Effect of Super-GDF9 on CAPA-IVM of COCs From Small Antral Follicles

CAPA-IVM (In Vitro Maturation) technology is an assisted reproductive method offering significant benefits in terms of safety and treatment costs, particularly for high-risk patients. These include individuals with ovarian hyperstimulation syndrome (OHSS), venous thrombosis, ovarian torsion, or polycystic ovary syndrome (PCOS). However, while the live birth rate in the CAPA-IVM group (35.2%) is comparable to conventional IVF (43.2%), the number of good-quality embryos and cumulative clinical pregnancy rates remain lower. Improving the CAPA-IVM culture process, particularly through the addition of growth factors found in follicular fluid, has shown promise in enhancing oocyte quality.

Growth differentiation factor 9 (GDF9) and Bone morphogenetic protein 15 (BMP15) play critical roles in follicular development, with their heterodimer structure demonstrating the most positive effects on cumulus-oocyte complexes (COCs). Recent studies have identified a potent variant, super GDF9, which is \>1000 times more effective than GDF9 and surpasses cumulin, a heterodimeric growth factor. Super GDF9 enhances cumulus cell expansion and oocyte developmental competence, closely mimicking in vivo maturation.

This study investigates the impact of supplementing super GDF9 during CAPA-IVM culture, aiming to improve outcomes of cumulus-oocyte complexes (COCs) from small follicles and ultimately enhance treatment success.

CAPA-IVM (In Vitro Maturation) technology is an assisted reproductive method offering significant benefits in terms of safety and treatment costs, particularly for high-risk patients. These include individuals with ovarian hyperstimulation syndrome (OHSS), venous thrombosis, ovarian torsion, or polycystic ovary syndrome (PCOS) - who typically present with a high number of antral follicles (constituting nearly 15% of all patients). Although the live birth rate following the first transfer in the CAPA-IVM group is 35.2%, which is not statistically different from the conventional IVF group at 43.2% (risk difference: -8.1%; 95% confidence interval: -16.6% to 0.5%), the number of good-quality embryos per cycle and the cumulative clinical pregnancy rate remain lower than in conventional IVF. Therefore, improving the CAPA-IVM culture process to achieve the optimal number and quality of oocytes is essential.

Concurrently, adding growth factors commonly found in follicular fluid to the culture medium represents a remarkable advancement in improving oocyte quality in CAPA-IVM. Some somatic compartments, such as expansion, metabolism, and apoptosis, are regulated by soluble growth factors, known as oocyte secretion factors (OSFs). Two OSFs, Growth differentiation factor 9 (GDF9) and Bone morphogenetic protein 15 (BMP15), have been identified as critical for follicular development and fertility in various species such as mice, sheep, and humans. During IVM culture, both the immature and mature forms of these factors as well as their homo- and heterodimer structures have been tested. Notably, the heterodimer structure has shown the most positive effects on cumulus-oocyte complexes (COCs) during IVM culture.

Although both growth factors exist in homodimeric forms, recent studies have found that the GDF9 and BMP15 heterodimer can also form a more potent growth factor called cumulin. BMP15 activates latent GDF9 in cumulin, leading to strong signaling in granulosa cells via type I receptors (ALK4/5) and SMAD2/3 transcription factors. Biomedically engineered cumulin has been proposed to noticeably improve embryo outcomes in mouse and porcine models. Recently, a modified version of wild-type GDF9, called super GDF9, has been demonstrated to be \>1000 times more potent than GDF9 and 4 times more activity than cumulin in SMAD2/3-responsive transcriptional assays in granulosa cells. Previous research has illustrated that adding super GDF9 to CAPA-IVM media in mice induces gene expression in the ovulatory cascade during CAPA-IVM maturation that closely resembles in vivo maturation. Super GDF9 effectively promotes cumulus cell expansion and enhances oocyte developmental competence in vitro. Hence, super GDF9 can potentially replace cumulin, which faces challenges in production and purification.

This study investigates the impact of supplementing super GDF9 during CAPA-IVM culture, aiming to improve outcomes of cumulus-oocyte complexes (COCs) from small follicles and ultimately enhance treatment success.

This study will recruit 300 COCs (an estimated 10 needed patients). 100 COCs will be allocated to the research arm (sGDF-9), while 200 COCs will be allocated to the control arm.

* Screening for eligibility

  * This study will be conducted at My Duc Hospital, Ho Chi Minh City, Vietnam.
  * Women who are potentially eligible will be provided information about the study at the time of IVM treatment indication.
  * Screening for eligibility will be performed on the day of the first visit when the IVM treatment is indicated.
  * Patients will be provided information about the study and informed consent documents. The investigators will obtain signed informed consent forms from all women before enrollment.
  * Eligible women will be scheduled to undergo oocyte pick-up procedures within 1-7 days from informed consent.
* Oocytes retrieval The oocyte pick-up procedure will be conducted according to the center's standard practices for CAPA-IVM cycles.

Cumulus-oocyte complexes (COCs) from small follicles after OPU will be divided into 2 groups:

* Group 1 (sGDF-9): donated COCs will be cultured in the CAPA and IVM steps, adding 50ng/ml Super-GDF9 during both steps in CAPA-IVM
* Group 2 (Control): The subject's remaining COCs will be cultured in the CAPA and IVM steps without adding Super-GDF9 during CAPA-IVM.

Groups 1 and 2: Collecting after the capacitation step: spent media and blank wells. Collecting after the maturation step: spent media, cumulus cell, and blank wells.

+ CAPA and Maturation culture: CAPA and Maturation culture will be performed routinely following current laboratory protocols. ICSI will be used to fertilize mature oocytes.

Eligibility Criteria:
Inclusion Criteria:

1. Women between the ages of 18 and 38 years (both inclusive)
2. BMI ≤ 32 kg/m2
3. PCOS women according to the Rotterdam criteria (2003)
4. Indicating CAPA-IVM treatment.
5. Serum AMH ≥ 4 ng/mL (28.57 pmol/L) at screening and having at least 24 antral follicles in two ovaries by transvaginal ultrasound at the time of CAPA-IVM indication
6. Willing to donate COCs for research purposes
7. Agreeing for frozen embryo
8. Signed informed consent before any study-related procedures

Exclusion Criteria:

1. Known endometrioma or grade 3-4 endometriosis according to ASRM classification
2. Uterine abnormalities
3. Couples with severe male factor (sperm concentration \<5 million/ml, motility \< 10%), surgical sperm retrieval.
4. Previous history of unexplained immature oocytes after IVF treatment
5. Cycles using donor oocytes