Research Design
This is a prospective, open, randomized control trial. Potentially eligible women will be given information about the study before invitro fertilization (IVF) treatment in four different tertiary hospitals: The International Peace Maternity and Child Health Hospital, Second Affiliated Hospital of Naval Medical University, The First Affiliated People's Hospital of Wenzhou Medical University, and Taizhou Hospital of Zhejiang province. Training was uniformly conducted before the start of the trial in order to ensure the consistency of procedures in all the centers. The progress is periodically checked by professionals to ensure the uniformity and consistency. The research project was approved by The International Peace Maternity and Child Health Hospital and was conducted according to the Declaration of Helsinki. its amendments will adhere to Good Clinical Practices. The study was registered on ClinicalTrials.gov (No: ChiCTR2000036527).
The flow chart of this study is displayed in Fig. 1. A schedule of enrollment, interventions, and assessment is provided as Table 1.
Inclusion criteria:
Patients who met the following inclusion criteria are included in the study:
1. Chinese women aged between 20 and 38 years
2. Meet Rotterdam PCOS standard diagnosis
Two of the following three criteria were met: (1) sparse ovulation or anovulation. (2) Hyperandrogenemia or clinical manifestations of hyperandrogenism. (3) Ultrasonography performed on 3 to 5 days after bleeding during the menstrual cycle or after progesterone withdraw show both ovaries had ≥12 small follicles with a diameter of 2 to 9mm and/or ovarian volume>10 mL
3. No IVF history
4. Willing to cooperate with the research plan and sign the informed consent.
Exclusion criteria used in this study:
1. Other causes of hyperandrogenism, such as thyroid disease congenital adrenal cortical hyperplasia
2. A history of three or more miscarriages, or combined with immunogenic infertility
3. Presence of uterine lesions: uterine malformation, adenomyosis, submucosal myoma or uterine adhesions
4. Chromosomal abnormalities and need to undergo preimplantation genetic testing (PGT)
5. Combined with major systemic diseases
6. Contraindications to estrogen-progestin therapy or the use of low-molecular-weight heparin
Randomization and blinding
PCOS women meeting the inclusion criteria will be recruited and randomized according to an online Central Randomization System, supported by shanghai clinical research center, on the day of progesterone transformation. Participants are assigned in a 1:1 ratio randomly to the study group (LMWH + Hormone Therapy) or the control group (Hormone Therapy). LMWH is given daily subcutaneous starting from the progesterone transformation day. The dynamic block method is used to develop the randomization scheme, and the multi-center electronic random system used to stratify the subjects within the block. The nature of the treatment interventions is open to treating physicians. However, this study will be blinded to embryologists, laboratory technicians, and nurses who conduct follow-ups until the completion of the statistical analysis of this study.
Sample size calculation
According to the previous data, the continuous pregnancy rate of female PCOS patients treated with LMWH within one week was 35%, while the continuous pregnancy rate of PCOS patients with IVF after routine pre-treatment was about 25%. According to the optimal efficacy experiment, α=0.05, β=0.20, and 10% loss of follow-up and shedding were set. According to the sample size formula, about 188 subjects in each group, and 376 subjects should be recruited.
Interventions
Participants will be subjected to an antagonist regimen of controlled ovulation stimulation, egg extraction and whole embryo cryopreservation. During the FET cycle,
the endometrium was prepared by hormone therapy. As endometrium thickness above 8mm, progesterone will be added preparing for embryos transfer. Clexane 4000 IU (0.4mL) is given injected daily from the progesterone transformation until hCG determination showing negative or transvaginal ultrasound verifying the presence of intrauterine pregnancy.
Adherence
Participants are fully informed that the trial does not involve the increase of patient visits times, Invasive examination, extra charges. The duration of LMWH administration ranges between 2 and 4 weeks for the study group. Clinic interventions will be dominated by one staff group with one fixed clinician for each participant. Adherence reminder system will enhance the relationship between participants and our staffs. Participants can consult the clinician in the clinic at each visit if any. Also, our staff will regularly contact the patients via WeChat to improve the adherence to the protocol.
