The Impact of Blastocyst Grade on Singleton Birthweight in Fresh Transplanted Cycles

Background: A retrospectively cohort study was performed to compare the birth weight of different blastocyst grades in fresh transplantation cycle and explore the related factors affecting the birth weight. Methods: The 1301 fresh cycles of single blastocyst transplantation and single live birth prole were analyzed,four groups were divided according to the grade of transplanted blastocyst. There are 170 cycles in group A with AA blastocyst grade, 312 cycles in group B with AB/BA blastocyst grade, 559 cycles in group C with BB/CA/AC blastocyst grade and 260 cycles in group D with BC/CB blastocyst grade. Comparison were made among four groups of birth weight, general conditions, fertilization rate, embryo rate, cleavage rate and D5,D6 blastocyst formation rate and other laboratory indicators. And then comparison were performed among the birth weight of different groups which were divided by the degree of blastocyst expansion, and the classication of inner cell mass(ICM) and the trophectoderm(TE). Results: The study shows that the birth weight of group A is signicantly higher than that of the other three groups (P < 0.05). And the high quality embryo rate and blastocyst rate of group A are signicantly higher than those of the other three groups (P < 0.05). What’s more, the clinical pregnancy rate, implantation rate and live birth rate of high grade blastocyst are higher, but there are no signicant difference in abortion rate. The birth weight of the degree of blastocyst expansion in grade 3 and below is signicantly lower than that of those with grade 3 and above (P < 0.05). The birth weight of grade A of ICM is signicantly higher than that of grade B (P < 0.05). The birth weight of grade B of TE is signicantly heavier than that of grade C (P < 0.05). Conclusions: Our results indicate that high grade blastocyst transplantation can achieve better pregnancy outcome. Different blastocyst grades affect birth weight, and low grade blastocyst transplantation is associated with a single birth weight loss. the process of in vitro fertilization and embryo transplantation(IVF-ET), even singletons live births can also increase the risk of adverse perinatal outcomes, including premature delivery, low birth weight and small gestational age problems, compared to their naturally conceived peer(1–3). In previous studies, more attention has been paid to the complications of infants born with ART, such as neonatal asphyxia, respiratory distress syndrome, intracranial hemorrhage, sepsis, etc(4), but there is a lack of information on birth weight around the world. Although the positive correlation between embryo quality and pregnancy outcomes has been well conrmed in many studies(5, 6), few studies research the impact of embryo quality on neonatal weight, especially the fresh transplanted blastocysts. Therefore, this study aims to compare the birth weight of different blastocyst grades in the fresh transplantation cycle of ART to explore the related factors affecting the birth weight of infants.


Background
With the development of assisted reproductive technology(ART), there are more than 5 million babies born by this way. However, in the process of in vitro fertilization and embryo transplantation(IVF-ET), even singletons live births can also increase the risk of adverse perinatal outcomes, including premature delivery, low birth weight and small gestational age problems, compared to their naturally conceived peer (1)(2)(3). In previous studies, more attention has been paid to the complications of infants born with ART, such as neonatal asphyxia, respiratory distress syndrome, intracranial hemorrhage, sepsis, etc(4), but there is a lack of information on birth weight around the world. Although the positive correlation between embryo quality and pregnancy outcomes has been well con rmed in many studies (5,6), few studies research the impact of embryo quality on neonatal weight, especially the fresh transplanted blastocysts. Therefore, this study aims to compare the birth weight of different blastocyst grades in the fresh transplantation cycle of ART to explore the related factors affecting the birth weight of infants.

Study object
The patients who received single blastocyst transplantation in the reproductive center of the First A liated Hospital of Zhengzhou University from January 2016 to December 2019 were selected as the research subjects, both couples have normal chromosomes; Exclusion of female uterine malformation, endometriosis, ovarian dysfunction and other related factors of female infertility. According to the level of transplanted blastocyst, it was divided into four groups: 170 cycles in group A with AA blastocyst grade, 312 cycles in group B with AB/BA blastocyst grade, 559 cycles in group C with BB/CA/AC blastocyst grade and 260 cycles in group D with BC/CB blastocyst grade. Four groups were compared with laboratory indexes such as birth weight, general conditions, fertilization rate, embryo rate, cleavage rate, D5 blastocyst formation rate and D6 blastocyst formation rate. Then blastocysts were divided into different groups according to the degree of blastocyst expansion, inner cell mass(ICM) development and trophectoderm(TE) appearance to compare the difference in birth weight.

