Human asthenozoospermia: Update on genetic causes, patient management, and clinical strategies

Abstract

Background

In mammals, sperm fertilization potential relies on efficient progression within the female genital tract to reach and fertilize the oocyte. This fundamental property is supported by the flagellum, an evolutionarily conserved organelle, which contains dynein motor proteins that provide the mechanical force for sperm propulsion and motility. Primary motility of the sperm cells is acquired during their transit through the epididymis and hyperactivated motility is acquired throughout the journey in the female genital tract by a process called capacitation. These activation processes rely on the micro‐environment of the genital tracts. In particular, during capacitation, a panoply of ion transporters located at the surface of the sperm cells mediate complex ion exchanges, which induce an increase in plasma membrane fluidity, the alkalinization of the cytoplasm and protein phosphorylation cascades that are compulsory for sperm hyperactivation and fertilization potential. As a consequence, both structural and functional defects of the sperm flagellum can affect sperm motility, resulting in asthenozoospermia, which constitutes the most predominant pathological condition associated with human male infertility.

Objectives

Herein, we have performed a literature review to provide a comprehensive description of the recent advances in the genetics of human asthenozoospermia.

Results and Discussion

We describe the currently knowledge on gene mutations that affect sperm morphology and motility, namely, asthenoteratozoospermia; we also specify the gene mutations that exclusively affect sperm function and activation, resulting in functional asthenozoospermia. We discuss the benefit of this knowledge for patient and couple management, in terms of genetic counselling and diagnosis of male infertility as a sole phenotype or in association with ciliary defects. Last, we discuss the current strategies that have been initiated for the development of potential therapeutical and contraceptive strategies targeting genes that are essential for sperm function and activation.

1. INTRODUCTION

In mammals, spermatozoa are highly specialized cells designed with the unique capacity to exit the body and fulfill their reproductive functions within another body. The development and functionality of such exceptional cells involve a series of intricate processes occurring within the male and female genital tracts, many of which are directly related to the acquisition of an essential feature: their capacity to move. ¹ , ²

The first of these processes occurs during the final stages of spermatogenesis, within the testis, where the developing germ cells undergo profound morphological changes, including their compaction in a hydrodynamic shape and the assembly of their flagellum, which constitutes the essential organelle sustaining their progression through the female genital tract to reach the oocyte. ³

Once produced within the testis and structurally differentiated, spermatozoa transit through different sections of the epididymis, and face a dynamic shift in the composition of the epididymal milieu that triggers signaling and energy metabolic pathways enabling the acquisition of their primary motility. ⁴ , ⁵ , ⁶ In particular, important signaling pathways regulating the balance between protein phosphorylation and dephosphorylation are associated with the acquisition of primary motility. ⁶ The sperm cells primed within the epididymal milieu are then stored in the caudal region of the epididymis where they remain in a quiescent state, until ejaculation.

Upon copulation and ejaculation within the female reproductive tract, sperm cells undergo an ultimate functional transformation, called capacitation, which confers them a state of hypermotility, characterized by a higher flagellar amplitude and lower beating frequency; such activation process enables their ascendence through the oviduct to reach and fertilize the oocyte. ⁷ Sperm capacitation is induced in response to stimuli from the female genital tract milieu and involves, in particular, a panoply of transmembrane ion channels and exchangers, which trigger downstream signaling pathways supporting the acquisition of novel biochemical and electrophysiological characteristics. ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹² In particular, capacitated sperm cells show cytoplasmatic alkalinization, increased membrane polarization and fluidity, and intense flagellar protein phosphorylation through the activation of protein kinase A (PKA)‐ and Calmodulin‐dependent kinase pathways. ¹³ , ¹⁴ , ¹⁵

As a consequence, the deficiency of any of the above intricate differentiation and maturation processes is expected to impact sperm motility. Hence, asthenozoospermia, defined as the reduction or the absence of sperm motility, constitutes the predominant pathological condition associated with human male infertility. Asthenozoospermia is diagnosed in 82% of infertile men with variable degrees of severity; it is often associated with quantitative and/or morphological defects (oligo‐ and/or teratozoospermia), but also observed in 19% of the cases as a pure phenotype. ¹⁶ The causes of asthenozoospermia are multiple but in most cases, it remains unexplained. ¹⁷ Among, the most common etiological factors of human asthenozoospermia, cryptorchidism (undescended testicle), varicocoele (dilation of a vein in the spermatic cord), genital infections, hormonal deregulation, exposure to toxic substances, certain treatments such as chemotherapy, life style, and genetic factors, have been reported. In this review, we will focus on the genetic causes associated with human asthenozoospermia, which only account for a minority of cases.

During the last decades, with the advances in DNA sequencing technologies, the exploration of pathogenic gene variants associated with male infertility has become easier, in terms of practical and financial aspects. In relation to asthenozoospermia, many of these recent advances have identified genes that are involved in flagellum formation (for reviews, see refs. ¹⁸ , ¹⁹ ); fewer gene variants were identified in transmembrane ion channels, ⁸ metabolic enzymes, and signaling pathways. ⁶ Altogether, the identified genes are related to many of the above‐described processes that are involved in the biogenesis of sperm flagellum together with the acquisition, maintenance, and hyperactivation of sperm motility.

