O-016 To be or not to be capacitated after sperm preparation: tyrosine phosphorylation as predictive indicator for capacitation status and male fertility potential

Abstract

Abstract Study question Can post-capacitation tyrosine phosphorylation (TP) levels distinguish “capacitated” from “non-capacitated” molecular profiles, and are these similarly distributed among infertile and fertile men? Summary answer TP levels after 3 hours of incubation post-capacitation robustly predicted “capacitated” and “non-capacitated” molecular profiles, with the latter being more common in the infertile group. What is known already TP is considered one of the most relevant intracellular signalling events in the capacitation process and in the regulation of sperm function. Despite the recognized general trend of increased TP post-capacitation, interindividual variability can be observed, with some samples showing phosphorylation levels like those before capacitation. Currently, no test is conducted on capacitated samples to verify whether they have indeed acquired a molecular pattern consistent with capacitation. Therefore, the objective of this study was to evaluate the predictive ability of post-capacitation TP levels measured at various time points in distinguishing molecularly “capacitated” patterns from “non-capacitated” ones. Study design, size, duration Prospective experimental study conducted from June 2021 to September 2023 with the aim of assessing TP levels in different populations before (B-cap) and after (A-cap) in vitro capacitation and after different incubation times (1h and 3h) under capacitating conditions. A total of 335 samples, including infertile patients (n = 111), tests for the inclusion in the sperm donation program (n = 57) and sperm donors (n = 167) were included. Participants/materials, setting, methods Semen samples were examined following WHO guidelines and in vitro capacitation was performed using density gradient centrifugation. Flow cytometry was used to assess global TP (%) (B-cap, A-cap, 1h and 3h). Receiver operating characteristic (ROC) curve analyses were performed to evaluate the discriminatory potential of post-capacitation TP levels (A-cap, 1h and 3h) against B-cap, aiming to distinguish between “capacitated” and “non-capacitated” molecular profiles. Chi-square test was used to compare proportions. P < 0.05 was considered statistically significant. Main results and the role of chance The dataset was randomly split into two subsets, one for model development and the other for subsequent performance evaluation. The areas under the curve (AUC) for A-cap, 1h and 3h were 73.8% (95% CI = 69.3%-78.3%), 84.5% (81.0%-88.0%), and 92.7% (90.4%-95.0%), respectively. Sensitivity and specificity values were calculated for the curve with the highest AUC, constructed with TP values from non-capacitated (B-cap) and 3-hours incubated (3h) samples. The selected cut-off point was 4.35% (sensitivity: 85.0%, specificity: 85.7%). The model exhibited robust overall performance in identifying molecularly “capacitated” and “non-capacitated” samples, with a sensitivity, specificity, false positive rate and false negative rate of 85.2%, 89.7%, 9.4% and 12.9%, respectively. Accuracy, positive predictive value and negative predictive value were 87.3%, 90.2%, and 84.5%, respectively. In infertile patients, using TP values at the third hour of incubation and the selected cut-off, the model identified 73.9% (82/111) of samples as molecularly “capacitated” and 26.1% (29/111) as “non-capacitated,” despite laboratory capacitation. In contrast, in the control group, the model identified 93.4% (156/167) of samples as “capacitated” and 6.6% (11/167) as “non-capacitated”. A significantly higher number of seminal samples were identified as “non-capacitated” in the infertile patient group compared to the proven fertile donor group (p < 0.001). Limitations, reasons for caution The model's performance demonstrates its utility and reliability in identifying samples with a molecular “non-capacitated” profile, even after in vitro capacitation. However, further refinement is possible by incorporating additional data. External validation is essential to confirm generalizability of the results. Wider implications of the findings The assumption that a molecular capacitation profile is acquired following in vitro sperm processing may be questioned. Our results suggest a potential link between the selected threshold and fertility, offering insights into male infertility aetiology and paving the way for personalized intervention strategies. Trial registration number NCT04962100