Analyzing the dynamic measurement of growth and development in children born after the transplantation of poor quality blastocyst embryos: a propensity matching study

Abstract Objective This study aimed to observe the dynamic measurement of growth and development in children (0–3 years) after transplantation of low-quality frozen-thawed single blastocysts. Methods This was a retrospective cohort study conducted from January 2016 to December 2019 at a single center. The follow-up data of the children were obtained from the Jiangsu Province Maternal and Child Database. A total of 350 singleton live births were included. Based on the blastocyst score, the live births were divided into good-quality blastocyst embryo (GQE) group (315 live births) and poor-quality blastocyst embryo (PQE) group (35 live births). To improve statistical efficiency and control for potential confounding factors, singletons conceived from PQEs were matched with a 3:1 ratio according to couple ages, BMI, occupation and women AMH levels. Ultimately, 32 children in the PQE group and 95 children (with one missing data) in the GQE group were included in the final analysis. Results After matching for parents’ age, BMI, occupation and maternal serum AMH level, there was no significant difference in growth and development of children between the PQE group and GQE group. However, the E2 level on trigger days, the rate of 2PN oocyte, and blastocyst formation rate in the PQE group were significantly lower than in the GQE group (p < .05). The number of embryo transfers (ETs) in the PQE group was higher than in the GQE group (p < .0001). The rate of cesarean section in the PQE group was significantly higher than in the GQE group (p < .05). The height (at 3 months) and head circumference (at 12 months) in the PQE group were lower than in the GQE group (p < .05). Conclusions Transplantation of PQEs did not affect the growth and development of offspring (0–3 years) compared to good-quality blastocysts. However, the oocyte and embryo development potential was lower in the PQE group than in the GQE group. These results provide clinical reference that the transfer of PQE could be acceptable for patients with only PQE embryos.


Introduction
Since the birth of the first "test tube baby" in 1978, assisted reproductive technology (Art) has become one of the most widely used technologies in the diagnosis and treatment of infertility [1].Over 10 million Art children have been born worldwide, accounting for more than 1% of all babies born each year through Art, and this trend is increasing [2].Art techniques include in vitro fertilization (iVF), intracytoplasmic sperm injection (icSi), freeze-thawing embryo transfer (Fet), assisted incubation (AH), preimplantation genetic diagnosis (PGD) and various other technologies.With the combination of techniques and the use of numerous drugs during ovulation induction, more and more researchers are concerned about the effects of Art technology on the short-term and long-term health of offspring [3].
early studies found no difference between Art children and those born naturally [4,5].However, recent studies have shown that Art technology can increase adverse perinatal outcomes such as pre-eclampsia, gestational hypertension, gestational diabetes, placental Growth and development; good-quality blastocyst embryo; poor-quality blastocyst embryo; embryo development potential; offspring abruption, postpartum hemorrhage, abnormal amniotic fluid levels and cesarean section rates [6][7][8][9][10].Furthermore, studies have reported that infants born from Fet cycles have a significantly higher risk of being large for gestational age (lGA) and having high birth weight (HBW) compared to those born from fresh cycles [11,12].However, most of these studies have focused on the perinatal period, and few have examined the shortand long-term health of offspring after birth.
With the advancement of the in vitro extension culture system, blastocyst culture has become the main embryo culture technology in many reproductive centers worldwide.Single blastocyst transplantation is favored by reproductive doctors due to its high implantation rate, low abortion rate and reduced rate of multiple births [13].Morphological scoring, based on the Gardner scoring criteria [14], is currently the main method for evaluating blastocysts in reproductive centers.Blastocysts with a low score or low quality (score c) are defined as secondary blastocysts.Several studies have shown a relationship between embryonic morphological quality (including cleavage embryos and blastocysts) and the success of pregnancy and live birth [15][16][17][18][19]. low morphologic grading often indicates lower developmental potential, and low-quality embryos have increased rates of abortion and aneuploidy [20,21].Patients with good-quality blastocysts are usually not considered low-score blastocysts, but some patients only have low-quality blastocysts and have no other option but to proceed with transplantation.
recent research has indicated that fresh low-quality embryos do not significantly differ from fresh good-quality embryos in terms of weight, gestational age and complications in neonates [22].However, the offspring of low-quality embryos transferred during Fet with single embryo have an increased risk of low birth weight and preterm birth (PtB) [23,24].Furthermore, a retrospective analysis of reproductive data in Australia showed a higher risk of congenital malformations in offspring from cycles involving the transfer of low-quality single blastocysts [25].
While many studies have focused on the health of children from birth to infancy, information about the long-term impact of embryo morphology grading on offspring health remains limited.A recent study involving the transfer of low-quality cleaved embryos found no significant abnormalities in metabolic and neurodevelopmental functions in 4-6-year-old offspring [26].However, there are still few studies examining the long-term health effects of children born from low-quality blastocysts.therefore, the objective of this study is to observe the growth and development of children aged 0-3 years who were born following the transplantation of low-quality freeze-thawed single blastocysts.

