Live birth after letrozole as an adjunct to follicle-stimulating hormone versus follicle-stimulating hormone alone for ovarian stimulation in in vitro fertilisation cycles—study protocol for a randomised controlled trial

This is an open-label randomised controlled superiority trial being performed in the Department of Obstetrics & Gynaecology, The University of Hong Kong, and the Centre for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital. Written informed consent was obtained from all participants before participation in the study.

Eligible women will be given information about the study during their first consultation. All recruited women provided written informed consent before participation. The trial was approved by the institutional review board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW16-014) and the Ethics Committee of Peking University Third Hospital (2016sz-066). The study was registered on Clinicaltrials.gov (NCT02912988).

The enrollment, interventions and evaluation during the study process are shown in Table 1. Figure 1 indicates a flowchart showing registration, allocation, intervention and follow-up of participants.

Infertile women undergoing IVF treatment at the Department of Obstetrics & Gynaecology, the University of Hong Kong, and the Centre for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, will be screened for eligibility. Eligible women will be counselled about the trial on the first day attending the centres for ovarian stimulation. Written informed consent will be obtained before joining the trial. Participants can decline to join the study at any time without any consequences for their clinical treatments.

Recruited women will be randomly allocated in a 1:1 ratio to the letrozole group or the control group according to a computer-generated randomisation list put in opaque sealed envelopes which are open consecutively for eligible participants after screening. Both investigators and participants are aware of the allocation of the intervention. The biostatistician performing the data analysis will be blinded to the group allocation.

A gonadotropin-releasing hormone (GnRH) antagonist regimen is used. Human menopausal gonadotropin (HMG; Menopur, Ferring Pharmaceuticals, Saint-Preix, Switzerland) or recombinant human follicle-stimulating hormone (FSH; Gonal-F, Merck Serono, Geneva, Switzerland) is administered at a dose of 150 to 225 IU per day based on body mass index and previous ovarian response on day 2–4 of the menstrual cycle, at the discretion of the physicians. Transvaginal scanning, serum luteinizing hormone (LH), oestrogen (E2) and progesterone (P4) will be performed regularly to monitor ovarian response. HMG or FSH doses will be adjusted according to ovarian responses.

GnRH antagonist 0.25 mg (Cetrotide, Merck Serono, Darmstadt, Germany, or Orgalutran, Merck Sharp & Dohme Ltd.) is started from day 5 of stimulation until the day of the trigger. When three or more follicles reach a diameter ≥ 17 mm, 250μg of rhCG (Ovidrel; Serono, Aubonne, Germany) or triptorelin 0.2 mg (Diphereline, Ipsen Beaufour) is to be given for final maturation. Oocyte retrieval is performed around 36 h after the trigger.

In the letrozole group, letrozole (Letroz, Sun Pharmaceuticals, Mumbai, India) 2.5 mg daily is started from day 5 of stimulation until the day before the trigger. The control group will not receive letrozole.

When the serum estradiol level on the day of hCG administration or before the day was ≥20000 pmol/L, 0.2 mg of triptorelin (Ferring International Center, Saint-Prex, Switzerland) will be given for agonist trigger. When the serum estradiol level on the day of hCG administration or before the day was 15,000–20,000 pmol/L, 0.2 mg of triptorelin (Ferring International Center, Saint-Prex, Switzerland) and 2000 IU of hCG (Livzon, Zhuhai, China) or 125 mg of recombinant hCG (Ovidrel; Merck Serono) will be given.

The freeze-all strategy to prevent ovarian hyperstimulation syndrome (OHSS) is advised in women with (1) E2 on HCG day ≥15,000 pmol/L, (2) retrieved oocyte number ≥ 20, and (3) E2 on HCG day ≥10,000 pmol/L and retrieved oocyte number ≥ 15.

Oocytes are cultured in Human Tubal Fluid medium and incubated in a humidified 37 °C incubator with 5% CO2, after oocyte retrieval immediately. The fertilisation method will be selected according to the parameters of the semen analysis.

Fertilisation will be considered normal when two pronuclei are present between 16 and 18 h after conventional insemination or intracytoplasmic sperm injection (ICSI). All zygotes will be cultured in a cleavage medium (G-1plus, Vitrolife, USA) for further 48–52 h after fertilisation. Cleavage stage embryos will be assessed according to the developmental stage and degree of cytoplasmic fragmentation.

