1. Study object
The patients who received single blastocyst transplantation in the reproductive center of the First Affiliated 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.
2. 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 identified 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% CO2 and 5% O2 for further cultured and matured.
3. 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 significantly 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.
4. 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 filled 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 significant.