
==== Front
BMC Pregnancy Childbirth
BMC Pregnancy Childbirth
BMC Pregnancy and Childbirth
1471-2393
BioMed Central London

6794
10.1186/s12884-024-06794-4
Research
The impact of blastocyst grade on singleton birth weight in fresh IVF‒ET cycles in ART: a retrospective study
Shi Senlin ssllove666@163.com

1234
Zhao Boya 1234
Hu Zhan 1234
Shi Qiongyao 1234
1 https://ror.org/056swr059 grid.412633.1 Reproductive Medical Center, the First Affiliated Hospital of Zhengzhou University, No. 1 Jianshe East Road, Zhengzhou, Henan Province 450052 People’s Republic of China
2 https://ror.org/056swr059 grid.412633.1 Henan Key Laboratory of Reproduction and Genetics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 China
3 https://ror.org/056swr059 grid.412633.1 Henan Provincial Obstetrical and Gynecological Diseases (Reproductive Medicine) Clinical Research Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 China
4 https://ror.org/056swr059 grid.412633.1 Henan Engineering Laboratory of Preimplantation Genetic Diagnosis and Screening, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 China
7 9 2024
7 9 2024
2024
24 58825 5 2024
29 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

The positive correlation between embryo quality and pregnancy outcomes has been confirmed in many studies, but there are few on the impact of embryo quality on neonatal weight, especially among neonates from fresh IVF‒ET cycles in ART. Therefore, this study aimed to compare the birth weights of infants from different blastocyst grades in fresh IVF-ET cycles and explore related factors affecting birth weight.

Methods

The main outcome measure was singleton birth weight. A total of 1301 fresh cycles of single blastocyst transplantation and single live birth profiles were retrospectively analyzed and divided into four groups according to blastocyst quality: the excellent group (grade AA), which included 170 cycles; the good group (grade AB/BA), which included 312 cycles; the average group (grade BB/CA/AC), which included 559 cycles; and the poor group (grade BC/CB), which included 260 cycles. The relationships among cystic cavity expansion, endocytic cell mass, ectodermal trophoblast cell grade, and birth weight were studied. Multiple linear regression analysis was performed to investigate the relationship between blastocyst quality and neonatal birth weight and logistic regression for the risk factors for low birth weight newborns.

Results

With decreases in the blastocyst quality, including ICM, TE quality, and embryo expansion stage, birth weight declined, and Z scores correspondingly decreased. After adjusting for confounders, the average and poor groups (P = 0.01 and P = 0.001, respectively) and blastocysts with TE grade C (P = 0.022) resulted in singletons with lower birth weight. Additionally, the poor group and blastocysts with Grade C TEs had a greater chance of leading to low birth weight infants compared with the other groups.

Conclusion

Our findings indicated that excellent and good-grade blastocyst transplantation could achieve better pregnancy outcomes and that average and poor-grade blastocyst transplantation, especially with grade C TEs, were associated with single birth weight loss. No association was found between the embryo expansion stage or ICM quality and neonatal birth weight.

Keywords

Blastocyst transplantation
Blastocyst grade
Birth weight
Fresh transplantation
Pregnancy outcome
Foundation of basic research for young teachers of Zhengzhou UniversityJC22862034 the Key Project of Medical Science and Technology Public Relations in Henan ProvinceSBGJ202102108 issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcIntroduction

The development of assisted reproductive technology (ART) has led to more than 5 million babies born via ART. However, with conception by in vitro fertilization and embryo transplantation (IVF-ET), the risk of adverse perinatal outcomes, including premature delivery, low birth weight and problems associated with small for gestational age, can be increased even among singleton live births compared with their naturally conceived peers [1–3]. Previous studies have focused on the complications of infants born via ART, such as neonatal asphyxia, respiratory distress syndrome, intracranial hemorrhage, and sepsis [4], but there is a lack of information on the effects of ART on birth weight worldwide. The positive correlation between embryo quality and pregnancy outcomes has been confirmed in many studies [5, 6]. It has been suggested that trophectoderm morphology predicts outcomes of pregnancy in a single-blastocyst transfer cycle [7], that TE quality could reflect embryo competence [8–10], and that advanced trophectoderm quality increases the risk of a large-for-gestational-age baby in single frozen–thawed blastocyst transfer cycles [11]. However, two other studies suggested that ICM quality might determine birth weight or gestational age [12, 13], although another study failed to report that ICM quality was related to the birth weight of infants [14]. Most of the above studies were conducted on patients undergoing frozen-thawed blastocyst transfer cycles. In addition, whether the ICM quality affects birthweight is still controversial, and whether the quality of TEs affects neonatal outcomes is still worth studying. Therefore, to explore the relevant factors affecting the birth weights of infants conceived via ART, this study aimed to compare the birth weights of infants that developed from differentially graded blastocysts that were transferred in fresh transplantation cycles.

