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.