Fresh embryo transfer or elective freezing of all embryos will be decided by physicians according to the conditions of women. All embryos will be cryopreserved without fresh transfer when the women are at risk of OHSS, have elevated serum progesterone level on the trigger day (> 1.5 ng/L or > 5 nmol/L) or had endometrial polyps or fluid in the uterine cavity. To reduce the risk of multiple pregnancies, up to two embryos will be transferred on day 2/3 and one blastocyst will be replaced on day 5. Excess cleavage stage embryos will be vitrified or further cultured to the blastocyst stage while excess blastocyst will be vitrified accordingly.

In women with fresh embryo transfer, the luteal phase is supported with vaginal progesterone gel (Crinone, Merck Serono) at a dose of 90 mg daily or oral progesterone (Duphaston, Abbott) at a dose of 10 mg three times daily from the day of oocyte retrieval until the day of serum or urine hCG test.

Transvaginal ultrasonography will be performed 2 weeks after a positive serum or urine pregnancy test to confirm intrauterine pregnancy. Scanning will be repeated later to confirm ongoing pregnancy, which is defined as a viable pregnancy with a foetal heartbeat at 10–12 weeks. Information regarding the outcome of the pregnancy and obstetrical and perinatal complications will be obtained through a review of obstetrical medical records and neonatal medical records. The live birth is defined by the live foetus in the uterine after 20 weeks of gestation.

We will collect the following information within 6 weeks after delivery, including antenatal information (pregnancy complications and foetal information), delivery information (gestational age, mode of delivery, placental weight and birth complications) and newborn information (foetal sex, birth weight and birth defects).

Adverse events (AE) are defined as any undesirable experience occurring to a subject during the trial. A serious adverse event (SAE) is any untoward medical event that results in death, is life-threatening (at the time of the event), requires hospitalisation or prolongation of existing inpatients’ hospitalisation, results in persistent or significant disability or incapacity, is a congenital anomaly or birth defect and is a new event of the trial likely to affect the safety of the subjects, such as an unexpected outcome of an adverse reaction.

SAE in this study includes moderate/severe OHSS, intraperitoneal haemorrhage or ovarian torsion after oocyte retrieval, ectopic pregnancy, severe preeclampsia, pregnancy complications leading to hospitalisation, stillbirth, birth defects and other serious medical events judged by researchers to meet the criteria of SAE.

The result will be analysed according to the intention-to-treat (ITT) principle. Per-protocol (PP) analysis will also be conducted as a sensitivity analysis. 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. For continuous variables, the normality distribution will be estimated by using frequency histograms and the Kolmogorov-Smirnov test initially. If the continuous variables are normally distributed, they will be presented as means with standard deviations (SDs). If the continuous variables are non-normally distributed, their medians and interquartile ranges (IQRs) will be reported. For categorical variables, we will present the proportion between each group. In addition, the recruitment number, those participants lost to follow-up, protocol violations and other relevant data will also be reported. A comparison between groups will be performed using the independent sample t-test, Mann-Whitney U test for continuous variables or Pearson chi-square test/Fisher’s exact test for categorical variables as appropriate. The primary outcome will be compared using Pearson’s chi-square test or Fisher’s exact test as appropriate. Categorical secondary outcomes will be compared between two groups using a similar approach as the primary outcome. Student’s t-test or Wilcoxon test will be used as appropriate for continuous secondary outcomes, such as birth weight, etc. The relative risks (RR) and absolute rate differences (ARD) and their 95% confidence interval (CI) between the two groups will be calculated. And the 99% CI of the ARDs will be used to evaluate if the letrozole group is superior to the control group. Multiple variable logistic regression models will be used to assess the treatment effect adjusting for other potential confounding variables that are unbalanced in the baseline.

The first participant was recruited in March 2018, and it is anticipated the recruitment will end by the end of April 2022. The follow-up is ongoing and the expected data collection will be completed in February 2023. This trial protocol is version 2.0, 20/11/2017. At the time of the manuscript preparation, we have recruited 890 women and the recruitment is currently ongoing. This study is externally peer-reviewed and does not receive any external funding.