Study design and participants
We performed a retrospective cohort study of 1434 FET cycles of PCOS from January 2017 to March 2020) in the fertility unit at a University Hospital. Patients in this present study had previously undergone treatment by IVF or intracytoplasmic sperm injection (ICSI) cycles. The study was approved by the Reproductive Ethics Committees of the Affiliated Hospital of Shandong University of TCM (ref approval no. SDTCM20201215). All participants provided written informed consent. Eligible patients included women with PCOS aged between 21 and 35 years, diagnosed by Rotterdam criteria21: oligo-or anovulation, clinical or biochemical evidence of hyperandrogenism, and polycystic ovarian morphology on ultrasonography (defined as an ovary that either contains ≥ 12 antral follicles or that has a volume > 10 cm3), with at least one embryo vitrified mainly at day 3, and for whom it was the first FET performed. The exclusion criteria were: (i) Body Mass Index (BMI) ≥ 30Kg/m2 at the time of embryo vitrification; (ii) Endometriosis; (iii) Preimplantation genetic diagnosis/screening cycle; (iv) History of recurrent pregnancy loss or recurrent implantation failure; (v) Uterine pathology; (vi) Cycles cancelled due to failure of embryo thawing and survival.
Controlled ovarian stimulation protocol
All Participants had undergone the IVF/ICSI treatment as clinically indicated. Furthermore, a flexible GnRH antagonist (GnRH-ant) (Cetrorelix; Merck Serono, Darmstadt, Germany) protocol was employed with 150-225 IU/day of recombinant FSH (Gonal-F, Merck-Serono, Lyon, France). Additionally, the doses of gonadotropin were determined based on the characteristics of individual patients. Thereafter, oocyte retrieval was conducted under ultrasound transvaginal guidance, 34-36 hours after triggering with GnRH-a (Triptoreline, Decapeptyl, Ipsen, France) or recombinant hCG (Ovitrelle®, 250μg, Merck), after which conventional IVF/ICSI were performed as previously described 22. The IVF/ICSI procedure had either been followed by a fresh embryo transfer and preservation of the redundant good embryos by vitrification or by a freeze-all strategy on clinical indication. Regular monitoring during controlled ovarian hyperstimulation (COS) treatment includes vaginal ultrasound (to assess endometrial thickness and follicle development) and blood hormone assays (including estradiol, progesterone and LH plasma levels).
The choice of embryos for vitrification was expected to focus on the inclusion of no less than six blastomeres with ≤ 20% fragmentation. Embryos that presented a fragmentation rate between 20% and 50% were vitrified only when they had reached the 8-cell stage on Day 3. The applied vitrification procedure has been described in detail before 22.
Endometrial preparation protocols
Women with PCOS were instructed to wait for spontaneous menses or prescribed with progestin to induce menses before endometrial preparation.23 The two endometrial preparation protocols used before the FET were the following:
i. Hormone replacement cycles
In hormone replacement cycles, 4 mg of oral estradiol valerate was administered starting on the second or third day of the menstrual cycle and continuing for five days. This was followed by 6 mg of oral estradiol for 6-8 days. When the endometrial thickness reached 7 mm and the serum progesterone level was below 1.5 ng/ml, we added vaginal supplementation with progesterone 90 mg daily (8% Crinone, Merck-Serono, Switzerland) prior to FET. The embryo was transferred according to its development stage at the time of freezing. The supplementation continued until a pregnancy test was performed. In case of a positive test, the patients were instructed to continue treatment until the 12thWG.24
ii. Stimulated cycles
In stimulated cycles, patients received a daily subcutaneous injection of Gonarfen (Merck Serono SA Aubonne Branch) (37.5–75 IU) from day 4 of the cycle onwards. The dose was adjusted according to the BMI, the ovarian reserve and any previous ovarian response to stimulation. A subcutaneous injection of hCG (5000 IU) or recombinant hCG (250 μg) was administered to induce oocyte ovulation, when the ovulation criteria were met (one dominant follicle ≥ 16 mm and peak plasma estradiol level > 200 pg/ml). These patients had no intercourse on ovulation day. The adequacy of the luteal phase was evaluated by measuring blood progesterone levels 3 days after ovulation had been triggered. If the progesterone level 3 days after ovulation triggering exceeded 3 ng/ml, FET was implemented (depending on the embryo’s development stage at the time of freezing). STC protocols for endometrial preparation were not supplemented with progesterone. 25
Study endpoints and definitions 26,27
Positive pregnancy was defined as a serum β-hCG level greater than 10 U/L in the 14 days after cleavage embryo transfer. The Patient underwent ultrasonographic monitoring to determine the number of gestational sacs and fetal viability at the 6th-7th week of gestation, i.e., clinical pregnancy, if the β-hCG assay yielded a positive result. Pregnancy loss was defined as clinically recognized spontaneous loss of pregnancy before the completion of twenty gestational weeks. Ectopic pregnancy, defined as a pregnancy in which implantation takes place outside the uterine cavity, diagnosed by ultrasound, surgical visualization or histopathology. Live birth, defined as the birth of at least one child with breath and heartbeat, irrespective of the duration of gestation. A birth weight of 3500 g or more can be used if gestational age is unknown. Furthermore, this pathological state in which the death of a fetus prior to the complete expulsion from its mother after 20 completed weeks of gestational age was diagnosed as stillbirth. As opposed to live birth, the fetus does not breathe or show any other evidence of life.
Statistical analysis
All data are evaluated using version 26.0 of SPSS program (SPSS Inc., Chicago, USA). Quantitative variables are expressed as means ± standard deviations (SD) and were analyzed using Student’s t-test, Independent-Samples Mann-Whitney U Test. Qualitative variables are expressed as frequencies and percentages and were analyzed using the χ2-test. P< 0.05 was considered statistically significant for the two groups of data tested. Furthermore, a propensity score matching (PSM) model was established to balance differences in baseline characteristics between the two groups.20 The propensity scores were calculated using binary logistic regression analyses based on the following patients’ characteristics: female age, infertility duration, body mass index (BMI), infertility type (primary or secondary), AMH, protocol of COS (Long GnRH-a protocol or GnRH-ant protocol), initial treatment (IVF or ICSI), Gn usage time, Gn dosage, oocytes retrieved, total number of embryos, good quality embryos, transferred embryos. Patients undergoing STC were matched with the HRC group using the nearest-neighbor random matching algorithm in a ratio of 1:1.