Materials and methods

Study subjects and patients

Patients who received single-blastocyst transplantation at the reproductive center of the First Affiliated Hospital of Zhengzhou University from January 2016 to December 2019 were selected as the research subjects. All women who met the following inclusion criteria were enrolled in the study: age ≤ 40 years old; BMI ≤ 30 kg/m2; underwent a single fresh blastocyst transfer and delivered a live-born baby from a singleton pregnancy at a gestational age greater than 24 weeks [15]. The key exclusion criteria were as follows: (1) babies born to women with pregnancy-induced hypertension, gestational diabetes, systemic lupus erythematosus or thyroid dysfunction; (2) patients with congenital uterine malformations, such as septate uterus or unicornuate uterus; (3) patients with environmental abnormalities, such as intrauterine adhesion and endometrial polyps; (4) patients with vanishing twin syndrome [16]. The quality of the blastocyst was grouped into four categories on the basis of inner cell mass (ICM) and trophectoderm (TE) scoring: excellent quality, AA; good quality, AB and BA; average quality, AC, CA and BB; and poor quality, BC and CB [17, 18]. According to the grade of the transplanted blastocyst, four groups were formed: 170 cycles in the excellent-quality group; 312 cycles in the good-quality group; 559 cycles in the average-quality group; and 260 cycles in the poor-quality group. The main outcome measure was singleton birthweight, and the Z score was used to calculate the birthweight adjusted for gestational age and newborn sex. This study conforms to the principles of the Helsinki Declaration and was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Approval Number: 2019-KY-39.

Ovulation induction program and oocyte collection

Controlled ovarian hyperstimulation was performed with Gn, oocyte retrieval was performed via transvaginal ultrasound-guided puncture 36–37 h after hCG injection, and the oocyte-corona-cumulus (OCCC) complexes were identified under an autopsy microscope and collected in G-MOPS Plus culture medium (Vitrolife, Sweden). After the OCCC of one side of the ovary was collected, the OCCC was washed three times with G-IVF Plus (Vitrolife, Sweden) medium and placed in a Petri dish with G-IVF Plus. The cells were subsequently placed into a three-gas incubator at 37 °C, 6% CO2 and 5% O2 for further culture and maturation.

Blastocyst culture and transplantation

Petri dishes for blastocyst culture were made the day before use, and D3 normal fertilized embryos, excluding frozen embryos, were transferred to medium containing 50 µl of G-2plus (Vitrolife, Sweden) for further culture. One embryo per droplet was cultured in a three-gas incubator until the blastocyst stage. Blastocyst formation was observed and recorded at Days 5 and 6. The blastocyst score was determined according to the Gardner scoring system, which is based on the blastocyst cavity, ICM development and TE appearance, the number of trophoblast cells and the density of the structure. The blastocyst grade was classified as follows: Grade 1 indicates an early blastocyst, that is, a blastocyst with a cavity volume of less than half of the total volume of the blastocyst; Grade 2 indicates a blastocyst cavity volume of more than half of the total blastocyst volume; Grade 3 indicates a blastocyst in the completely dilated stage, where the blastocyst cavity occupies almost the whole blastocyst; Grade 4 indicates an expanded blastocyst, where the volume of the blastocyst cavity is significantly larger than that of the early blastocyst and the zona pellucida is thinner; Grade 5 indicates a hatching blastocyst, which breaks out of the zona pellucida; and Grade 6 indicates a blastocyst that has completely emerged from the zona pellucida. A indicates many cells that are tightly packed, B indicates a low number of cells that are loosely grouped, and C indicates very few cells. With respect to trophoblast cells, A denotes many cells distributed around the blastocyst, B indicates a low number of cells that are loosely grouped, and C indicates very few cells.

Blastocyst transfer

Fresh blastocysts were transferred to ET dishes (G-2 plus Vitrolife, Sweden) for transfer on Day 5. Before transplantation, the blastocysts were transferred to another dish filled with 1 ml of EmbryoGlue (Vitrolife, Sweden). On Day 35 after transplantation, B-ultrasonography of the gestational sac was performed to determine whether a clinical pregnancy was present.

