 8-cell group (p < 0.01); however, the proportions were not significantly different among the 8-cell, 9-cell, and ≥ 10-cell groups (p ≥ 0.01). Furthermore, as the number of blastomeres in D3 embryos increased, the proportions of blastocysts with an expansion stage of 4 and an ICM grade of A increased; these proportions were significantly higher in the ≥ 10-cell group than in the 8-cell group (p < 0.01); however, these proportions were significantly lower in the 5-cell group than in the 8-cell group (p < 0.01) (Table 2).

Table 2

Quality of transferred blastocysts developed from D3 embryos with different numbers of blastomeres
	≤ 5 cells (n = 225)	6 cells (n = 236)	7 cells (n = 431)	8 cells (n = 814)	9 cells (n = 217)	≥ 10 cells (n = 351)	P value
Blastocyst quality n (%)
High quality	85(37.8)*	110(46.6)*	234(54.3)*	524(64.4)	132(60.8)	250(71.2)	0.000
low quality	140(62.2)*	126(53.4)*	197(45.7)*	290(35.6)	85(39.2)	101(28.8)	0.000
Days of frozen embryo n (%)
Day 5	92(40.9)*	137(58.1)*	306(71.0)*	690(84.8)	188(86.6)	312(88.9)	0.000
Day 6	133(59.1)*	99(41.9)*	125(29.0)*	124(15.2)	29(13.4)	39(11.1)	0.000
Expansion n (%)
3	72(32.0)*	55(23.3)	100(23.2)	151(18.6)	35(16.1)	27(7.7)*	0.000
4	146(64.9)*	174(73.7)	320(74.2) *	644(79.1)	177(81.6)	308(87.7)*	0.000
5	5(2.2)	5(2.1)	9(2.1)	14(1.7)	5(2.3)	13(3.7)	0.502
6	2(0.9)	2(0.8)	2(0.5)	5(0.6)	0(0.0)	3(0.9)	0.808
Inner cell mass n (%)							0.000
A	6(2.7)*	3(1.3)*	16(3.7)*	70(8.6)	15(6.9)	47(13.4)*	0.000
B	162(72.0)	16369.1)*	344(79.8)	637(78.3)	164(75.6)	259(73.8)	0.021
C	57(25.3)*	70(29.7)*	71(16.5)	107(13.1)	38(17.5)	45(12.8)	0.000
Trophectoderm n (%)							0.000
A	5(2.2)	6(2.5)	14(3.2)	54(6.6)	14(6.5)	31(8.8)	0.003
B	134(59.6)*	174(73.7)	293 (68.0)	576(70.8)	156(71.9)	262(74.6)	0.000
C	86(38.2)*	56(23.7)	124(28.8)	184(22.6)	47(21.7)	58(16.5)	0.000

Data are presented as number(percentage). *P < 0.01 represent signifcant diferences in the analysis according to Bonferroni adjustment (using the 8-cell embryo group as reference) .

Pregnancy outcomes of vitrified–thawed single blastocyst transfer with different numbers of D3 blastomeres

The association between clinical outcomes and the number of D3 blastomeres is shown in Table 3. The LBR was 28.4%, 36.4%, 42.5%, 46.1%, 45.2%, and 58.1% in the ≤ 5-cell, 6-cell, 7-cell, 8-cell, 9-cell, and ≥ 10-cell groups, respectively. Significant differences in the LBR were observed between the groups (p < 0.001). Furthermore, significant differences in the clinical pregnancy and miscarriage rates were observed between the groups (p < 0.05). The clinical pregnancy rate increased as the number of D3 blastomeres increased (p < 0.001). Significant differences in the miscarriage rate were observed among the groups (p < 0.05). The miscarriage rate decreased as the number of D3 blastomeres increased; the miscarriage rates in the ≤ 5-cell, 6-cell, 7-cell, 8-cell, 9-cell, and ≥ 10-cell groups were 23.3%, 18.6%, 14.0%, 15.9%, 13.6%, and 8.9%, respectively. The LBR and clinical pregnancy rate in the ≥ 10-cell group were significantly increased compared with those in the 8-cell group (p < 0.01); however, they were significantly decreased in the ≤ 5-cell group (p < 0.01). Nonetheless, no significant differences in PTM, VPTM, birth weight, and rate of congenital malformations were observed between the groups (p > 0.05).