Herein, we have performed a literature review to provide a comprehensive description of the recent advances in the genetics of human isolated asthenozoospermia. We describe the currently known gene mutations that affect sperm morphology and motility in humans, namely, asthenoteratozoospermia, together with gene mutations that especially affect sperm function, resulting in functional asthenozoospermia. We also discuss the clinical translation of these genetic findings for patient and couple management, in terms of counselling and diagnosis of male infertility as a sole phenotype or in association with ciliary defects. Lastly, based on this gain of knowledge, we discuss the current strategies that have been initiated for the development of potential therapeutical and contraceptive strategies targeting proteins essential for sperm functional activation.

2. GENETICS OF ISOLATED ASTHENOZOOSPERMIA DUE TO SEVERE SPERM MORPHOLOGICAL DEFECTS

2.1. Background: structure of the sperm flagellum

Spermatozoa constitute one of the most highly differentiated cell types of the body and comprise two main compartments: the head and the tail, each fulfilling specific functions that are essential for fertilization. The head contains the haploid paternal DNA, highly compacted within the nucleus, and the acrosome, required to digest the zona pellucida surrounding the oocyte. ²⁰ The flagellum, also called tail, corresponds to a peculiar type of motile cilia, which are organelles that protrude at the surface of nearly all quiescent mammalian cell types ²¹ and are built on a microtubule‐based cytoskeleton, called the axoneme. Their structural distinctions allowed a simplistic functional classification in two principal types, namely, motile (9 + 2 pattern) and non‐motile cilia (9 + 0 pattern). In motile cilia, the axoneme is composed of nine peripheral microtubules doublets connected to a central pair (CP) of microtubules by T‐shaped extensions called radial spokes (RSs) (Figure 1). ²² One microtubule of each doublet also harbors inner‐ and outer‐dynein arms (IDA and ODA), which are multi‐protein complexes acting as ATP‐dependent motor elements and driving ciliary beating ²³ , ²⁴ (Figure 1). Motile cilia also contain, nexin‒dynein regulatory complexes (N‐DRC), which connect the peripheral doublets to one another and play an important role in coordinating dynein arm activity. ²⁵ Such cilia are present as multiple entities on the surface of epithelial cells lining the respiratory tract, the oviduct and brain ventricles, where they regulate fluid flow and clearance. ²⁶ Among motile cilia, the sperm flagellum is unique as it harbors a set of specific peri‐axonemal structures, which are the mitochondrial sheath, the outer dense fibers and the fibrous sheath, acting as a scaffold for signaling molecules and glycolytic enzymes ²⁷ , ²⁸ (Figure 1).

FIGURE 1.

Structure of sperm flagella and motile cilia (9 + 2). (A) Schematic representation of the structure of cellular appendages such as the flagellum (on the left) and motile cilia (on the right), which share the axoneme as a common cytoskeletal backbone. A transversal axonemal section is depicted in the middle and shows the classical 9 + 2 microtubular organisation and the additional protein complexes regulating its bending. In addition, the centriole is show at the base of motile cilia, while spermatozoa display unique periaxonemal structures of the flagellum, namely, the annulus, the outer dense fibers, and the fibrous sheath. (B) Structure of the sperm flagellum of human spermatozoa visualized by transmission electron microscopy (TEM). Upper panel: longitudinal section showing the annulus at the junction of the midpiece (with the mitochondria) and the principal piece (with the fibrous sheath). Lower panel: transversal section through the midpiece showing the mitochondria, the outer dense fibers, and axonemal microtubules.

Asthenozoospermia is evidenced in many infertile men with no other symptomatology (i.e., isolated asthenozoospermia) and overall, it constitutes the most frequent sperm defect observed in infertile men. ¹⁶ Accordingly, asthenozoospermia can be associated with a wide range of sperm tail morphological defects, such as abnormal mitochondrial sheath, abnormal head‐tail or midpiece (MP)‒principal piece (PP) junction, abnormal tail bending or coiling, irregular tail caliber or abnormal residual cytoplasm. ²⁹ , ³⁰ Here, we will describe the genetic causes associated with severe cases of asthenozoospermia, namely, “multiple morphological abnormalities of flagella” (MMAF) and the genetic causes so far associated with mild morphological defects. All causal genes listed below have been exclusively identified in asthenozoospermic men with no other symptomatology (i.e., isolated asthenozoospermia); these are distinct from causal genes mutations associated with syndromic asthenozoospermia (see Chapters 4.2 and 4.3).

2.2. Multiple morphological anomalies of flagella—MMAF phenotype

The most severe form of sperm tail morphological defects consists in the total or near absence of the tail, a phenotype called “short tails” or “stump tails” ³¹ , ³² , ³³ , ³⁴ and more recently termed as MMAF, which is associated with a condition of extreme asthenozoospermia with nearly zero progressive sperm motility. ³⁵ MMAF is characterized by a mosaic of sperm cells with absent, short, irregular, and coiled flagellum, that can be easily evidenced during semen routine analysis by means of optic microscopy (Figure 2A). At the ultra‐structural level, evaluated by transmission electron microscopy, MMAF is associated with a severe disorganization of both axonemal and peri‐axonemal structures and has been exhaustively described (see refs. ³¹ , ³² , ³⁶ , ³⁷ ) (Figure 2A). In particular, the sperm heads are often attached to large cytoplasmic bags with unassembled microtubule and peri‐axonemal elements in due place of the flagella. When present, the flagellum shows a disorganized axonemal structure lacking the CP and/or peripheral microtubule doublets, and the dynein arms can also be absent (for reviews, see refs. ³⁶ , ³⁷ ). In addition, the longitudinal columns and the fibrous sheath are frequently abnormal; the latter being evidenced with increased thickening, which led to an additional appellation for this phenotype, namely, “dysplasia of the fibrous sheath.” ³¹ , ³² , ³⁸ , ³⁹

FIGURE 2.