Statistical analysis

SPSS 21.0 software was used for statistical analysis. Independent sample t tests were used for statistical analysis of the general data of the patients, which are presented as the means ± standard deviations. The laboratory indicators of embryo development and pregnancy outcome were tested using the chi-square test, and the data are presented as the “rate”. Multiple linear regression analyses were performed to assess the impact of morphological grade and general situation on birth weight, gestational age, and Z score. Multivariate logistic regression analysis was performed to evaluate the associations between blastocyst quality and neonatal outcomes. Covariates were selected among potential risk factors for IVF singleton birth weight based on existing knowledge. All potential risk factors whether or not significant between-group differences were observed, including embryo expansion stage, ICM grade, TE grade, blastocysts quality and some basic characteristics of both parents. Furthermore, embryo expansion stage 3, ICM grade A, TE grade A and excellent quality blastocyst were taken as a reference group in multivariable analyses. Statistical significance was defined as P < 0.05.

Results

Comparison of pregnancy outcomes at different blastocyst grades

After excluding 6 cases of chronic maternal diseases, 29 cases of uterine malformations, 38 cases of intrauterine adhesions and endometrial polyps, and 1 case of vanishing twin syndrome, 2521 women (300 in the excellent group, 581 in the good group, 1073 in the average group, and 567 in the poor group) who fulfilled the inclusion criteria were available for analysis, with no loss to follow-up. A total of 1193 unborn and 27 twin deliveries were excluded. There were significant differences in the clinical pregnancy rate, implantation rate, and live birth rate among the four groups (P < 0.05), but there were no differences in the abortion rate among them (P > 0.05). The data are presented in Table 1.

Table 1 Comparison of pregnancy outcomes between different grades of blastocysts

Fresh	Excellent	Good	Average	Poor	X2	P	
ET cycles (n)	300	581	1073	567	-	-	
Clinical pregnancy rate [n (%)]	196 (65.33)	377 (64.89)	675 (62.91)	319 (56.26)	11.766	0.008	
Implantation rate [n (%)]	192 (64)	369 (63.51)	665 (61.98)	313 (55.20)	11.117	0.011	
Live birth rate [n (%)]	170 (56.67)	312 (53.70)	559 (52.10)	260 (45.86)	11.709	0.008	
Abortion rate [n (%)]	20 (10.2)	54 (14.32)	89 (13.19)	48 (15.05)	2.758	0.431	
Note Four categories were grouped on the basis of ICM and TE scoring: excellent quality: AA; good quality: AB/BA; average quality: AC/CA/BB; and poor quality: BC/CB

General characteristics and birth weights of infants in the blastocyst groups

A total of 170, 312, 519, and 260 infants born in the excellent, good, average and poor groups, respectively, were studied. No differences were observed in terms of female age, infertility years, BMI, FSH, LH, E2, AMH, endometrial thickness on the day of transplantation, gestational age, or birth sex among the four groups. However, the E2 level on the day of hCG trigge, mean birth weight and Z scores and proportion of low birth weight infants varied significantly according to blastocyst quality. The relevant data are listed in Table 2.

Table 2 Baseline characteristics and neonatal outcomes according to blastocyst growth parameters