Study design and participant
this cohort study recruited children who were delivered following the transplantation of low-quality blastocysts at the changzhou Maternal and child Health Hospital reproductive center between January 2016 and December 2019.the follow-up data of the children were obtained from the Jiangsu Province Maternal and child Database.infertile women who underwent Fet resulting in a singleton live birth were included in the study.
Out of the 3955 patients who underwent Fet between 2016 and 2019, we excluded patients who had transferred cleavage embryos or two blastocyst embryos (n = 2635).the remaining patients (n = 1320) who underwent Fet with singleton pregnancies after blastocyst embryo transplantation were included.We further excluded patients who had no pregnancy or had experienced abortion (n = 691), gave birth to twins (n = 109), were over 40 years old (n = 36), had chromosomal abnormalities in either partner (n = 87), had a BMi ≥30 kg/m 2 (n = 47) or had missing data on the children (n = 8).
Based on the blastocyst scores, the patients were divided into two groups: the good-quality blastocyst embryo (GQe) group, consisting of 315 live births, and the poor-quality blastocyst embryo (PQe) group, consisting of 35 live births.to improve statistical efficiency and control for potential confounding factors, singletons conceived by PQes were matched to the GQe group at a 3:1 ratio based on couple ages, BMi, occupation and women's anti-Mullerian hormone (AMH) levels.Ultimately, 32 children in the PQe group and 95 children (with one missing data) in the GQe group were included in the final analysis.See Figure 1 for a flowchart illustrating the study design.
the study and its protocols were approved by the ethics committee of the changzhou Maternity and child Health care Hospital (17020490718).Written informed consent was obtained from the infertile couples participating in the study.

Embryo quality assessment
Blastocyst scoring was performed by two experienced embryologists in a double-blind manner according to Gardner rules in our center, that is, grade 1: early blastocyst, where the blastocele is less than half the volume of the embryo; grade 2: blastocyst, where the blastocele is greater than or equal to half of the volume of the embryo; grade 3: full blastocyst, where the blastocele completely fills the embryo; grade 4: expanded blastocyst, where the blastocele volume is larger than that of the early embryo and the zona pellucida is thinning; grade 5: hatching blastocyst, which the trophectoderm has started to herniate through the zona pellucida; and grade 6: hatched blastocyst, in which the blastocyst has completely escaped from the zona pellucida.the development of the inner cell mass (icM) and trophectoderm was also assessed.the icM grading was as follows: (A) many cells that are tightly packed; (B) several cells that are loosely grouped; or (c) very few cells.the trophectoderm grading was as follows: (A) many cells forming a tightly knit epithelium; (B) a few cells; or (c) very few cells forming a loose epithelium.A blastocyst with a score of c (trophectoderm or icM) was defined as low quality, and a blastocyst with a score of 4AA was defined as good quality.

Follow-up children
the couples' treatment information, including basic details, ovulation stimulation processes, embryo development, embryo transfer (et) and endometrial thickness, was collected and obtained from the electronic medical records system of the reproductive center.information regarding women's delivery and fetal development was collected and extracted from the Women and children System of Jiangsu Province.this system automatically collects pregnancy and delivery information of women from hospitals and gathers physical examination data of children from community service stations through the Health information System (HiS).By accessing these systems, we collected comprehensive data on the treatment and outcomes of the couples undergoing embryo transplantation, as well as the delivery and development of the children born from the low-quality blastocysts.
All questionnaires were recorded to see how the children had developed at certain times (0, 1, 3, 6, 8, 12, 18, 24, 30 and 36 months old) according to a uniform scale after being tested by a professional physician.the questionnaire was prepared based on the Maternal and child Health Handbook in china.the answers included the children's weight, height, head circumference, fontanel area and number of teeth, which were compared with those published as national averages.High birthweight (HBW) is defined as 4000 g or more.low birthweight (lBW) is less than 2500 g.Preterm birth was recorded when the baby was born alive before 37 weeks' gestation.SGA and lGA were defined when the birthweight was less or more than 2 standard deviations from the mean reported by the Ministry of Health, labor and Welfare (3rd, 10th, 25th, 50th, 75th, 90th and 97th percentiles), as well as a range of developmental parameters.lBW/HBW, PtB and SGA/lGA were also analyzed based on the definition given by the china Society of Obstetrics and Gynecology (china Society of Obstetrics and Gynecology, 2018).

