Functional heterogeneity of sperm binding to the ZP2 N terminus across normospermic and infertile phenotypes

Abstract

Background

Human gamete recognition is mediated by the N-terminal domain of the zona pellucida protein ZP2. Conventional semen analysis often fails to identify functional subpopulations within heterogeneous ejaculates, particularly in men with defective spermiogenesis. This study sought to evaluate if a recombinant ZP2^39–154 peptide can serve as a high-stringency selection tool to identify and enrich for morphologically superior and binding competent sperm.

Methods

Sperm samples from 10 normospermic and 12 infertile participants (oligozoospermia, asthenozoospermia, and teratozoospermia) were incubated with agarose beads conjugated to recombinant ZP2^39–154. Binding efficiency was quantified using confocal microscopy. Morphometric analysis, including nuclear area and flagellum length, was performed on bound sperm and compared to sperm bound to native human zonae pellucidae.

Results

Normospermic individuals exhibited nearly four-fold inter-individual heterogeneity in ZP2^39–154 binding efficiency (2.88 to 9.04 sperm/bead), which did not correlate with standard concentration, motility, or morphology metrics. In the infertile cohort, ZP2^39–154 beads identified a reservoir of binding-competent sperm even in severe cases of oligoasthenoteratozoospermia. While the global functional reservoir was reduced in defective spermiogenesis, the top-tier binding events frequently overlapped with the normospermic range. Furthermore, the ZP2^39–154 significantly enriched for morphologically normal sperm, increasing the proportion from a 1–5% baseline in raw semen to as high as 90% on the beads. Notably, 70–94% of bound sperm in the infertile group remained structurally atypical, suggesting molecular binding competence can be preserved despite gross morphological defects.

Conclusion(s)

Recombinant ZP2^39–154 beads provide a standardized, high-throughput functional proxy for the human zona pellucida. This assay transcends the diagnostic limitations of light microscopy by identifying physiologically competent gametes, offering a robust tool for targeted sperm recovery in assisted reproduction.

Introduction

Human fertilization is governed by highly specific molecular interactions, chief among them the recognition and binding of the sperm to the zona pellucida (ZP), an extracellular matrix surrounding the ovulated egg [1]. In humans, the zona pellucida is composed of four distinct glycoproteins [2] (ZP1, ZP2, ZP3, and ZP4). Studies in transgenic mice have definitively identified the N-terminal domain of the ZP2 glycoprotein as the necessary and sufficient for taxon-specific human gamete recognition [3]. As sperm binding to this zona domain is important for zona penetration [3, 4], and ultimate gamete fusion, evaluating this interaction may serve as a critical functional readout of a sperm physiological competence to finally fertilize an egg [1].

The acquisition of such binding competence strictly depends upon the successful completion of spermiogenesis. During this terminal phase of spermatogenesis, round spermatids undergo extensive cellular differentiation and morphological remodeling to emerge as highly polarized spermatozoa [5]. This remarkable differentiation process involves the precise assembly of the flagellum, which confers motility to the fertilizing sperm and an appropriate morphology to the sperm head, both required for zona interaction [5]. Normal spermiogenesis ultimately yields sperm capable of navigating the female reproductive tract and binding the oocyte; conversely, developmental errors during this process precipitate severe morphological defects that directly impair the sperm’s fertilization capacity [6].

When spermiogenesis is defective, it manifests clinically as distinct male fertility phenotypes. These conditions are broadly classified by standard semen analysis criteria [7]: oligozoospermia is characterized by a reduced sperm count (fewer than 15 million (M) sperm/ml); asthenozoospermia is defined by reduced motility (less than 40% normal motility, or fewer than 32% progressive motile sperm); and teratozoospermia is identified by an abnormal sperm shape (less than 4% normal morphology).

At the molecular level, defects in spermiogenesis and sperm morphology arise from a highly diverse and heterogeneous genetic landscape [5, 6, 8]. Evidence from gene-edited mouse models [9] and human genetic studies [6] indicates that both monogenic and polygenic variants [10] can disrupt spermiogenesis, ultimately leading to oligozoospermia, asthenozoospermia, teratozoospermia, or combinations thereof. These clinical classifications, group together patients whose conditions may arise from mutations in genes with distinct and non-overlapping roles during spermiogenesis [6]. This genetic variability exposes the limitations of relying solely on subjective, morphology-based clinical semen parameters (such as Kruger’s strict criteria) for diagnosing infertility and selecting gametes [11]. Such traditional parameters may overlook the presence of binding, competent sperm subpopulations hidden within the generally compromised ejaculates of men with defective spermiogenesis.

