Estradiol 8 vs 12 mg for Endometrial Preparation in HRT-FET

Study Methods 7.1 Study population Women undergoing assisted reproductive technology and scheduled for their first autologous programmed HRT-FET cycle will be screened for eligibility. Randomization will occur at the start of the FET cycle; ovarian stimulation, oocyte retrieval, fertilization, embryo culture, and vitrification from the antecedent IVF/ICSI cycle will have occurred before trial entry and will not constitute part of the randomized intervention.

7.2 Inclusion criteria

• Women aged 18-40 years.
• Body mass index <30 kg/m².
• First autologous programmed HRT-FET cycle.
• Endometrial preparation planned using oral estradiol valerate only.
• Vitrified embryos derived from a previous autologous IVF or ICSI cycle.
• Planned transfer of vitrified-warmed day-5 blastocyst(s).
• Normal uterine cavity documented within the preceding 12 months.
• Baseline transvaginal ultrasonography on cycle day 2 or 3 showing no ovarian cyst, dominant follicle, or intracavitary pathology.
• Ability and willingness to provide written informed consent.
• Ovulatory-factor infertility, including polycystic ovary syndrome, will be eligible provided all other criteria are fulfilled.

7.3 Exclusion criteria

• Contraindication to estrogen treatment, including current or previous breast cancer or endometrial cancer, deep venous thrombosis, pulmonary embolism, stroke, or allergy to the study medication.
• Irregular unexplained vaginal bleeding.
• Untreated thyroid disorder or hyperprolactinemia.
• Uncontrolled diabetes mellitus or hypertension.
• Significant renal, hepatic, or severe systemic disease.
• Uterine abnormality, untreated hydrosalpinx, or untreated intracavitary uterine pathology detected by ultrasonography or hysteroscopy.
• Donor-oocyte, donor-embryo, gestational-carrier, or PGT-A cycle.
• Natural or modified-natural FET cycle; transdermal, vaginal, or mixed-estrogen regimen; or use of letrozole, gonadotropins, or GnRH agonists for endometrial preparation.
• Premature ovarian insufficiency.
• Stage III-IV endometriosis.
• Recurrent implantation failure or recurrent miscarriage.
• Abnormal parental karyotype.
• Active smoking.
• Anticipated or documented difficult embryo transfer.
• Refusal or withdrawal of consent. 7.4 Baseline assessment Before treatment initiation, all eligible participants will undergo standardized personal, menstrual, obstetric, infertility, medical, surgical, and family history taking, followed by general and gynecological examination. Routine investigations will be obtained according to unit practice. Serum thyroid-stimulating hormone and prolactin will be assessed to exclude relevant endocrine disorders. Baseline transvaginal ultrasonography will be performed on cycle day 2 or 3 to document endometrial thickness and exclude ovarian cysts, a dominant follicle, or uterine-cavity pathology.

Characteristics of the antecedent IVF/ICSI cycle and embryo cohort, including ovarian stimulation characteristics, oocyte yield, fertilization variables, embryo development, and embryo quality, will be abstracted from clinical records for baseline comparability only and will not be considered outcomes of the randomized estradiol intervention.

7.5 Randomization and allocation concealment Eligible participants will be randomized in a 1:1 ratio. Randomization will be stratified by study center using separate computer-generated permuted-block schedules prepared by an independent statistician who will have no role in recruitment, clinical care, embryo transfer, outcome assessment, or the primary statistical analysis. Allocation concealment will be maintained using sequentially numbered, opaque, sealed envelopes prepared and stored by independent personnel. After eligibility confirmation and written informed consent, the next envelope will be opened in sequence by the designated study coordinator, who will assign the corresponding coded medication pack.

7.6 Blinding and study medication The trial will be double-blind and placebo-controlled. The active study medication will be commercially available estradiol valerate (Progynova® 2-mg coated tablets; Bayer Weimar GmbH und Co. KG, Weimar, Germany). All participants will receive six tablets daily according to the same administration schedule. Women allocated to the 12 mg/day group will receive six active 2-mg estradiol valerate tablets daily, whereas women allocated to the 8 mg/day group will receive four active 2-mg estradiol valerate tablets plus two matched placebo tablets daily.