Phenotype and genetics of asthenoteratozoospermia, with a focus on the multiple morphological anomalies of the sperm flagella (MMAF) phenotype. (A) Sperm structural and ultrastructural defects observed in the ejaculate of patients showing the MMAF phenotype, by means of optical microscopy and transmission electron microscopy (TEM), respectively. Spermatozoa mostly display absent, short, coiled, abnormal calibre flagella, as well as impaired mitochondrial sheath and excessive retained cytoplasm. (B) Causal genes, listed by protein function, associated to non‐syndromic asthenoteratozoospermia in humans, namely, the MMAF phenotype—either validated (black) or putative (gray) mutations—or other sperm morphological defects (blue).

Although morphological and ultra‐structural defects associated with MMAF condition have been thoroughly described for several decades, ³¹ , ³² , ³⁴ , ⁴⁰ , ⁴¹ , ⁴² the genetic etiology of this phenotype was only recently investigated. Such task was initiated by work performed in 2014 by Khelifa et al., who described DNAH1, encoding for an axonemal dynein of the IDAs preferentially expressed in the testis, as the first MMAF‐associated gene inducing male infertility. ³⁵ Since then, the development of next‐generation sequencing and the increasing number of phenotypically well‐characterized cohorts from different ethnicities, contributed to a burst in the identification of MMAF‐associated genes. Remarkably, the number of MMAF‐identified genes nearly reached 40 in less than 10 years (Figure 2B) and accounts for 50% to over 60% of the MMAF cases in different cohorts. ⁴³ , ⁴⁴ Several reviews in the last years have provided comprehensive description of the genetic factors associated with the MMAF phenotype (for reviews, see refs. ¹⁸ , ³⁷ , ⁴⁵ , ⁴⁶ , ⁴⁷ ). In Figure 2B, an update of the non‐syndromic MMAF genes so far identified in humans is provided. ⁴⁸ , ⁴⁹ , ⁵⁰ , ⁵¹ , ⁵² , ⁵³ , ⁵⁴ , ⁵⁵ , ⁵⁶ , ⁵⁷ Table S1 provides the mutations type and the allele frequency for each causal gene.

As one could expect, most of the identified MMAF‐associated genes encode proteins related to axonemal and peri‐axonemal structures of the sperm flagellum (for reviews, see refs. ¹⁸ , ³⁷ , ⁴⁵ , ⁴⁶ , ⁴⁷ ). In particular, numerous mutations in gene coding for components of the inner and outer dynein arms and their associated axonemal complexes were identified ( CFAP57 , CFAP61 , CFAP65 , CFAP70 , CFAP91 , CFAP135 , CFAP206 , CFAP251 , DNAH12 , DNAH17 , DNHD1 , LRRC23 , and ZMYND12 ) (Figure 2B). The second main category of genes identified with mutations in MMAF patients is related to the processes of intraflagellar transport (IFT) and assembly of axonemal complex ( CCDC38 , CFAP53 , CFAP58 , TTC21A , TTC29 , and WDR19 ) (Figure 2B). IFT is a highly specialized molecular motor‐driven process, which ensures the selective transport of proteins from the proximal to the distal part of the growing organelle, and its importance for cilia is well established. ⁵⁸ Recent work on IFT knockout (KO) mouse models, ⁵⁹ , ⁶⁰ , ⁶¹ , ⁶² together with the advances made in the genetics of the MMAF phenotype, clearly confirms the essential role of IFT for the biogenesis of mammalian sperm flagellum. In addition, consistently with the importance of centrioles, which are essential to initiate the assembly of the axoneme in both cilia and flagella, several MMAF genes encode centrioles‐associated proteins ( CEP128 and DZIP1 ) (Figure 2B). A few additional MMAF genes code for components of peri‐axonemal structures ( AKAP3‐4 , FSIP2 , and ODF2 ) and for enzymatic proteins such as the deubiquitinating enzyme USP26 and the Ser/Thr kinase STK33. Lastly, if most of the MMAF proteins correspond to well‐defined components of the core axoneme and peri‐axonemal structures, the exact functions and molecular mechanisms of a substantial proportion of proteins remain unclear (ARMC3, CCDC9, CCDC34, CCDC146, and RABL2B) (Figure 2B); such genes constitute a valuable source to decipher the cellular and molecular mechanisms governing the assembly, stabilization, and function of mammalian sperm flagellum.

2.3. Sperm annulus and other flagellar defects

2.3.1. Mutations in genes encoding for components of the sperm annulus

The annulus is an electron‐dense ring structure located at the junction between the MP and the PP (Figure 1). It is mainly composed of Septin polymers (SEPT2, 4, 6, 7, and 12) and the Testis Anion Transporter 1 (SLC26A8). ⁶³ , ⁶⁴ The contribution of the annulus in the organization of the flagellar MP is established based on the study of mouse models, but its precise function in the process of sperm flagellum biogenesis is not well understood yet. ⁶³

In humans, bi‐allelic mutations in SEPT4 ⁶⁵ and SLC26A8 ⁶⁶ were shown to result in asthenozoospermia with a complete loss of the annulus structure and a thinning of the MP/PP junction (Figure 2B). The mitochondrial sheath was also severely disorganized and almost absent in patients carrying mutations in SLC26A8. Frequent bending of the sperm flagella at the MP/PP junction was also associated with SEPT4 mutations. Most of these observations were confirmed in mouse models deficient for these proteins, ⁶⁷ , ⁶⁸ , ⁶⁹ supporting the importance of SEPT4 and SLC26A8 for the integrity of the annulus. To note, despite severe asthenozoospermia, patients with SEPT4 and SLC26A8 bi‐allelic mutations were able to conceive by means of in vitro fertilization procedures (intracytoplasmic sperm injection, ICSI). ⁶⁵ , ⁶⁶