In previous studies, we showed that he human ZP2^39–154 peptide is capable of selecting human sperm with higher competence to bind and cross transgenic mouse zonae pellucidae expressing human ZP2 [4]. In the present study, we used the recombinant ZP2^39–154 peptide [3, 4] to evaluate the binding ability of human sperm across different participants and discovered that every ejaculate presents subpopulations of functionally and morphologically superior sperm that are not predicted by conventional semen analysis. Furthermore, we show that this assay effectively enriches binding-competent and morphologically-normal sperm even in samples from participants affected by defective spermiogenesis, establishing the recombinant ZP2^39–154 peptide as a robust tool for targeted recovery of high-quality sperm.

Materials & methods

Inclusion/exclusion criteria and IRB protocols

Participants were recruited from the reproductive medicine unit at Sidra Medicine. The study focused on men, whose age spanned 29–50 years old (age of controls 30–50; age of infertile men 29–49), presenting with abnormal semen parameters consistent with oligozoospermia, asthenozoospermia or teratozoospermia, either in isolation or in combination, as determined by routine clinical semen analysis performed according to the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (6th edition) [7, 12]. Infertile patients affected by obstructive and non-obstructive azoospermia were excluded from the study. Normospermic control participants were defined based on semen parameters within WHO reference ranges [7]. All participants provided written informed consent prior to inclusion in the study. The study was approved by the Institutional Review Board and the Institutional Biosafety Committee of Sidra Medicine.

Semen collection and analysis

Semen samples were obtained by masturbation into sterile containers following a period of sexual abstinence of 2–5 days. Samples were allowed to liquefy at 37 °C for 30 min and were analyzed within 30–60 min of collection. Semen analysis was performed according to WHO Laboratory Manual for the Examination and Processing of Human Semen (6th edition) [7], including assessment of sperm concentration, motility, and morphology using a Makler chamber. Following initial evaluation, sperm samples were washed by a density gradient (SpermGrad, VitroLife, USA) and resuspended in (Human Tubal Fluid supplemented with human serum albumin (HTF/HSA; G-IVF PLUS, VitroLife, USA).

Recombinant ZP2 peptide generation and bead conjugation

cDNA encoding the N-terminus of ZP2 (amino acids 39–154; ZP2^39–154) was cloned into a baculovirus expression vector under the control of the polyhedrin promoter and fused to a secretion signal peptide to enable extracellular expression. A C-terminal 6×His tag was included in frame to facilitate purification. Recombinant bacmid DNA, generated following transformation into DH10Bac Escherichia coli, was used to transfect insect cells for the production of recombinant baculovirus particles. Sf9 cells were subsequently infected according to manufacturer protocols to express the recombinant ZP2^39–154 peptide [3, 4, 13]. Secreted recombinant ZP2^39–154 was purified by immobilized metal affinity chromatography using IMAC Sepharose High-Performance resin (Cytiva, USA) according to the manufacturer’s instructions, and protein integrity was assessed by SDS–PAGE as previously described [4, 12]. Plasmid generation and recombinant protein expression were carried out by GenScript (USA).