Fresh	Excellent	Good	Average	Poor	X2	P		
Cycle number(n)	170	312	559	260				
Female age (years)	29.14 ± 3.78	29.40 ± 3.36	29.25 ± 3.92	29.43 ± 3.87	1.59	0.19		
Female BMI (kg/m 2 )	23.24 ± 3.29	23.25 ± 3.24	23.45 ± 3.31	23.43 ± 3.39	1.06	0.37		
Paternal age (years)	31.25 ± 3.41	31.43 ± 3.54	31.28 ± 3.62	31.51 ± 3.07	1.48	0.16		
Paternal BMI (kg/m2)	24.11 ± 3.02	24.06 ± 3.18	24.22 ± 3.16	24.46 ± 3.38	1.14	0.24		
Infertility duration (years)	3.73 ± 2.78	3.41 ± 2.67	3.53 ± 2.38	3.83 ± 2.85	1.24	0.29		
Female FSH (mIU/ml)	6.13 ± 1.46	5.77 ± 1.57	6.11 ± 1.57	6.16 ± 1.86	2.508	0.057		
Female E2 (pg/ml)	55.90 ± 94.67	46.26 ± 83.93	64.21 ± 245.78	78.96 ± 324.29	2.213	0.14		
Female LH (mIU/ml)	6.57 ± 4.54	6.66 ± 4.79	6.42 ± 4.25	7.68 ± 3.96	2.368	0.069	
Female AMH (ng/ml)	5.64 ± 3.37	5.86 ± 3.13	5.48 ± 3.29	5.45 ± 3.50	0.475	0.70		
E2 level on the day of hCG trigge(pg/ml)	5180.68 ± 2344.71	4685.97 ± 2184.53	4307.28 ± 1977.89	4075.72 ± 1994.51	12.09	0.000a*		
Endometrial thickness (mm)	12.40 ± 2.69	12.54 ± 2.33	12.45 ± 2.55	12.47 ± 2.41	0.120	0.95		
Gestational age (weeks)	38.94 ± 1.45	38.82 ± 1.37	38.83 ± 1.33	38.63 ± 1.42	1.976	0.116		
Percentage of male births (%)	57.65 (98/170)	59.94 (187/312)	57.96 (324/559)	59.23 (154/260)	0.113	0.990		
Birthweight (g)	3512.69 ± 506.76	3401.24 ± 521.99	3395.26 ± 528.62	3343.58 ± 522.09	5.584	0.001a*		
Z scores	0.72 ± 0.86	0.54 ± 1.00	0.53 ± 0.99	0.36 ± 1.02	2.27	0.011a*		
Birthweight < 2500 g (%)	1.18 (2/170)	1.92 (6/312)	5.90 (33/559)	6.15 (16/260)	13.710	0.003b*		
Note a: One-way ANOVA, b: Pearson chi-square test

Comparison of laboratory conditions among different blastocyst grade groups

After grouping, the indicators of embryo development were compared among the groups.The 2PN fertilization rate and cleavage rate in the excellent group were significantly greater than those in the poor group (P < 0.05); the high quality embryo rate and D6 blastocyst formation rate in the excellent group were significantly higher than those in the other three groups (P < 0.05). Compared with the other three groups, the poor group presented a lower rate of blastocyst formation (P < 0.05). These data are shown in Table 3.

Table 3 Embryo development conditions among different blastocyst grade groups

Fresh	Excellent	Good	Average	Poor	X2	P	
Cycle number (n)	170	312	559	260			
2PN fertilization rate

[n (%)]

	2378 (82.54)D	3972 (81.79)	6880 (80.94)	2889 (78.99)	15.98	0.001	
2PN cleavage rate

[n (%)]

	2362 (99.33)d	3919 (98.67)	6804 (98.90)	2841 (98.34)	11.92	0.008	
High quality embryo rate[n (%)]	1920 (81.29)B, C,D	3062 (77.09)	5205 (76.50)	1948 (68.57)	125.53	0.000	
Blastocyst formation rate [n (%)]	1359 (71.64)	2188 (71.36)	3601 (68.37)	1297 (61.53)A, B,C	67.77	0.000	
D5 blastocyst formation rate [n (%)]	1205(63.50)D	1928(62.88)	3138(59.58)	1023(48.53)	9.69	0.021	
D6 blastocyst formation rate [n (%)]	154(8.14)	260(8.48)	463(8.79)	274(13.00)A, B,C	24.53	0.000	
High score blastocyst rate[n (%)]	756(39.85)	1056(34.44)	1334(25.33)	80(3.80)	558.03	0.000*	
D5 high score blastocyst ratio[n (%)]	715(37.69)	996(32.49)	1255(23.83)	0	156.14	0.000*	
D6 high score blastocyst ratio[n (%)]	41(2.16)	60(1.96)	79(1.50)	80(3.80)B, C	24.56	0.000	
Note *: There were significant differences between groups after correction (P < 0.008)

“A”, “B”, “C”, and “D” indicate that significant differences were detected between the two groups after correction (P < 0.008)

“d” indicates that significant differences were detected between the two groups after correction (P < 0.008). 2PN fertilization rate = number of 2PN oocytes/ number of retrieved oocytes for IVF or number of 2PN oocytes/number of MII oocytes for ICSI; high quality embryo rate = number of high quality embryos/number of cleavaged 2PN embryos; blastocyst formation rate = number of blastocysts /number of embryos for cultivating blastocysts, high score blastocyst rate = number of high score blastocysts / number of embryos for cultivating blastocysts

