P-270 Assisted gamete treatment to pinpoint acquired meiotic maturity and overcome oocyte activation deficiency contributed by both gametes
Authors: Vyas N , Xie P , Cheung S , Rosenwaks Z , Palermo G 
Published: June 2022

Abstract

Study question
How can we treat couples with complete and persistent fertilization failure with ICSI linked to a combination of oocyte- and sperm-related oocyte activation deficiency (OAD)?

Summary answer
By targeting spindle presence, we optimized oocyte response to chemical activation and enhanced fertilization. Genomic assessment confirmed gamete contribution.

What is known already
Total fertilization failure occurs in 1-3% of all intracytoplasmic sperm injection (ICSI) cases. In sperm-factor OAD, the lack of phospholipase C zeta (PLCζ) prevents the spermatozoon from initiating downstream calcium oscillation in the oocyte. In these cases, assisted gamete treatment (AGT), which exposes gametes to calcium ionophore, has been adopted to artificially trigger the influx of calcium ions and has been shown to effectively improve fertilization. However, AGT is limited to triggering an intracytoplasmic calcium influx and still requires optimal ooplasmic maturity.

Study design, size, duration
Over the past 17 months, we identified couples with compromised PLCζ and reported persistent fertilization failure with ICSI despite AGT treatment. We then devised a treatment plan comprising an extended in vitro culture (IVC) to pinpoint meiotic oocyte maturity confirmed by the presence of a meiotic II spindle and followed by AGT post-ICSI. Genomic assessment was also carried out.

Participants/materials, setting, methods
Two couples with recurrent and total fertilization failure even after AGT were included. PLCζ expression was assessed using immunofluorescence on ≥ 200 cells/specimen with a 30% threshold. In the follow-up cycles, IVC was extended for at least 8 hours between retrieval and ICSI. Metaphase II spindles were visualized by Oosight®. AGT was performed by exposing both spermatozoa and oocytes to calcium ionophore. NGS was performed on spermatozoa to identify gene mutations involved in fertilization.

Main results and the role of chance
We identified 2 couples (couple A: 37-year-old female, 39-year-old male; couple B: 32-year-old female, 33-year-old male) with the following semen parameters: average volume of 2.6 ml, concentration of 82.0x106/ml, 44% motility, and normal morphology of 3%. The oocyte maturation rate was 76.3% (45/59) but resulted in zero fertilized out of a total of 45 MII oocytes injected. In-house PLCζ assessment revealed a deficiency of oocyte activation factor at 12.9%. AGT treatment alone failed to enhance fertilization on a subsequent cycle, resulting in 0% (0/8) and 5.6% (1/18) fertilization rates for couples A and B, respectively. Couple A then underwent 3 ICSI cycles with extended IVC and AGT; upon examination of nuclear maturity, 91.4% (32/35) of oocytes displayed normal metaphase II spindle and achieved an overall fertilization rate of 43.8% (14/32). To date, 12 blastocysts were cryopreserved. In couple B, 27 oocytes out of 34 retrieved presented normal metaphase II spindles after extended IVC; ICSI with AGT yielded a fertilization rate of 63.0% (17/27). All 17 zygotes were cryopreserved. Overall, our treatment improved fertilization to an overall rate of 52.5% (31/59, P  &lt;0.00001). Genomic assessment of spermatozoa identified gene mutations involved in fertilization (ADAM15, ADAM30) and calcium channel activity (CATSPER1).

Limitations, reasons for caution
Assisted gamete treatment can enhance fertilization in cases of deficiency in PLCζ. However, chemical activation requires a responsive ooplasm that has reached meiotic maturity. These rare cases require precise diagnoses and tailored treatment techniques to address each aspect of sperm- and/or oocyte-factor OAD.

Wider implications of the findings
Our study has demonstrated the usefulness of extended IVC by targeting spindle presence to enhance chemical responses to AGT. Our findings show that although calcium ionophore can trigger the release of intracellular calcium and allow fertilization, a fully mature ooplasm is required.

Trial registration number
N/A