To compare the efficacy of SANDWICH (delayed start GnRH antagonist) protocol with conventional antagonist stimulation protocol in terms of IVF outcomes in POSEIDON poor responders: A randomized controlled trial

Research HypothesisThis study is undertaken to validate our research hypothesis that implementation of Sandwich (delayed start antagonist)protocol will improve outcomes in IVF/ICSI cycles in POSEIDON poor responders as it has advantages of both GnRH antagonist protocol & long GnRH agonist protocol. Sandwich protocol results in better synchronised follicular response leading to lesser cycle cancellation rate and more number of mature oocytes. It gives the opportunity to use dual trigger and requires less total gonadotrophin requirement compared to long GnRH agonist protocol.

Aim: To compare the efficacy of sandwich (delayed start GnRH antagonist) protocol with conventional antagonist stimulation protocol in terms of IVF outcomes in POSEIDON poor responders.

**Primary objective:**To compare the efficacy of sandwich (delayed start GnRH antagonist ) protocol with conventional antagonist stimulation protocols  in POSEIDON poor responders in terms of  oocyte yield.

Secondary objectives: To compare the efficacy of sandwich (delayed start GnRH antagonist) protocol with conventional antagonist protocol in POSEIDON poor responders in terms of

•         Total dose of gonadotrophins required

•         Duration of controlled ovarian stimulation

•         Peak serum estradiol (E2) & progesterone (P4) on the day of trigger

•         FORT/Follicular output rate

•         Endometrial thickness on the day of OPU

•         Number of mature oocytes (MII) retrieved

•         Fertilization rate

•         Cleavage rate

•         Total number of embryos

•         Embryo grading

•         Cycle cancellation rate

•         Clinical pregnancy rate per embryo transfer

•         Live birth rate (LBR)

•         Miscarriage rate

Materials and methods - Study design: Open label, randomized controlled trial, parallel design allocation where Sandwich(delayed start antagonist)  protocol (Study group A) will be compared individually with  conventional GnRH antagonist protocol (Study group B).This study will be registered in the Clinical Trial Registry of India (CTRI).

Study population: Infertile women undergoing IVF/ICSI cycles

Place of study: ART (Assisted Reproductive Technology) centre, Department of Obstetrics and Gynecology, AIIMS, New Delhi.

Duration of study: From date of ethical clearance to July 2026

Study groups:

1)Study group A: Infertile women undergoing Sandwich(delayed start antagonist    protocol)

2)Study group B: Infertile women undergoing conventional  GnRH antagonist protocol

**Sample size:**patients (45 patients in each study group)

Sample size calculation: primary objective of this study is to compare the oocyte yield between the Sandwich (delayed start antagonist protocol) and the conventional GnRH antagonist protocol in POSEIDON poor reponders. Previous study by Al-Jeborry et al(2020)(28) reported the average number of oocyte yield from GnRH antagonist protocol in POSEIDON poor responders  as 2.8±2.32 . With a significance level of 0.05, power of 80% and an estimated 10% loss to follow up, a minimum of 45 subjects per group is required if we assume that there will be improvement of 1.5 units.A total 90 patients will be enrolled for present study.

Statistical analysis : The data will be presented using descriptive statistics. Qualitative data will be displayed as frequency and percentages, while quantitative data will be shown as either mean ± SD for normally distributed data, or median (Min-Max) for non-normally distributed data. Categorical variables will be analyzed using either the Chi square or Fisher exact test. Differences between groups will be observed using the independent T test or One-way ANOVA, assuming normality of data, or the Mann Whitney U test or Kruskal Wallis test if normality of data cannot be assumed. Other appropriate analyses will be performed as needed to meet the objectives at the time of analysis. Statistical significance will be defined as a p value of <0.05.

Inclusion criteria:

• Unexpected poor responders who belong to

POSEIDON 1 (Age < 35 years, AMH ≥ 1.2ng/ml and / or AFC ≥ 5, Previous ovarian response yielding <4 oocytes in subgroup 1a and 4-9 oocytes in subgroup 1b)

POSEIDON 2 (Age ≥ 35 years, AMH ≥ 1.2ng/ml and / or AFC ≥ 5, Previous ovarian response yielding <4 oocytes in subgroup  2a and 4-9 oocytes in subgroup 2 b)

• Expected poor responders who belong to

POSEIDON 3 ( Age < 35 years ,  AMH <1.2ng / ml and / or AFC < 5 ) and

POSEIDON 4 ( Age  ≥   35 years ,  AMH <1.2ng / ml and / or AFC < 5 )

Exclusion criteria:

• Anatomical and pathological abnormalities in the uterus

• Endocrine disorders such as thyroid dysfunction, hyperprolactinemia and diabetes mellitus

• h/o tumour in hypothalamus and pituitary, Hypogonadotropic hypogonadism

• Azoospermia in male partner

• Endometriosis, h/o previous adnexal surgeries

• Recurrent miscarriage and recurrent implantation failure

• BMI ≥

Recruitment: Infertile women attending the out-patient department and IVF Clinic will be screened according to the inclusion and exclusion criteria. Eligible candidates will be informed about the study and informed written consent will be taken from the couple after explaining the detailed plan, purpose and duration of the study in their own understandable language. The couples refusing to participate in the trial shall be treated at par with all other ongoing IVF/ICSI cycles at the center. Their decision to refuse participation in this study shall not affect their IVF treatment in any way.