Outcome measures
The primary outcome will be the ongoing pregnancy rate resulting from the FET cycles, which is defined by the presence of a gestational sac with a fetal heartbeat after 12 weeks of gestation. Secondary outcomes include clinical pregnancy rate, live birth rate, and early pregnancy loss rate. The clinical pregnancy is defined as the presence of a gestational sac at 6–7 weeks of gestation when visualized by transvaginal ultrasound, and clinical pregnancy rate refers to the percentage of clinical pregnancy cycles assessed as clinical pregnancy in the number of transplant cycles. Live birth rate is defined as the percentage of birth cycles with at least one surviving birth to transplant cycles in the number of transplant cycles. Early pregnancy loss is defined as loss of the embryo before 12 weeks of gestation, and early pregnancy loss rate refers to the percentage of early pregnancy loss cycles in the number of clinic pregnancy cycles.
Safety assessment
All subjects will be followed up by medical personnel including a chief physician throughout this study. Although LMWH is the preferred anticoagulant drug for pregnant women, we do not rule out that its use could cause some side effects. The commonly reported adverse reactions of LMWH use was blooding, and less common adverse effects include heparin-induced thrombocytopaenia, osteoporosis, and spontaneous fractures. Patients were well informed of the potential risks in advance. Follow-up will be performed in the outpatient department at weeks 2, 4, 6, 8, and 10 after FET to record any local and/or systemic reactions during medication and any adverse events defined as below.
Adverse events: Any discomfort or worsening of existing discomfort during the study period (including ovulation induction cycle, FET cycle, and follow-up), whether associated with the study intervention or not. Discomfort may be symptomatic (e.g., abdominal pain, abdominal distension, chest distress), physical signs (e.g., mobility dullness positive, adnexal mass) or abnormal examination (e.g., laboratory examination, electrocardiograph). Serious adverse events: adverse events occurring during the study period that meet one or more of the following criteria: 1) Severe IVF complications, including severe ovarian hyperstimulation syndrome (OHSS), complete ovarian torsion, and intraperitoneal bleeding. Severe OHSS is diagnosed when ovarian enlargement of ≥ 12 cm is observed and when there is clinical evidence of ascites and/or hydrothorax or breathing difficulties with or without hemoconcentration, severe hypoproteinemia, abnormal liver function, coagulation abnormalities, or diminished renal function. 2) Serious complications of LMWH: thrombocytopenia, damage of liver and kidney function. 3) Pregnancy-related complications: massive bleeding during pregnancy, shock during pregnancy, et.al. Adverse events will be reported in the appropriate section of the case report form. It is important that these records include the duration, extent, relationship to the study intervention and/or course of intervention, and any combination therapy.
Data collection and monitoring
Baseline characteristics of patients were collected, including age, BMI, parity, education levels, etc. Also, detailed IVF process and outcomes recorded in time. Once a patient is enrolled, a staff team will be prespecified for the follow-up and data collection. Meanwhile, the auditor will conduct data verification and quality check from time to time. The investigator and the monitor work together to ensure information of case report form (CRF) is accurate and clear. Data corrections or additions to CRF must be made by qualified researchers, signed with initials and dated. Data editing, input, and validation are performed continuously and rapidly in the study. Any missing, impossible, or inconsistent data in the CRF will be asked of the investigator via the Data Query Form and then archived for each subject until data cleansing is complete. Clinical research integration platform is used for data entry and management.
The informed consent is compliance with the relevant data protection and privacy laws. Participants will authorize the collection, use, and publication of the study data by the investigator and those who need the information for the study. Written informed consent was obtained from patients before study enrollment.
Statistical analysis
The study data are collected and managed by nonclinical staff who are responsible for data management in each clinical center. Baseline characteristics will be described by descriptive analysis and the balance among groups or subgroups will be assessed by analysis for different kinds of data. Continuous variables with normal distribution are presented as means with standard deviations (SDs), and continuous variables with non-normally distributed as medians and interquartile ranges (IQRs). Categorical variables are presented in the form of n (%). Results will be analyzed according to the intention to-treat (ITT) principle and per-protocol (PP) analysis as a sensitivity analysis. The differences between the two groups are detected using Pearson chi-square test/Fisher’s exact test for categorical variable or the independent sample t-test, Mann-Whitney U test for continuous variables. Statistics will be run using SPSS version 21 software (SPSS, Inc., Chicago, IL). P values of less than 0.05 considered to be statistically significant.