Imaging and quantification of sperm binding and morphometric analysis

Washed sperm samples were processed to obtain the motile fraction, and 1 × 10⁵ progressive motile sperm, (quantified as above), were incubated with discarded native human zonae or agarose beads conjugated to recombinant ZP2^39–154 under IVF conditions (HTF/HSA, 5% CO₂, 37 °C). After 2 hrs co-incubation, sperm–bead complexes were gently washed with pin HTF/HSA to remove unbound sperm, and samples were fixed in 2% paraformaldehyde (PFA) in PBS for 1 h at room temperature or overnight at 4 °C, washed, and stained. To assess sperm binding to the beads conjugated with the ZP2^39–154, or with discarded human oocytes, and sperm morphological features, samples were stained with α-tubulin-Alexa Fluor 488, wheat germ agglutinin (WGA)-Alexa Fluor 633, and Hoechst 33,342 (ThermoFisher Scientific). Stained samples were mounted in ProLong Glass Antifade (Invitrogen, USA) and imaged using a Zeiss LSM 780 confocal microscope (Zeiss, Germany). Confocal z-stacks were acquired and projected into single-plane images using maximum intensity projection. Morphometric parameters, including nuclear area, acrosomal area, and flagellum length, and sperm binding data were quantified using ZEN lite software (Zeiss, Germany). To determine the morphological signatures of sperm bound to native human zonae pellucidae or ZP2^39–154, sperm nucleus area (µm²) and flagellum length (µm) were quantified from maximum intensity projections acquired via LSM780 confocal microscopy, using the ZEN lite software (Zeiss, Germany). Morphological thresholds were defined by the 5th and 95th percentiles of sperm bound to native human zonae to ensure physiological selection. By utilizing this functionally validated sperm population, our criteria for ‘normality’ are based on physiological competency rather than solely clinical observations.

Statistics

Statistical differences in sperm binding efficiency across the 10 normospermic participants were evaluated using non-parametric methodology, as the majority of the datasets violated the assumption of normality (p < 0.05 via Shapiro-Wilk test). To determine if significant variance existed between the cohorts, a Kruskal-Wallis H-test was performed on the distribution of sperm counts per bead (n = 50 beads per participant). Following the identification of a significant global effect, post-hoc pairwise comparisons were conducted using Mann-Whitney U tests. To control the family-wise error rate and minimize Type I errors arising from the 45 unique pairwise combinations, a Bonferroni correction was applied to all resulting p-values. All statistical computations and data visualizations, including the generation of median-centered box plots and descriptive metrics (mean ± SD), were executed using the SciPy and Seaborn libraries in a Python-based environment. A corrected alpha level of 0.05 was used as the threshold for statistical significance. To measure functional heterogeneity of sperm binding, we analyzed the distribution of binding events across 50 discrete ZP2-peptide beads per participant. Beyond standard measures of central tendency, we employed a subpopulation analysis to identify the frequency and density of high-affinity binding events within samples from infertile patients. A Kolmogorov-Smirnov test was used to compare the cumulative distribution functions (CDFs) of binding across phenotypes, determining if the ‘top-tier’ binding-competent subpopulation in infertile samples overlapped with the normospermic range. Numerical thresholds for ‘normal’ morphology were established based on the 5th and 95th percentiles of the native human zonae-bound sperm population to reflect physiological selection stringency. Differences in morphological distributions between groups were assessed using the Mann-Whitney U test. The proportion of ‘normal’ sperm within the infertile cohort was also calculated on a per-patient basis to evaluate the efficiency of the ZP2-bead assay as a functional enrichment tool.

Results

Inter-individual heterogeneity in sperm binding among normospermic participants

Washed sperm from 10 normospermic individuals were incubated with beads conjugated with the recombinant ZP2^39–154, and 2 h after incubation, washed sperm were fixed, stained and imaged by confocal microscopy (Fig. 1A). Quantification of this sperm- ZP2^39–154 binding assay revealed profound inter-individual heterogeneity in binding efficiency among 10 normospermic participants. While all individuals met standard clinical criteria for normospermia, their functional capacity to adhere to the ZP2^39–154-beads varied by nearly four-fold. The global analysis confirmed that these differences were statistically significant (Kruskal-Wallis, p = 9.75 × 10^{− 43}), suggesting that conventional semen parameters may not fully capture the molecular competency of sperm interaction with the ZP2 N terminus [3]. Post-hoc pairwise analysis identified a distinct pattern of binding affinity. High-performing participants, such as Participant 5 (8.16 ± 3.52 sperm/bead) and Participant 9 (9.04 ± 3.00 sperm/bead) exhibited significantly higher binding efficiency compared to ‘low-binding’ participants, like Participant 8 (3.64 ± 1.27 sperm/bead) or Participant 10 (2.88 ± 1.87 sperm/bead)(Bonferroni-adjusted p < 0.001). Of the 45 pairwise comparisons, 26 reached the threshold of significance, highlighting that even within a normospermic population, sperm-ZP2^39–154 interaction remains a highly variable functional phenotype (Fig. 1B).

