The goal of this clinical trial is to learn whether the sudden administration of vaginal micronized progesterone during the late follicular phase can trigger an LH surge and reactivation of oocyte meiosis in healthy oocyte donors aged 18 to 33 undergoing a mild ovarian stimulation cycle. The main question it aims to answer is:

• Does progesterone administered in the late follicular phase induce an LH surge and subsequent oocyte meiosis reactivation?

Participants will:

• Undergo a mild ovarian stimulation with 75 IU of follitropn alfa
• Start vaginal micronized progesterone (400 mg every 12 hours) once at least one follicle reaches 15 mm
• Have blood samples collected to measure hormone levels before and after progesterone administration
• Undergo oocyte retrieval 35-36 hours after the progesterone trigger

This is a low-intervention, single-center pilot study including approximately 10 oocyte donors.

Ovulation is triggered by the sudden surge of gonadotropins, FSH and mainly LH, which occurs during the late follicular phase. This LH surge creates the environment for follicular eruption by increasing the activity of the proteolytic enzymes that weaken the ovarian wall leading to follicle rupture. However, the stimuli leading to the LH surge arises are not clear yet. The current scientific paradigm postulates that high levels of estradiol at the end of the follicular phase may, by positive feedback, stimulate the sudden release of gonadotropins. However, several factors seem to contradict this theory. Ovarian stimulation cycles with high doses of gonadotropins in high responders leads to high levels of estradiol in an early phase without incurring in ovulation until the late follicular phase. Thus, there may be a narrow window in the late follicular phase in which ovulation may occur. As such, the sustained high levels of estrogen in the late follicular phase could be responsible for the LH surge. Nevertheless, studies using high dose exogenous estradiol in late follicular phase showed that there was no impact on gonadotrophin surge or ovulation. On the other hand, studies based on ovarian stimulation cycles with letrozole, with much lowers serum estrogen levels, showed that the ovulatory peak occurs in the same way, even in the presence of very low or decreasing levels of estradiol. Interestingly, patients treated with letrozole may present higher levels of LH at peak. Hence, the role of estrogens in inducing the gonadotrophin peak is yet unclear.

Progesterone may eventually have a role in the physiological induction of ovulation. Long-standing studies have shown that an increase in serum progesterone levels precedes the LH peak and supports the action of the LH on the follicle rupture. Evidence has long shown that elevated serum progesterone levels during controlled ovarian stimulation are associated with a considerable risk of earlier ovulation. Likewise, previous studies have shown that the administration of intramuscular progesterone during the late follicular phase leads to the onset of an endogenous LH peak, both following controlled ovarian stimulation and in a natural cycle. Also supporting these theories is a new protocol of endometrial preparation for frozen embryo transfer presented by our research group. This strategy, in which, in an advanced follicular phase, after establishing adequate endometrial thickness on ultrasound, exogenous administration of vaginal micronized progesterone is started, simulating a second phase of the cycle, with endogenous production of estrogens and exogenous administration of progesterone. Curiously, in the first obstetric assessment the presence of an adnexal mass is frequently been noted, probably corresponding to a corpus luteum. If so, exogenous progestins may have acted as a possible trigger of ovulation.

Contrary to these theories is the fact that the exogenous administration of progestins for pituitary inhibition has been used for decades as a method of contraception. Thus, there may be a narrow window of time in the late follicular phase during which the pituitary gland is sensitive to the positive feedback effect of progesterone, leading to sudden release of gonadotropins. This may eventually be explained by the effect of the rising levels of estradiol during the follicular phase, which may prime the hypothalamus for a progesterone-induced gonadotropin surge. Another possibility is that sustained use of exogenous progestins may lead to desensitization of pituitary receptors, while in the presence of low serum progesterone levels, the receptors maintain sensitivity and the sudden increase of this hormone has a positive feedback effect on the pituitary gland.

Progestins may be administered by various routes. Vaginal micronized progesterone is a convenient form of administration and results in rapid and efficient absorption, with peak serum concentrations achieved within four to six hours.

With this study, the investigators intend to evaluate whether the sudden administration of exogenous progesterone may lead to LH peak, with resulting reactivation of oocyte meiosis.

Inclusion Criteria:

• Informed Consent Form dated and signed correctly prior to the performance of any study procedure.
• Oocyte donors aged >17 and <34 years old in the moment of the recruitment.
• Patients with regular menstrual cycles (between 25 to 35 days long).
• Generally healthy women with no history of endocrine disorders (e.g., polycystic ovary syndrome (PCOS) or significant systemic disease).
• Body Mass Index (BMI) between 18.5 and 28.

Exclusion Criteria:

• Presence of ovarian cyst(s) detected on the baseline (initial) ultrasound examination.
• Untreated endocrine conditions (polycystic ovary syndrome (PCOS), uncontrolled thyroid dysfunction and hyperprolactinemia).
• Hypogonadotropic or hypergonadotropic hypogonadism.
• Known hypersensitivity or clinical contraindication to the administration of vaginal progesterone or its excipients. This includes, but is not limited to: active deep vein thrombosis or thromboembolic disorders, known or suspected estrogen- or progesterone-dependent neoplasia, undiagnosed abnormal genital bleeding, porphyria, or severe hepatic dysfunction. Additionally, patients with anatomical abnormalities of the vagina that preclude the proper insertion or absorption of the vaginal insert are excluded.
• Any condition contraindicating vaginal ultrasound and/or vaginal oocyte retrieval.These include: active pelvic inflammatory disease (PID), acute vaginal or cervical infection, or presence of pelvic malignancies. Patients with significant anatomical distortions or vascular anomalies in the pelvic region that, in the investigator's opinion, would make oocyte retrieval technically unfeasible or pose an unacceptable risk of hemorrhage or injury to adjacent organs, are also excluded.
• Use of hormonal contraceptives (oral, patch, or ring) or GnRH analogues within the month preceding the study. Note: Participants who have discontinued these or other prohibited medications for a period of at least 30 days are eligible for rescreening starting at Visit 1.
• Participation in any other ongoing clinical trial or use of investigational drugs within the last 30 days.