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.
Patients undergoing assisted reproductive technology cycles when ICSI is indicated.
Patients with a minimum of 3 MII oocytes after denudation.
Maternal age 18-43 years old.
PGT-A cycles with only trophectoderm biopsies on day 5/6/7.
PGT-M/SR cycles.
Fresh and frozen motile ejaculated / FNA sperm.
IVF inseminated oocytes.
Parallel
Group 1: Oocyte Activation (OA) CultActive
Other interventionOocytes are cultured immediately after injection in a pre-calibrated OA-CultActive dish for 15 min in CO2 incubator, then injected oocytes are rinsed well in culture dish/ Embryoscope slide in GT-culture medium. Then transferred to the numbered droplet or well.
Group 2: Oocyte Activation (OA) Ionomycin
Other interventionOocytes are placed immediately after injection in a pre-calibrated OA-Ionomycin dish (dish 1) for 7 - 10 min in CO2 incubator, then they are rinsed well and placed in another culture dish (dish 2) for 25 min in CO2 incubator. Then MIIs are exposed again in OA-Ionomycin dish for 10 min, then they are rinsed well and placed into culture dish/ Embryoscope slide in GT-culture medium. Then transferred to the numbered droplet or well.
Group 3: Control
Other interventionOocytes are cultured as per routine practice after injection in culture dish/ Embryoscope slide in routine culture GT-culture medium.
Fertilization rates and abnormal fertilization.
The primary endpoint is the proportion of metaphase II (MII) oocytes that achieve normal fertilisation (2 pronuclei; 2PN) following insemination or ICSI, compared across study groups. Additionally, the incidence of abnormal fertilisation (e.g., 0PN, 1PN, ≥3PN) will be evaluated. Fertilisation assessment will be performed at the standard time point of 16-20 hours post-insemination/ICSI.
Oocyte Degeneration Rate
The proportion of metaphase II (MII) oocytes that undergo degeneration following ICSI across the study groups. Degeneration will be assessed at the standard post-ICSI evaluation time point, and expressed as the percentage of injected oocytes exhibiting morphological signs of degeneration.
Usable Blastocyst Rate
The proportion of normally fertilised oocytes that develop into blastocysts deemed suitable for vitrification or transfer based on established morphological and developmental criteria. The rate will be calculated as the number of usable blastocysts divided by the total number of fertilised oocytes (2PN) within each study group.
Blastocyst Quality at Time of Biopsy
Blastocyst quality will be assessed using the modified Gardner blastocyst grading scale, which evaluates blastocyst expansion stage, inner cell mass quality, and trophectoderm quality at the time of biopsy. The blastocyst expansion stage is scored from 1 to 6, where higher scores indicate a more expanded or hatched blastocyst. The inner cell mass and trophectoderm are graded from A to C, where A indicates the best quality, B indicates intermediate quality, and C indicates the lowest quality. Blastocyst quality scores will be compared across study groups to determine the impact of Ca²⁺ ionophore treatment on blastocyst development.
Blastocyst Ploidy on Day 5, 6, or 7
Assessment of chromosomal status of biopsied blastocysts using preimplantation genetic testing (PGT). Ploidy results (euploid, aneuploid, mosaic) will be evaluated and compared across study groups to determine the influence of Ca²⁺ ionophore treatment on chromosomal integrity and embryo competence.
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