Fig. 1.

Functional characterization and quantification of sperm binding to ZP2^39–154 across a normospermic cohort. (A) Sperm from normospermic participants (n = 10) binding to the recombinant ZP2^39–154 peptide; top, confocal z-projection images of beads carrying a recombinant ZP2^39–154 peptide after staining with Hoechst (top) or Hoechst, plus mAb⁴⁸⁸ against alpha-tubulin, and WGA⁶³³. (B) Quantification of sperm binding to recombinant ZP2^39–154 across a cohort of ten clinically normospermic individuals. Box plots represent the distribution of sperm counts per bead (n = 50 beads per participant). The horizontal line within each box indicates the median, while white circles denote the mean binding value. Box boundaries represent the interquartile range (IQR, 25th to 75th percentiles), and whiskers extend to 1.5 x IQR. Statistical significance across the cohort was determined by a Kruskal-Wallis H-test (p = 9.75 × 10^− 43). Pairwise comparisons (Mann-Whitney U with Bonferroni correction) identified significant differences between high-affinity and low-affinity binders (p_adj < 0.001)

Infertile men present high-binding sperm subpopulations but exhibit a reduced pool of binding-competent sperm

To investigate the binding ability and heterogeneity of sperm from infertile participants with defective spermiogenesis, we assessed binding using beads conjugated with the recombinant ZP2^39–154, as done for the normospermic participants (Fig. 1A). Washed sperm from 12 individuals diagnosed with defective spermiogenesis leading to oligozoospermia, asthenozoospermia, teratozoospermia, or combinations thereof (Table 1) were incubated with ZP2^39–154-coated beads for 2 h. Samples were then fixed, stained, and imaged by confocal microscopy (Fig. 2A). Sperm from infertile men with defective spermiogenesis exhibited a lower overall mean binding density (2.90 ± 3.12) compared to normospermic controls (5.97 ± 3.50). Despite this reduction, distribution analysis revealed the presence of a functionally competent sperm subpopulation across all samples and clinical diagnoses (Fig. 2B). Notably, even participants with severe oligoasthenoteratozoospermia retained sperm capable of interacting with the recombinant ZP2^39–154 (Fig. 2A). In oligozoospermic samples, where sperm count was ≤ 2.0 M/ml%, more than 56% of beads supported at least one binding-competent sperm. Furthermore, the top 10% of binding events in oligoasthenoteratozoospermia samples (8.0 sperm per bead) exceeded the mean binding observed in normospermic controls (5.97 ± 3.50)(Figs. 1B and 2B). Consistently, high-affinity binding events (Rank 1–10; Fig. 2B) in oligoasthenoteratozoospermic and teratozoospermic samples overlapped with the normospermic range, further supporting the presence of ZP2^39–154 binding-competent sperm subpopulations. To assess maximal functional capacity, we applied a successional counting strategy prioritizing beads with positive binding events and generated functional capacity curves for each phenotype (Fig. 2B). While high-binding events (arbitrarily defined as > 10 sperm per bead) were observed in both groups, defective spermiogenesis samples displayed a significantly reduced functional reservoir, defined as the number of beads capable of supporting binding. Normospermic samples maintained a stable binding plateau across all 50 analyzed beads, whereas oligozoospermic and oligoasthenoteratozoospermic samples showed rapid depletion of binding-competent sperm, reaching zero by approximately 30 and 45 beads, respectively. This decline, particularly evident in samples with low sperm count (0.5-2 M/ml), highlights exhaustion of the binding-competent sperm reservoir. Together, these findings indicate that sperm from infertile men can be characterized not only by reduced average binding, but also by a diminished pool of ZP2^39–154-competent sperm.

Table 1.

