Hydrogel-mEV-SKAP2: A promising biotherapeutic product for male fertility decline

Sperm quality refers to the functional characteristics of spermatozoa, encompassing their concentration, morphological normality, and progressive motility, which collectively determine their potential to achieve fertilization. Declines in sperm quality are generally termed as oligozoospermia, teratozoospermia, or asthenozoospermia. Multifaceted causes include complex interactions between diverse etiologies including environmental endocrine-disrupting chemicals, smoking, obesity, medical conditions/treatments (e.g., varicoceles, infections, chemotherapy), and genetic predisposition. Diminished sperm quality is associated with reduced fertilization rates, increased embryonic aneuploidy, and higher rates of early pregnancy loss, irrespective of chronological age. While overt symptoms like erectile dysfunction or anejaculation are easily recognizable, subclinical declines in sperm quality frequently develop before noticeable symptoms appear. This delayed manifestation may narrow the opportunity for effective intervention once fertility concerns arise. Critically, there is a lack of effective pharmacological intervention, particularly targeted biological agents, to reverse or substantially repair sperm quality.

Widely accepted treatment approaches for asthenoteratozoospermia encompass lifestyle modifications (e.g., smoking cessation, weight optimization), empirical medical therapies (e.g., antioxidant supplementation, hormonal treatments such as gonadotropins or selective estrogen receptor modulators), and surgical correction of identifiable anatomical abnormalities (notably varicocele repair). These interventions are often combined with assisted reproductive technology (ART), including intrauterine insemination, in vitro fertilization, and intracytoplasmic sperm injection, depending on different etiologies. However, it is worth noticing that treatment efficacy varies significantly among individuals, influenced by factors such as the severity of sperm dysfunction, the duration of infertility, and coexisting medical conditions.

Therefore, highly effective targeted biological agents for this condition are warranted. Previous studies have found that polytetrafluoroethylene (PTFE) can cause a decrease in sperm motility.1 Using a PTFE exposure model, SKAP2 was identified as a target that specifically binds to haploid sperm.2 Inhibition of SKAP2 was found to impair sperm head morphology, disrupt the “9 + 2” microtubule structure of sperm flagellum, cause acrosome loss, and consequently reduce sperm motility. Furthermore, in mice and human sperm samples, milk-derived extracellular vesicles (mEVs) were utilized as natural carriers to load functional SKAP2 protein (mEV-SKAP2). These vesicles were precisely delivered to damaged sperm via microinjection by efferent duct or in vitro co-incubation. The results showed a significant improvement in sperm motility, normalization of sperm morphology, and a reduced rate of DNA fragmentation. In vivo, the fertility of male mice receiving this treatment was substantially restored.

We aimed to develop a targeted biologic agent for asthenoteratozoospermia that not only repairs decreased sperm motility caused by diverse etiologies but also meets the demand for convenient use in household settings. To this end, we built upon the mEV-SKAP2 hydrogel, with manufacturing details as follows. Firstly, the SKAP2 gene fragment was ligated with the vector PEGX-6P-1 using a ligase enzyme. Following plasmid construction, the resultant plasmid served as a template for transforming competent BL21 (DE3) cells to induce protein expression. Secondly, the mEVs were loaded with SKAP2 through sonication using an ultrasonic system (Sonics & Materials, Newtown, CT). The mEVs and SKAP2 were mixed at a 1:1 (mass/mass) ratio in phosphate-buffered saline, with the final concentration of mEVs adjusted to 0.8 μg/μL. Sonication parameters were set at 20% amplitude, with 6 on/off cycles (10 s each) and a 2-min cooling interval between cycles. Thirdly, a hydrogel was prepared containing 4% trehalose, 2% carboxymethyl cellulose, and a final SKAP2 concentration of 20 μg/mL. This formulation has been validated in multiple sperm damage models, including lead exposure, phthalate exposure, heat-induced injury, and aging-related impairment. As an innovative intervention for male reproductive health, this hydrogel application holds significant potential, providing the first targeted repair strategy for asthenoteratozoospermia in clinical practices.

The study results of this agent are of great value in clinical guidance, including offering precise targeted treatment options for asthenoteratozoospermia patients and optimizing the efficacy of fertility interventions. Furthermore, the mEV-SKAP2 hydrogel application has offered a clinical solution for family reproductive health management, particularly for couples planning ART. By restoring sperm motility and morphology, it may further enhance success rates for both natural conception and assisted reproduction. We believe this targeted biological agent will break through the limitations of traditional treatments for asthenoteratozoospermia that rely on empirical interventions and lack specific targeted approaches.