Study design and population
This retrospective cohort study was performed at the Reproductive Medical Center of the Third Affiliated Hospital of Zhengzhou University between June 2017 and September 2019. Women who undergone first autologous PGT-A due to advanced age (≥38years) or had a history of more than two unsuccessful IVF-ET cycles or recurrent spontaneous abortion and then followed by single euploid FET cycles were enrolled. Finally, only 232 cycles were found to be eligible for inclusion in the data analysis, as shown in Figure 1. This study was performed in accordance with the Code of Ethics in the Declaration of Helsinki and was approved by the Ethics Review Committee of our hospital (protocol number 2021-WZ-010).
Ovarian stimulation protocol
Each female patient underwent a standard ovarian stimulation, trigger of oocyte maturation, oocyte retrieval, fertilization, embryo culture and transfer. The Gonadotropin (Gonal-F, Merck Serono, Switzerland) started injection from the second or third day of the menstrual cycle, dosage (150-300IU) was adjusted based on patient's age, basal antral follicle count (AFC), body mass index (BMI), basal follicle stimulating hormone (FSH)and ovarian reserve. The response to stimulation was assessed by performing transvaginal ultrasounds and measuring serum estradiol levels. GnRH antagonist (Cetrotide, Merck Serono, Switzerland) 0.25mg was usually injected for pituitary suppression when follicle diameter is 12 ~ 14 mm or Gonadotropin has been used for 5 ~ 6 days. GnRH agonist 0.2 mg (Dophereline, Ipsen Pharma Biotech, France) was used to trigger the final oocyte maturation. Untrasound guided oocyte retrieval was performed 33-36 hours after the trigger.
Blastocyst evaluation was performed prior to embryo biopsy. Blastocysts were graded according to the Gardner and Schoolcraft grading system, and the score was dependent on blastocyst expansion, ICM development and trophectoderm TE appearance. The degree of expansion included the following six grades:(1) a nonexpanded embryo with the blastocele filling <50%; (2) the blastocele filling >50% of the embryo; (3) a full blastocyst with a blastocoele filling the embryo; (4) an expanded blastocyst with a blastocoele volume larger than that of the full blastocyst, with a thinning zona; (5) a hatching blastocyst with the TE starting to herniate through the zona; and (6) a hatched blastocyst, with the blastocyst completely escaping from the zona. In our center, for blastocysts with an expansion score ≥4, the development of the ICM and TE was then evaluated and the ICM grade should at least B. The ICM was graded as follows: (A) tightly packed, with many cells; (B)loosely gathered, with several cells; and (C) very few cells. The three TE grades were (A) many cells forming a cohesive epithelium, (B) few cells establishing a loose epithelium and (C) very few large cells. The quality of the blastocyst was grouped into three categories based on ICM and TE scoring: good quality: AA, AB and BA; average quality: BB; and poor quality: AC and BC. Embryo grading was performed by the same team of four highly trained embryologists and each with five years of experience, which minimized the difference in human judgment. Then the embryos were biopsied on day 5 or day 6 based on the time of blastulation. The zona pellucida was perforated by use of a Saturn laser system (Research Instruments, Singapore) to opening of 6–9 mm, and a biopsy pipette was used to aspirate 3–5 herniated TE cells. Then the washed TE cells were placed in 0.2mL PCR tubes containing 5μL phosphate-buffered saline solution (PBS). All selected embryos were screened for 24 chromosome aneuploidy with NGS, as described in Zimmerman et al. Finally, three different outcomes were considered after the PGT-A testing: euploid and aneuploid and mosaic. After the biopsy, the blastocyst were vitriﬁcated using Cryotop® (Kitazato Corporation, Shizuoka, Japan). The vitrified-warmed procedure has been described in detailed previously.
Embryos that were screened by NGS to be euploid were transferred in FET cycles. In general, women with regular ovulatory cycles underwent natural FET cycles and the artificial cycles were applied for women with irregular menses, ovulation dysfunction or thinner endometrial thickness. After five days ovulation and/or when endometrial thickness was ≥7 mm, which were all monitored by vaginal ultrasound, only single selective frozen-thawed euploid blastocyst was transferred and then all patients provided for conventional luteal support.
Outcome measures and statistical analysis
All statistical results were calculated with SPSS 25.0 statistical software (IBM, United States). Using Empower Stats software to draw the smooth curve fitting diagram between maternal age and live birth rate and to analysis the threshold effect. LBR after the transfer of euploid embryos are we mainly discussed measure in this study. The secondary outcome measures are pregnancy rate (PR) and early spontaneous abortion rate (SAB). The LBR was defined as the number of live births divided by the sum of embryos transferred cycles included in the cohort. The PR was defined as the percentage of the intrauterine gestational sac with fetal heartbeat by all transferred embryos at 4 weeks after blastocyst transfer. The SAB was considered to be the proportion of clinical pregnancies (a fetal heartbeat was seen on scan) that did not progress in the first-trimester spontaneous abortion.
All cycles were divided into three groups according to the women’s age (<30 ,30-34 and ≥35 years). The outcomes measure, embryos data and the baseline demographic characteristics were all compared among the three age groups. Categorical variables were compared with the Chi-square (X2) and Fisher’s exact tests. Continuous variables were tested for normality, and they were expressed as mean ± standard deviation, and parametric data were compared using the analysis of variance (ANOVA) test. The odds ratio (OR) with 95% confidence interval (CI) were calculated and controlled for confounding factors. P<0.05 was considered to be statistically significant.