Table 3

Pregnancy outcomes of vitrified–thawed single blastocyst transfer with different numbers of D3 blastomeres
	≤ 5 cells (n = 225)	6 cells (n = 236)	7 cells (n = 431)	8 cells (n = 814)	9 cells (n = 217)	≥ 10 cells (n = 351)	P value
Clinical pregnancy, n (%)	86(38.2)*	113(47.9)	214(49.7)	452(55.5)	118(54.4)	225(64.1)*	0.000
Miscarriage, n (%)	20(23.3)	21(18.6)	30(14.0)	72(15.9)	16(13.6)	20(8.9)	0.023
Live birth, n (%)	64(28.4)*	86(36.4)*	183(42.5)	375(46.1)	98(45.2)	204(58.1)*	0.000
Preterm deliveries (<37 weeks)	3/64(4.7)	4/86(4.7)	5/ 183(2.7)	10/375(2.7)	6/98(6.1)	2/204(1.0)	0.166
Very preterm deliveries(<32 weeks)	0/64(0.0)	0/86 (0.0)	2/183(1.1)	4/375(1.1)	1/98(1.0)	1/204 (0.5)	0.839
Birth weight	3318.75(3219.91,3417.59)	3195.58(3103.86,3287.30)	3514.86 (3177.32,3852.41)	3281.56 (3233.39,3329.73)	3213.11 (3109.49,3316.74)	3415.11 (3107.19,3723.02)	0.369
<1500g	0/64(0.0)	0/86(0.0)	1/183(0.5)	1/375(0.3)	1/98(1.0)	1/204(0.5)	0.869
<2500g	0/64(0.0)	5/86(5.8)	5/183(2.7)	21/375(5.6)	8/98(8.2)	8/204(3.9)	0.125
>4000g	3/64(4.7)	4/86(4.7)	15/183(8.2)	22/375(5.9)	4/98(4.1)	10/204(4.9)	0.679
congenital malformations n (%)	1/86(1.2)	2/113(1.8)	1/214(0.5)	6/452(1.3)	1/118(0.8)	1/224(0.4)	0.771

Data are presented as number (percentage) or mean (95% CI).*P < 0.01 represent signifcant diferences in the analysis according to Bonferroni adjustment (using the 8-cell embryo group as reference) .

Pregnancy outcomes of high-quality and low-quality blastocyst frozen embryo transfer cycles

As shown in Table 2, the quality of blastocysts obtained from D3 embryos with different numbers of blastomeres was significantly different. Because the quality of blastocysts is an important factor that determines the success rate of IVF, we demonstrated the association between pregnancy outcomes and D3 cell number in high- and low-quality blastocyst transfer in Table 4. The table shows that in the high-quality blastocyst frozen embryo transfer groups, significant differences in the clinical pregnancy rate and LBR were observed among the groups (p < 0.01), and the LBR was higher in the ≥ 10-cell group but lower in the ≤ 5-cell group than in the 8-cell group (p < 0.01). No significant differences in the miscarriage rate and the rate of congenital malformations were observed among the six groups (p > 0.05). In the low-quality blastocyst frozen embryo transfer groups, significant differences in the clinical pregnancy rate and LBR were observed among the groups (p < 0.05). However, no significant differences were found when each group was compared with the 8-cell embryo group comparisons according to Bonferroni adjustment. Furthermore, the miscarriage rate showed a gradual descending trend with rates of 29.4%, 26.7%, 22.5%, 20.9%, 17.1%, and 7.4% for the ≤ 5-cell, 6-cell, 7-cell, 8-cell, 9-cell, and ≥ 10-cell groups, respectively; however, no statistically significant difference was observed (p > 0.05).