Relationships between blastocyst expansion stage, ICM grade, TE grade and birth weight

As the ICM, TE quality, and embryo expansion stage decreased, the birth weight declined (3470.21 ± 515.02 and 3381.37 ± 526.74 for ICM grades A and B, respectively, P = 0.003; 3441.52 ± 526.13 and 3335.04 ± 527.88 for TE grades A and C, respectively, P = 0.012; and 3427.96 ± 533.57 and 3365.10 ± 571.65 for expansion stages > 3 and ≤ 3, P = 0.041). The incidence rate of low birth weight babies was affected by the blastocyst expansion stage and the ICM grade. It is interesting that there is a relationship between blastocyst level and E2 level on the day of hCG trigge. The E2 levels in the ICM grade A group and TE grade A group were significantly higher than those in the other groups.The neonatal outcomes according to the blastocyst growth parameters are presented in Table 4.

Table 4 Relationships between blastocyst expansion stage, ICM grade, TE grade, and birth weight

Fresh	Blastocyst expansion grade	X2	P	ICM grade	X2	P	TE grade	X2	P	
≤ 3	>3	A	B	A	B	C	
n	289	1012	-	-	476	825	-	-	153	866	282	-	-	
Female age (years)	29.20

± 3.96

	29.14

± 3.77

	2.11	0.85	28.95 ± 3.64	29.27 ± 3.91	2.33	0.15	28.46 ± 2.76	29.20 ± 3.05	29.47 ± 3.10	7.55	0.023	
Infertility duration (years)	3.69

± 2.50

	3.61

± 2.66

	0.054	0.64	3.60 ± 2.74	3.63 ± 2.54	0.11	0.87	3.44 ± 1.89	3.61 ± 1.89	3.76 ± 2.10	1.88	0.39	
Female BMI (kg/m 2 )	23.47

± 3.53

	23.31

± 3.25

	3.27	0.49	23.11 ± 3.25	23.48 ± 3.34	0.53	0.053	23.02 ± 2.69	23.40 ± 2.75	23.38 ± 2.81	2.68	0.26	
Female basal FSH (mIU/ml)	6.18

± 1.68

	6.04

± 1.58

	0.41	0.21	6.00 ± 1.52	6.12 ± 1.66	0.23	0.20	5.78 ± 1.16	6.12 ± 1.14	6.15 ± 1.30	6.67	0.036	
Female basal E2 (pg/ml)	65.91 ± 274.68	60.66 ± 213.06	0.051	0.98	49.48 ± 88.79	69.01 ± 274.60	3.47	0.063	56.04 ± 92.39	58.13 ± 208.36	77.83 ± 321.29	0.835	0.434	
Female basal LH (mIU/ml)	7.05

± 6.65

	6.65

± 5.97

	1.28	0.32	6.47 ± 4.37	6.91 ± 6.94	3.88	0.17	6.92 ± 3.40	6.45 ± 2.94	7.57 ± 4.40	1.03	0.60	
Female AMH (ng/ml)	5.29

± 3.27

	5.62

± 3.36

	0.14	0.19	5.69 ± 3.32	5.49 ± 3.35	0.68	0.38	5.88 ± 2.45	5.54 ± 2.49	5.41 ± 2.67	4.33	0.12	
E2 level on the day of hCG trigge(pg/ml)	4433.81 ± 2165.62	4471.12 ± 2092.99	0.046	0.987	4884.97 ± 2280.28	4234.26 ± 1982.35	28.90	0.000	5104.05 ± 2325.69	4454.67 ± 2073.92	4093.07 ± 2015.34	12.69	0.000	
Endometrial thickness (mm)	12.37

± 2.45

	12.48

± 2.55

	0.60	0.50	12.43 ± 2.57	12.47 ± 2.51	0.25	0.79	12.62 ± 1.80	12.42 ± 2.09	12.46 ± 1.94	1.13	0.53	
Birth weight (g)	3365.10 ± 571.65	3427.96±

533.57

	1.738	0.041	3470.21 ± 515.02	3381.37 ± 526.74	8.73	0.003	3402.68 ± 492.13	3441.52 ± 526.13C	3335.04 ± 527.88	4.46	0.012	
Birth weight < 2500 g	6.57 (19/289)	2.87 (29/1010)	8.659	0.003	1.26 (6/476)	4.48 (37/825)	9.82	0.002	2.61 (4/153)	3.0 (26/866)	4.61 (13/282)	1.98	0.372	