Percentage of morphologically normal sperm in raw semen or bound to the recombinant ZP2^39–154

Participant	Clinical diagnosis	Raw semen normal morphology (%)	Sperm bound to the ZP239–154 normal morphology (%)
Participant 01	Normospermia	4	85
Participant 02	Normospermia	4	73.2
Participant 03	Normospermia	5	60
Participant 04	Normospermia	5	72
Participant 05	Normospermia	4	85
Participant 06	Normospermia	4	64
Participant 07	Normospermia	4	46
Participant 08	Normospermia	4	56
Participant 09	Normospermia	4	90
Participant 10	Normospermia	4	62
Participant 11	Oligozoospermia	4	30
Participant 12	Asthenozoospermia	4	20
Participant 13	Teratozoospermia	3	30
Participant 14	Teratozoospermia	2	24.4
Participant 15	Teratozoospermia	3	15.2
Participant 16	Teratozoospermia	4	8
Participant 17	Oligoteratozoospermia	2	22
Participant 18	OAT	3	30
Participant 19	OAT	1	24
Participant 20	OAT	3	14
Participant 21	OAT	2	6
Participant 22	OAT	1	6

OAT, oligoasthenoteratozoospermia

Fig. 2.

ZP2-bead binding capacity and functional heterogeneity across participants with defective spermiogenesis. (A) Same as in 1 A, for sperm from participants diagnosed with oligozoospermia (Oligo), asthenozoospermia (Astheno), teratozoospermia (Terato), oligoteratozoospermia (Oligoterato) or oligoasthenoteratozoospermia (Oligoasthenoterato)(n = 12). (B) Sperm binding curve illustrates sperm counts per bead, rank-ordered from highest to lowest density for each clinical phenotype (n = 50 beads per participant)

Comparative analysis of ZP2-binding efficiency and clinical semen profiles in normospermic and men with defective spermiogenesis

Semen analysis of the 10 normospermic samples revealed a high degree of functional variation in ZP2-binding capacity that was not reflected in conventional semen parameters. Participants displayed normal semen parameters [7], with mean concentrations of 99.7 ± 21.6 M/mL and motilities of 64 ± 4%. However, no significant correlation was observed between binding efficiency and concentration (Spearman’s ρ = -0.298, p = 0.403), motility (ρ = -0.043, p = 0.906), or morphology (ρ = 0.138, p = 0.705). For instance, Participant 10 exhibited superior concentration and motility (110 M/ml; 63%) yet demonstrated the lowest binding density (2.88 sperm/bead). Conversely, Participant 9 showed the highest binding capacity (9.04 sperm/bead) despite a lower concentration (74 M/ml). These findings suggest that the ZP2-bead assay measures a discrete molecular competency of human sperm that is independent of, and potentially more sensitive than, standard clinical assessments. A similar lack of predictive value was observed in the cohort of 12 infertile participants. Despite the clinical presentations of oligo, astheno, and teratozoospermia (or combinations of these conditions), standard semen metrics remained poor predictors of individual binding performance. For example, participants with severe oligozoospermia (0.5-9 M/ml) frequently demonstrated functional reservoirs and binding-competent subpopulations comparable to those with significantly higher sperm counts (> 60 M/ml). These data suggest that the ZP2-bead matrix identifies a discrete molecular competency that is not represented by conventional semen thresholds, providing a functional assessment that transcends the descriptive limits of standard light microscopy.

Sperm bound to ZP2^39–154 show comparable morphology to sperm bound to native zonae pellucidae

Our data indicates that a specific fraction of sperm, originating from both normospermic or infertile participants, maintains the ability to bind to the N-terminal domain of ZP2. While clinical paradigms suggest that binding to the zona may serve as a surrogate for superior morphology [14–16], a systematic and rigorous quantification of this relationship has been historically limited by the paucity of human oocytes available for research [15, 17]. To address the limitations of these conventional methods and the scarcity of native oocytes, we tested the ZP2^39–154 to quantify whether this binding interaction specifically targets a morphologically superior subpopulation of sperm. To quantify the morphological features of sperm capable of binding to a zona matrix, we first compared the morphometric profiles of normospermic sperm bound to human oocytes against those bound to the ZP2^39–154. Under these criteria, a sperm was classified as morphologically ‘normal’ if it simultaneously met the thresholds for nucleus area (11.28–13.56 µm²) and flagellum length (40.10–47.95 μm). Our analysis of sperm bound to the ZP2^39–154 revealed no statistically significant differences with populations of sperm bound to native zonae, in either the nucleus area (12.46 ± 0.76 µm² vs. 12.51 ± 0.92 µm²; p = 0.856) or flagellum length (42.97 ± 2.25 µm² vs. 42.90 ± 2.93 µm²; p = 0.785). These data suggest that the recombinant ZP2^39–154 may support binding of a specific and morphologically consistent sperm subpopulation.