Heterozygous mutations in genes encoding for components of the annulus have also been associated with asthenozoospermia and male infertility. This is particularly the case for the SEPT12 c.589G>A, p.Asp197Asn mutation, which causes oligo‐asthenozoospermia. ⁷⁰ This mutation affects the GTP‐binding domain of the protein and is likely acting with a dominant negative effect, preventing the formation of the Septin polymer and precluding the formation of the entire annulus structure. In this case, spermatozoa from the patient also exhibited loss of the annulus, thinning of the MP and flagellum bending at the MP/PP junction, which are overall characteristics of annulus deficiency ⁷⁰ , ⁷¹ (Figure 2B). Heterozygous mutations in SLC26A8 were also reported to be associated with asthenozoospermia and mild flagellar defects. ⁷² In vivo, these mutations caused a reduction in SLC26A8 protein amount in spermatozoa from the patients. In vitro, the mutations were also shown to induce protein degradation and reduced SLC26A8 and CFTR protein amounts, precluding physical interaction between the two proteins. ⁷¹ By contrast, another study failed to confirm the pathogenicity of heterozygous SLC26A8 mutations in asthenoteratozoospermic patients. ⁷³ These observations highlight the importance of considering each type of mutation independently; the consequences of different mutations in the same gene being likely to vary depending on the type of mutation (e.g., loss of function vs. dominant negative effect) and overall, the location of the mutation site (functional protein domain).

2.3.2. Mutations in genes encoding for axonemal components

As described above, most mutations in genes coding for axonemal proteins result in a MMAF phenotype. However, in certain cases, the phenotype is less severe and falls into the wider category of asthenoteratozoospermia.

This is, for example, the case of impairing Tektin‐T, a spermatozoa‐specific Tektin involved in microtubule assembly and stability. A heterozygous mutation was found in a patient with asthenozoospermia and displaying from 15% to 40% of spermatozoa with neck angulation, irregular arrangement of the mitochondria ultra‐structural defects of the axoneme ⁷⁴ (Figure 2B).

2.3.3. Mutations in genes involved in mitochondrial function

Mutations in ARMC12 , encoding for a protein identified in the mitochondrial peripheral membrane in mice, ⁷⁵ were implicated in humans as a cause of sperm MP defects and asthenozoospermia. ⁷⁶ Hence, bi‐allelic mutations of ARMC12 were identified in three patients and associated with the near absence of mitochondria and MP, as well as a folding of sperm flagella leading to axonemal microtubules dispersion ⁷⁶ (Figure 2B).

2.3.4. Mutations in genes encoding proteins with functional properties

Regulators of gene transcription

Mutations in genes involved in mRNA regulation, TAF7L , and mRNA stability, TENT5D , were also identified in asthenozoospermia associated with mild flagellar morphological defects (Figure 2B). Both TAF7L and TENT5D are X‐linked genes with testis‐specific isoforms expressed in sperm cells. TENT5D mutations mainly result in defects located at the sperm head (multiple, isolated heads, and DNA fragmentation) ⁷⁷ , ⁷⁸ , ⁷⁹ and TAF7L mutations induce DNA compaction defects. ⁸⁰ In both cases, some flagellar defects were also observed: multiple and coiled flagella (TENT5D) and irregular caliber (TENT5D and TAF7L). Lastly, a mutation in EIFAG1 , ⁸¹ encoding for a protein that regulates the recruitment of mRNAs to ribosomes, was also associated with severe asthenozoospermia. Spermatozoa from the patient showed abnormal mitochondrial sheath and mitochondria, abnormal fibrous sheath, as well as a loss of the CP and microtubules doublets (DMT) (Figure 2B). Altogether, these studies indicate that alterations in genes involved in the regulation and recruitment of mRNAs during spermatogenesis can subsequently lead to flagellar defects causing asthenozoospermia, possibly by deregulating the expression of genes that are essential for flagellum biogenesis.

Ion channels and transporters

Biallelic missense variants in KCTD19 , encoding the potassium channel tetramerization domain containing 19, were also identified as causative of male infertility because of oligoasthenoteratozoospermia. ⁸² Although no structural function is established for KCTD19 protein, its mutations are associated with severe anomalies of the sperm head (enlargement of the head, decondensed nucleus, and aneuploidy). The sperm cells also presented multiple and abnormally sized flagella or, in some cases, lack flagella, which altogether likely account for the observed asthenozoospermia ⁸² (Figure 2B).

In addition, a heterozygous mutation was identified in SLC26A3 gene, member of the SLC26 family of anion exchangers (Figure 2B). Patients showed asthenozoospermia with a low percentage of morphologically normal spermatozoa, even though the authors did not specify the details of the structural anomalies (ref. PMID: 29079751). Similarly to SLC26A8, SLC26A3 physiologically cooperates with the CFTR chloride channel and the described mutation was shown to impact the functional activity of this latter without affecting its expression nor interaction with SLC26A3.

