Materials and Methods
We performed a retrospective cohort study between Jan 2015 and Oct 2018 at the Northwest Women and Children’s Hospital, China. This study was approved by the institutional research ethics review board (2019013).
We included women aged 36 years or older who received IVF ovarian stimulation cycles and who had at least two blastocysts of any grade available for transfer. Couples undergoing treatment with preimplantation genetic testing (PGT) or donor oocytes were excluded. Patients were included in the study only once. Demographic and IVF cycle characteristics data were obtained from our assisted reproductive center. The results were analyzed per IVF/ICSI cycle, including both fresh and frozen embryo transfers. Given the retrospective nature of the work, no specific consent was required from the patients. Figure 1 shows the flowchart of patient selection.
Included women were divided into two comparison groups based on the number of blastocysts used
in their fresh transfer. The women in the eSBT group had one single-blastocyst stage embryo transferred in the fresh cycle, and one or more blastocysts cryopreserved. In the DBT group, all women had two blastocysts transferred in the fresh IVF/ICSI cycle. The subsequent frozen-thawed embryo transfer cycles were a combination of single- and double- blastocyst transfers, more commonly the latter.
Laboratory procedures
For a full description of the IVF protocols, luteal phase support, and laboratory procedures please refer to our previous publication.19 If two good-quality cleavage-stage embryos were found on day 3, embryos were transferred to blastocyst-stage media and cultured through day 5-6, at which time they were re-evaluated for blastocyst formation. The blastocyst quality was assessed according to the criteria of Gardner and Schoolcraft.20
Good-quality blastocysts were ≥3Bb. All other blastocysts including early blastocysts were graded as fair-quality. If two blastocysts were transferred, the quality of the best embryo was used for analysis. The embryo transfer was performed on Day 5 of development under ultrasound guidance. All remaining blastocysts viable were vitrified.
Transfer strategies and patient education
All patients were extensively counseled on the chances of pregnancy and risk of multiple gestation pregnancies before ART treatment. The updated pregnancy and livebirth outcomes were posted on the wall in the participants’ waiting area, with the rationale of single embryo transfer in our institution. Information on the pregnancy rate and complications associated with multiple pregnancy were reiterated for a second time regarding their blastocyst quality and embryo transfer options. At our institution, a mandatory single-blastocyst transfer policy was implemented for young patients (≤35 years) with good-prognosis. However, older patients were encouraged to accept eSBT which was offered as the primary recommendation for their transfer, with DBT presented as our secondary recommendation, as long as there was no medical contraindication for a multiple pregnancy. The final decision on the number of blastocysts to transfer was made by the couples after collaboration with the embryologist, physicians. ‘
Frozen embryo transfer (FET) cycles
The subsequent FET cycles were a combination of single- and double- blastocyst transfers. In the eSBT group, single blastocyst transfers were performed in women with at least one good quality cryopreserved blastocyst or those did not want to have multiple pregnancies. If a patient had no good quality blastocyst left or failed in the first two transfer attempts, we usually performed a DBT. In the DBT group, we usually performed double- blastocysts transfers unless a patient with only one blastocyst left.
FETs were done in either a natural cycle or an artificial cycle by the use of estradiol as described in detail in previous publication.21 FET was scheduled at 5 days after ovulation or 6 days of progesterone supplementation. All embryo transfers were performed using transabdominal ultrasound guidance. Luteal support was continued until 10 weeks of pregnancy.
Outcome measures
The primary outcome was cumulative live birth rates. Secondary outcomes were clinical pregnancy, implantation and miscarriage rate in initial fresh cycles as well as the cumulative multiple live births rates. We also compared rates of low birthweight (<2.5 kg), preterm birth (<37 weeks), cesarean section delivery and congenital anomaly. The cumulative live-birth rate within a cycle was defined as the probability of a live birth from an ovarian stimulation encompassing all subsequent fresh and frozen embryo transfers from that stimulation. Live birth was defined as a living birth after 24 weeks of gestation. Multiple birth was defined as a live birth of multiple infants divided by all live births. The implantation rate was defined as the number of gestational sacs divided by the number of embryos transferred. we defined multiple births as low birthweight if the outcomes applied to any of the live babies.
Statistical analysis
All statistical analyses were performed using SPSS version 25.0 (IBM Corp., USA). Categorical data were presented by the number of cases and corresponding percentage and continuous data were presented as the mean value ± SD. Categorical data and continuous data that did not show a normal distribution were analyzed by Pearson’s chi-squared test/Fisher’s exact test or Kruskal-Wallis test as appropriate. A binary logistic regression model was used to assess the influence of single- versus double-blastocyst transfer on the odds of cumulative live births. Time to live birth for the two groups was estimated by Kaplan-Meier method.P -values <0.05 were considered to indicate statistically significance.
Subgroup analyses
To further investigate the effect of number of blastocyst transfer on cumulative LBR between different subgroups of women, we performed analyses split by certain characteristics. The subgroups were woman’s age (36-37,38-39,>40 years old) and blastocyst quality (high and fair). The age of 36 and 38 were chosen as thresholds for stratification, given the embryo transfer guidelines released by the American Society for Reproductive Medicine in 2017, recommending that good prognosis patients under 38 should have a single-embryo transfer.5,8 The threshold of age 40 years reflected the most commonly used threshold in legislation and guidelines.22 Given the quality of blastocyst has been shown to be associated with live birth.20,23 The analysis was divided by the quality of blastocysts into good and fair quality blastocysts, as graded by morphological examination. When two blastocysts were transferred, the higher grade was included in the analysis. For each subgroup, we generated new inverse probability of treatment weights within each imputed dataset. We then used these to weight a logistic regression model to assess the odds of cumulative live births.