Statistical analysis
Statistical analyses were performed using SPSS software version 26.0 (iBM, Armonk, NY).Quantitative variables with normal distribution were expressed as mean ± standard deviation, and Student's t-test was used to compare means.Quantitative variables with abnormal distribution or heterogeneous variance were expressed as M (P25, P75), and the median was compared by Mann-Whitney's U-test.chi-square test was used to compare the differences between the two groups.Fisher's exact test was used to compare differences in rates when the expected count was <5 or the total sample size was <40.p < .05 was considered statistically significant.Propensity score matching (PSM) was used to screen a group of patients so that baseline parameters in the good quality embryo group were very similar to those in the poor quality embryo group.the caliper width of the PSM was 0.2 of the propensity score logit SD. the nearest neighbor match ratio is 1:3. the SD values of independent variables before and after PSM were calculated.An absolute value of SD <10% is considered an equilibrium.Spearman's analysis was used to analyze the correlation between embryo quality and progeny development.p < .05 was considered statistically significant [27].lMS method was used to plot the growth curves of children's height, weight and head circumference from 1 month to 2 years with the percentiles (P3, P10, P25, P50, P75, P90 and P97) as reference.

Baseline characteristics of the two groups
thirty-two children born from PQe group and 95 children from GQe group were included in this study.there was no significant difference in maternal age, BMi, AMH, occupation and infertility type between the two groups.the male basic characteristics (age, BMi and occupation) were similar in the two groups (table 1).

Stimulation cycle characteristics of the two groups
type of controlled stimulation protocol, Gn dose, number of oocyte retrieved, Mii oocyte rate, number of et and embryo state were similar in the two groups.However, e2 level on the trigger days, the rate of 2PN oocyte and blastocyst formation rate in PQe group were significantly lower than in the GQe group (p < .05)(table 2).

Neonatal characteristics of the two groups
there were no significant difference in gestational age, PtB, birth weight (low birth weight), height, macrosomia, congenital anomaly, Apgar score and gender ratio

Analysis of correlation between sperm source (GQE or PQE group) and children development (height, weight, BMI, head circumference, fontanel area, no. of teeth)
there is no correlation between the children growth and development (height, weight, BMi, head circumference and fontanel area) and the blastocyst embryo score (GQe and PQe) in different months old (p > .05)(Figure 2).

Children growth development curve of the two groups (height, weight and head circumference)
the children (0, 1, 3, 6, 8, 12, 18, 24, 30 and 36 months old) height, weight and head circumference of PQe group and GQe group are shown in Figure 3 and there was no statistical significance between the two groups at the same time point (p > .05). each parameter was calculated, displayed on a line graph and compared with the reported data (3rd, 10th, 25th, 50th, 75th, 90th and 97th percentiles).

