Methods
Study population
Records were reviewed for all women who underwent fresh or frozen autologous IVF/intracytoplasmic sperm injection (ICSI) cycles at the Assisted Reproduction Center of Northwest Women’s and Children’s Hospital, Xi’an, China. A cohort of women with positive serum β- human chorionic gonadotropin (β-hCG) after embryo transfer between May 2014 and April 2019 were included. Demographical traits, cycle characteristics, clinical and laboratory data were extracted from electronic medical records. If a clinical intrauterine pregnancy resulted, the maximum number of fetal sacs in early pregnancy, the pregnancy and perinatal outcome were included. Figure 1 shows the flowchart of patient selection and an overview of the treatment.
The diagnosis of PCOS was re-evaluated according to the Rotterdam criteria with satisfying at least two of the three criteria:11 oligo-/anovulation (a cycle length >35 days or variation between consecutive menstrual cycles of >10 days); clinical or biochemical hyperandrogenism; ultrasound diagnosis of polycystic ovary morphology.12Patients with other causes of hyperandrogenism and ovulation dysfunction (congenital adrenal hyperplasia, Cushing’s syndrome and androgenic-secreting tumors) were excluded. Exclusion criteria also included patients with recurrent pregnancy loss, uterine malformations, treatment with preimplantation genetic testing (PGT) and those involving donor sperms and oocytes.
The baseline characteristics were the female age, body mass index (BMI), maternal underlying medical conditions, gravidity and parity, history of prior spontaneous pregnancy loss as well as basal hormone levels. Age (years) and BMI (kg/m2) were also categorized for the clarity of data analysis. Three age subgroups were formed: age <30 years, 30-34 years and ≥35 years. BMI subgroups were: <25 kg/m2 (normal), 25-29.9 kg/m2 (overweight), ≥30 kg/m2(obesity). Infertility diagnosis (ovulation dysfunction, male and tubal factor, unexplained and multiple diagnoses) were included as categorical variables. Cycle characteristics included the insemination type (conventional IVF, ICSI or a combination of IVF and ICSI), fresh versus frozen cycle, the number and quality of transferred embryos.
Stimulation and embryos transfer protocol
For a full description of the IVF protocols, luteal phase support, and laboratory procedures please refer to our previous publication.13 Embryos were cultured to day 3 or day 5 depending on the number of embryos of good morphological quality on day 3. The strategy of the number of embryos transferred changed gradually during the study period. From 2014 to July 2018, one or two of the best quality embryos were transferred into the uterus on day 3 or 5. Since Sep 2018, offering transfer of single-embryo is the routine in clinical care. If two embryos were transferred, the quality of the best embryo was used for analysis. Embryonic cleavage and morphologic appearance were assessed as described previously.14 In case of high risk of OHSS or those with elevated serum progesterone levels on the day of ovulation trigger embryos were electively cryopreserved at the physician’s discretion and after discussion with the patient. Embryos that were cryopreserved in ‘freeze-all’ cycles and supernumerary embryos which were vitrified, were transferred in artificially supplemented cycles or in natural cycles. The vitrification, warming procedure, endometrial preparation and embryos transfer procedures was done according to standard protocols.15 If pregnancy was achieved, luteal phase support was continued until 10 weeks’ gestation.
Pregnancy assessment and outcomes
Pregnancy was defined as a serum β-hCG level greater than 20 IU/L 14 days after cleavage embryo transfer or 12 days after blastocyst transfer. If the β-hCG assay yielded a positive result, the patient underwent ultrasonographic monitoring to determine the number of gestational sacs and fetal viability at the 6th-7th week of gestation. A biochemical pregnancy loss was defined as a pregnancy without the intrauterine gestational sac that resolved spontaneously. Clinical pregnancy was defined as an intrauterine gestational sac visible by means of transvaginal ultrasound coincident with a positive serum β-hCG concentration. The ongoing pregnancy was defined by presence of fetal heart beat on ultrasound scan at 12 weeks’ gestation. Live birth was defined as delivery of a live-born infant after 24 weeks’ gestational age.
The primary endpoint of interest was clinical pregnancy loss which was defined as a pregnancy ending before 24 weeks of gestation, which were further categorized based on gestation length: early pregnancy loss (≤13 weeks), late pregnancy loss (13-24 weeks).16,17 We also examined the rate of pregnancy loss stratified by plurality of the pregnancy, defined as the number of fetal sacs on early ultrasound. Pregnancies with two and more than two fetal sacs in the ultrasound were combined in the analysis because of low numbers. Vanishing twins was defined as pregnancy with two intrauterine gestational sacs at 6-7 weeks’ gestation but that eventually delivered one infant. Fetuses dying after ≥24 gestational weeks are registered as stillborn. Ectopic pregnancies and hydatid moles were excluded from the pregnancy loss analyses due to their different etiology.
Maternal complications in the analysis included gestational diabetes mellitus (GDM) diagnosed via the 75 g 2-hour oral glucose tolerance test,18 hypertensive disorders of pregnancy, including gestational hypertension or pre-eclampsia, placental abruption and placenta previa, premature rupture of the membranes (PROM), macrosomia (birth weight >4,000g). Neonatal outcome variables included gestational age at delivery, preterm birth (PTB; <32 and <37 weeks), low birthweight (<1,500 and <2,500 g) and macrosomia (>4,000 g). With the intention to eliminate the impact of multiple pregnancies on maternal and newborn outcomes, we restricted the analysis to only singletons.
Statistical analysis
All statistical analyses were performed using SPSS version 21.0 (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. Binary logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (CI) of pregnancy loss and to evaluate the effect of potential confounders. Female age and BMI were recorded as either continuous or categorical variables. Female age, BMI, number of embryo transferred, a history of previous pregnancy loss and comorbidities (hypertension and diabetes), were considered as a covariate potential confounders for the hypothesized relationships. P -values <0.05 were considered to indicate statistically significance.