P-260 A more physiological approach to support oocyte activation in cases with fertilization failure
Authors: Wu E , Xie P , Cheung S , Rosenwaks Z , Palermo G 
Published: June 2023

Abstract

Study question
Can we use a physiological compound to trigger oocyte activation in couples undergoing ICSI with history of fertilization failure due to sperm activating factor deficiency?

Summary answer
In a pilot sibling oocyte study, oocyte activation using recombinant human phospholipase C-zeta (rhPLCζ) yielded a comparable fertilization compared to conventional method using ionomycin.

What is known already
ICSI overcomes most forms of male gamete defects, however partial or total fertilization failure still occurs regardless of adequate semen parameters and optimal oocyte maturity. This has been attributed to the lack of a cytosolic factor identified as PLCζ. To address this, various approaches of assisted oocyte activation have been proposed, including gamete exposure to chemical or electrical stimuli. The most popular approach remains to be ionomycin. Despite its apparent safety, this stimulus is unnatural and may be aggressive on the oocytes. Therefore, we propose a more physiological approach by utilizing a recombinant form of this specific enzyme.

Study design, size, duration
In the past 2 months, couples with poor fertilization at their initial ICSI cycles were identified and counseled for assisted oocyte activation (AOA) in the subsequent cycle after confirmation of a PLCζ deficiency. Oocytes were separated into 2 equal cohorts activated either by ionomycin or co-injection with recombinant human rhPLCζ during ICSI. Fertilization rates between ionomycin-activated and rhPLCζ-activated cohorts were compared, as well as with the couples’ history cycles.

Participants/materials, setting, methods
Six couples with suboptimal fertilization outcomes consented for the study (IRB 0712009553) and were offered AOA with their subsequent ICSI cycles. Conventional AOA was carried out by exposing post-ICSI oocytes to 50 mM ionomycin. The rhPLCζ had a confirmed &gt;95% purity. Our physiological rhPLCζ-activation method was performed by co-injecting spermatozoa with 0.4pL of a rhPLCζ (5mg/mL in glycerol) during ICSI.

Main results and the role of chance
Couples (n = 6, female age: 36.2 ± 6yrs, male age: 38.0 ± 7yrs) had negative infertility workups with normal semen parameters (3.3 ± 3 mL volume, 42.1 ± 35 x106/mL concentration, 30.8 ± 23% motility, 2.0 ± 1% normal morphology). They underwent 7 unsuccessful ICSI cycles with 20.0% (12/60) fertilization rate and yielded a 91.7% cleavage rate, of those, 3 were transferred at cleavage stage but did not yield clinical pregnancy. A total of 2 blastocysts were obtained but were all aneuploid.
In their subsequent treatment cycles, overall AGT achieved a significantly improved fertilization rate of 51.6% (32/62) (P&lt;0.001). We then compared the efficiency between the two AGT methods in equally distributed sibling oocyte cohort. Although mathematically insignificant, we observed a trend that rhPLCζ-activation outperforms ionomycin-activation in total activation (67.9% vs 50.0%) as well as fertilization (57.1% vs 47.0%). The new rhPLCζ-activation method yielded a comparable embryo cleavage rate at 87.5% compared to the conventional method at 81.3%. Although fresh embryo transfer at cleavage stage did not yield pregnancy rate yet, rhPLCζ-activated cohort had one euploid blastocyst while the ionomycin-activated cohort did not yield euploid blastocyst.

Limitations, reasons for caution
While we observed that a more physiological rhPLCζ-activation yields a comparable fertilization compared to the conventional ionomycin method, the study is preliminary, and a confirmatory clinical outcome is not yet available.

Wider implications of the findings
This is the first attempt to use recombinant sperm-specific protein to mimic physiological oocyte activation for patient treatment. If this data is confirmed in a larger series, this may represent a way to normalize fertilization in cases of sperm activating factor deficiency even in different sources of spermatozoa.

Trial registration number
N/A