Ovulation induction program and oocyte collection
Controlled hyperovulation was performed with gonadotropin (GN), then oocyte retrieval was preformed by transvaginal ultrasound-guided puncture 36-37 hours after HCG injection, and the ovum crown-cumulus complex (OCCC) was identi ed under an autopsy microscope and collected in G-MOPS Plus culture medium (Vitrolife, Sweden). When OCCC of one side ovary were collected, washing three times with G-IVF Plus (Vitrolife, Sweden) medium and placing in a petri dish with G-IVF Plus. Then it was put into a three-gas incubator at 37℃, 6% CO 2 and 5% O 2 for further cultured and matured.

Blastocyst culture and transplantation
Petri dishes for blastocyst culture were made the day before, D3 normal fertilized embryos, except for frozen embryos, were transferred to medium containing 50ul of G-2plus (Vitrolife, Sweden) for further culture, one embryo per droplet was cultured in a three-gas incubator until blastocyst stage. The blastocyst formation was observed and recorded at day 5 and 6. Blastocyst score was made according to Gardner scoring system, which was based on the grade of blastocyst expansion, as well as morphology of ICM and TE, the number of trophoblast cells and the density of the structure. Blastocyst grading: grade 1: early blastocyst, that is, blastocyst cavity volume is less than half of the total volume of blastocyst; Grade 2: blastocyst cavity volume is more than half of total blastocyst volume; Grade 3: blastocyst in complete dilated stage, the blastocyst cavity almost occupies the whole blastocyst; Grade 4: the expanded blastocyst, the volume of the blastocyst cavity was signi cantly larger than that of the early blastocyst, and the zona pellucida was thinner. Grade 5: Inhatching blastocysts, which are breaking out of the zona pellucida; Grade 6: The blastocyst has completely emerged from the zona pellucida. ICM: A indicates a large number of cells and a close bound; B indicates that the number of cells is small and the combination is loose; C means very few cells. Trophoblast cells: A denotes a large number of cells that distributed around the blastocyst; B refers to a small number of cells and a loose combination of cells; C means very few cells.

Blastocyst transfer
Fresh blastocysts were transferred to ET dishes (G-2 plus Vitrolife, Sweden) for transfer within day 5. Before transplantation, blastocysts were transferred to another dish lled with 1ml Embryoglue (Vitrolife, Sweden). On the 35th day after transplantation, B-ultrasonography of the gestational sac was performed to determine whether a clinical pregnancy was possible.
5. Statistical analysis SPSS 21.0 software was used for statistical analysis. The independent sample t test was used for statistical analysis of the general data of patients, represented by means±SDs. The laboratory indicators of embryo development and pregnancy outcome were tested by chi-square test, and the datas were represented by rate. P<0.05 was considered statistically signi cant.

General situation and birth weight of different grades of blastocyst group
A total of 170,312,559 and 260 infants respectively with the grade of AA AB/BA BB/AC/CA BC/CB blastocysts were transplanted in this study.There were no differences in female age, infertility years, Body mass index (BMI), Follicle-stimulating hormone(FSH),Luteinizing hormone( LH), Anti-Mu¨ llerian Hormone(AMH), intimal thickness on transplantation day, gestational age and birth sex among the four groups, but the grade of blastocyst transplant to AA's birth weight of infants is signi cantly higher than the other three groups, and infants of low birth weight in group A and group B were signi cantly lower than that of group C and D, the difference is statistically signi cant. Relevant data are shown in Table 1.  Table 2. After abortion, Single-nucleotide polymorphism (SNP) detection cycle was counted, a total of 75 cycles, and 42 cycles is abnormal. Statistical analysis showed that there was no signi cant difference in the distribution of abnormal embryonic karyotype between different groups. The data showed that 30 cases were trisomy, 2 cases were triploid, 6 cases were monomers and 4 cases were structural abnormality. Details were shown in Table 3.

