Controlled ovarian hyperstimulation (COH) is an important step during in vitro fertilization (IVF). Its aim optimally is to recruit 10-15 oocytes. When deciding on the actual treatment, different stimulation protocols, various stimulating agents and wide range of gonadotropin dose can one choose from. Prior to the decision on the actual stimulation protocol and gonadotropin (Gn) dose the patient's expected response to stimulation is assessed primarily using ovarian reserve markers. Most medications used during stimulation exert their effect in a dose-dependent manner hence with a higher Gn dose one would expect a better response, more oocytes. More oocytes could translate into more embryos and potentially a higher pregnancy rate. The currently available evidence however does not support this practice as randomized controlled trials (RCT) have failed to show that the use of higher Gn dose results in higher pregnancy, live-birth rates. These studies however identified patients based on different criteria, compared different stimulation protocols and various Gn doses. There are only two RCTs that compared cumulative live birth rates (fresh + frozen embryo transfers) and they identified poor responders based on different criteria and used different drug regimens. Therefore, the aim of our study is to compare cumulative IVF clinical pregnancy rates using a lower and a higher gonadotropin dose among poor responders identified based on universally accepted criteria.

Background: IVF is started after proper evaluation based on an appropriate indication for the treatment. The first phase of the treatment is the stimulation with the aim to recruit sufficient number of oocytes. In general, 10-15 oocytes are aimed for. This many oocytes should be sufficient for the embryology laboratory procedures and should allow the provider to choose the embryo or embryos for transfer from a larger cohort. More oocytes are associated with more side effects and fewer oocytes limit the efficacy of the laboratory procedures. Stimulation is individualized and usually age, ovarian reserve markers, weight and response to previous stimulation are taken into consideration when decisions are made. Based on ovarian reserve markers patients are grouped as expected normal responders, hyper-responders or poor responders. A poor responder is a patient with low ovarian reserve or low number of oocytes in the ovaries. Their care can be challenging. Different pre-stimulation interventions and various stimulation approaches have been explored in this subset of patients. Typically, providers will prescribe higher gonadotropin doses and expect the retrieval of more oocytes resulting in more transferable embryos. Studies so far however have not supported this practice. In expected poor responders most studies have not shown the collection of more oocytes with higher dose gonadotropins. Those that did find more oocytes described an additional 1-2 oocytes only. None of the studies so far have reported improved pregnancy outcome, including cumulative outcome with more gonadotropins though. The literature is confounded by the various definitions of the poor responder patient, the use of various stimulation protocols in the active treatment versus control groups, the use of oral agents as active treatment and the different clinical endpoints tested (oocyte number, clinical pregnancy, live birth, cumulative live birth). Therefore, we plan to study patients identified as poor responders based on established criteria (POSEIDON criteria). We will use the same stimulation protocols, drug types in the experimental and control groups. We will study clinically meaningful outcomes, live birth rate and cumulative live birth rate.

Design: Investigator initiated prospective, randomized, multi-center, non-blinded trial to compared higher and lower gonadotropin in poor responder patients undegoing IVF-ICSI treatment identified based on Posedion III-IV criteria

Participants: The investigators will enroll patients who, based on their initial evaluation, reproductive history or prior fertility treatment, have a proper indication to undergo in vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI) treatment. A basic infertility evaluation includes: 1.) hormonal evaluation of ovarian function, including ovarian reserve testing (anti-Müllerian hormone [AMH] level, antral follicle count [AFC]), 2) transvaginal ultrasound evaluation of female internal genital organs, 3) evaluation of the uterine cavity (hysterosalpingogram, saline sonohysterogram or hysteroscopy), 4) cervical cancer screening, 5) cervical culture for Chlamydia, 6) serologic testing for HIV, hepatitis B, C, syphilis (both partners), 7) semen analysis, 8) male andrological exam, 9) if needed male hormonal +/- genetic testing. IVF-ICSI treatment is started for advanced maternal age, diminished ovarian reserve (DOR), unexplained infertility after failed intrauterine inseminations, male factor infertility, endometriosis.

The investigators plan to recruit:

- infertile patients between the ages of 18-42 who require IVF-ICSI treatment and are identified as poor responders based on: a) AMH: 0.3-1.2 ng/ml or AFC <5, b) AMH>1.2 ng/ml or AFC≥5 but the retrieval of ≤ 4 oocytes during previous IVF treatment. (POSEIDON category III-IV, Alviggi et al. Fertility Sterility 2016)

Methods: prospective, multicenter (5 centers), randomized controlled trial comparing a higher or lower gonadotropin (Gn) dose treatment. In both the low and high groups two different drug regimens (follitropin alpha + hp human menopausal gonadotropin or follitropin delta + hp human menopausal gonadotropin) will be used. 150 IU follitropin alpha was shown to be equally effective to 10 mcg follitropin delta. Therefore, the investigators expect similar response (oocyte yield) with the two regimens within the high and low Gn dose groups but the study design still will allow the investigators to compare the two follitropin medications too, which is a secondary aim.

