Live birth following an innovative artificial oocyte activation protocol using double calcium stimulators

Intracytoplasmic sperm injection (ICSI) achieves an average fertilization rate of about 70%, yet total fertilization failure occurs in roughly 3% of cycles, often due to oocyte activation deficiency. Physiological fertilization triggers intracellular Ca2+ oscillations that activate the oocyte. Artificial oocyte activation (AOA) using mechanical, electrical, or chemical Ca2+ stimulators can remedy activation deficiency, but single-stimulator chemical AOA (e.g., ionomycin, calcimycin, or strontium chloride [SrCl2]) is not effective for all patients. This study explored whether combining two Ca2+ stimulators could overcome repeated fertilization failure in a couple for whom conventional AOA had been ineffective.

Chemical AOA is widely used clinically, with ionomycin/calcimycin (Ca2+ ionophores) and SrCl2 being common choices. SrCl2 effectively induces prolonged Ca2+ oscillations and supports development in mice but has variable and often limited efficacy in humans, possibly due to lower ATP levels in human oocytes that limit Ca2+ signaling strength. Single transient Ca2+ increases (e.g., ionomycin) may be insufficient for full oocyte activation and optimal embryo development, whereas cumulative Ca2+ input appears critical. Prior animal studies suggest combining Sr2+ with ionomycin can increase activation rates versus either alone. Clinical reports support safety of AOA with ionomycin or SrCl2 compared with ICSI alone, but data on combined use in humans are limited.

Design: Before-after case study in a couple with a 10-year history of primary infertility and repeated fertilization failure, including prior failures with conventional IVF, ICSI, and ICSI+AOA (SrCl2) at other clinics. Ethics approval obtained; informed consent provided (ChiCTR2200055839). Patient details: Female 32 years, male 33 years. Semen: volume 3 mL, concentration 21.25×10^6/mL, progressive motility 20.69%, morphology 1% normal, low chromatin maturity, abnormal polymorphonuclear elastase. Female evaluations were normal. Ovarian stimulation: GnRH antagonist protocol. AOA media: Prepared 1 day prior: 10 mmol/L SrCl2 and 10 mmol/L ionomycin; drops equilibrated under oil at 37°C, 6% CO2, 5% O2. ICSI and AOA protocols tested on sibling MII oocytes at 37°C, 6% CO2, 5% O2:

• SrCl2: exposure immediately after ICSI for 1 h.
• Ionomycin: exposure for 10 min following 1 h post-ICSI manipulation.
• Combined SrCl2+ionomycin: SrCl2 immediately after ICSI for 1 h, followed by ionomycin for 10 min. Embryo assessment: Fertilization checked on day 1 post insemination; embryo quality assessed on day 3; embryos cryopreserved; frozen embryo transfer (FET) with hormone replacement endometrial preparation and luteal support (vaginal progesterone gel and estradiol). Time-lapse monitoring used in cycle 2. Pregnancy monitored by serum hCG and ultrasound; obstetric and neonatal outcomes recorded.

Cycle 1: 16 oocytes retrieved, 13 MII injected by ICSI. Post-ICSI AOA allocation: SrCl2 (4), ionomycin (5), combined (4). Outcomes day 1: SrCl2 0/4 fertilized; ionomycin 1/5 fertilized (developed to poor-grade embryo, discarded day 3); combined 2/4 fertilized (both fair-grade, cryopreserved). Subsequent FET of the two fair-grade embryos did not result in pregnancy. Cycle 2: 14 sibling MII oocytes injected and equally divided: ionomycin (7), combined (7). Outcomes: Ionomycin 1/7 fertilized (fair-grade embryo on day 3); combined 5/7 fertilized, yielding 5 day-3 embryos (2 optimal, 3 fair-grade). Two optimal embryos from the combined protocol were thawed and transferred in the next cycle. Serum hCG was 922.62 mIU/mL at 13 days post-transfer; ultrasound at 32 days post-FET showed a gestational sac with fetal heartbeat. Pregnancy and delivery were uncomplicated; a healthy baby was born with no apparent abnormalities. Comparative efficacy: SrCl2 alone was ineffective in this patient; ionomycin alone yielded low fertilization and poorer embryo quality; the combined SrCl2+ionomycin protocol markedly improved fertilization rate and embryo quality and achieved live birth.

This case demonstrates that in a patient with repeated fertilization failure, SrCl2 alone did not provide sufficient Ca2+ signaling to activate oocytes, consistent with reports that human oocytes may not mount robust Ca2+ oscillations to Sr2+ due to lower ATP and other species-specific factors. Ionomycin alone, which elicits a single prolonged Ca2+ transient, modestly improved fertilization but did not support high-quality embryos or pregnancy. The combined protocol likely compensates for Ca2+ signal insufficiency by delivering both oscillatory-like input (Sr2+) and a strong transient (ionomycin), increasing cumulative Ca2+ exposure to surpass the activation threshold needed for downstream events and improved embryo development. The successful live birth provides initial human evidence supporting the efficacy and short-term safety of combined Ca2+ stimulators for AOA when conventional single-stimulator AOA fails.

The first reported live birth using an innovative AOA protocol combining SrCl2 and ionomycin is presented. Compared with single-stimulator AOA, the combined approach substantially improved fertilization and embryo quality in a patient with prior fertilization failures, culminating in a healthy live birth. The likely mechanism is compensatory enhancement of Ca2+ signaling to meet activation requirements. Future research should evaluate efficacy and safety in larger cohorts, optimize dosing and timing parameters, and investigate long-term offspring outcomes.

Single-patient before-after study limits generalizability and prevents causal inference. Prior treatment history and small oocyte numbers constrain statistical interpretation. SrCl2 efficacy may vary among patients; outcomes may depend on specific dosing and timing. Safety data for the combined protocol are limited to a single uneventful pregnancy and delivery; long-term child follow-up and larger safety studies are needed. Publication does not report comprehensive karyotyping or extended pediatric outcomes.