Table 4

Pregnancy outcomes of high-quality and low-quality blastocyst frozen embryo transfer cycles
	≤ 5 cells	6 cells	7 cells	8 cells	9 cells	≥ 10 cells	P value
High-quality blastocysts(n)	85	110	234	524	132	250
Clinical pregnancy, n (%)	35(41.2)*	68(61.8)	134(57.3)	318(60.7)	77(58.3)	171(68.4)	0.001
Miscarriage, n (%)	5(14.3)	9 (13.2)	12((9.0)	44(13.8)	9(11.7)	16(9.4)	0.605
Live birth, n (%)	30(35.3)*	57(51.8)	122(52.1)	270(51.5)	66(50.0)	154(61.6)*	0.002
Genetic disorder/malformations n (%)	0(0.0)	1(1.5)	0(0.0)	5(1.6)	0(0.0)	1(0.6)	0.505
Low-quality blastocysts(n)	140	126	197	290	85	101
Clinical pregnancy, n (%)	51(36.4)	45(35.7)	80(40.6)	134(46.2)	41(48.2)	54(53.5)	0.031
Miscarriage, n (%)	15(29.4)	12(26.7)	18(22.5)	28(20.9)	7(17.1)	4(7.4)	0.087
Live birth, n (%)	34(24.3)	29(23.0)*	61(31.0)	105(36.2)	32(37.6)	50(49.5)	0.000
congenital malformations (%)	1(1.8)	1(2.3)	1(1.3)	1(0.7)	1(2.4)	0(0.0)	0.855

Data are presented as number (percentage). *P < 0.01 represent signifcant diferences in the analysis according to Bonferroni adjustment (using the 8-cell embryo group as reference) .

Results of logistic regression analysis of the clinical pregnancy rate and LBR

The results of logistic regression analysis of the clinical pregnancy rate and LBR after adjusting for some confounding variables are shown in Table 5. The ≥ 10-cell group was associated with higher LBR than the 8-cell group (aOR, 1.612; 95% CI, 1.230–2.112; p = 0.001). Conversely, the LBR was significantly lower in the ≤ 5-cell group (aOR, 0.627; 95% CI, 0.442–0.891; p = 0.009). The clinical pregnancy rates were similar to the LBR in the ≤ 5-cell and ≥ 10-cell groups. Moreover, age, infertility duration, endometrial thickness, blastocyst expansion stage of 4, ICM, and TE grade also significantly affected the clinical pregnancy rate and LBR. Scarred uterus only affected the LBR (aOR, 0.744; 95% CI, 0.577–0.959; p = 0.022).

Table 5

Results of logistic regression analysis of the clinical pregnancy rate and LBR
	Clinical pregnancy rate			Live birth rate
	aOR	95% CI	P‑value	aOR	95% CI	P‑value
D3 blastomeres number
≤ 5 cells	0.693	0.498,0.965	0.030	0.627	0.442,0.891	0.009
6 cells	0.909	0.666,1.239	0.545	0.810	0.588,1.117	0.199
7 cells	0.916	0.716,1.171	0.483	0.989	0.770,1.269	0.929
8 cells	Reference			Reference
9 cells	0.976	0.715,1.332	0.879	1.010	0.739,1.382	0.948
≥ 10 cells	1.364	1.037,1.793	0.026	1.612	1.230,2.112	0.001
Maternal age at transfer(years)	0.967	0.944,0.991	0.007	0.962	0.939,0.986	0.002
Infertility duration (years)	0.965	0.934,0.997	0.031	0.956	0.924,0.990	0.010
Type of infertility
Primary infertility	Reference			Reference
Second infertility	0.958	0.787,1.165	0.665	0.961	0.788,1.174	0.699
Maternal BMI(kg/m2 )	0.996	0.966,1.027	0.792	0.996	0.965,1.027	0.787
Scarred uterus, n (%)
NO	Reference			Reference
Yes	0.790	0.619,1.010	0.060	0.744	0.577,0.959	0.022
Stimulation protocol, n (%)
Agonist	Reference			Reference
Antagonist	1.158	0.956,1.404	0.133	1.127	0.963,1.427	0.227
Fertilization protocol, n (%)
IVF	Reference			Reference
ICSI	0.913	0.719,1.159	0.456	0.998	0.783,1.272	0.986
Number of oocytes retrieved	1.014	0.996,1.033	0.132	1.016	0.997,1.035	0.091
FET preparation
Natural cycle	Reference			Reference
Hormonal replacement cycle	1.009	0.797,1.278	0.941	0.980	0.770,1.247	0.867
Mild Stimulation cycle	0.956	0.619,1.478	0.840	1.062	0.679,1.662	0.793
Endometrium thickness on the day of embryo transfer (mm)	1.048	1.007,1.091	0.020	1.054	1.013,1.097	0.010
Blastomere symmetry of day 3 embryo
even	Reference			Reference
uneven	0.902	0.746,1.091	0.287	0.843	0.696,1.021	0.081
Fragmentation of day 3 embryo
3	Reference			Reference
2	0.870	0.658,1.151	0.329	0.765	0.584,1.002	0.051
1	0.861	0.662,1.120	0.265	0.751	0.564,1.000	0.050
Blastocyst expansion
3	Reference			Reference
4	1.395	1.111,1.752	0.004	1.350	1.067,1.709	0.012
5	0.923	0.490,1.739	0.803	0.848	0.438,1.643	0.625
6	0.582	0.170,1.996	0.390	0.522	0.133,2.045	0.351
Inner cell mass
A	3.013	1.935,4.691	0.000	3.686	2.392,5.681	0.000
B	1.701	1.318,2.195	0.000	2.316	1.765,3.040	0.000
C	Reference			Reference
Trophectoderm
A	1.610	1.041,2.488	0.032	1.538	1.005,2.354	0.048
B	1.479	1.188,1.839	0.000	1.557	1.246,1.946	0.000
C	Reference			Reference
Days of frozen embryo
D5	Reference			Reference
D6	0.779	0.618,0.981	0.034	0.849	0.668,1.078	0.179