Discussion
the present study has shown that there is no significant difference in the growth and development of children born from the transfer of poor-quality blastocysts compared to those born from good-quality blastocysts.However, it is important to note that the embryo development potential of patients with poor-quality blastocysts is significantly lower than that of patients with good-quality blastocysts.in addition, the study has found that consistent with previous findings, the cesarean rate among the group receiving poor-quality blastocysts is significantly higher than the group receiving good-quality blastocysts.Furthermore, the levels of e2 (estradiol) at the time of HcG administration were significantly lower in the poor-quality blastocyst group compared to the good-quality blastocyst group.it has also been observed that children born from the poor-quality blastocyst group had lower height measurements at 3 months and head circumferences at 12 months compared to those from the good-quality blastocyst group.Morphological evaluation remains the primary method for assessing embryo quality due to its noninvasive, easy-to-perform, rapid and inexpensive nature [27].However, it does have certain limitations compared to newer technologies.For instance, it relies on the subjective judgment of embryologists and does not provide continuous observation of the dynamic development of embryos.As a result, some scholars believe that it may not accurately identify the developmental potential of embryos.Although newer technologies like time-lapse monitoring [28] and noninvasive metabolism detection [29] are promising for embryo assessment, there is currently no solid evidence to support their accuracy and effectiveness in  16 [16,17] 16 [16, 16.] .65820 [20,20] 20 [20,20] .926--determining the developmental potential of embryos.
Nevertheless, recent studies have shown that the rates of good-quality embryos and blastocyst formation are significantly lower in the group receiving poor-quality blastocysts.these findings suggest that low-score embryos indeed have lower quality and limited developmental potential.Furthermore, studies have indicated that low-score embryos have lower clinical pregnancy and implantation rates.the study has indicated that the low-quality blastocysts did not have a detrimental effect on the growth and development of offspring aged 0-3 years compared to the good-quality blastocysts.Although there were lower height measurements at 3 months and head circumferences at 12 months in the low-quality blastocyst group, these differences were within the normal range and did not persist as the children continued to develop, suggesting that they may not be clinically significant.currently, it is still unclear whether poor embryo quality affects the development of offspring.Some studies suggest that the methylation levels of good-quality blastocysts are similar, while those of low-quality blastocysts are variable and different from those of good-quality blastocysts [30].these changes in epigenetic modifications could theoretically impact fetal growth patterns.However, more research shows that the human endometrium can serve as a biosensor for embryo quality.the endometrial stromal cells undergo decidualization and can distinguish between good-quality and low-quality embryos, responding selectively with the secretion of certain growth factors and cytokines necessary for embryo development.these changes may have favorable effects on placental and fetal growth in low-quality embryos [31].Both Nakagawa et al. [32] and Bouillon et al. [33] believed that neonates born from low-quality et did not show any abnormal characteristics compared with neonates born from good-quality et.this could be attributed to a process of mutual selection, mutual adaptation and co-development between the low-quality embryos and the endometrium.
the study has also revealed that the rate of cesarean section in the low-scoring blastocyst group is higher than in the high-scoring blastocyst group, potentially due to couples in the low-quality blastocyst  group valuing their children more highly due to the lower rate of implantation.Previous research with a large sample size has shown that low-graded blastocysts are associated with higher rates of cesarean section and a higher likelihood of a female baby [34,35]. in our study, the ratio of girls in the low-quality blastocyst group was higher, although not statistically significant, which may be related to the sample size.the increased ratio of female babies after the transfer of PQes could be explained by the higher survival ability of female embryos within the mother's body.
the study has also observed that the e2 (estradiol) levels in the poor quality blastocyst group were lower than in the good quality group.A large cohort study has confirmed a significant association between serum e2 levels on the day of HcG administration and the clinical pregnancy and live birth rates of iVF/icSi.A decrease of about 10% in serum e2 levels is associated with a 50% decrease in clinical pregnancy and live birth rates [36].these results suggest that serum e2 levels may reflect the development of oocytes and embryos. in our study, we also found significantly lower e2 levels on the day of HcG administration in the poor-quality blastocyst group compared to the good-quality blastocyst group, and significantly lower rates of good-quality embryos and blastocyst formation.therefore, it can be concluded that patients with low e2 levels on the day of HcG administration tend to have low-quality oocytes and are more likely to produce low-quality embryos.
this study has several strengths.it is a matched cohort study that only included singletons, which eliminates potential confounding effects caused by multiple births.the growth and development of offspring were dynamically assessed, providing a theoretical basis for subsequent research.the study also used statistical methods, including propensity matching, to control for confounding factors of patient characteristics.Additionally, the study relied on detailed data measured by professional doctors using standardized scales, ensuring the reliability and authenticity of the retrospective results.
However, there are limitations to be considered.the sample size of the study was limited, mainly due to the proportion of patients with PQes and the rate of single births.Although the sample size was appropriate compared to the information on Art single births in our center, further verification with a larger multi-center sample is needed.Despite thorough consideration of confounding factors, residual confounding factors may still exist, such as children's lifestyle and sleep quality.Additionally, due to follow-up time limitations, the study only followed up until the age of three and lacked indicators for intelligence, language and social development.More comprehensive, long-term follow-up studies are needed for further validation [10,37].
in conclusion, the study has shown that the transfer of PQes does not affect the growth and development of offspring aged 0-3 years compared to the transfer of good-quality blastocyst embryos.However, the oocyte and embryo development potential in the poor-quality blastocyst group is lower than in the good-quality blastocyst group.these findings may provide clinical guidance that the transfer of poor-quality blastocysts could be acceptable for patients who only have poor-quality embryos.

Figure 2 .
Figure 2. analysis of correlation between the blastocyst quality and the children growth and development (1, 3, 6, 8, 12, 18, 24, 30 and 36 months).blue is positive correlation, red is negative correlation.the numbers in the squares represent the correlation coefficients (r).

Figure 3 .
Figure 3. the growth and development speed curve of the children according to the blastocyst quality (GQe and PQe).children's growth curve (1, 3, 6, 8, 12, 18, 24, 30 and 36 months) of the height, weight and head circumference in the PQe group and the GQe group.(a) height, (b) weight and (c) head circumference.the blue line represents PQe group, the red line represents GQe group and the lines from bottom to top represent 3%, 10%, 25%, 50%, 75%, 90% and 97%, respectively.Data are shown as box and whisker plots representing the median, inter quartile range and range.

Table 1 .
baseline characteristics of patients in two groups (GQe group and PQe group).
in the PQe group and GQe group.therate of the cesarean section in the PQe group was significantly higher than the GQe group (p < .05)(table3).

Table 2 .
clinical characteristics of patients in two groups (GQe group and PQe group).

Table 3 .
neonatal outcomes of singleton gestation of patients in two groups (GQe group and PQe group).

Table 4 .
children growth and development of patients in two groups (GQe group and PQe group).