Factors influencing neonatal birth weight according to linear regression analysis

Multiple linear regression analyses are presented in Table 5. Infants born to the average poor-quality group weighed 118.03 g (95% CI: -206.39 to -29.66, P = 0.01) and 170.43 g (95% CI: -269.98 to -70.88, P = 0.001) less than those born to the excellent-quality group in terms of the mean birth weight. Singletons from the TE Grade C group weighed 132.30 g (95% CI: -245.91 to -18.70, P = 0.022) less than those from the TE Grade A group. However, the above birth weight difference remained statistically significant in terms of gestation-adjusted Z scores.

Table 5 Linear regression analysis of singleton birth weights

	Birth weight	T	P	Gestational age (weeks)	T	P	Z scores	T	P	
β(95% CI)	β(95% CI)	β(95% CI)	
Embryo expansion stage	3	Reference			Reference			Reference			
4	25.98

(-41.81 to 93.78)

	0.75	0.45	0.41

(-0.87 to 1.69)

	0.63	0.53	-0.013

(-0.11 to 0.067)

	-0.43	0.67	
5	55.01

(-101.87 to 211.89)

	0.69	0.49	0.058

(-2.92 to 3.03)

	0.038	0.97	0.004

(-0.19 to 0.21)

	0.13	0.90	
6	586.96

(-431.64 to 1605.56)

	1.13	0.26	0.43

(-18.89 to19.75)

	0.044	0.97	0.006

(-1.17 to 1.43)

	0.20	0.84	
ICM grade	A	Reference			Reference			Reference			
B	-22.74

(-92.69 to 47.22)

	-0.64	0.52	0.11

(-1.21 to 1.44)

	0.17	0.87	-0.023

(-0.12 to 0.058)

	-0.68	0.49	
TE grade	A	Reference			Reference			Reference			
B	-64.62

(-153.66 to 24.42)

	-1.42	0.16	-0.078

(-1.77 to 1.61)

	-0.091	0.93	-0.091

(-0.24 to 0.012)

	-1.17	0.23	
C	-132.30

(-245.91 to -18.70)

	-2.29	0.022	-0.28

(-2.43 to 1.88)

	-0.25	0.80	-0.18

(-0.44 to -0.15)

	-2.94	0.03	
Blastocysts quality	Excellent	Reference			Reference			Reference			
Good	-86.97

(-183.32 to 9.38)

	-1.77	0.077	-0.006

(-1.96 to 1.69)

	-0.15	0.88	-0.113

(-0.29 to -0.054)

	-1.72	0.085	
Average	-118.03

(-206.39 to -29.66)

	-2.62	0.01	-0.005

(-1.78 to 1.57)

	-0.12	0.90	-0.14

(-0.29 to -0.071)

	-2.20	0.02	
Poor	-170.43

(-269.98 to -70.88)

	-3.36	0.001	-0.013

(-2.19 to 1.58)

	-0.32	0.75	-0.22

(-0.49 to -0.23)

	-2.57	0.015	
Female age	-2.34

(-9.78 to 7.62)

	-0.52	0.60	0.026

(-0.17 to 0.22)

	0.26	0.79	-0.041

(-0.019 to 0.005)

	-1.19	0.23	
Female BMI	27.20

(16.93 to 37.02)

	5.26	0.00	-0.04

(-0.27 to 0.19)

	-2.35	0.021	0.051

(0.027 to 0.070)

	2.74	0.011	
Paternal age	-2.17

(-8.64 to 7.24)

	-0.49	0.54	0.019

(-0.13 to 0.21)

	0.22	0.65	-0.039

(-0.012 to 0.004)

	-1.21	0.19	
Paternal BMI	22.10

(18.93 to 36.12)

	1.22	0.13	0.02

(-0.18 to 0.29)

	0.15	0.21	0.042

(0.021 to 0.006)

	1.14	0.22	
Infertility duration	-9.26

(-23.00 to 2.64)

	-1.40	0.16	-0.041

(-0.33 to 0.25)

	-0.28	0.78	0.093

(0.007 to 0.041)

	1.46	0.15	
Female basal FSH	-15.54

(-34.79 to 9.43)

	-1.36	0.17	-0.13

(-0.64 to 0.37)

	-0.52	0.61	0.000

(-0.029 to 0.029)

