This study aims to investigate the effectiveness and safety of assisted oocyte activation (AOA) using Ca²⁺ ionophores in cases of total fertilization failure (TFF) due to oocyte activation deficiency (OAD). The study will be conducted in two phases: Phase I will compare the fertilization rates of oocytes exposed to two types of Ca²⁺ ionophore treatments- Ionomycin solution and commercially available CultActive-against a control group without calcium ionophore treatment. Phase II will assess whether an additional injection of CaCl2, prior to exposure to the chosen ionophore from Phase I, further improves fertilization outcomes. The goal is to identify the optimal AOA protocol for improving fertilization rates and to evaluate the safety and efficacy of this approach in relation to ploidy, with a focus on ensuring normal pre-implantation embryo development.

The introduction of ICSI has significantly improved fertilization rates worldwide from 15% to 70-80% between 1996 and 2019. Despite its use, total fertilization failure (TFF) still occurs in 1-5% of cases Oocyte activation deficiency (OAD) is a primary cause of TFF, preventing mature oocytes from undergoing activation and successful fertilization. This deficiency, which can originate from either the sperm or the oocyte, is estimated to contribute directly to 40% of ICSI failures , with potentially higher indirect contributions.

As infertility and ICSI cycles continue to rise, so does the frequency of TFF and OAD. While deficiencies in oocyte-derived factors are more challenging to address and likely involve pathways downstream of sperm-induced activation, the absence of oocyte activation due to deficient sperm-derived signals has been partially overcome through assisted oocyte activation (AOA) approaches. During fertilization, oocyte activation is initiated by sperm-specific phospholipase C zeta (PLCζ), which induces Ca²⁺ oscillations within the oocyte. A disruption in this mechanism is a leading cause of fertilization failure in mammals. Some male etiologies imply deficiencies in sperm PLCζ. In particular, cases of globozoospermia, where sperm lack phospholipase C zeta (PLCζ) and fail to initiate the necessary Ca2+ oscillations for fertilization, have been addressed and proven efficient with AOA. Assisted oocyte activation (AOA) approaches have been proposed as a clinical intervention and have demonstrated some success in compensating for sperm factor deficiencies, by replicating this Ca²⁺ release using mechanical, electrical, or chemical stimulation by Ca²⁺ ionophores. However, concerns regarding its non-physiological nature and incomplete understanding of Ca²⁺ signaling in fertilization limit its widespread implementation.

Ca2+ ionophores are chemical compounds that facilitate the transport of Ca²⁺ ions (Ca²⁺) across biological membranes, bypassing the cells' natural Ca²⁺ signaling mechanisms, therefore they have been applied as AOA biochemical approach. The two most used types of Ca²⁺ Ionophores, are a commercially available Ca2+ Ionophore, A23187 (also known as calcimycin, and is commercially available by Gynemed), a carboxylic antibiotic that binds and freely transports Ca2+ across all biological membranes, and Ionomycin, which is far more specific and potent for Ca²⁺ compared to A23187 and can activate and indirectly stimulate gene expression due to the activation of various Ca²⁺-dependent signaling pathways. Ca2+ signalling is not only essential for nuclear processes such as fertilization mechanisms and cortical granules release, but also for cytoplasmic events such as cytoskeletal rearrangement, mitochondrial function and energy production, and a role in oxidative balance. Although several studies have been published, including a Cochrane review, there have been few randomized controlled trials (RCTs) involving sibling oocytes at the MII stage. Many studies have either included in vitro-matured oocytes or were of retrospective nature, which complicates the interpretation of conclusions regarding the efficacy of the optimal method. Of note, two RCTs using sibling MIIs applied A23187 with no differences in fertilization rates, and only one in Ca²⁺ using Ionomycin which resulted in better fertilization outcomes. Use of CaCl2 in combination to Ca²⁺ ionophore seems to improve outcomes in relation to fertilization without impacting birth characteristics and congenital malformations of the 47 children born. However, safety studies involving preimplantation genetic testing for aneuploidy (PGT-A) on embryos derived from AOA have not been conducted, with only one retrospective report available. Given that for some couples using AOA would mean a last resource to obtain available embryos and for some a significant increase on the availability of embryos, it is crucial to assess both the safety and efficacy by analyzing ploidy and identifying the optimal protocol in this context.

Inclusion Criteria:

1. Patients undergoing assisted reproductive technology cycles when ICSI is indicated.
2. Patients with a minimum of 3 MII oocytes after denudation.
3. Maternal age 18-43 years old.
4. PGT-A cycles with only trophectoderm biopsies on day 5/6/7.
5. BMI<35.
6. Fresh and frozen immotile ejaculated sperm.
7. Fresh and frozen TESE sperm (motile and immotile).
8. Globozoospermia.

10. Couples undergoing ICSI due to poor fertilization history (≤30%), or previous fertilization failure.

Exclusion Criteria:

1. PGT-M/SR cycles.
2. Fresh and frozen motile ejaculated / FNA sperm.
3. IVF inseminated oocytes.

upon request