The results of this study provide evidence that the number of blastomeres in D3 embryos was independently associated with the LBR in vitrified–thawed single blastocyst transfers. Compared with 8-cell embryos, blastocysts arising from fast-developing embryos (≥ 10 cells) resulted in higher LBR, whereas lower LBR was observed in slow-developing embryos (≤ 5 cells), regardless of the quality of blastocysts. Moreover, the miscarriage rate in vitrified–thawed single blastocyst transfer was significantly affected by the number of blastomeres in D3 embryos, which provided an effective indicator of blastocyst selection in IVF.

Currently, time-lapse embryo imaging[16], preimplantation genetic testing[17], and culture media analysis[18] have been used to evaluate embryo quality. Nevertheless, morphological scoring remains the most widely used criterion owing to its inexpensive and noninvasive nature. Of all morphological characteristics typically assessed in cleavage-stage (D3) embryos, the number of blastomeres is believed to be the single most important indicator of embryo viability [19]. Furthermore, the most widely accepted morphological scoring criterion of D3 embryos was the Istanbul consensus, which claimed that an optimal D3 embryo would have eight equally sized blastomeres, whereas embryos cleaving slower or faster are likely to be abnormal[3]. National data from the Society for Assisted Reproductive Technology Clinic Outcome Reporting System (SART CORS) also reported that LBR is positively associated with increased cell numbers up to 8 but was reduced in embryos with over 8 cells[20]. A low blastomere number in D3 embryos commonly indicates poor prognosis. Studies have confirmed that the transfer of slow-developing embryos results in low implantation rates and LBR [21, 22]. In this regard, several possible reasons have been proposed. The primary reason is that slow-developing embryos have higher aneuploidy rates and reduced likelihood of releasing euploid blastocysts [12, 13]. Other reasons, such as fewer surviving blastomeres, may also lead to developmental arrest or unexplained delays[23]. Some studies have also explained that the poor clinical outcomes of D3 embryos with ≤ 6 blastomeres might be partly due to embryo–endometrium asynchrony in ET cycles[22]). Our findings are in line with the general consensus regarding the decreased developmental potential of slow-developing embryos.