ZP2^39–154 identifies and enriches for normal sperm subpopulations

Raw semen samples from fertile individuals typically exhibit a baseline of only 4–5% normal morphology [11], a figure that is frequently even lower in samples from infertile individuals [7]. While standard density gradient centrifugation is estimated to eliminate portions of morphologically abnormal cells, the resulting population remains notably heterogeneous [18, 19]. Thus, as we identified a ZP2^39–154 binding-competent sperm subpopulation across all samples with defective spermiogenesis (Fig. 2A), we proceeded to investigate the specific morphological traits of the ZP2^39–154 bead-bound sperm from the 10 normospermic or the 12 infertile participants. Within the normospermic group, on average, 71.4 ± 15.2% of the sperm that successfully bound to the ZP2^39–154 met the physiological criteria of the standard nuclear area and flagellar length values defined by zonae-bound sperm (Fig. 3A and B). On the other end, sperm from men with defective spermiogenesis presented significantly larger and more variable nuclei compared to the normospermic controls (16.60 ± 4.09 µm² vs. 12.51 ± 0.92 µm²; p = 2.49 × 10^{− 76}), accompanied by a slightly, yet significantly shorter and more heterogeneous flagella (42.13 ± 5.86 μm vs. 42.90 ± 2.93 μm; p = 6.8 × 10^{− 4}). Yet, by applying the stringent numerical thresholds derived from the native zonae-bound sperm population, we found that ZP2^39–154 identifies subpopulations of morphologically normal sperm even in the defective spermiogenesis cohort, with 6–30% meeting the criteria for both nuclear area and flagellar length combined (Fig. 3A and B). While most bound sperm in the infertile cohort remained structurally atypical compared to the normal sperm bound to zonae, our assay demonstrates a clear capacity to enrich for a functionally and morphologically superior subpopulation.

Fig. 3.

Morphological heterogeneity of ZP2^39–154-bound sperm across normospermic and infertile participants. (A) Distribution of nucleus area for 22 individual participants, including 10 normospermic (Participants 1–10; blue) and 12 infertile individuals (Participants 11–22; red). Red dashed lines indicate physiological thresholds, representing the 5th and 95th percentiles of sperm bound to human zonae. Box plots represent the median and interquartile range (IQR), with whiskers extending to 1.5 x IQR. Individual data points (sperm) are overlaid as a jittered strip plot to visualize population density. (B) Same as in A, for the distribution of flagellum length

Discussion

Our study demonstrates that the recombinant ZP2^39–154 peptide may serve as a high-stringency selection tool, able to identify a functional subpopulation of sperm with superior binding competence and morphology across both normospermic and defective spermiogenesis groups. In addition, we show that while overall binding density is reduced in men with defective spermiogenesis, these individuals retain a reservoir of binding-competent sperm that cannot be predicted by conventional semen analysis.

The historical scarcity of human oocytes for research has severely limited our ability to characterize the morphological patterns of sperm capable of sperm-zona interactions with statistical rigor [15–17]. By demonstrating that sperm bound to recombinant ZP2^39–154 are morphologically comparable to those bound to native human zonae, we provide a validated, high-throughput alternative to traditional hemizona assays. This standardization is important for moving andrology from descriptive to functional. Beyond diagnosis, the ability of this assay to enrich the proportion of morphologically normal sperm from a baseline of 1–4% in raw semen to as high as 90% suggests its immediate utility as a sperm-processing technology for assisted reproduction.

A most striking finding of our study is the profound inter-individual heterogeneity in ZP2^39–154 binding efficiency among clinically normospermic men. Despite meeting all standard WHO reference ranges for concentration, motility, and morphology [7], these individuals exhibited nearly a four-fold variance in their functional capacity to adhere to the ZP2 N-terminus. This lack of correlation, where even a participant with superior concentration and motility demonstrated the lowest binding density, suggests that ZP2^39–154 binding measures a discrete molecular competency independent of conventional semen parameters. Consequently, relying solely on descriptive, population-level metrics may overlook significant functional deficiencies at the individual sperm level, reinforcing the need for assays that go beyond simple observation by light microscopy.