Lower-dose group:

• 150 IU follitropin alpha + 75 IU highly purified human menopausal gonadotropin (hpHMG)
• 10 mcg follitropin delta + 75 IU hpHMG

Higher-dose group:

• 225 IU follitropin alpha + 150 IU hpHMG
• 15 mcg follitropin delta + 150 IU hpHMG

Treatment protocol:

• The stimulation will get started on day 2 or 3 of the spontaneous menstrual cycle, or on the 5th day after oral contraceptive pill use or after luteal estradiol pretreatment.
• During the stimulation ultrasound +/- serum hormone measurements will be used to monitor response. The first ultrasound is scheduled for day 5 or 6 of stimulation. At the time of the ultrasound blood test for serum estradiol is planned too. At the time of the last scan serum estradiol and progesterone levels will be measured.
• According to the study protocol the patient will continue with the assigned medication dose throughout her treatment. Dose increase is not allowed. If there is evidence for hyper-response [estradiol level > 4000 pmol/l on day 6 or more than 15 follicles over 10 mm any time during the stimulation] the dose can be reduced.
• As soon as two or more follicles reach >17 mm in diameter human chorionic gonadotropin (hCG) or gonadotropin-releasing hormone agonits [GnRH a] trigger injection will be used and 35-36 hours later transvaginal ultrasound guided follicle aspiration will be performed.
• The luteal phase will be supported by vaginal progesterone starting on the day after the retrieval.
• IVF or ICSI fertilization will be performed based on the semen parameters or previous fertilization records.
• The day after the retrieval the success of fertilization will be checked and the embryo(s) will be transferred 2-5 days after the retrieval. Embryo(s) will be transferred transcervically using soft transfer catheters under ultrasound guidance using the afterload technique.
• Surplus good quality embryos will be cryopreserved using vitrification. Elective cryopreservation (no transfer in the fresh cycle) will be performed if: 1) risk of ovarian hyperstimulation syndrome, 2) serum progesterone level over 1.5 ng/ml [5.5 nmol/l] prior to the oocyte collection, 3) any complications between the retrieval and planned fresh transfer (bleeding, infection, illness).
• 12-14 days after the transfer serum human chorionic gonadotropin (HCG) measurement will determine whether implantation has occurred. If the test is positive in 2-3 weeks a vaginal ultrasound will be done to determine the size, location and number of gestational sacs. Viable pregnancies will be referred to a formal prenatal care around week 8-9 of pregnancy. Delivery and perinatal outcome data will be collected by phone call after the delivery.
• If the fresh IVF cycle is not successful but embryos have been cryopreserved then the patient will undergo a frozen embryo transfer treatment cycle either in her own cycle, or in a minimally stimulated or completely artificial cycle.

The care of the patients will not differ from the care of non-study patients in terms of potential medication dose, the number of clinic visits, retrieval and embryology procedures as well as prenatal care.

Randomization: Randomization will be performed according to a computer generated list. (www.randomizer.org) The planned sample size is 700 patients (350 in both the low- and high-dose groups).

Sample size calculation: In order to determine the sample size, the investigators calculated with a 20% pregnancy rate in a patient population that fits the inclusion-exclusion criteria. The investigators expect higher oocyte yield in the higher dose group that should result in more available embryos and therefore more frozen embryo transfers. The investigators calculate that this could increase the cumulative pregnancy rate by 50%. The investigators also believe that about 20% of the patients will drop out for various reasons. Therefore, 350 participants are needed in both arms of the study.

Statistical analysis: After the enrollment of the first 350 patients, a planned interim analysis will be performed to decide whether the planned sample size is sufficient to achieve our goal. Logistic regression analysis using a generalized, mixed linear model will be used to assess the impact of various Gn doses on pregnancy rates. Chi-square test will be used to test significance and OR will be calculated.

Data will be collected for the following parameters:

• Age
• Cycle day 3 follicle stimulating hormone (FSH), estradiol level
• anti-Müllerian hormone (AMH)
• antral follicle count (AFC)
• Indication for IVF: male, tubal, unexplained, endometriosis, diminished ovarian reserve
• Smoking (yes-no)
• Body mass index
• Cycle number (fresh + frozen together)
• Estradiol level on day 6
• Estradiol + progesterone level at the last scan
• Number of follicles >10 mm at the end of stimulation
• Stimulation duration
• Endometrial thickness
• Gn dose (daily, total)
• Trigger mechanism (HCG vs. GnRh agonist)
• Sperm parameters
• Oocyte number
• Mature (MII) oocyte number
• Proportion of mature oocytes (MII/ oocyte number)
• IVF vs. ICSI fertilization
• Number of fertilized (2 pronuclei (PN)) oocytes
• Fertilization rate per oocyte
• Fertilization rate per mature oocyte
• The number of good quality embryos (morphology better than score "6B2" on day 3, or score >2BB on day 5) and proportion of good morphology embryos among all embryos
• Number of transferred embryos
• Number of cryopreserved embryos
• Pregnancy rate (positive hCG)
• Clinical pregnancy rate (sac with viable embryo in it)
• Cumulative pregnancy rate (fresh + frozen embryo transfers)
• Pregnancy/ neonatal outcome

Inclusion Criteria:

• Expected poor responder based on:

  1. AMH: 0.3-1.2 ng/ml or AFC <5,
  2. AMH>1.2 ng/ml or AFC≥5 but the retrieval of ≤ 4 oocytes during previous IVF treatment.

• motile sperm with normal morphology obtained from the ejaculate of testicular biopsy

• no more than 3 previous failed IVF cycles (if the patient had 2 or more previous cycles cancelled for poor response she cannot be included)

• BMI: 18-35 kg/m2

• regular 24-35 day cycles

• intact uterine cavity

• indication for in vitro fertilisation treatment (tubal factor, male factor, low ovarian reserve, endometriosis, unexplained infertility)

• age 18-421 yrs

Exclusion Criteria:

• presence of hydrosalpinx
• positive HIV, hepatitis B, C screening tests
• planned preimplantation genetic testing of the embryos
• planned elective cryopreservation
• lack of consent
• not meeting the inclusion criteria

4 weeks if the pregnancy test is negative: from start of the study drug administration up to the pregnancy test. 8 weeks if the pregnancy test is positive: from start of the study drug administration up to the first ultrasound confirming the pregnancy.

SAE were colected for based on standard definitions.