3. GENETICS OF ISOLATED ASTHENOZOOSPERMIA DUE TO SPERM FUNCTIONAL DEFECTS

3.1. Background: sperm functional maturation

Sperm progression within the female genital tract to reach and fertilize the oocyte relies on the integrity of the flagellum together with functional activation processes, which will confer spermatozoa a set of specific cytoplasmic and membranous hallmarks that are essential for fertilization potential. It is well established that these functional activation processes rely on the micro‐environment of both the male and female genital tracts (Figure 3A). In particular, during capacitation within the female genital tract, a panoply of ion transporters located at the surface of the sperm cells mediate complex ion exchanges, which induce an increase in plasma membrane fluidity, alkalinization of the cytoplasm, together with cAMP‐ and calcium‐dependent protein phosphorylation cascades that are compulsory for sperm hyperactivation and fertilization potential ¹³ (Figure 3A). In addition, sperm cells must adapt their metabolic status to the nutrients available during their journey through the male and female genital tracts. Relative to somatic cells, spermatozoa also require exceptionally high amounts of ATP to sustain the mechanical and signaling processes necessary for sperm motility and hyperactivation ⁸³ (Figure 3A); their bioenergetics have been exhaustively investigated through the prism of glycolysis and mitochondrial oxidative phosphorylation. ⁸⁴ , ⁸⁵ It is thus predictable that, similar to structural and morphological defects of the flagella, the deregulation of the signaling and/or energy metabolic pathways in sperm cells will result in asthenozoospermia. However, the genetic etiology of such phenotype of functional asthenozoospermia remains scarcely documented, probably because of the difficulty to discriminate purely functional defects from mixed origin defects in patients. In recent years, the genetic analyses of a few cases of patients mainly characterized by functional asthenozoospermia (i.e., lack of major morphological defects) allowed the identification of a dozen of causative gene mutations, which will be described in this chapter. Figure 3B summarizes the subcellular localization and function of the proteins encoded by these genes; the mutation types and frequencies are listed in Table S2.

FIGURE 3.

Physiological relevance of sperm functional activation and genetics of functional asthenozoospermia. (A) As a response to external stimuli within the male and female genital tracts, spermatozoa undergo a series of molecular changes mainly involving ion fluxes and phosphorylation pathways essential for the acquisition of motility and the fertilization potential. (B) Subcellular localization of the causal genes associated to human functional asthenozoospermia.

3.2. Defects in signaling pathways regulating sperm motility and capacitation

In parallel to sperm flagellar defects, asthenozoospermia can derive from exclusive functional defects. In these cases, some patients may also display little morphological and structural defects of the flagella, which although remain limited as compared to the extreme phenotypes we discussed above (i.e., MMAF phenotype or annulus defects). In particular, flagellar coiling or bending together with sperm membrane fragility can constitute secondary effects induced by osmotic stress and/or deregulation of ion channels and transporters. ⁸⁶ , ⁸⁷ , ⁸⁸

The so far identified causal genes mainly code for ion channels, signaling proteins and metabolic actors in consistence with the importance of signal transduction and energy production for the acquisition of sperm motility and capacitation.

3.2.1. Mutations in genes encoding for ion channels and transporters

Calcium currents regulate several aspects of sperm maturation and are mediated by many actors such as the multi‐subunit cation channel CATSPER, polycystic kidney disease (PKD) and voltage‐dependent anion selective (VDAC). ⁸ , ⁸⁹

CATSPER2 was the first ion channel‐encoding gene whose deletions were associated with syndromic human male infertility: congenital dyserythropoietic anemia type I and deafness‐infertility syndrome in French and Iranian men, respectively. ⁹⁰ , ⁹¹ A heterozygous CATSPER2 mutation was described for a Chinese patient with normal sperm parameters but altered sperm hyperactivation and penetration in the egg zona pellucida. ⁹² Iranian men affected by oligoasthenoteratozoospermia were also identified with missense mutations in CATSPER1 . ⁹³ Lastly, a pathogenic in‐frame deletion in the auxiliary subunit CATSPERε was detected in a patient with idiopathic infertility ⁹⁴ (Figure 3B).

In addition to CATSPER mutations, pathogenic variants in PKD1‐2 were proposed for autosomal dominant polycystic kidney disease patients presenting oligoasthenozoospermia. ⁹⁵ Missense variants and indels in VDAC3 were also identified in asthenozoospermic patients ⁹⁶ and abnormal hypermethylation profile of VDAC2 promoter was observed in cases of idiopathic asthenozoospermia ⁹⁷ (Figure 3B).

As mentioned in Chapter 2.3, mutations were identified in the anion transporters SLC26A3 and SLC26A8 genes. SLC26A8 displays a specific expression in the testis while SLC26A3 is expressed in the gastrointestinal and the male reproductive tract. For both SLC26A3 and SLC26A8 genes, heterozygous mutations were identified in asthenozoospermic patients with moderate morphological defects of sperm flagella ⁷² , ⁹⁸ (Figure 3B). These mutations were shown to impact SLC26/CFTR physical and functional cooperation, therefore likely altering ion homeostasis and the functional activation of sperm motility. ⁷² , ⁹⁸ These pathogenic mutations are in line with the male sterility phenotype observed in the corresponding KO mouse models and the absence of PKA‐dependent phosphorylation events when KO spermatozoa were capacitated in vitro. ⁹⁹ , ¹⁰⁰

Additional ion channels were recently associated with human infertility. The spermatozoa‐specific cation/proton antiporter SLC9C1/sNHE was described with a homozygous splicing variant affecting the cyclic nucleotide‐binding domain in an asthenozoospermic patient from sub‐Saharan Africa (Guinea) ¹⁰¹ (Figure 3B). A homozygous missense variant in the testis‐specific and pH‐sensitive K+ channel hSLO3 was also recently identified in a Chinese patient affected by asthenozoospermia with impaired acrosome structure, mitochondrial dysfunction, and membrane potential alteration during capacitation ¹⁰² (Figure 3B).

