Subjects
A total of 286 FETs patients from December 1st, 2020 to June 30th, 2021 at the Center for Reproductive Medicine, Affiliated Hospital of Jining Medical University (Shandong province, China) were included in the present work. The experimental protocols were authorized by the ethics committee of Affiliated Hospital of Jining Medical University and written informed consent was obtained from all participants. Study data, including age and body mass index (BMI), etc., were collected from the clinical database. Patients with embryo cryopreservation prior to IVF/ICSI cycle were included in the present study. Patients with possible confounding comorbidities (autoimmune diseases, diabetes mellitus or the intake of confounding medication, uterine malformations, and leiomyomas, adenomyoma, endometrial polyps, intrauterine adhesions, endometrial tuberculosis history) or those with a history of recurrent abortion or implantation failure were excluded from the study.
Endometrial Preparation Protocols
The participants were assigned into different groups of endometrial preparation based on the clinician’s experience and patient’s characteristics. Four protocols were available for endometrial preparation as follows: 1) the natural cycle, 2) the ovulation induction cycle, 3) the hormone replacement treatment (HRT) cycle and 4) the HRT with gonadotropin-releasing hormone agonist (GnRHa) pretreatment (HRT + GnRHa).
The natural cycle was the first choice for patients with regular menstrual cycles, in which ultrasound monitoring was initiated on cycle days 10-12 to assess the dominant follicle and the endometrium. When the follicle became mature, human chorionic gonadotropin (HCG, Merck Serono, China) was administered to trigger ovulation. The timing of embryo transfer was determined based on the stage of embryonic development on the day of embryo freezing.
In the ovulation induction cycle, participants were administered with human menopausal gonadotropin (HMG, Livzon Pharmaceutical, China) at a dose of 75 IU/day from day 3 to day 5 of the menstrual cycle. The dose of HMG was adjusted according to the follicular development monitored by ultrasound and serum sex steroid measurements. Besides, once the dominant follicle reached 18 mm or more in diameter, HCG at a dose of 8,000-10,000 IU was adopted to trigger ovulation.
For the HRT cycle, the participants were orally administered with estradiol valerate tablets (Progynova, Bayer, Germany) at a dose of 3–12 mg/day from the 2nd to 5th day of the cycle, and such regimen lasted for at least 7 days. The starting dose was determined according to the patient's previous endometrium. When the endometrial thickness was≥7 mm, progesterone at a dose of 40 mg/day was given to transform the endometrium. As for HRT + GnRHa cycles, GnRH agonist was initiated on day 1 of the menstrual cycle. Moreover, on day 1 of the next menstrual cycle, estrogen stimulation was started as HRT cycles without GnRH agonist.
According to the patient’s age and previous IVF cycles on the 4th (cleavage-stage embryo) or 6th day (blastocyst) after ovulation or progesterone injection, one or two thawed embryos were transferred using a soft-tipped Wallace catheter under ultrasound guidance. Luteal support with progesterone was given to all patients after embryo transfer. Such regimen was continued until 10 weeks of gestational age if a gestational sac and embryonic heartbeat at 4–6 weeks after embryo transfer was observed by transvaginal ultrasound.
Enzyme-linked immunosorbent assay (ELISA)
Samples for a fasting blood test were collected from all participants. The specimens were subjected to centrifugation at 3,000 rpm for 10 min, followed by serum collection and storage at −80°C prior to further analysis. The contents of serum periostin were measured by Human Periostin ELISA Kit (MM-13746H2), which was purchased from Jiangsu Enzyme Immune Co., Ltd, China.
Assessment of embryo quality and vitrification of embryos
Embryo morphology was assessed based on the developmental speed, degree of fragmentation, and evenness of the cleavage sphere. Cleavage-stage embryos with at least seven blastomeres and fragmentation < 20% were regarded as high-quality embryos. The blastocyst was graded by using the Gardner scoring system, and embryos graded 3BB or greater were regarded as embryos of good quality[24]. The timing of FET was determined according to the stage of embryonic development, and the embryos were vitrified and synchronized with the duration of progesterone exposure of the endometrium. The number of embryos transferred was determined according to the patient’s age and previous IVF cycle, and a maximum of two embryos was allowed to be transferred.
The vitrification and thawing were carried out as previously described [25]. In brief, embryo vitrification was conducted using a Cryotop carrier system, and dimethylsulfoxide-ethylene glycol-sucrose was used as cryoprotectants. Embryos were sequentially transferred to the diluted solution to thaw (1 mol/L to 0.5 mol/L to 0 mol/L sucrose).
Definition of Clinical Outcomes
American Society for Reproductive Medicine (ASRM) 2017 consensus definitions [26] were used to assess the clinical outcomes. Clinical pregnancy was defined as a pregnancy diagnosed by ultrasonographic visualization of one or more gestational sacs or definitive clinical signs of pregnancy. The patients with the above-mentioned pregnancy were assigned to the pregnant group. When pregnancy was diagnosed only by the detection of HCG in serum (14 days after embryo transfer) or did not develop into a clinical pregnancy, it was defined as biochemical pregnancy. Consequently, the non-pregnant (blood β-HCG< 10 mIU/mL) patients or those with biochemical pregnancy were assigned to the non-pregnant group.
Statistical Analysis
Statistical analysis was performed with the statistical packages R (The R Foundation; http://www.r-project.org; version 3.4.3) and Empower (R) (www. empowerstats.com, X&Y Solutions, Inc., Boston, Massachusetts). Continuous data were presented as the means ± standard deviations, and the categorical data were expressed as N (%). P <0.05 was considered statistically significant. To assess the risk factors of pregnancy outcome, variables were analyzed by multivariate logistic regression. The predictive accuracy of serum periostin levels on the day of FET on the pregnancy outcome was investigated according to the areas under the receiver operator characteristic (ROC) curves (AUC).