	0.006	0.99	
Female basal E2	-0.007

(-0.13 to 0.12)

	-0.11	0.91	0.00

(-0.003 to0.003)

	-0.083	0.93	0.025

(0.000 to 0.000)

	0.76	0.45	
Female basal LH	0.38

(-5.68 to 5.67)

	0.13	0.89	-0.011

(-0.14 to 0.12)

	-0.17	0.86	-0.040

(-0.012 to 0.003)

	-1.09	0.28	
Female AMH	-1.23

(-11.95 to 9.27)

	-0.23	0.82	0.091

(-0.15 to 0.33)

	0.74	0.46	0.054

(-0.003 to 0.025)

	1.51	0.13	
E2 level on the day of hCG trigge	-0.055

(-0.031 to -0.003)

	-1.66	0.098	-0.028

(0.00 to 0.00)

	-1.00	0.32	0.044

(0.00 to 0.00)

	1.58	0.11	
Endometrial thickness	7.95

(-6.51 to 18.41)

	1.23	0.22	0.023

(-0.05 to 0.52)

	1.62	0.11	-0.030

(-0.024 to 0.009)

	-0.91	0.36	

Factors influencing neonatal birth weight according to logistic regression analysis

The logistic regression analysis results in Table 6 show the factors influencing neonatal birth weight. Compared with the other groups, the average group(adjusted odds ratio (aOR): 10.29, 95% CI: 1.39–76.04, P = 0.022), the poor group (adjusted odds ratio (aOR):10.89, 95% CI: 1.44–82.15, P = 0.021) and the TE Grade C group (adjusted odds ratio (aOR):11.45, 95% CI: 1.36–96.36, P = 0.025) had a higher chance of having low birth weight infants.

Table 6 Risk of abnormal birthweight in singleton pregnancies following fresh blastocyst transfer

	Birth weight < 2500 g	P	
aOR (95% CI)	
Embryo expansion stage	3	Reference		
4	0.88 (0.47 to 1.65)	0.69	
5	1.00 (0.19 to 5.06)	0.99	
6	0.00	1.00	
ICM grade	A	Reference		
B	0.62 (0.31 to 1.25)	0.18	
TE grade	A	Reference		
B	5.02 (0.63 to 39.74)	0.13	
C	11.45 (1.36 to 96.36)	0.025	
Blastocysts quality	Excellent	Reference		
Good	6.77 (0.88 to 52.35)	0.067	
Average	10.29 (1.39 to 76.04)	0.022	
Poor	10.89 (1.44 to 82.15)	0.021	
Female age	0.93 (0.87 to 1.01)	0.071	
Female BMI	0.96 (0.88 to 1.04)	0.32	
Paternal age	0.94 (0.87 to 1.05)	0.35	
Paternal BMI	1.02 (0.99 to 1.09)	0.12	
Infertility duration	1.14 (1.03 to 1.26)	0.062	
Female basal FSH	1.13 (0.93 to 1.36)	0.22	
Female basal E2	1.00 (1.00 to 1.00)	0.10	
Female basal LH	0.97 (0.91 to 1.03)	0.34	
Female AMH	1.02 (0.94 to 1.12)	0.60	
E2 level on the day of HCG trigge	1.00(1.00 to 1.00)	0.29	
Endometrial thickness	1.04 (0.94 to 1.16)	0.45	

Discussion

TE quality was associated with single birth weight loss

In this large retrospective study, we found that TE quality was associated with birth weight, especially TE Grade C, which was a contributing factor to low birth weight in infants. Thus, we speculated that the birth weight of infants was influenced mainly by the TE grade. Ebner et al. [14]. have suggested that TE is the only relevant parameter that significantly affects the outcome of pregnancy, which is consistent with our results. In fact, the mechanism by which TE quality impacts birth weight remains unclear, but TE is related to birth weight, possibly via its effect on the growth and development of the placenta. During the implantation process, TE cells recognize and interact with endometrial cells [19, 20]. Changes in the intercellular interactions between embryonic and maternal cells may alter the uterine microenvironment, which may further affect placental formation and fetal growth. TE eventually develops into the placenta, the weight and thickness of the placenta are closely related to newborn health and birth weight, and placental dysfunction has adverse effects on fetal growth [21, 22].