For a detailed description of the above mutations in genes encoding for ion channels and transporters, and additional genetic information provided from KO mouse models, see recent reviews, refs. ⁸ , ⁸⁹

3.2.2. Mutations in genes encoding proteins/enzymes involved in sperm signaling pathways

The signal transduction pathways of sperm cells, in particular those mediated by cAMP and calcium, have been mostly characterized by means of KO mouse models and the use of inhibitors of key enzymes involved in those pathways. In humans, a frameshift variant in ADCY10 , coding for the soluble adenylyl cyclase (sAC) responsible for the activation of the cAMP‐PKA, was detected in two related patients with asthenozoospermia (Figure 3B). These two patients also displayed a few morphological defects together with absorptive hypercalciuria. ¹⁰³ Although the variant was not validated at the transcript and protein levels using semen samples from the patients, their sperm motility defects were rescued by incubation with a cAMP permeant analog, thus strongly supporting the pathogenicity of the identified variants in ADCY10. Very recently, an infertile patient displaying functional asthenozoospermia and in vitro capacitation failure was described with a homozygous splicing mutation in IQCH , which encodes a Calmodulin‐associated protein potentially involved in sperm motility regulation ¹⁰⁴ (Figure 3B).

3.2.3. Mutations in genes involved in cytoskeletal dynamics

As discussed above, mutations in cytoskeletal or axonemal proteins usually determine severe forms of teratozoospermia that dramatically alter sperm flagellum structure and prevent proper sperm motility. In some cases, mutations in such genes were observed in asthenozoospermic individuals with a very limited proportion of spermatozoa showing morphological defects of the flagella. This is the case of TEKT3 , for example, which codes for the mitochondrial‐associated protein Tektin 3, and for which bi‐allelic mutations were described in oligoasthenozoospermic patients without sperm flagellar defects but acrosomal hypoplasia. ¹⁰⁵ Similarly, mutations in KIF9 were associated with isolated functional asthenozoospermia in humans ¹⁰⁶ and the phenotype was confirmed by a KO mouse model showing an asymmetric waveform pattern of sperm motility. ¹⁰⁷ Lastly, homozygous nonsense variants in IQUB , encoding a protein with IQ motif and a ubiquitin‐like domain, were identified in a patient with immotile spermatozoa. ¹⁰⁸ This patient also showed defective RS, which led to the hypothesis of the existence of an IQUB/Calmodulin/RSPH3/ERK protein network required for proper assembly of the axonemal RS complex ¹⁰⁸ (Figure 3B).

3.2.4. Additional clues from mutant mouse models—signaling pathways

Additional candidate genes for human functional asthenozoospermia can be suggested from studies on KO mouse models. Several gene invalidations were performed in mice to test their impact on male infertility. In particular, mice models with mutations in genes encoding for ion channels ( CatSperδ , ¹⁰⁹ Nha1‐2 , ¹¹⁰ Pmca4 , ¹¹¹ Cnnm4 , ¹¹² and Atp1a4 ¹¹³ , ¹¹⁴ ), signaling proteins ( Gsk3 , ¹¹⁵ Pp1c , ¹¹⁶ Pp1γ1‐2 , ¹¹⁷ calcineurin subunits , ¹¹⁸ PkaCα , ¹¹⁹ and Iqcf1 ¹²⁰ ), regulators of post‐translational modifications ( Ttll3‐8 ¹²¹ and αTAT1 ¹²² ), and regulators of oxidative stress ( Gpx4 ¹²³ ) have all been reported with a detrimental impact on sperm motility. Genetic analyses by whole exome sequencing of asthenozoospermic individuals are required to confirm the involvement of those genes in the regulation of sperm motility and capacitation in humans.

3.3. Defects in sperm energy metabolic pathways

Sperm motility is a highly energy‐demanding process. Spermatozoa are equipped with several metabolic pathways ensuring the production of sufficient energy levels to support their progression within the female genital tract. Although there is increasing evidence suggesting that impairment of sperm energy metabolic pathways can cause functional asthenozoospermia, to date, only a few causal gene mutations have been identified and formally validated in humans.

3.3.1. Mutation in sperm mitochondrial genome

Glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) have long been considered as the main energy‐producing pathways in human sperm cells. ¹²⁴ OXPHOS consists of successive redox reactions through the electron transport chain (ETC) coupled to ATP synthesis. ETC includes five enzymatic complexes, located in the inner mitochondrial membrane. The protein subunits constituting these complexes are encoded by either genomic DNA or mitochondrial DNA (mtDNA). Thus, large‐scale deletions in mtDNA have been pointed out as risk factors for asthenozoospermia. This includes the frequent 4977‐bp deletion as well as 4866, 7345, 7436, and 7599‐bp deletions, which correspond to the removal of 30%‒45% of the mitochondrial genome. ¹²⁵ , ¹²⁶ , ¹²⁷ Importantly, these deletions result in the complete loss or truncation of crucial genes encoding for ETC protein complexes including MT‐ND3 , MT‐ND4 , MT‐ND4L , MT‐ND5 , MT‐ND6 (complex I subunits), MT‐CYB (complex III subunit), MT‐COIII (complex IV subunit), MT‐ATPase6 , and MT‐ATPase8 (complex V subunits). These massive genome deletions are likely to impact OXPHOS and induce energy metabolism defects. As a matter of fact, several reports indicate the association between mtDNA large‐scale deletions and low sperm motility. ¹²⁵ , ¹²⁷ , ¹²⁸ Moreover, these mutations were found at higher frequency in asthenozoospermic patients. ¹²⁶ , ¹²⁷ , ¹²⁹ , ¹³⁰