Comparison of laboratory conditions of different grades of blastocysts groups
2PN fertilization rate in group A is signi cantly higher than that in group D (P < 0.000); 2PN cleavage rate in group A is signi cantly higher than that in group D (P < 0.001); The high quality embryo rate and D6 blastocyst formation rate in group A are signi cantly higher than those in the other three groups (P < 0.000). The blastocyst formation rate of group A, B and C are signi cantly higher than that of group D (P < 0.000). The ratio of high quality blastocysts and D5 high quality blastocysts are signi cantly different between the groups (P < 0.000). Dates are shown in Table 4. Note: *: There were signi cant differences between groups, after correction P < 0.000; "A","B","C","D" indicate that signi cant differences were detected between the two groups,after correction P < 0.000; "d" indicate that signi cant differences were detected between the two groups,after correction P < 0.001;

Relationship between the grade of blastocyst expansion, ICM, TE level and birth weight
The birth weight of infants with blastocysts grade 3 and below is signi cantly lower than that of infants with blastocysts grade 3 and above (P < 0.05), while the former is signi cantly higher than the later in low birth weight, (P < 0.05). The birth weight of infants with level A of ICM is signi cantly higher than that of blastocysts with level B, and the incidence rate of low birth weight infants of group A is signi cantly lower than that of group B, with statistical signi cance (P < 0.05). The birth weight of infants with level B of TE is signi cantly higher than that of level C, P < 0.05. Seen Table 5.

Discussion
Our study suggests that the grade of transplanted blastocysts is related to the birth weight of infants, and the birth weight of the grade AA blastocysts is signi cantly higher than that of other grades, and the incidence of low birth weight infants is signi cantly lower than that of other grades of blastocysts. At the same time, the grade of blastocyst expansion, ICM development and TE appearance all have a certain in uence on the birth weight of infants, especially the blastocyst level above 3, the ICM level of the A blastocyst can better avoid the birth of low birth weight infants, this is consistent with previous research results (7). However, Ebner et al. believe that TE is the only relevant parameter that signi cantly affects the outcome of pregnancy, and is related to the birth weight by affecting the growth and development of the placenta(8). We believe that this difference may be due to the different grouping standards used in different studies, and the differences in the ratings of embryos by operators of different pro ciency. In addition, the effect of transplanted blastocyst quality on pregnancy outcomes is different (5,9). The results of this study suggests that high-grade blastocyst transplantation can obtain better pregnancy outcome, but it has no signi cant effect on abortion rate.
Previous studies have shown that multiple pregnancy rate was obviously reduced by single blastocyst transplant (5,10), thereby complications during pregnancy and the negative impacts of perinatal outcome were reduced (11), meanwhile, the clinical pregnancy rate and singletons live birth rate were improved (12), therefore, the proportion of blastocyst embryo transfer is increasing. Many studies have shown that high quality blastocyst transfer can achieve a better clinical pregnancy rate and live birth rate (13). Akamine Kozue et al, however, think that blastocyst quality has no signi cant effect on neonatal outcome, including birth weight (14). This difference may be related to its small sample size and single. Studies have suggested that the selection of transplanted blastocysts is very important for single blastocyst transplantation without affecting the embryo implantation rate, clinical pregnancy rate, live birth rate and other indicators (15,16).
The causes of low grade blastocysts leading to birth weight loss are unclear. Some studies believe that the changes in deoxyribonucleic acid(DNA) methylation of poor quality embryos are uncontrollable (17), so epigenetic changes may play an important role. In addition, multiple exposures were involved in the in vitro culture stage of ART, such as changes in DNA methylation caused by embryo operation, culture and implantation, which may also had adverse effects on placenta development and fetal growth (17,18). Studies have also shown that when embryos are damaged in development, they are more active in metabolism, stress and other aspects than normal embryos, producing more reactive oxygen species and adenosine triphosphate(ATP). Therefore, metabolic disorder and imbalance in vivo may also be the reason why low grade blastocyst transplantation leads to lower birth weight (19). Another cause of birth weight loss due to low grade blastocyst transplantation may be related to maternal uterine factors, In vitro studies have shown that the endometrium has the function of a biosensor to identify embryo quality (20), that is, after embryo implantation, it can actively transmit information to facilitate implantation by improving the internal uterine environment, decuvialized endometrium stromal cells can receive and identify high quality and inferior embryos, and respond to them by producing different growth factors or cytokines, but the response to inferior embryos is greater than that of high quality embryos, more 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 placenta formation and subsequent embryo development and fetal growth (21).
Conclusions this study believes that in the fresh transfer cycle, the blastocyst level has a greater impact on the pregnancy outcome and the birth weight. The blastocyst grade is also related to laboratory indicators such as the rate of high quality embryos and the rate of high quality blastocysts. In the selection process of blastocyst transplant, paying attention to the size of the blastocyst cavity and ICM is bene cial to reduce the birth of low birth weight infants.