The proposed study will adopt a pilot cohort methodology consisting of 12 weeks of intervention and follow-up monitoring of participants for primary and secondary outcomes. Secondary outcomes will be compared with retrospective data from the tNFET control group.

The study population will consist of 20 women undertaking FET cycles at the Public Fertility Care of The Royal Women’s Hospital (RWH), Melbourne, Australia. The recruitment process will commence at this location, whereby clinicians and nursing teams will be invited to screen the patients they interact with for eligibility for the study. These staff members will be trained on appropriate screening according to the study protocol and will be delegated by the principal investigator. Eligible patients will then be approached for study by team members who are not directly involved in potential participants’ clinical care and, therefore, will not have had any clinical interactions with the participant. All patients will be provided with a patient informed consent form (Multimedia Appendix 1) to ensure they are familiar with the protocol requirements and understand their rights as participants. Additionally, they will be provided with a patient information document created by the research team (Multimedia Appendix 2) to assist with patient education surrounding the use of the P4 pessary during the study.

Inclusion criteria consist of women with regular cycles defined as 21-35 days, women younger than 40 years at the start of a cycle, and women whose BMI ranges from 18 to 35, inclusively.

The exclusion criteria include anovulatory women, women who are 40 years or older at the start of the cycle, and women who have preexisting contraindications to exogenous P4 supplementation, such as those with liver disease or thromboembolic disease. The exclusion criteria will also include the use of additional LPS (eg, subcutaneous), women with uterine pathology, including congenital malformations of the female reproductive tract, endometrial polyps, intrauterine adhesions, adenomyosis, and leiomyoma. Regarding the retrospective element of the study, patients for whom inclusion criteria information is lacking will not be included in the study.

The control group will consist of retrospective data from 20 women, matched against inclusion and exclusion criteria, within the Public Fertility Services of the RWH data bank. These controls would have undergone tNFET at the service provider within the preceding 12 months of the study start date.

All participant data will be stored securely in the database of the RWH Reproductive Services Unit (RSU), ensuring the security of patients’ confidential information. Furthermore, on completion of the study, all patient information will be securely stored within the hospital server with restricted access for a minimum of 15 years, with custodial responsibilities given to the principal investigator.

The primary outcome will be the presence of corpus luteum with a characteristic “ring of fire” appearance on the transabdominal ultrasound on the day of embryo transfer.

The secondary outcomes consist of the number of clinic visits required per patient undergoing this protocol, the biochemical pregnancy rate defined as the detection of βhCG in serum or urine [14], and the clinical pregnancy rate defined as the ultrasonographic visualization of one or more gestational sacs [14].

This is a pilot prospective cohort study without randomization.

Participants will undergo active treatment for 2-8 weeks, with a follow-up period of up to 12 weeks or gestation. For a patient with a 28-day menstrual cycle, on days 10-12 (Figure 1), a transvaginal ultrasound will be performed to evaluate for an appropriate endometrial thickness of 7.0 mm and a mean follicle diameter of 14.0 mm. If these ultrasound criteria are met, the patient will undergo blood tests for estrogen, LH, and P4 on the same day; should levels of P4 be under 5 nmol/L, exogenous P4 supplementation via vaginal pessary will then commence on the same day, with FET scheduled 120-125 hours post P4 administration. P4 pessary, ORIPRO (Orion Laboratories Pty Ltd T/A Perrigo Australia, Balcatta, Western Australia), at a dose of 200 mg twice daily, will be the only form of LPS for patients undergoing P4mNFET. Following FET, serum hCG will be taken 10 days later. If positive, LPS via P4 pessary at 200 mg twice daily will continue to 8 weeks’ gestation with a pregnancy scan between 6-7 weeks. If negative, the patient will be advised to cease LPS. In the instances where P4 levels are greater than 5 nmol/L, the patient will be removed from the study.

The control group will consist of patients, matched to the inclusion and exclusion criteria, who would have undergone tNFET at the RWH RSU. Patients undertake an ultrasound on days 10-12 of their cycle for those with a 28-day cycle. Upon detection of a dominant follicle of 18 mm or more and an endometrial thickness of 7 mm or more, serum LH and P4 are taken. An LH surge is defined as >25 IU/L in the context of P4 <5 nmol/L. Ovulation is defined as a P4 >5 nmol/L. LPS, in the form of a vaginal ORIPRO 200 mg pessary (Orion Laboratories Pty Ltd T/A Perrigo, Australia), is commenced on day 1 post ovulation, and embryo transfer is carried out 6 days post LH surge.

Given the qualitative and binary nature of the primary outcome, no statistical testing is proposed for this. Secondary outcomes will undergo t tests and Mann-Whitney U tests for data that are normally and not normally distributed, respectively. Should there be significant variation in the key baseline variables between the study and the control group, a multivariate analysis will be performed.

Safety oversight for the study will be carried out under the direction of the independent safety monitor working within the framework of the Data and Safety Monitoring Board Charter to ensure an objective assessment of the safety and efficacy of the study. AEs deemed secondary to the administration of the pessary will be monitored and reported accordingly by either the investigators or the independent safety monitor, from administration to the end of follow-up. Relevant physical examinations or investigations will then be carried out to ensure the effects of the AE are managed accordingly. The primary investigator will record the AE appropriately into the patient’s medical record or study shadow file.

On completion of the study, primary and secondary outcomes will be disseminated to investigators, ensuring the confidentiality of patient information. Findings will be synthesized and communicated to colleagues of the RWH RSU and Human Research Ethics Committee, as well as in further peer-reviewed publications related to the RCT that may be conducted following the completion of this cohort study. Additionally, findings will be communicated confidentially in potential presentations at national and international conferences.

We have designed a pilot cohort nonrandomized study to assess the feasibility of a novel P4mNFET protocol that circumvents the need to monitor for an endogenous LH surge to pinpoint ovulation to schedule FET. This protocol, therefore, may combine the advantages of the tNFET, AFET, and mNFET into one protocol that provides greater flexibility for the timing of transfer with fewer requirements for surveillance and no impact on pregnancy outcomes.

As with any study of this kind, some challenges are anticipated with patient recruitment. The research team aims to mitigate this via appropriate counseling and use of our patient informed consent form, which is in a question-and-answer format.

The second challenge may be the accuracy of the retrospective data for the control group. This is an inherent issue with all retrospective studies. However, it is anticipated that key secondary outcomes will be accurately reported.

As with any study, there are limitations to acknowledge. First, cohort studies lack the same level of rigorous methodology as an RCT. However, based on the results of this pilot, we aim to proceed with a noninferiority RCT to compare this novel protocol to tNFET. Second, the retrospective nature of the control group may be associated with issues related to data entry.

The implications of this study are that it may inform the future of FET. If primary outcomes are met such that the presence of the corpus luteum is maintained with P4mNFET, then a noninferiority RCT will be carried out to formally compare the two methods. Moving forward, this P4mNFET protocol may provide clinics the opportunity to perform FET with greater scheduling flexibility and comparable outcomes to established methods while potentially leading to cost-saving and increased efficiency.