Stimulation is a key step of in vitro fertilization (IVF). Typically, injectable gonadotropins are used for stimulation, and their dose is individually determined to avoid hypo- as well as hyper-response. Despite the individualization some patients respond with a lower-than-expected number of oocytes. If the low response is unexpected based on the baseline parameters or if an unusually high dose of gonadotropins is needed to achieve a proper response we talk about "hypo-response". In such cases if the first treatment fails and a repeat attempt is planned typically even more gonadotropins, the combination of luteinizing hormone (LH) with follicle stimulating hormone (FSH) or the use of the more potent recombinant preparations are considered. The benefits of these approaches however have not been studied properly in hypo-responders. The studies have used various criteria to identify hypo-responders, have used various gonadotropin doses and have evaluated different outcome parameters. Live birth was only studied in one trial.

It is also known that in a different cycle the same patient is likely to have a slightly different response to the same type and dose of drugs. Therefore, the question arises whether a hypo-responder in one treatment is expected to have hypo-response again if the treatment is similarly carried out in a different cycle. Do we need to change/ increase the gonadotropin dose if based on age and ovarian reserve otherwise we would expect a normal response? Furthermore, if we consider a change should we increase the dose of FSH or should we combine it with LH?

Therefore the aim of this randomized controlled trial is compare an unchanged medication regimen to increased dose of FSH vs the combination of FSH and LH in hypo-responder patients identified based on POSEIDON (Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number) criteria (Gr 1 and 2: retrieval of 9 or fewer oocytes in patients with an anti-Müllerian hormone (AMH) level ≥ 1.2 ng/ml or antral follicle count (AFC) ≥ 5 and age <35 years [Group (Gr) 1] or ≥35 years [Gr2]). Hypo-responder patients will be randomized to:

1. Same gonadotropin dose as in previous treatment (recombinant(r) FSH) ['control group']
2. The same dose as in the previous cycle but in the form of FSH + LH combination (rFSH:rLH 2:1 ratio) ['additional LH group']
3. A dose increase of 75 international unit (IU) compared to the dose in the previous treatment. ['higher dose FSH group'] The primary outcome parameter to study is live clinical pregnancy. In addition, baseline demographic, stimulation and further clinical outcomes (pregnancy rate, miscarriage rate, live birth rate) will be compared.

The key step of an IVF treatment is controlled ovarian stimulation. The aim of stimulation is to recruit an optimal number of oocytes. The outcome of the fresh cycle improves if the collected oocyte number increases up to 10-15 oocytes. Cumulative success however keeps on rising even over 20 oocytes.

In most of the IVF cycles injectable gonadotropins are used to induce multi-follicular development. The type and dose of the gonadotropins are individually determined based on age, ovarian reserve markers, weight and previous response to treatment. Based on these parameters patients are grouped as expected low-, normal- or high-responders.

Despite a seemingly adequate stimulation regimen some patients respond with fewer than the expected number of follicles/ oocytes. Such patients are called hypo-responders. According to the POSEIDON classification these patients belong to POSEIDON groups 1 and 2 (Gr 1 and 2: retrieval of 9 or fewer oocytes in patients with an anti-Müllerian hormone (AMH) level ≥ 1.2 ng/ml or antral follicle count (AFC) ≥ 5 and age <35 years [Gr1] or ≥35 years [Gr2]). Other definitions to identify hypo-responders are: 1) use of >2500 IU gonadotropins during stimulation, 2) lack of follicle growth during stimulation, 3) lack of estradiol rise during stimulation. In these cases, we expect a proper response, but it either does not happen, or we need to administer high doses of gonadotropins to achieve the desired result. The exact cause of this phenomenon is not known but receptor polymorphism is one mechanism that could interfere with appropriate signal transduction. Another hypothesis is that these patients are less sensitive to LH and despite adequate serum LH levels they suffer from a reduced LH effect.

If a hypo-responder patient needs to undergo a second treatment, usually either the gonadotropin dose is increased from the onset of the stimulation, or the FSH preparation is combined with LH, or the more potent recombinant preparations are favored. LH could play a dual role; it increases androgen synthesis in the theca cells which in turn increases the FSH sensitivity and aromatase activity of the granulosa cells. In addition, mid-size follicles express LH receptors and therefore LH can directly support their development.

According to one meta-analysis, if treatment outcome is compared with rFSH vs rFSH+rLH in the general, non-selected population then with rFSH more oocytes can be collected while the rFSH+rLH combination results in more pregnancies. Another meta-analysis focusing on poor responders found more oocytes and higher clinical pregnancy rates after rFSH+rLH treatment but no difference in the live birth rates.