Another key finding of our analysis is that the ability to bind the ZP2^39–154 domain is not strictly reserved for sperm with normal morphological criteria. While the ZP2^39–154 peptide significantly enriches for morphologically normal sperm, a measurable fraction of sperm with structural abnormalities retained binding competence. Specifically, in the cohort with defective spermiogenesis, 70–94% of the sperm that successfully adhered to the ZP2^39–154 remained atypical according to the stringent thresholds derived from native zonae-bound populations. This suggests that the molecular machinery required for gamete recognition can remain functional even when the global processes of cellular remodeling and flagellar assembly are disrupted.

Nonetheless, our data reveals that men diagnosed with defective spermiogenesis still present subpopulations of functionally competent sperm. While these cohorts displayed a reduced global functional reservoir and faster depletion of binding-competent cells compared to controls, the ‘top-tier’ binding events in even the most severe cases of defective spermiogenesis frequently overlapped with the normospermic range. This suggests that the structural disruptions characteristic of defective spermiogenesis do not uniformly impair every cell in the ejaculate.

A translational implication of our study is the decoupling of visual morphology from binding capacity. Historically, clinical selection has relied on Kruger’s strict criteria, assuming that only sperm from semen with normal morphology are able to bind the zona and eventually fertilize an egg in vivo. However, we found that many sperm capable of ZP2^39–154 binding did not meet these rigid structural thresholds, yet they remained functionally competent for binding. This suggests that the molecular machinery required for zona recognition is preserved even in cases of compromised spermiogenesis. By using our ZP2^39–154 beads as a selection tool, we shifted the focus from subjective visual assessment to a physiological readout of binding competence, potentially expanding the pool of viable sperm available for assisted reproduction [20].

In conclusion, our findings establish the recombinant ZP2^39–154 peptide as a robust functional proxy for the human zona pellucida, enabling the identification and recovery of a physiologically superior sperm subpopulation that transcends the diagnostic limitations of standard semen analysis. This work serves as a compelling proof-of-concept, demonstrating the molecular heterogeneity that traditional semen analysis misses. Future research, involving larger and more diverse patient cohorts, is essential to correlate ZP2-bead selection density with clinical outcomes such as fertilization rates, blastocyst development, and live birth rates.

Abbreviations

CDF

Cumulative Distribution Function

HSA

Human Serum Albumin

HTF

Human Tubal Fluid

ICSI

Intracytoplasmic Sperm Injection

IQR

Interquartile Range

IVF

In Vitro Fertilization

OAT

Oligoasthenoteratozoospermia

PFA

Paraformaldehyde

SDS-PAGE

Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

WGA

Wheat Germ Agglutinin

WHO

World Health Organization

ZP

Zona Pellucida

Author contributions

MAA conceived the study, designed the experimental framework, performed the statistical analyses, and drafted the manuscript. MAAta coordinated patient recruitment and executed the bead-binding experiments. FAA, JA, and FC were responsible for participant recruitment and the contribution of clinical biospecimen samples. MAAbdulla, AS, MAAta, DH, MSEsteves and SG performed confocal microscopy acquisition and image processing. AS provided specialized assistance with high-resolution imaging. All authors have reviewed, refined, and approved the final version of the manuscript.

Funding

This work was supported by intramural funding from Sidra Medicine (Grant No. SDR400185) to Matteo Avella.

Data availability

All data generated or analyzed during this study are included within the published manuscript and its accompanying figures and tables.

Declarations

Ethics approval and consent to participate

This research was performed in accordance with the protocols approved by the Institutional Review Board at Sidra Medicine (IRB 1923874). Prior to their inclusion, all individuals involved in the study provided written informed consent.

Consent for publication

Every participant signed an informed consent for the publication of this study’s findings and all associated images.

Competing interests

The authors declare that they have no competing interests. There are no financial relationships, equity holdings, or consultancies that could be perceived as influencing the objectivity of this research.