Besides these large‐scale deletions, several point mutations have been directly identified in mtDNA of asthenozoospermic patients. The m.11696G>A and m.11994C>T variants in the MT‐ND4 gene were associated with decreased sperm motility in infertile patients. ¹³¹ , ¹³² In addition, several mutations were identified in the mitochondrial genes encoding complex IV subunits, namely MT‐COI , ¹³³ , ¹³⁴ , ¹³⁵ MT‐COII , ¹³⁶ and MT‐COIII ¹³⁷ , ¹³⁸ ; all these mutations were found in asthenozoospermic patients, but not in normozoospermic infertile men nor in fertile men (Figure 3B).

3.3.2. Mutations in genes involved in energy production pathways

Bi‐allelic variants in AK9 were identified in five unrelated patients exhibiting asthenozoospermia without any structural defects of sperm flagella ¹³⁹ (Figure 3B). AK9 is an adenylate kinase predominantly expressed in the testis, which catalyzes the interconversion of two molecules of ADP into one molecule of ATP and one molecule of AMP (2 ADP ↔ 1 ATP + 1 AMP). Thus, AK9 plays an important role in the homeostasis of sperm nucleotides. A metabolomic analysis revealed a decrease in ATP levels in sperm cells from patients carrying mutations in AK9. Additionally, the levels of glycolytic intermediates were also reduced, indicating abnormal glycolysis in the spermatozoa from AK9‐mutated patients. ¹³⁹ This hypothesis was further confirmed by the characterization of the Ak9‐KO mouse model, which exhibited reduced sperm motility, decreased ATP levels and glycolysis‐related metabolites. ¹³⁹

A heterozygous mutation in GALNTL5 (polypeptide N‐acetylgalactosaminyltransferase‐like protein 5) was identified in an asthenozoospermic patient ¹⁴⁰ (Figure 3B). GALNTL5 is specifically expressed in the testis of humans and mice. In mice, heterozygous mutation of Galntl5 impaired sperm motility, decreased the level of some glycolytic enzymes, disrupted protein loading to the acrosome, and induced aberrant localization of the ubiquitin‒proteasome machinery. ¹⁴⁰

Last, a homozygous missense variant (c.1097G>A) in GFPT2 was described in three infertile patients from two related consanguineous families presenting severe asthenozoospermia and teratozoospermia (Figure 3B). This variant was subsequently identified at the homozygous status in an infertile patient with idiopathic asthenozoospermia. ¹⁴¹ GFPT2 encodes a glutamine‐fructose‐6‐phosphate transaminase, responsible for the first reaction of the hexosamine biosynthesis pathway. This pathway generates important substrates for post‐translational modifications of proteins (glycosylation processes). Because fructose‐6‐phosphate is the first substrate of the hexosamine biosynthesis pathway and one of the first products of glycolysis, the GFPT2 pathway is also closely associated with glucose metabolism. In this case, the patient was reported to have poor sperm morphology but no proper spermocytograms were provided, precluding a formal categorization of the patient (asthenoteratozoospermia vs. functional asthenozoospermia).

3.3.3. Additional clues from mutant mouse models—sperm energy metabolism

Several pieces of evidence arising from mouse models suggest additional candidate genes responsible for energy metabolism defects in the context of functional asthenozoospermia. Cyt cT encodes the testis‐specific isoform of cytochrome c, an electron shuttle between complex III and IV of the ETC. Although Cyt cT ^−/− mice were fertile in vivo, their spermatozoa exhibited decreased motility and ATP levels, and were less efficient when performing in vitro fertilization. ¹⁴² Likewise, the invalidation of the testis‐specific glycolytic enzymes, Gapds (glyceraldehyde‐3‐phosphate dehydrogenase, spermatogenic), Pgk2 (phosphoglycerate kinase 2) and Ldhc (lactate dehydrogenase C), reduced the fertility potential of male mice. Gapds ^−/− and Ldhc ^−/− mice were both infertile and exhibited a pronounced decrease in sperm motility, associated with a drop in ATP levels in sperm cells. ¹⁴³ , ¹⁴⁴ Pgk2 ^−/− mice showed a similar phenotype although less severe, probably because of alternative pathways that allow bypassing the glycolytic step catalyzed by PGK2. ¹⁴⁵ Interestingly, diminished protein amounts of these three enzymes were observed in spermatozoa from asthenozoospermic patients. ¹⁴⁶ Last, defects in the long‐chain fatty acid triglyceride metabolism also resulted in male infertility. Indeed, mice deficient for Atgl (adipose triglyceride lipase), the enzyme catalyzing the first reaction of lipolysis, exhibited impaired spermatogenesis and decreased sperm motility. ¹⁴⁷

3.4. Other functional defects

A few genes involved in other sperm physiological functions have been related to human functional asthenozoospermia; some of these genes are not directly expressed by the sperm cells but have paracrine effects. DEFB126 encodes a sperm surface‐coating protein, specifically expressed in the epididymal epithelium. DEFB126 was shown to prevent the recognition of spermatozoa by the female immune system and to facilitate sperm progression into the cervical mucus. Several mutations in DEFB126 were identified in Chinese infertile patients and associated with asthenozoospermia ¹⁴⁸ (Figure 3B).