However, only a few properly designed studies have compared the various treatment strategies among hypo-responders. Lisi et al. have supplemented with rLH from day 7 onwards the second stimulation of those patients who required >2500 IU gonadotropin in their first attempt. Clinical pregnancy rate was higher in the rLH supplemented cycles. Based on these findings the authors designed a quasi-randomized trial during which every 3rd patient was given rLH supplementation from day 7 onwards. Among those who eventually needed >2500 IU gonadotropin those who were supplemented with rLH had higher implantation rates. There were however only 8 such cases. Ferraretti et al., have studied patients who had stagnation of follicle growth and estradiol production between days 7-10 of their treatment. The participants either received a higher FSH dose or received the combination or rFSH+rLH or the combination of rFSH + human menopausal gonadotropins (hMG). With rFSH alone the number of oocytes, the estradiol level and the clinical pregnancy rate were lower, but the live birth rate did not differ among the three groups. De Placido et al., in a study with a similar design to Ferraretti et al. found more oocytes in the increased rFSH dose arm but clinical outcome remained comparable in the three arms. In a study by Ruolvo et al., among women who needed >3000 IU gonadotropins previously the combination of rFSH with rLH resulted in more pregnancies. Finally, Yilmaz et al. reported improved implantation and pregnancy rates in hypo-responders with the combination of rFSH+rLH. Conforti et al., in a meta-analysis of trials in hypo-responders found that with rFSH+rLH an additional 2 oocytes can be collected and the live birth rate increases.

It is also known from daily practice that the same patient can respond differently to the same medication type and dose in a different cycle. Folliculogenesis is a long process lasting 3-6 months and most of this process is independent of gonadotropins. Those follicles that enter the final 2-3 weeks of this process become responsive to FSH and LH. The size of these groups of follicles varies from cycle to cycle. Therefore, the question arises whether a hypo-responder in one treatment is expected to have a similar hypo-response if the treatment is carried out without alterations. Do the providers need to change/ increase the gonadotropin dose if otherwise based on age and ovarian reserve one would expect a normal response? Furthermore, if adjustments are considered should the dose of FSH be increased or should a mixture with LH be used? In order to try to answer these question the investigators plan to perform a randomized controlled trial.

The aim of this randomized controlled trial is compare unchanged medication protocol to increased dose of FSH vs the combination of FSH and LH in hypo-responder patients identified based on POSEIDON criteria.

Inclusion criteria:

• indication for IVF- intracytoplasmic sperm injection (ICSI)
• age 18-40 years
• <9 oocytes in a previous IVF cycle and having an AMH ≥1.2 ng/ml and/or AFC ≥5 (POSEIDON 1,2)
• The use of ≤225 IU FSH (either rFSH alone or the combination of rFSH+human Menopausal Gonadotropin (hMG)) in the previous cycle
• regular 24-35 day cycles
• Intact uterine cavity
• motile sperm from ejaculate or testicular biopsy Exclusion criteria
• not meeting the inclusion criteria
• the use of rFSH:rLH (2:1 ratio) in the previous cycle
• the use of clomiphene citrate or aromatase inhibitor in the previous cycle
• >225 IU gonadotropin in the previous cycle
• >3 failed previous treatments
• patient with recurrent miscarriages
• presence of a hydrosalpinx
• positive human immunodeficiency virus (HIV) or hepatitis screening test
• planned preimplantation genetic testing of the embryos
• planned elective cryopreservation
• lack of consent Randomization: An online randomization list will be accessible from all participating centers by the local primary investigator using a personal link (www.randomizer.org). The other participating providers will receive the assignment from the local principle investigator (PI) once the patient has signed the consent form.

Sample size: Considering the pilot nature of the study the investigators plan to enroll 50 patients per treatment arm, 150 in total. If one treatment proves to be superior to the others, then the results could serve as the basis for a larger properly powered study.

Study sites: 4 IVF centers will participate: Dunamenti REK Istenhegyi IVF Center, Dunamenti REK Gyor IVF Center, Dunamenti REK Tapolca IVF Center, Reproductive Medicine Institute University of Szeged.