However, the development potential of fast-developing embryos remains controversial. Recently, some questions regarding the current consensus on embryo assessment have been raised. Some studies have hypothesized that fast-cleaving embryos have a similar or even higher developmental competence than 8-cell embryos [13, 21, 23, 24]. Luna et al.[24]demonstrated that fast-cleaving embryos (> 10 cells) not only reach the blastocyst stage at a rate similar to that of intermediate-cleaving embryos (8 cells) but also have a significantly greater likelihood of reaching better blastocysts. Pons et al.[13]reported that > 11-cell embryos can produce euploid blastocysts. Euploid blastocysts derived from > 11-cell and 8-cell embryos exhibit similar LBR in preimplantation genetic testing. Extended culture provides an opportunity to screen high-quality embryos and eliminate embryos with chromosomal abnormalities or poor developmental potential[25]. A pertinent question regarding blastocysts with similar morphological quality is that whether the number of blastomeres in D3 embryos affects their developmental potential. Currently, few studies have focused on this issue, and the conclusions are inconsistent, particularly for blastocysts arising from fast-developing D3 embryos. Wu et al. [11] analyzed the relationship between cell number in D3 embryos and pregnancy outcomes in vitrified–thawed single blastocyst transfer cycles and found that the clinical pregnancy rate and LBR were similar between the > 8-cell and 8-cell groups, and a low D3 cell number (≤ 5 cells) was associated with decreased LBR after blastocyst transfer in young women. Nevertheless, other studies have shown that the clinical pregnancy rate and LBR of blastocyst transferred in the > 8-cell group increased significantly compared with those in the < 8-cell or 8-cell group[26]. Time-lapse imaging research also indicated that a higher cell number was associated with higher blastocyst formation rates. The implantation rate and LBR exhibited an increasing trend from 36.4% (5–6 cells) to 62.5% (> 10 cells) and from 27.3% (5–6 cells) to 57.0% (> 10 cells) as the cell number increased in normal division embryos[23]. The results of our study to some extent agree with those of the aforementioned studies, and the clinical outcomes significantly increased as the number of blastomeres in D3 embryos increased. Transferred blastocysts derived from fast-developing embryos can result in higher clinical pregnancy rates and LBR.

The reason why blastocysts arising from fast-developing D3 embryos increase LBR remains unclear. We compared the developmental potential of D3 embryos with different numbers of blastomere using extended culture and found that the blastocyst formation and available blastocyst rates increased in embryos with cell numbers up to 8 but decreased in embryos with over 8 cells. However, the high-quality blastocyst rate increased in the ≥ 10 cell group compared with that in the 8-cell group. These results indicate that 8-cell embryos have the greatest potential to develop into blastocysts; however, fast-developing embryos (≥ 10 cells) could form blastocysts that exceed morphological quality in extended culture. Our data also indicate that fast-developing embryos (≥ 10 cells) have a higher proportion of 4-stage expanded blastocysts and are more likely to form blastocysts with the desired ICM and TE grades (ICM A and TE A and B). Our findings are consistent with those of other studies[23, 24]. Kong et al.[23]applied time-lapse imaging technology to monitor embryos and found that embryos with high cell numbers resulted from accelerated cell cycles, which were associated with a higher developmental potential. The number of D3 blastomeres of > 9 is associated with significantly increased aneuploidy rates[12]. Therefore, we speculated that some aneuploid fast-developing embryos (≥ 10 cells) were eliminated through extended culture and euploid embryos may possess vigorous growth ability that is likely to produce more high-quality euploid blastocysts [23], as the euploid rate may be positively related to the rate of high-morphology-quality blastocysts[27]. However, the exact mechanism underlying this phenomenon remains unclear.

Our study showed that as the number of blastomeres increased, the miscarriage rate significantly decreased, particularly in the low-quality blastocysts group. The miscarriage rate in the ≥ 10-cell group was lower than that in the 8-cell group; however, the difference was not significant (p = 0.026). This trend may be attributed to the relatively small number of samples included in the ≥ 10 cell group. Larger studies are required to confirm this finding. Several studies[15, 26] have reported that the miscarriage rates have no statistically significant differences between > 10- and 8-cell-derived blastocysts in the frozen embryo transfer group; however, comparable reduced miscarriage rates (4.3% vs. 13.5%; p = 0.04) were observed in the ≥ 10-cell and 8-cell groups during ET cycles [14]. Furthermore, some studies have reported that blastocysts arising from good-quality D3 embryos with high cell numbers are associated with lower miscarriage rates[28, 29]. These observations to some extent agree with the results of our study. Because chromosomal abnormality is one of the main reasons for early spontaneous abortions[28], morphological evaluation along with preimplantation genetic testing should be performed to verify our outcomes. Furthermore, in this study, no association was observed between the number of D3 blastomeres and neonatal outcomes. This result agrees with the findings of Chen L et al.[30]. However, Zhao et al.[15] found that blastocysts derived from fast-cleaving D3 embryos were associated with improved neonatal outcomes, including high mean birth weight, low rates of infants with LBW, and low rates of PTM. Further studies are required to determine whether the rapid developmental speed of fast-cleaving embryos affects neonatal outcomes.

