Comparing FET Guided by ERA vs Standard Timing in Patients With Recurrent Implantation Failure

Trial design This is a multicenter randomized double‐blind controlled trial evaluatingthe clinical effectiveness of ERA‐guided frozen embryo transfer in improving thepregnancy rate of infertile women with RIF. All participants will first undergo a biopsycycle to obtain an endometrial biopsy for ERA and will then be randomly assigned into theintervention or control group. At the transfer cycle, participants in the interventiongroup will have frozen embryo transfer timed according to the ERA result while those inthe control group will have frozen embryo transfer according to standard timing.Screening, recruitment and consent Women with RIF will be recruited from 10 hospitalsacross China. Recurrent implantation failure is defined as failure to achieve pregnancyafter three embryo transfer of both fresh and frozen embryos.3 The inclusion andexclusion criteria is summarized in Table 1.Table 1. Inclusion and exclusion criteria Inclusion criteria 1. Women with RIF 2. Women aged < 40 years 3. Body mass index of women between 18.5 (inclusive) and 30 (exclusive) kg/m² 4. Women with at least one high quality frozen blastocyst (BB grade or above) andplanning to undergo a single blastocyst transfer 5. Women who will give written informed consentExclusion Criteria 1. Women with recurrent pregnancy loss (3 or more biochemical or spontaneousmiscarriages) 2. Either partner with known chromosomal abnormalities including balancedtranslocations 3. Women undergoing preimplantation genetic testing 4. Women with endometrial thickness < 7 mm in the IVF cycle 5. Women with a confirmed diagnosis of stage III‐IV endometriosis or adenomyosisaffecting uterine cavity morphology 6. Women with a confirmed diagnosis of antiphospholipid syndrome 7. Pathologies affecting the uterine cavity, including polyps, submucosal fibroids,intramural fibroids > 4 cm, or hydrosalpinxEligible women will be counselled for the nature, purpose, procedures, expected duration,potential risks and benefits, and any possible discomforts of the trial duringrecruitment. Participation is voluntary. All participants will be required to sign awritten informed consent and no study‐related procedure will be performed without anyconsent.Demographic data of all participants will be recorded, which includes their age, height,weight, body mass index, smoking and education level. Their medical history and physicalexamination will also be recorded, including the indication for IVF, details of previousembryo transfer, obstetrics history, previous medical, surgery or infection history. Theresults of laboratory tests will also be recorded if available, which include basicendocrine tests such as serum anti‐mullerian hormones level and thyroid function tests.Biopsy cycle All participants will go through a biopsy cycle in a hormonal treatmentcycle to obtain an endometrial sample for ERA testing. Oral estradiol (Estrofem, NovoNordisk A/S, Bagsværd Denmark) 2mg three times a day will be started on the first orsecond day of their menstrual cycle for 10‐15 days, with a maximum of 22 days. Thentransvaginal ultrasound will be performed to measure the endometrial thickness and bloodwill be taken for serum estradiol and progesterone levels. When the endometrial thicknessis ≥ 7mm, serum estradiol level is > 150 pg/ml (or 500 pmol/ml) and serum progesteronelevel is < 1 ng/ml (or 3.18 nmol/L), oral dydrogesterone (Duphaston, Abbott, Illinois,U.S.) 10mg three times a day and vaginal progesterone gel (Crinone 8%, Merck KGaA,Darmstadt, Germany) 90mg daily will be started within 24 hours in addition to oralestradiol.An endometrial biopsy will be performed in the out‐patient clinic after usingprogesterone for 5 consecutive days (120 + 3 hours) as the starting of progesterone P +0. The endometrial sample will be sent to the central laboratory (Hangzhou Yizhen MedicalLaboratory, China) for ERA testing.Sample collection and delivery An endometrial biopsy will be performed with a pipelleprovided in the ERA kit under aseptic technique. The plunge will be withdrawn to create anegative pressure and a piece of endometrial tissue will be collected from the fundus tothe internal cervical os. A tissue sample of around 50 to 70 mg will be collected.The endometrial sample will be immediately transferred to a cryotube containing 1.5ml ofRNA‐later stabilizer liquid, which prevents the RNA from degradation during transport tothe laboratory. Excessive blood or mucus within the sample will be removed to avoid RNAdegradation. The collection tubes containing endometrial samples will be shaken to ensurethorough mixing of RNA solution with the endometrial samples. After labelling, theendometrial samples in the collection tube will be stored at 4℃ in the refrigerator forat least 4 hours and then transported at room temperature to the central laboratory forERA.Endometrial receptivity analysis RNA‐Seq high‐throughput sequencing technology will beused to detect the expression of nearly 250 genes related to