P-096 Prediction of fertilization success by voltage-dependent intracellular Ca2+ dynamics in non-normozoospermic human semen samples undergoing in vitro fertilization: preliminary results
Authors: Matamoros Volante A , Torres Rodriguez P , Ramírez-Dominguez L B , Soriano M , Carrasquel-Martinez G , Flores D , Jiménez I , Maldonado I , Villar L , Agarwal A 
Published: June 2023

Abstract

Study question
Does the change in intracellular calcium levels evoked by artificial membrane depolarizations predict fertilization success in a non-normozoospermic semen sample undergoing assisted reproductive techniques?

Summary answer
The increase in the intracellular calcium after artificial plasma membrane depolarization correlates negatively with fertilization rates in conventional IVF but not with ICSI

What is known already
Sperm capacitation-related features, like the regulation of the intracellular calcium concentration ([Ca2+]i) and the hyperpolarization of the plasma membrane potential (Vm), have been proposed as predictors of success during artificial reproduction techniques (ART). Intriguingly, upon Vm depolarization, sperm exhibit a rapid and transitory increase in the [Ca2+]i controlled, likely, by the activation of the sperm-specific calcium channel CatSper. The regulation of CatSper activity is essential for sperm function and is crucial for fertilization. Therefore we wondered if the Vm depolarization-dependent rise in the [Ca2+]i can be used to predict fertilization success rates in non-normozoospermic semen samples of men undergoing ART.

Study design, size, duration
This prospective study employed unidentified semen samples from 29 non-normozoospermic men (37.8 ± 5.6 years old) undergoing fertility treatment from March 2022 to the present at CITMER Reproductive Medicine, Mexico City. Frozen semen samples and procedures with less than 3 oocytes were excluded. Ten normozoospermic semen samples were analyzed as a control. The depolarization-dependent [Ca2+]i changes were evaluated by flow cytometry under four different Vm conditions: resting (variable among samples), –64, –46, and –32 mV.

Participants/materials, setting, methods
Semen samples were obtained on the same day as oocyte retrieval. Sperm cells were washed and incubated in capacitating HTF media for further use in conventional IVF or ICSI procedures. When available, the surplus cells were stained with vitality, Ca2+, and Vm-sensitive fluorescent dyes. Fluorescence changes were evaluated by AccuriC6 flow cytometer. The kinetical analysis of the transitory [Ca2+]i increase as well as the statistical comparisons were performed using custom-made R programming language code.

Main results and the role of chance
Analysis of resting Vm showed that normozoospermic semen samples presented more depolarized Vm than non-normozoospermic controls (–75.7 ± 6.9 vs –67.3 ± 10.5 mV, respectively, p = 0.02). The former group of semen samples was either used for conventional IVF (n = 11) or ICSI (n = 14). The semen samples in these last categories showed a tendency to present a more depolarized Vm than non-normozoospermic samples (–69.9 ± 8.8 and –66.6 ± 11.3 mV, respectively). No differences were seen in the resting Ca2+ levels. We then induced a Vm hyperpolarization by adding the K+ ionophore, valinomycin followed by increasing concentrations of extracellular K+. Each depolarizing stimulus evoked a fast and transitory increase in the [Ca2+]i. We then calculated seven different kinetic parameters of such responses: basal Ca2+ level, increase and decrease velocity, maximum Ca2+ reached, response durability, and area under the curve (AUC). The basal Ca2+ level reached after depolarizing stimuli correlated negatively (R =  –0.64, p = 0.03) with the conventional IVF fertilization ratio (number of zygotes/total number of mature oocytes) when the Vm was clamped either to –64 and –46 mV. A negative and significant correlation (R =  –0.76, p = 0.006) was seen for the AUC analysis at –64 mV. No relevant correlations were found in the ICSI samples.

Limitations, reasons for caution
This is an in vitro study, and caution must be taken when extrapolating these results in vivo. The preliminary nature of the results implies a necessity to increase the number of analyzed semen samples (in progress).

Wider implications of the findings
Further research into the molecular regulation of the CatSper channel could offer promising alternatives for clinicians to appropriately choose between IVF and ICSI for each couple. This protocol is fast and simple to perform in a laboratory equipped with a flow cytometer or any fluorescence-based reader.

Trial registration number
NA