ORIGINAL RESEARCH article
Front. Endocrinol.
Sec. Reproduction
Annexin A2 defines late apoptotic membrane states in human spermatozoa
Provisionally accepted
- 1CONICET Rosario, Rosario, Argentina
- 2Universidad Nacional de Rosario, Rosario, Argentina
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Male factor infertility contributes to nearly half of all infertility cases, and optimizing sperm selection remains critical for improving assisted reproductive technologies (ARTs) outcomes. Conventional semen preparation techniques, such as swim-up and density gradient centrifugation, efficiently select motile and morphologically normal spermatozoa but fail to discriminate cells with DNA damage or apoptotic features. Magnetic-Activated Cell Sorting (MACS) using Annexin A5 (ANXA5) has been implemented to remove apoptotic sperm based on phosphatidylserine (PS) exposure; however, recent evidence indicates that ANXA5 is ubiquitously present on the surface of human sperm, even in the absence of apoptotic stimuli. In this study, we evaluated the potential of Annexin A2 (ANXA2), a Ca²⁺-dependent phospholipid-binding protein with high PS affinity but absent from the sperm surface, as an alternative apoptotic marker. Recombinant ANXA2 was cloned, expressed, purified, and conjugated with fluorescein isothiocyanate (FITC). Human spermatozoa were treated with betulinic acid (100 μM, 30 min) to induce apoptosis and analyzed by fluorescence microscopy and flow cytometry. Betulinic acid treatment significantly increased acrosomal alterations and DNA fragmentation, accompanied by activation of caspase-3/7. ANXA2-FITC preferentially bound to spermatozoa exhibiting disrupted membranes, localized mainly in the subacrosomal region and midpiece, correlating with propidium iodide (PI)-positive cells. Quantitatively, ANXA2-FITC– positive sperm increased by 21.7% following betulinic acid treatment, paralleling caspase activation but differing from ANXA5, which also labeled early apoptotic (A5⁺/PI⁻) cells. These results indicate that ANXA2 binds selectively to late apoptotic or necrotic spermatozoa, distinguishing them from early apoptotic-like cells. The specific association of ANXA2 with membrane-disrupted domains suggests its potential use as a molecular probe for identifying structurally compromised sperm. Overall, these findings expand the understanding of annexin–membrane interactions in human spermatozoa and support ANXA2 as a promising marker for refining sperm selection strategies in ARTs.
Keywords: Annexin A2, Annexin A5, Apoptosis, Human sperm, membrane disorder
Received: 12 Nov 2025; Accepted: 31 Jan 2026.
Copyright: © 2026 Teijeiro and Vanzetti. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Juan Manuel Teijeiro
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