High-grade blastocyst transplantation can achieve better pregnancy outcomes

Our study suggested that the transplanted blastocyst grade was related to birth weight and that the birth weight of infants from AA blastocysts was significantly greater than that of infants from average and poor-grade blastocysts. The mechanisms underlying the association between low-grade blastocysts and low birth weight are unclear. Some studies have suggested that changes in the DNA methylation of poor-quality embryos are uncontrollable [23]; thus, epigenetic changes may play an important role. In addition, multiple exposures occur in the in vitro culture stage of ART, such as changes in DNA methylation caused by embryo handling, culture, and implantation, which may also have adverse effects on placental development and fetal growth [23, 24]. Studies have also shown that when embryos are damaged during development, there is increased activity in terms of metabolism, stress, and other aspects, leading them to produce more reactive oxygen species and ATP compared with normal embryos. Therefore, metabolic disorders and imbalances in vivo may also explain why low-grade blastocyst transplantation leads to lower birth weight [25]. Another cause of low birth weight due to low-grade blastocyst transplantation may be related to maternal uterine factors. In vitro studies have shown that the endometrium functions as a biosensor to assess embryo quality [26]; that is, after embryo implantation, it can actively transmit information to facilitate implantation by improving the internal uterine environment. Decidualized endometrial stromal cells can receive and identify high-quality and inferior embryos and respond to them by producing different growth factors or cytokines; however, the response of inferior embryos is greater than that of high-quality embryos, and greater energy is required to participate in the repair of developmental damage. These reactions and changes may have an impact on the uterine environment of embryo implantation, thus affecting placental formation and subsequent embryo development and fetal growth [20].

The necessity and transplantation strategy for single blastocyst transfer

Previous studies have shown that the multiple pregnancy rate is clearly lessened by a single-blastocyst transplant [5, 14], thereby reducing complications during pregnancy and negative impacts in terms of perinatal outcomes [27]. Moreover, the clinical pregnancy rate and singleton live birth rate improve [28]; therefore, the proportion of blastocyst embryo transfers is increasing. Many studies have shown that high-quality blastocyst transfer can improve the clinical pregnancy rate and live birth rate [18]. Akamine et al. [29]. however, reported that blastocyst quality has no significant effect on neonatal outcomes, including birth weight. This difference may be related to the small sample size and single-center nature of that study. Several studies have suggested that the selection of transplanted blastocysts is very important for single-blastocyst transplantation, in which the embryo implantation rate, clinical pregnancy rate, live birth rate and other indicators are unaffected [12, 30]. Based on the results of the present study, we believe that single blastocyst transfer is necessary, as it can not only reduce the rates of multiple pregnancies and various complications during the perinatal period but also achieve higher implantation rates and lower miscarriage rates. In the process of selecting blastocysts, we recommend prioritizing high-grade blastocysts for transplantation. For blastocysts of the same grade, such as BB/AC/CA or BC/CB, we recommend prioritizing blastocysts with higher TE grades for transplantation.

In conclusion, high-grade blastocyst transplantation could achieve better birth weight, and TE quality was associated with singleton birth weight loss. For blastocysts of the same grade, we recommend prioritizing blastocysts with higher TE grades for transplantation.

Acknowledgements

The authors sincerely acknowledge the staff of Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University for their diligent work, and all the patients for their selfless participation.

Author contributions

Each author is expected to have made substantial contributions to the conception. Senlin Shi designed the study. Senlin Shi, Boya Zhao and Qiongyao Shi were responsible for the study subjects’ enrollment and the collection of data. Zhan Hu and Boya Zhao re-analyzed the data and made significant contributions in article revision.

Funding

The study was supported by the Foundation of Basic Research for Young Teachers of Zhengzhou University (JC22862034 to Senlin Shi) and the Key Project of Medical Science and Technology Public Relations in Henan Province (SBGJ202102108 to Senlin Shi).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Human Ethics and Consent to participate declarations

We confrm that all methods were carried out in accordance with relevant guidelines and regulations in the Declaration of Helsinki. Written informed consent was obtained from all participants for data use prior to enrollment. This study was approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University(Approval Number: 2019-KY-39).

Competing interests

The authors declare no competing interests.

Abbreviations

ART Assisted reproductive technology

IVF-ET In vitro fertilization and embryo transplantation

ICM Intracellular mass

TE Trophectoderm

Gn Gonadotropin

OCCC Oocyte-corona-cumulus complex

BMI Body mass index

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Senlin Shi, Boya Zhao and Zhan Hu have contributed equally to this work and share first authorship.
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