Comparison of Two Types of Calcium (Ca²⁺) Ionophore Treatments for Oocyte Activation in Cases of Suboptimal Fertilization Potential ((Ca²⁺))

May 6, 2026 updated by: Noreen, ART Fertility Clinics LLC
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.

Study Overview

Status

Not yet recruiting

Conditions

Intervention / Treatment

Detailed Description

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.

Study Type

Interventional

Enrollment (Estimated)

20

Phase

  • Not Applicable

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Contact

Study Contact Backup

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

  • Adult

Accepts Healthy Volunteers

Yes

Description

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.

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Primary Purpose: Other
  • Allocation: Randomized
  • Interventional Model: Parallel Assignment
  • Masking: None (Open Label)

Arms and Interventions

Participant Group / Arm
Intervention / Treatment
Other: Group 1: Oocyte Activation (OA) CultActive
Oocytes 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.
Other: Group 1: Oocyte Activation (OA) CultActive
Oocytes 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.
Other: Group 2: Oocyte Activation (OA) Ionomycin
Oocytes 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.
Other: Group 2: Oocyte Activation (OA) Ionomycin
Oocytes 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.
Other: Group 3: Control
Oocytes are cultured as per routine practice after injection in culture dish/ Embryoscope slide in routine culture GT-culture medium.
Other: Group 3: Control
Oocytes are cultured as per routine practice after injection in culture dish/ Embryoscope slide in routine culture GT-culture medium.

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Fertilization rates and abnormal fertilization.
Time Frame: 16-20 hours post-insemination/ICSI
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.
16-20 hours post-insemination/ICSI
Oocyte Degeneration Rate
Time Frame: 24 hours post-ICSI
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.
24 hours post-ICSI

Secondary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Usable Blastocyst Rate
Time Frame: 7 days post fertilization
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.
7 days post fertilization
Blastocyst Ploidy on Day 5, 6, or 7
Time Frame: From enrollment to the end of treatment at 4 week
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.
From enrollment to the end of treatment at 4 week
Blastocyst Quality at Time of Biopsy
Time Frame: 7 days post fertilization

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.
7 days post fertilization

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

ART Fertility Clinics LLC

Investigators

  • Study Director: BARBARA LAWRENZ, ART Fertility Clinics LLC

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Estimated)

April 30, 2026

Primary Completion (Estimated)

December 1, 2026

Study Completion (Estimated)

December 31, 2026

Study Registration Dates

First Submitted

April 30, 2026

First Submitted That Met QC Criteria

April 30, 2026

First Posted (Actual)

May 6, 2026

Study Record Updates

Last Update Posted (Actual)

May 11, 2026

Last Update Submitted That Met QC Criteria

May 6, 2026

Last Verified

March 1, 2026

More Information

Terms related to this study

Keywords

Additional Relevant MeSH Terms

Other Study ID Numbers

  • 2503-ABU-008-NH

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

UNDECIDED

IPD Plan Description

upon request

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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