The matched placebo tablets will be prepared specifically for the trial by the Drug Manufacturing Unit, Faculty of Pharmacy, Cairo University, Cairo, Egypt. They will contain no estradiol valerate and will be formulated to resemble the active Progynova® 2-mg tablets in external appearance. Quality-control procedures will include visual inspection for consistency of tablet appearance, assessment of tablet-weight uniformity, confirmation of the absence of active estradiol valerate, inspection of packaging and labeling to ensure indistinguishability between active and placebo medication packs, and appropriate stability and storage checks.

Active and placebo tablets will be dispensed in identical, sequentially numbered medication packs according to the concealed randomization code. Personnel involved in the preparation and coding of the study medication will have no role in participant recruitment, clinical assessment, embryo transfer, outcome evaluation, or statistical analysis. Participants, treating physicians, sonographers, embryologists, outcome assessors, and statisticians will remain blinded to treatment allocation unless emergency unblinding is clinically required.

7.7 Endometrial preparation and cycle monitoring Oral estradiol valerate (Progynova® 2-mg coated tablets) will be started on cycle day 2. The randomized daily dose will be continued for 10 days, after which endometrial reassessment will usually be performed on cycle days 10-12. Endometrial thickness will be measured by transvaginal ultrasonography in the mid-sagittal plane as the maximal double-layer thickness.

On the reassessment day and before the first progesterone dose, serum progesterone and serum estradiol will be measured in the morning. Serum LH may also be measured according to local monitoring practice. Progesterone will be initiated only if endometrial thickness is at least 7 mm and serum progesterone is <1.0 ng/mL. A pre-progesterone serum progesterone concentration ≥1.0 ng/mL will be considered biochemical evidence of premature ovulation or spontaneous luteinization. If LH is measured, an LH rise >20 IU/L will be considered supportive but will not be used as the sole cancellation criterion.

If endometrial thickness remains <7 mm at reassessment, the allocated estradiol regimen will be continued for an additional 3-5 days followed by repeat ultrasonography. A cycle will be cancelled if adequate endometrial development is not achieved after the permitted extension, if premature ovulation/spontaneous luteinization occurs, or if no embryo survives warming for transfer. The reason for cancellation will be recorded prospectively.

7.8 Luteal-phase support After the cycle-continuation criteria are met, luteal-phase support will be initiated using vaginal progesterone pessaries (Cyclogest®) 400 mg twice daily together with intramuscular progesterone (Prontogest® 100 mg/2 mL ampoules) 100 mg every other day. If 400-mg vaginal pessaries are unavailable, the same vaginal dose will be administered as two 200-mg pessaries twice daily. In participants who achieve pregnancy, oral estradiol valerate and progesterone support will be continued until completion of 12 weeks of gestation unless clinically contraindicated.

7.9 Embryo warming, grading, and transfer Only vitrified-warmed day-5 blastocyst transfer cycles will be included. Embryos will be warmed on the day of transfer using the IVF laboratory standard operating procedure, and post-warming survival will be confirmed before transfer. Blastocyst morphology will be graded by experienced embryologists using the Gardner and Schoolcraft system. Day-5 blastocyst transfer will be performed after five completed days of progesterone exposure under transabdominal ultrasound guidance using a soft embryo-transfer catheter. The number of blastocysts transferred will be determined according to patient age, embryo quality, and unit policy and will be recorded prospectively.

7.10 Follow-up and treatment adherence Medication adherence will be assessed by participant self-report and direct questioning at follow-up visits regarding tablet intake, missed doses, and tolerability. Serum beta-human chorionic gonadotropin (β-hCG) will be measured 14 days after embryo transfer. Participants with a positive β-hCG will undergo transvaginal ultrasonography at 6-8 weeks after embryo transfer and will subsequently be followed until pregnancy loss or delivery to ascertain ongoing pregnancy and live-birth outcomes.

