GnRH for Luteal Support - Mechanism of Action

Luteal phase support is essential in IVF treatment to overcome luteal phase deficiency and improve pregnancy outcomes, yet the optimal protocol remains unclear. In recent years, GnRH agonists have emerged as a promising option for luteal support, administered either by subcutaneous injection or intranasal spray, with the latter offering a convenient and non-invasive route. Previous studies demonstrated that GnRH agonist supplementation during the luteal phase improves pregnancy and live birth rates, including a prospective randomized study from our group showing significantly higher positive βhCG rates compared with standard progesterone support. Although the exact mechanism remains uncertain, proposed explanations include stimulation of LH secretion, direct effects on the endometrium and embryo, and modulation of placental βhCG production. Evidence also suggests a luteotrophic effect through maintenance of corpus luteum function, reflected by higher progesterone levels and continued pregnancy progression after treatment discontinuation. However, important limitations remain, including variable patient response, possible underlying endocrine or receptor-related factors, and a potential association with ovarian hyperstimulation syndrome (OHSS). Therefore, this study aims to investigate the mechanism of action of GnRH agonists as sole luteal support and identify predictors of treatment success or failure.

The study is a prospective observational study, which will be conducted among women undergoing IVF treatments based on GnRH antagonist protocol with a fresh embryo transfer (ET) at the IVF unit in Shaare Zedek medical center.

About 800 egg retrieval procedures take place at our unit every year, not including pre-implantation genetic testing (PGT) and oocyte cryopreservation cycles. Approximately 80% of the cycles use the antagonist protocol. It is estimated that in 400 of them, patients are administrated with GnRH agonist for luteal support.

During the visit to the clinic, the women's demographic and clinical data will be collected.

All patients will undergo ovarian stimulation based on GnRH antagonist protocol: Ovarian stimulation with gonadotropins (recombinant FSH and\\or hMG depending on patients' age, BMI, and basal serum FSH levels) will begin within the first 2-3 days of the menstrual period. When the leading follicle reaches a diameter of \>12mm, treatment with daily injections of GnRH antagonist (0.25 mg Orgalutran or 0.25 mg Cetrotide) will be added and continued until the day of ovulation induction. Once sonography will demonstrate three or more follicles at size ≥17mm, ovulation triggering will be administrated. The stimulation for egg maturation will be performed by either recombinant hCG (250 mcg Ovitrelle), GnRH agonist (0.2 mg Decapeptyl), or a combination of the two -dual triggering (250 mcg Oviterelle plus 0.2 mg Decapeptyl). The stimulation type will be chosen according to the decision of the attending physician, usually based on the estimated risk of OHSS (based on laboratory and sonographic markers).

The eggs will be retrieved 36 hours after the ovulation triggering and fertilized by IVF or by intracytoplasmic sperm injection (ICSI). Fertilized embryos will be incubated until the day of ET. During incubation time and before ET, the quality of embryos will be evaluated and graded according to the accepted criteria in the laboratory.

Following egg retrieval, patients will receive luteal phase support with either intranasal GnRH agonist - spray of 200 mcg Nafarelin (Synarel) twice a day for two weeks or vaginal progesterone. After two weeks, at βhCG examination day, Synarel treatment will be stopped, regardless of the test results and progesterone treatment will continue until 8-10 weeks pregnancy.

Blood samples will be collected for each patient at the six following stages:

1. Ovum pick-up day 2. Day of ET 3. 7 days after ovum pick-up (mid-luteal phase) 4. 12 days after ET 5. 14 days after ET In addition, follicular fluid from the OPU of each woman will be centrifuged and tested as well.

Laboratory markers will include LH, FSH, βhCG, Estradiol and Progesterone. IL-6. Relaxin, IL-8, VEGF and PEDF will be analyzed by enzyme-linked immunosorbent assay (ELISA) using commercial kits. All samples will undergo appropriate freezing for future testing.

Study variables will include demographic characteristics (age, BMI, obstetric and infertility history), IVF cycle parameters (stimulation protocol, hormonal response, oocyte and embryo characteristics), blood and follicular fluid biomarkers (including LH, FSH, βhCG, steroid hormones, relaxin, IL-6, IL-8, VEGF, and PEDF), and pregnancy outcomes such as positive βhCG, clinical pregnancy, miscarriage, live birth, OHSS, and vaginal bleeding.

• Level of blood markers along the luteal phase [Time frame: 2 weeks]