CAPA-IVM Culture With Low Oxygen Tension

Capacitation in-vitro maturation (CAPA-IVM) has recently been advanced in culturing oocytes from the germinal vesicle (GV) stage. This approach is a modified version of conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), following mild stimulation or no controlled ovarian stimulation occurred. Specifically, IVM can be indicated for patients diagnosed with polycystic ovary syndrome (PCOS), a higher number of secondary follicles (constituting nearly 15% of total patients), and treat a range of patients with the risks of ovarian hyperstimulation, venous thromboembolism or ovarian torsion. Additionally, CAPA-IVM helps shorten treatment time, is less expensive, and upgrades patient convenience without multiple follow-up examinations. The live birth rate after the first embryo transfer in the CAPA-IVM group was 35.2%, which was not statistically significantly different from the IVF group at 43.2% (risk difference -8.1%; 95% confidence interval from -16.6% to 0.5%). However, the number of high-quality embryos in each cycle and the cumulative clinical pregnancy rate in CAPA-IVM were still lower than in cIVF.

Moreover, further investigation should be considered due to the lack of high-quality evidence of concurrent reports. Therefore, improving the oocyte maturation conditions in CAPA-IVM to harvest the optimal number of oocytes and the highest possibility of embryo development is essential. Many studies conducted on both animal and human models have demonstrated that the effectiveness of CAPA-IVM depends on various factors. Among these, the environmental culture conditions such as oxygen (O2) concentration play a crucial role in producing healthy mature oocytes. O2 is a vital physical and chemical component of the fallopian tube, uterus and ovarian follicle, it is closely related to metabolic activity, oocyte maturation, and early embryo development. Recent research suggested that O2 concentration may significantly regulate oocyte maturation and early embryo development through hypoxia-inducible factor (HIF). A consensus on the O2 concentration index in the IVM culture environment has not been reached. Oocyte-embedded culture systems have been commonly used in two O2 concentrations, 5% and 20% worldwide. In the human body, cumulus-oocyte complexes (COCs) mature in conditions with low O2 concentrations ranging from 2% to 9%.

Conversely, COCs are exposed to an atmospheric O2 concentration of 20% during IVM manipulation and culture. Although the concentration of 5% mimics the most proper environment in the fallopian tube and uterus, the 20% O2 is widely applied in IVM techniques. The use of high concentrations facilitates a better progression of differentiation processes and increases the maturation rate of oocytes. However, some referential frames indicated that a 20% O2 may pose a risk of reactive oxidative stress (ROS), leading to an imbalance in the ratio of pro-oxidants to antioxidants, resulting in cellular damage. Furthermore, real-time respiration analysis of oocytes cultured at 5% O2 is similar to in vivo-developed oocytes but induced cellular activity and oxygen consumption at 20% O2. The impact of atmospheric O2 concentration (20%) and low O2 concentration (5%) during CAPA-IVM culture in mice shown in the study of Vrije Universiteit Brussel (VUB) - Belgium that the respiratory capability of COCs cultured at 5% O2 was relatively similar to COCs developing and maturing in vivo.

Nonetheless, COCs cultured at 20% O2 increased respiratory activity and oxygen consumption remarkably. The study observed that pre-IVM culture of COCs at 20% O2 caused developmental disruptions. Also, the result was unfavorable if mouse COCs were cultured at the IVM step with 5% O2. Based on these analyses, the researchers hypothesized that a 5% O2 was the optimal culture condition for the pre-IVM step, while a 20% O2 was more relevant to the IVM culture step. Combining these findings with results from VUB and characteristics of the differentiation process in CAPA-IVM oocytes, this study is divided into two main groups, including 5% pre-IVM and 20% IVM versus 20% pre-IVM and IVM) and demonstrates whether this hypothesis should be applied CAPA-IVM in human. The enhancement of the CAPA-IVM culture system leads to improved treatment efficiency of this technique and provides various benefits for patients undergoing assisted reproductive technology.

Study procedure:

Screening for eligibility

• This trial will be conducted at My Duc Hospital, Ho Chi Minh City, Viet Nam.
• Women who are potentially eligible will be provided information about the trial 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 related to the study together with the informed consent documents. Signed informed consent forms will be obtained by the investigators from all women before the enrolment.

Oocytes will be divided into 2 groups:

Group 1 (includes 2 subgroups: 1A and 1B): Air Oxygen Concentration CAPA-IVM culture T = Total number of oocytes after OR and there are two subgroups.

The number of oocytes is divided below:

If T is an even number:

• Number of oocytes in Group 1A: One oocyte.
• Number of oocytes in Group 1B: T1B = (T-2)/2.

If T is an odd number:

• Number of oocytes in Group 1A: One oocyte.
• Number of oocytes in Group 1B: T1B = ([T-1]-2)/2. One oocyte remainder of the first patient will be assigned to group 1B, and the remainder of the next patient will be assigned to group 2B. Continuing to do so sequentially for the next remainder.

Group 2 (includes 2 subgroups: 2A and 2B): Low Oxygen Concentration CAPA- IVM culture T = Total number of oocytes after OR and there are two subgroups.

The number of oocytes is divided below:

If T is an even number:

• Number of oocytes in Group 2A: One oocyte.
• Number of oocytes in Group 2B: T2B = (T-2)/2.

If T is an odd number:

• Number of oocytes in Group 2A: One oocyte.
• Number of oocytes in Group 2B: T2B = ([T-1]-2)/2. One oocyte remainder of the first patient will be assigned to group 1B, and the remainder of the next patient will be assigned to group 2B. Continuing to do so sequentially for the next remainder.

Group 1A, 2A: Collecting after capacitation: oocyte and cumulus cell.

Group 1B, 2B: Collecting after capacitation: spent media, blank well. Collecting after maturation: spent media, cumulus cell, blank well.

Inclusion criteria

• 18-42 years of age
• Diagnosed with polycystic ovary syndrome according to the Rotterdam criteria (2003)
• Indicating CAPA-IVM treatment
• Having at least 40 antral follicles in two ovaries by transvaginal ultrasound at the time of CAPA-IVM indication
• Agreeing to have frozen embryo transfer
• Agreeing to participate in the trial

Exclusion criteria

• Cycles with oocyte donation, preimplantation Genetic Testing (PGT)
• Couples with severe male factor (sperm concentration <5 million/ml, motility < 10%), surgical sperm retrieval
• Previous history of unexplained immature oocytes after IVF treatment
• Uterine abnormalities