7.11 Treatment-adverse effects and safety monitoring Treatment- adverse effects will be collected prospectively using a structured checklist at follow-up during endometrial preparation, at the progesterone-initiation visit, and at the pregnancy-test visit. Participants will also be invited to report additional symptoms spontaneously. The checklist will include headache, epigastric pain, vomiting, dermatitis, breast tenderness, bloating, mood changes, nausea, and leg cramps. Clinically important adverse events will be evaluated and managed according to standard clinical practice, and study medication may be discontinued if continuation is considered unsafe.

7.12 Data collection and quality control Data will be recorded on standardized case-report forms and transferred to a secure study database using unique study identifiers. Source documents and database entries will be checked for completeness, plausibility, and consistency. Access to identifiable information will be restricted to authorized investigators. Any protocol deviations, cycle cancellations, withdrawals, and reasons for discontinuation will be documented.

1. Study Outcomes 8.1 Primary outcome Clinical pregnancy rate will be the primary outcome and will be defined as the proportion of randomized participants in whom one or more gestational sacs are visualized by transvaginal ultrasonography at 6-8 weeks after embryo transfer.

8.2 Secondary outcomes

• Endometrial thickness on the day of progesterone initiation: maximal double-layer thickness measured by transvaginal ultrasonography immediately before progesterone initiation.
• Serum estradiol concentration on the day of progesterone initiation: morning serum estradiol level obtained before the first progesterone dose.
• Cycle cancellation: cancellation before embryo transfer because protocol continuation criteria are not met or no embryo survives warming.
• Biochemical pregnancy: positive serum β-hCG above the local laboratory threshold 14 days after embryo transfer.
• Implantation rate: number of gestational sacs divided by number of embryos transferred.
• Ongoing pregnancy: viable intrauterine pregnancy with fetal cardiac activity at or beyond 12 completed weeks of gestation.
• Miscarriage: spontaneous loss of an intrauterine clinical pregnancy before 24 completed weeks of gestation.
• Live birth: delivery of at least one live-born infant after 24 completed weeks of gestation.
• Multiple pregnancy: visualization of more than one gestational sac or more than one fetal heartbeat.
• Birth plurality: reported as no live birth, singleton live birth, or multiple live birth.
• Treatment-related symptoms: frequency of symptoms collected with the structured symptom checklist described above.

1. Sample Size The sample size will be calculated for a two-arm parallel superiority trial using clinical pregnancy rate as the primary outcome. The assumed clinical pregnancy rate in the 8 mg comparator group will be 34.1%, based on the fixed-dose group reported by Hizkiyahu et al. [9]. The trial will be powered to detect a clinically meaningful absolute increase of 10 percentage points, from 34.1% to 44.1%, with oral estradiol valerate 12 mg/day.

Using a two-sided alpha of 0.05 and 80% power for comparison of two independent proportions, 373 women will be required per group. After allowing approximately 10% for cycle cancellation or attrition, at least 414 women per group (828 total) will be required. To ensure the minimum analyzable sample and balanced recruitment across centers, the investigators will aim to randomize approximately 850 women (about 425 per group). The calculation will be performed using MedCalc Statistical Software (version 19.5.3 or later equivalent).

The study will be powered for superiority of 12 mg/day over 8 mg/day. It will not be designed as an equivalence or non-inferiority trial.

1. Statistical Analysis Plan The primary analysis will follow the intention-to-treat principle and will include all randomized participants in the groups to which they were allocated. Cancelled cycles will remain in the denominator for biochemical pregnancy, clinical pregnancy, ongoing pregnancy, and live birth and will be considered as not achieving these outcomes. A per-protocol analysis excluding major protocol deviations and cancelled cycles will be performed as a supportive sensitivity analysis.

Continuous variables will be summarized as mean ± standard deviation or median with interquartile range, according to their distribution. Categorical variables will be presented as number and percentage. Approximately normally distributed continuous variables will be compared using Welch's t-test, markedly skewed variables using the Mann-Whitney U test, and categorical variables using the chi-square or exact test, as appropriate. Baseline characteristics will be assessed primarily descriptively.

