Ovarian stromal fibrosis resulting from each ovulation and due to aging is characterized by a decrease in hyaluronic acid concentrations, loss of synthesis and migration proteins CD63 and CD81, decreased miRNA125, miRNA21, miRNA132 and miRNA199. Also, enzymatic alterations affecting kinases and mitochondrial sirtuins SIRT3-5 with increased oxygen free radicals preventing the normal maintenance and development of folliculogenesis, leading to the inability to reproduce at advanced ages and accelerating the onset of pathologies secondary to the decrease of ovarian estrogens. Methodology: An observational, prospective, randomized, comparative, double-blind, analytical pilot study in 30 women between 38 and 46 years of age, with diminished ovarian reserve and who refused the egg donation procedure. Three groups were designated: one for autologous exosomes, one for PRP and one with physiological solution.

METHODOLOGY AND STUDY DEVELOPMENT

This study is an observational, prospective, randomized, comparative, double-blind, and analytical investigation conducted at a fertility clinic in Caracas, Venezuela, from January 2024 to September 2024. The study involved an ovarian biostimulation or bioregeneration procedure performed on 30 patients aged 38 to 46 years with low ovarian reserve who wished to conceive using their own eggs and declined egg donation.

Patient selection criteria were based on parameters indicative of decreased ovarian reserve at the study's onset. These included hormonal levels measured on the third day of each patient's menstrual cycle: Follicle-Stimulating Hormone (FSH) greater than 12 mIU/mL, Estradiol less than 35 pg/mL, and Anti-Müllerian Hormone (AMH) less than 0.7 ng/mL. Additionally, patients had to present a total antral follicle count of fewer than five follicles across both ovaries, confirming low ovarian reserve or ovarian resistance, alongside a documented refusal to use donor eggs.

An intraovarian injection protocol was implemented based on three different treatment groups:

Autologous Exosomes obtained from the patient's platelets, Activated Platelet Growth Factors and Physiological Solution (control group).

This procedure was performed over four cycles, once per month, on days 7, 8, or 9 of menstruation. Patients were randomly assigned to one of the three groups using a randomized list managed by a trained nurse who determined allocation based on the order of arrival. The groups were distributed as follows:

Group 1: Patients receiving autologous exosomes derived from their own platelets.

Group 2: Patients receiving activated platelet growth factors. Group 3: Patients receiving a physiological solution as a placebo.

Inclusion and Exclusion Criteria

Inclusion Criteria:

• Female patients aged 38 to 46 years.
• Patients desiring to conceive.
• Patients with a Body Mass Index (BMI) between 23 and 30.
• Patients diagnosed with ovarian failure, based on the specified parameters.
• Patients refusing egg donation.
• Patients who provided informed consent after fully understanding the study.
• Patients with no active ovarian or other oncological pathologies, endometriomas, or suspected tumors.
• Patients with a platelet count exceeding 250,000 and no history of hematological diseases.
• Patients not undergoing anticoagulant therapy or presenting active infections.

Exclusion Criteria:

• Patients outside the specified age range (38-46 years).
• Patients unwilling to conceive.
• Patients with a BMI outside the 23-30 range.
• Patients without ovarian failure or who declined to participate and did not provide informed consent.
• Patients with oncological diseases or suspected tumors.
• Patients with a platelet count below 250,000.
• Patients with a history of hematological diseases, ongoing anticoagulant therapy, or active infections.

Following the initial evaluation, informed consent was obtained, and baseline laboratory tests were conducted. Blood samples were collected from each patient on the third day of their menstrual cycle before the first biostimulation to measure FSH, LH, Estradiol, AMH, and antral follicle count in each ovary. These same tests were repeated after the fourth biostimulation and before initiating fertility procedures.

All blood samples were frozen and analyzed simultaneously at a clinical laboratory in Caracas to minimize bias. Patients were advised to follow a diet free of dairy and processed carbohydrates for five days before and after each procedure. Additionally, they were encouraged to consume unflavored gelatin twice daily to enhance platelet quality.

The procedures were performed under controlled sedation in the clinic's operating room. Peripheral blood was collected from all patients using a PRP kit with separator gel, ensuring identical collection and processing protocols across all groups. Blood samples were drawn from the cubital vein using a vacutainer system under gentle compression (avoiding tourniquet use) and centrifuged at 270g for 10 minutes.

Treatment Groups:

Autologous Exosome Group: PRP was processed using the EXOSMART kit to isolate platelet exosomes. A concentrate of 6 cc containing 5-6 trillion exosomes per milliliter was obtained, and 3 cc were injected into each ovary.

Platelet Growth Factor Group: Activated PRP was prepared, and 3 cc were injected into each ovary.

Control Group: Patients received 3 cc of physiological solution injected into each ovary.

Injections were performed transvaginally using a follicular aspiration needle guided by transvaginal ultrasound. The procedures were conducted during the early follicular phase (days 7-9) under surgical sedation.

Inclusion Criteria:

• Female patients between 38 and 46 years of age.
• Patients with a desire to become a mother
• Patients with Body Mass Index (BMI) between 23 and 30.
• Patients with proven ovarian insufficiency, according to the parameters mentioned above.
• Patients who refuse egg donation.
• Patients with full willingness to participate in the study and who have understood and signed the informed consent.
• Patients with both ovaries without oncological pathologies, active endometriomas or suspicious ovarian or other tumors.
• Patients with platelet count over 250 thousand.
• Patients with no history of hematological diseases.
• Patients without treatment with anticoagulants.
• Patients with no active infection at the time.

Exclusion Criteria:

• Female patients outside the age range of 38 to 46 years old.
• Patients who are not willing to become mothers.
• Patients with a BMI outside the range of 23 to 30
• Patients without ovarian insufficiency
• Patients who do not wish to participate in the procedure and have not signed the informed consent form.
• Patients with suspected or diagnosed oncological diseases.
• Patients with platelet count of less than 250 thousand
• Patients with a history of hematological diseases.
• Patients with anticoagulant treatment.
• Patients with any type of active infection.

Women aged 38 to 46 years

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