According to the results of logistic regression analysis, we observed that the number of D3 blastomeres was an independent predictor of live birth in the presence of other confounders. Other parameters for morphological assessment (e.g., fragmentation, symmetry of D3 embryos, expansion stage, ICM, and TE of blastocysts) may also contribute to the outcomes of IVF. The present results showed that fragmentation and symmetry of D3 embryos were not significantly associated with the clinical pregnancy rate and LBR; however, the expansion, ICM, and TE grades were closely related to the clinical pregnancy rate and LBR. Studies have produced conflicting results regarding which morphology parameter is most closely related to pregnancy outcomes. Some researchers have declared that the TE grade is the best predictor of IVF success for single blastocyst transfers [31, 32], whereas others suggested ICM grade as an optimal predictor of pregnancy outcomes, particularly the clinical pregnancy rate and LBR[33, 34]. Moreover, some researchers recommend assessing the degree of blastocyst expansion to predict the clinical pregnancy rate and LBR [35, 36]. Recently, some studies have shown that both TE and ICM morphological grades were positively associated with pregnancy outcomes[11, 37], which is consistent with the findings of our study.

Because of the retrospective nature of this study, some limitations should be considered. First, this retrospective study was conducted in a single center, although all embryos were evaluated by two embryologists to reduce inter-embryologist variability. Second, embryos with the highest quality among 8-cell embryos (1 or 2 embryos) were frozen on day 3, which may have caused bias in the results of blastocysts derived from 8-cell embryos. The study strengths were its large sample size to demonstrate the association between the number of blastomeres in D3 embryos and the clinical pregnancy rate and LBR in vitrified–thawed single blastocyst transfer cycles. Furthermore, multiple regression models were established to control for various confounding factors. Furthermore, the number of D3 blastomeres was divided into more details, which exactly showed the trends of pregnancy outcomes in different cell number groups after single blastocyst transfers.

In conclusion, our study showed that the number of D3 embryo blastomeres is an important indicator for selecting blastocysts during vitrified–thawed single blastocyst transfer cycles. The transfer of single blastocysts arising from D3 embryos with ≥ 10 blastomeres is expected to reduce the miscarriage rate and improve LBR, particularly when low-quality blastocysts are transferred. Further prospective studies with a larger sample size are warranted to confirm our findings.

LBR

live birth rate

ART

assisted reproductive technology

ICM

inner cell mass

trophectoderm cell

BMI

body mass index

aOR

adjusted odds ratio

CIs

confidence intervals

hCG

human chorionic gonadotropin

VPTM

very preterm birth

PTM

preterm birth

VLBW

very low birth weight

LBW

low birth weight

HBW

high birth weight

IVF

in vitro fertilization.

Acknowledgments

We gratefully acknowledge the staff of the Reproductive Medicine Center in Guangdong Provincial Fertility Hospital for their support and cooperation.

Authors’ contributions

W.Z. supervised the entire study, including the procedures, conception, design, and completion. S.L. and Y.H. were responsible for data collection. L.H conducted the statistical analyses. Y.T. ,C.M. and Y.C. performed embryo culture and transfer. R.J. and G.S. supervised the project administration. All authors approved the final manuscript after critical revisions for intellectual content.

Funding

This study was supported by grants from the Innovation Team Project of the Guangdong Provincial Reproductive Science Institute (Guangdong Provincial Fertility Hospital) (C-02) and the Guangdong Provincial Medical Research Fund (A2020604).

Ethics approval and consent to participate

This study was approved by the ethics committee of the Reproductive Medicine Centre of Guangdong Provincial fertility Hospital, and all participants had signed the informed consent during treatment.

Consent for publication

Not applicable.

Data availability

Data available on request from the corresponding author.

Competing interests

The authors declare that they have no competing interests.

Author details

1.NHC Key Laboratory of Male Reproduction and Genetics, Guangdong Provincial Reproductive Science Institute (Guangdong Provincial Fertility Hospital), Guangzhou, China.

2.Reproductive Medicine Center, Guangdong Provincial fertility Hospital, Guangzhou, China.

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No competing interests reported.

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Increased blastomere number in D3 embryos is associated with higher live birth rates in vitrified– thawed single blastocyst transfer cycles

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