The primary outcome, clinical pregnancy rate, will be compared between treatment groups using the intention-to-treat population. Treatment effects will be reported as absolute risk difference, risk ratio, and odds ratio with 95% confidence intervals. A supportive multivariable logistic-regression analysis will adjust for prespecified pre-randomization covariates, including study center, maternal age, infertility type, and infertility cause. Post-randomization variables will not be included in the principal adjusted treatment-effect model.

Secondary outcomes, including cycle cancellation, biochemical pregnancy, ongoing pregnancy, miscarriage, live birth, multiple pregnancy, endometrial thickness, serum estradiol concentration, and treatment-related symptoms, will be compared between groups using appropriate statistical tests and reported with 95% confidence intervals. Denominators will be clearly specified for outcomes applicable only to participants reaching a particular reproductive stage.

Implantation analyses will account for clustering of embryos within participants when more than one embryo is transferred. Treatment-related symptom analyses will be considered supportive secondary analyses, and no formal adjustment for multiple comparisons will be planned.

The extent and pattern of missing data will be described. No imputation will be performed if primary-outcome data are complete or nearly complete; if clinically important primary-outcome data are missing, appropriate sensitivity analyses will be undertaken.

All tests will be two-sided, and P<0.05 will be considered statistically significant for the primary outcome. Analyses will be performed using validated statistical software, with the final software and version documented in the statistical report.

1. Ethical Considerations The study will be conducted in accordance with the principles of the Declaration of Helsinki and applicable institutional requirements. Recruitment at each center will begin only after approval by the relevant institutional ethics committee.

Written informed consent will be obtained from every participant before randomization. The consent process will explain the study purpose, procedures, treatment alternatives, foreseeable risks and potential benefits, confidentiality protections, voluntary nature of participation, and the right to withdraw at any time without affecting clinical care. Participants will not be required to provide a reason for withdrawal.

Confidentiality will be maintained by using coded study identifiers and limiting access to identifiable data. Study information will be stored securely and used only for approved research purposes. Any publication will present aggregate or anonymized data so that individual participants cannot be identified.

Both estradiol doses will use a medication routinely used for programmed FET endometrial preparation. Participants will be monitored for clinically relevant estrogen-related risks and treatment-related symptoms. Any serious or unexpected safety concern will be managed according to standard clinical practice and reported to the ethics committee when required.

1. Funding and Conflicts of Interest The study will be investigator-initiated and will not receive a specific grant from public, commercial, or non-profit funding agencies. Study-related routine clinical care will be delivered through the participating institutions. Investigators will disclose any potential conflicts of interest; none are anticipated at protocol preparation.
2. Time Plan The study is expected to be completed within approximately 36 months following ethical approval and completion of site preparation. Participant recruitment and randomization will commence after obtaining all required ethical and institutional approvals and will continue until the target sample size is achieved.

Recruitment, endometrial preparation, and embryo transfer will be conducted during approximately the first 24-27 months of the study period. Pregnancy follow-up will occur concurrently and will continue through delivery for participants who achieve pregnancy. The remaining study period will be allocated to completion of live-birth follow-up, data verification, resolution of missing or inconsistent data, database lock, and final statistical analysis.

The proposed timeline may be adjusted according to the actual recruitment rate and duration of pregnancy follow-up, while maintaining the planned study procedures and outcomes.

Study milestone Final verified date Event Cairo University ethics approval 16 April 2022 Ethics approval obtained before recruitment Kafr El-Sheikh University ethics approval 23 April 2022 Ethics approval obtained before recruitment First participant enrolled/randomized 25 April 2022 Study recruitment commenced on 25 April 2022 Last participant randomized 16 February 2025 Recruitment and randomization were completed on 16 February 2025 Last embryo transfer 2 March 2025 Embryo transfers were completed on 2 March 2025 Primary completion date 29 April 2025 Final primary-outcome assessment was completed on 29 April 2025 Final live-birth follow-up 21 January 2026 Live-birth follow-up was completed on 21 January 2026 Study completion date 21 January 2026 Final participant follow-up for secondary outcomes was completed on 21 January 2026 Database verification/final statistical analysis 26 February 2026 Final statistical analysis was completed in February 2026 ClinicalTrials.gov submission 2 April 2026 Trial was retrospectively registered on 2 April 2026