Randomization: Eligible candidates will be randomized into Sandwich(delayed start antagonist) protocol or  GnRH antagonist protocol  group. Mixed block randomization method will be used. Computer generated random number list will be generated by using nQuery software and the participants will be allocated into one of the three groups. Allocation of the patients into one of the two groups will be done by sequentially numbered opaque sealed envelope. The envelopes will be prepared by a person not associated with the conduct of the study. The envelopes will be sequentially opened. This randomization and allocation to the two groups will be done by a part of the team of investigators who are not involved in the treatment procedure of the participants. This randomization and allocation process will ensure blinding and minimize bias in the study.

Intervention: Before a patient is recruited for IVF, a detailed clinical history including menstrual history, previous obstetric history including history of pregnancy losses, history of prior treatment taken for infertility, details of previous IVF cycles (if available) will be recorded on a predesigned proforma. Detailed examination including general physical examination and gynecological examination will be done.

She will undergo baseline blood investigations including serum luteinizing hormone (LH), follicle stimulating hormone (FSH) levels, anti-mullerian hormone (AMH), thyroid stimulating hormone (TSH) and prolactin levels. Baseline transvaginal ultrasound (TVS) will be done on day 2-5 of the menstrual cycle to see for antral follicle count (AFC) and 3D

ultrasound on day 14-16 of the menstrual cycle for uterine cavity evaluation, endometrial thickness and to rule out any other pelvic pathology. If any uterine pathology is suspected, the participants will undergo hysteroscopy. Semen analysis and semen culture of the partner will also be done prior to recruitment.

Patient will undergo stimulation protocol as per randomization.

In the Study Group A (Sandwich protocol), patients will receive Tab.Estradiol valerate (Tab Estrabet) 2mg bd  from midluteal phase (day 21) of previous cycle till the onset of menses/ the day of starting GnRHantagonist. GnRH antagonist (Injection Cetrotide 0.25mg/d) will be started on day2 and continued for 7 days. Gonadotropins {Injection recombinant FSH (Gonal F) subcutaneously and Injection Human Menopausal Gonadotrophin(HMG )intramuscularly} will be initiated from the next day in the ratio of 2:1 after confirmation of downregulation (Serum estradiol < 50 pg/ml, no dominant follicle & endometrial thickness <4mm on TVS). The dose of gonadotrophin will be decided depending on patient’s age, BMI (body mass index), AMH (Anti mullerian hormone), AFC(Antral follicle count) and/or ovarian response in previous cycles (if available). The dose will be adjusted based on ovarian response. GnRH antagonist (Injection Cetrorelix) 0.25 mg/day subcutaneously will be started when the lead follicle is  â‰¥13mm and will be continued up to & including the day of trigger. Recombinant LH 75IU will be added from the day of starting GnRH antagonist till the time of trigger.

In the Study Group B (GnRH Antagonist protocol), patients will receive patients will receive Tab.Estradiol valerate (Tab Estrabet) 2mg bd from midluteal phase (day 21) of previous cycle till the onset of menses/the day of starting GnRH antagonist. Gonadotropins {Injection recombinant FSH (Gonal F) subcutaneously and Injection Human Menopausal Gonadotrophin (HMG )intramuscularly} will be initiated from the next day in the ratio of 2:1 after confirmation of downregulation (Serum estradiol < 50 pg/ml, no dominant follicle & endometrial thickness <4mm on TVS). The dose of gonadotrophin will be decided depending on patient’s age, BMI (body mass index), AMH (Anti mullerian hormone), AFC(Antral follicle count) and/or ovarian response in previous cycles (if available). The dose will be adjusted based on ovarian response. GnRH antagonist (Injection Cetrorelix) 0.25 mg/day subcutaneously will be started when the lead follicle is  ≥13mm and will be continued up to & including the day of trigger. Recombinant LH 75IU will be added from the day of starting GnRH antagonist till the time of trigger.

For both the protocols, the criteria for final follicular maturation trigger will be at least 2 follicles with diameter ≥17mm.Dual trigger((Injection recombinant hCG 250mcg subcutaneously and Leuprolide 1mg subcutaneously) will be used. Oocytes will be retrieved with flushing 34-36 hours post ovulation trigger Routine IVF or ICSI will be performed, as appropriate. Freshly ejaculated sperm samples will be prepared by double density gradient centrifugation and each oocyte will be inseminated with 55,000 sperm or ICSI performed depending on the patient profile.Then both the gametes will be incubated at 37 °C, 5% O2 and 6% CO2 in the conventional incubator. Day 3 fresh   double embryo transfer will be performed under ultrasound guidance if <3 embryos are obtained. If ≥3 good grade embryos are obtained, we will consider day 5 fresh blast transfer.  Luteal phase support will be maintained until 12 weeks of gestation or until the day of pregnancy test if negative.

Follow up:

Beta hCG and urine pregnancy test will be performed on day 16 in our laboratory and progesterone will be continued up to 12 weeks of gestation in a normal intrauterine pregnancy and discontinued in case of a biochemical pregnancy or no pregnancy. Clinical pregnancy rates will be calculated as per transvaginal ultrasound performed at 6-7 weeks showing one or more gestational sacs with fetal cardiac activity. Clinical pregnancy rate includes ectopic pregnancy as well.