P-094 Dynamics of progesterone-induced intracellular Ca2+ changes in sperm predicts zygote formation rate by IVF: Preliminary results
Authors: Trevino C , Torres-Rodríguez P , Ramírez-Domínguez L B , Soriano M , Carrasquel-Martinez G , Flores D , Jiménez I , Maldonado I , Villar L , Agarwal A , Matamoros-Volante A 
Published: June 2023

Abstract

Study question
Can the overall intracellular Ca2+ dynamics evoked by progesterone stimulation predict the fertilizing potential of a non-normozoospermic semen sample under fertility treatment?

Summary answer
Some kinetic parameters of the transient intracellular Ca2+ increase evoked by progesterone stimulation correlate with fertilization rates in conventional IVF but not with ICSI.

What is known already
Stimulation with female hormone progesterone induces the activation of the sperm-specific ion channel CatSper which in turn produces a fast increase in the intracellular Ca2+ concentration ([Ca2+]i). After reaching a maximum [Ca2+]i, different Ca2+ clearance mechanisms become active producing a transient followed by a plateau. We investigated whether the [Ca2+]i dynamics upon progesterone stimulus may be used to assess the fertilizing potential of sperm samples from men undergoing assisted reproduction.

Study design, size, duration
This prospective study employed unidentified semen samples from 39 non-normozoospermic men (age 38 ± 6 years old) undergoing fertility treatment from March 2022 to the present at CITMER Reproductive Medicine, Mexico City. Frozen semen samples and ART procedures with less than 3 oocytes were excluded. Ten normozoospermic semen samples were also analyzed. The progesterone-dependent [Ca2+]i changes were evaluated using time-lapse flow cytometry.

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 and Ca2+-sensitive fluorescent dyes. Fluorescence changes were evaluated by AccuriC6 flow cytometer. Progesterone-induced [Ca2+]i transient as well as the correlations with the fertilization rates were calculated using custom-made R programming language code.

Main results and the role of chance
The progesterone (1 µM) stimulation evoked a fast and transitory increase in the [Ca2+]i. We then calculated six different kinetics parameters of such responses: basal Ca2+ level, increase and decrease in velocity, maximum Ca2+ level, total response duration, and the area under the curve (AUC). There were no significant differences in any of these parameters between normozoospermic donors (and non-normozoospermic patients. The former group of semen samples were divided into either conventional IVF (n = 17) or ICSI (n = 21). In IVF but not ICSI samples, the duration, and the area under the curve of the progesterone response tended to be higher than in normozoospermic (2.39±0.7 min, p = 0.03, and 113±30 AUF*min, p = 0.18, respectively). Furthermore, both parameters showed a tendency to correlate negatively with the conventional IVF (R= –0.45, p = 0.07 and R= –0. 5, p = 0.04, respectively) but not with ICSI fertilization ratio (number of zygotes/total number of mature oocytes). These results suggest that cells with a better Ca2+ recovery mechanism are well suited for IVF. Interestingly, we observed no difference between the magnitude of the [Ca2+]i increase after progesterone stimulus among donors, IVF, or ICSI samples, as was previously reported. Differences in the concentration of progesterone could explain such results.

Limitations, reasons for caution
The preliminary nature of the results implies a necessity to increase the number of analyzed semen samples (in progress). It is well known that progesterone induces variable responses even among the same donor cells. Our population-based analysis may be underestimating such single cells variations.

Wider implications of the findings
Current protocols for sperm analysis fail to successfully predict the fertilizing capability of semen samples from men seeking reproductive treatment. Further research into the molecular regulation of Ca2+ dynamics could offer promising alternatives for clinicians to appropriately choose between IVF and ICSI for each couple.

Trial registration number
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