endometrial receptivity. Theendometrial implantation window of the analyzed samples will be obtained bybioinformatics analysis.Transfer cycle Participants will be requested to have the frozen embryo transfer within1‐3 months of the biopsy cycle. The frozen embryo transfer is performed in a hormonaltreatment cycle using the same endometrial preparation regimen as the biopsy cycle. Thetiming of embryo transfer will be based on their group allocation. For participants inthe intervention group, the timing of frozen embryo transfer will be arranged accordingto the ERA result. For those in the control group, frozen embryo transfer will bearranged according to standard timing, i.e. after 5 days of consecutive progesterone use.The best quality blastocyst will be thawed and transferred first.Details of the transfer cycle will be recorded, including endometrial thicknessmeasurement, serum progesterone level, start date and time of progesterone, embryotransfer date and time, fertilization method and the Gardner grading of the embryo.Follow‐up After 14 days of frozen embryo transfer, participants will have blood tests forserum beta human chorionic gonadotropin (β‐hCG), estradiol and progesterone level. Thestudy ends if the participants are not pregnant.If the participant is pregnant, follow‐up will continue until one week postpartum.Ultrasound findings on the number of fetuses and fetal heart status will be recorded at 5weeks and 10‐12 weeks post‐embryo transfer. Antenatal complications, delivery details andneonatal information will also be recorded. The study ends after one week postpartum orif pregnancy loss occurs during the follow‐up period.Randomization and blinding Randomization Randomization will be performed in the HangzhouYizhen Medical Laboratory, China after receipt of the endometrial samples and beforeproceeding to ERA. Women will be randomly assigned to either the interventional group orthe control group in a 1:1 ratio. Randomization will be stratified according to thecenters and the age of women (<35 vs ≥35 years) using a random number table.16 Each womanwill be included and randomized only once in the study.Blinding This is a double‐blind study and neither the participants nor researchers willbe aware of the group allocation. Researches in each participating centre will benotified by the laboratory of the recommended time of embryo transfer for eachparticipant. The report will state the following: "it is recommended that this womanundergoes frozen blastocyst transfer at P+X hours."Unblinding Unblinding will occur when the participant has blood tests for β‐hCG,estradiol and progesterone levels 14 days after embryo transfer. Both participant andresearchers will be informed of the group assignment. Participants in the control groupwho do not conceive can opt to replace the remaining frozen blastocysts in subsequentcycles based on the ERA results.Study outcome Primary outcome The primary outcome is the ongoing pregnancy rate definedas pregnancy with fetal pulsation at 10‐12 weeks.Secondary outcome Secondary outcomes are the endometrial receptivity status, thebiochemical pregnancy rate, clinical pregnancy rate, biochemical miscarriage rate, earlymiscarriage rate, ectopic pregnancy rate and live birth rate. Their definitions arelisted in Table 2. Obstetrics outcomes include the incidence of gestational diabetesmellitus, preeclampsia, placenta previa, placental abruption and the mode of delivery.Neonatal outcomes include the gestational age, birth weight, Apgar scores, preterm rate,malformation rate and neonatal mortality rate.Sample size The primary outcome measure is the ongoing pregnancy rate, which is expectedto be 35% in the intervention group and 25% in the control group. With a two‐sided testlevel α = 0.05 and power (1‐β) = 80%, using PASS15 software and based on Fisher's exacttest, the calculated sample size is 660 women (330 in the intervention group and 330 inthe control group). Considering a 10% dropout rate, a total sample size of 734participants is required (367 in the intervention group and 367 in the control group).Data analysis Full analysis set, per protocol set and safety analysis sets will beanalyzed. Full analysis set refers to those who have received at least one test and atleast one evaluation on effectiveness. Per protocol set refers to all cases in the fullanalysis set who have completed the protocol‐specified cycles and had no major protocolviolations. Safety set refers to those who received at least one test after enrollmentand had at least one safety evaluation.Count data will be presented as frequencies and percentages, while measurement data willbe presented with mean, standard deviation, median, maximum and minimum. The primary andsecondary outcomes among different groups will be directly compared.The ongoing pregnancy rates among three groups will also be compared: transfer withadjusted timing based on ERA‐nonreceptive results, transfer with standard timing based onERA‐receptive results and transfer with standard timing in women with ERA‐nonreceptiveresults.