Outcome parameters:

• primary outcome: live clinical pregnancy at week 10-12 of gestation
• secondary outcomes: pregnancy rate, clinical pregnancy (intrauterine gestational sac), live birth rate (birth a live newborn after week 24 of gestation), miscarriage rate, birth weight, week of delivery

Definitions:

• Biochemical pregnancy: hCG>10 IU/l
• Clinical pregnancy: intrauterine gestational sac
• Ongoing pregnancy: embryo with heartbeat at week 10-12
• Early miscarriage: pregnancy loss prior to week 12
• Late miscarriage: pregnancy loss week 12-24
• Live birth: delivery after 24 weeks gestation
• Preterm delivery: delivery prior to week 37 gestation
• Very preterm delivery: delivery before week 32 gestation
• Low birth weight: < 2500 g
• Very low birth weight: < 1500 g

Treatment arms:

1. Same gonadotropin dose as in previous treatment (recombinant(r) FSH) ['control group']
2. The same dose as in the previous cycle but in the form of FSH + LH combination (rFSH:rLH 2:1 ratio) ['additional LH group']
3. A dose increase of 75 IU compared to the dose in the previous treatment. ['higher dose FSH group']

Treatment plan:

1. The patient meeting the inclusion or exclusion criteria is seen for an ultrasound during the cycle preceding the IVF treatment and is randomized according to the online list after consent has been given.
2. Stimulation is started on day 2 or 3 of the menstrual cycle and is continued for 5 days. On day 6 the patient is seen for transvaginal ultrasound to assess the size and number of follicles and undergoes blood test for estradiol and LH. If the provider decides to increase the dose of the medication, then it can be increased by max. 75 IU every 2 days to a maximum of 300 IU/day.
3. Once the largest follicles reach 12-14 mm in diameter 0.25 mg cetrorelix (Gonadotropin-releasing hormone (GnRH) antagonist) is started.
4. At the time of the last follicle scan serum estradiol, progesterone and LH levels are measured again.
5. Once the largest follicle reaches >17 mm in diameter the trigger injection (recombinant human chorionic gonadotropin (rHCG) or the combination of rHCG + GnRH agonist) is administered or if the provider considers the risk of ovarian hyperstimulation syndrome (OHSS) to be high pure GnRH agonist trigger (with elective cryopreservation) can be administered.
6. 35-36 hours after the trigger transvaginal oocyte retrieval is performed. Fertilization by IVF or ICSI depending on the semen parameters and patient history.
7. Vaginal progesterone supplementation from the day after the retrieval.
8. 3-5 days after the retrieval 1-2 embryos/blastocysts are transferred transcervically. Surplus good morphology embryos are cryopreserved using vitrification.
9. 12-14 days after the transfer serum beta HCG test to confirm pregnancy.
10. If the test is positive in another 3+/-0.5 weeks ultrasound to confirm intrauterine implantation.
11. Second ultrasound to confirm live ongoing pregnancy around week 10.
12. Luteal support up to week 8-9 of gestation.
13. Follow up until delivery to collect perinatal outcome parameters.
14. If the first transfer fails but frozen embryos are available frozen embryo transfer (FET) can be planned.

Parameters to be collected:

• age
• number of previous embryo transfers
• indication for IVF
• body mass index
• smoking history
• day 6 estradiol + LH levels
• estradiol, LH, progesterone at last scan
• endometrial thickness at last scan
• number of oocytes
• number of mature oocytes
• method of fertilization
• number of fertilized oocytes
• number of embryos
• number of embryos transferred
• number of embryos cryopreserved
• embryo quality (Gardner score in case of blastocysts)
• result of the initial hCG
• clinical pregnancy
• live clinical pregnancy
• miscarriage
• live birth
• week of delivery
• birth weight
• adverse events: 1) elevated progesterone prior to retrieval (>1.5 ng/ml), 2) elective cryopreservation for OHSS, 3) OHSS, 4) other (complications during retrieval)

Statistical methods:

Statistical analysis: Baseline demographic, IVF treatment related, and clinical outcomes will be compared in the three different stimulation protocol groups. Continuous variables will be shown as mean +/- standard deviation (SD), while categorical variable as number and percent. ANOVA and Mann-Whitney U test will be used for the analysis.

Inclusion Criteria:

• indication for IVF-ICSI
• age 18-40 years
• <9 oocytes in a previous IVF cycle and having an AMH ≥1.2 ng/ml and/or AFC ≥5 (POSEIDON 1,2)
• The use of ≤225 IU FSH (either rFSH alone or the combination of rFSH+hMG) in the previous cycle
• regular 24-35 day cycles
• Intact uterine cavity
• motile sperm from ejaculate or testicular biopsy

Exclusion Criteria:

• not meeting the inclusion criteria
• the use of rFSH:rLH (2:1 ratio) in the previous cycle
• the use of clomiphene citrate or aromatase inhibitor in the previous cycle
• >225 IU gonadotropin in the previous cycle
• >3 failed previous treatments
• patient with recurrent miscarriages
• presence of a hydrosalpinx
• positive HIV or hepatitis screening test
• planned preimplantation genetic testing of the embryos
• planned elective cryopreservation
• lack of consent

Patient data will be shared upon reasonable request.