An over‐expression of SEMG1 , which is specifically expressed in the seminal vesicles to prevent premature capacitation, ¹⁴⁹ was also shown to be associated with decreased sperm motility in several cohorts of asthenozoospermic patients ¹⁵⁰ , ¹⁵¹ (Figure 3B). Consistently, a proteomic study highlighted higher levels of the encoded protein, Semenogelin 1 (Sg1), in semen samples from men presenting with reduced sperm motility. ¹⁵² Taken together, these data suggest that the increased expression of SEMG1 could result in Sg1 accumulation in spermatozoa from asthenozoospermic patients. Sg1 has a role in semen coagulation and is the main protein secreted by the seminal vesicles; it has been hypothesized that the overexpression of SEMG1 can impact semen viscosity and subsequently alter sperm motility. ¹⁵⁰ , ¹⁵¹

In addition, oxidative stress has been identified as a cause of sperm dysfunction. ¹⁵³ Nitric oxide, produced by nitric oxide synthase (NOS), is considered an important element of oxidative stress in the testis. An intron variant in NOS3 was associated with an increased risk of oligoasthenozoospermia. ¹⁵⁴ In the same line, glutathione‐S‐transferases (GST) are key enzymes, which fulfill essential antioxidant functions and are involved in cellular detoxification processes. GST locus polymorphisms were evidenced as a risk factor for male infertility. In particular, the null‐genotype of GSTM1 or GSTT1 was associated with an increased risk for oligoasthenozoospermia, which was even higher in men carrying the combination of both null‐genotypes ¹⁵⁵ (Figure 3B).

A truncating deletion in NSUN7 , encoding an RNA cytosine methyltransferase, was identified in asthenozoospermic individuals (Figure 3B). This nonsense mutation was observed at a higher frequency in asthenozoospermic patients as compared to healthy control men and oligozoospermic patients in Iranian men. ¹⁵⁶ Consistently, Nsun7‐mutated mice are infertile and exhibit a decrease in sperm progressive motility associated with a rigid flagellar MP. ¹⁵⁷

4. GENETIC SCREENING AND PATIENT MANAGEMENT

4.1. Patient management and assisted reproductive technologies

From a clinical point of view, the major advances made in the genetics of asthenozoospermia and overall, of male infertility, are a clear benefit for patients consulting for infertility. In a first instance, the genetic screening of large cohorts of asthenozoospermic patients, such as MMAF patients, has proven to be efficient in clearly specifying the molecular cause of the infertility for 30%‒60% of the patients, depending of the cohorts. ⁴³ , ⁴⁴ Such genetic diagnosis is significant for the patients and contributes to a better acceptance of their pathological condition. Importantly, the identification of a clear genetic etiology indicates that the use of assisted reproductive technologies (ARTs) will inevitably transmit the genetic defects to the offspring as most of the mutations identified so far are homozygous. Therefore, it is important that the patients and affected couples are informed of the necessity to perform such prenatal genetic diagnosis before starting ART procedures.

In addition, based on the knowledge obtained from genetic screening, it is theoretically possible to associate a particular gene mutation to a specific phenotype of asthenozoospermia, and ultimately establish genotype–phenotype correlations to provide a prognosis for ART, and in particular ICSI. These studies have been mainly performed for asthenozoospermic patients with a MMAF phenotype, for whom the largest cohorts have been established so far. The majority of the studies indicate that the fertilization and embryo rates generated from ICSI with spermatozoa from MMAF patients are similar to the rates obtained for other infertile patients and that they result in live births. ¹⁵⁸ , ¹⁵⁹ , ¹⁶⁰ , ¹⁶¹ , ¹⁶² , ¹⁶³ Some exceptions have been noted but would need to be confirmed on larger sample sizes (see, for instance, refs. ¹⁶⁴ , ¹⁶⁵ ). Overall, most of the studies performed so far, indicate that ICSI is an efficient clinical treatment for this phenotype of infertility, in contrast to phenotypes such as globozoospermia or macrozoospermia. ¹⁵⁸ , ¹⁶⁶

4.2. Diagnosis of asthenozoospermia associated with primary ciliary dyskinesia

In humans, any abnormality in cilia formation, structure, and function, during organogenesis or at the adult stage, can result in severe pathological conditions, which form a family of diseases with pleiotropic clinical features called ciliopathies. ¹⁶⁷ Among these, primary ciliary dyskinesia (PCD) has been exhaustively documented as it is related to defects in motile cilia (9 + 2 pattern), which are structurally similar to the sperm flagella. PCD is mainly characterized by chronic airway infections, rhinosinusitis, and bronchiectasis in consistence with the presence of motile cilia lining the surface of the respiratory airways and essential for mucus clearing (Figure 4A). Most males affected by PCD are infertile principally because of severe or total asthenozoospermia. ¹⁶⁸ However, while over 50 PCD causative gene mutations have been identified, ¹⁶⁹ semen analysis of PCD patients is not constantly performed and the diagnosis of male infertility for PCD patients remains elusive. ⁴⁶ , ¹⁷⁰ In this chapter, we will therefore highlight the genetic defects identified so far and associated with asthenozoospermia in the context of PCD.

FIGURE 4.

Ciliopathies and male infertility. (A) Phenotypical spectrum listing the frequently observed symptoms for primary cilia dyskinesia (PCD), on the left, and other ciliopathies, such as Bardel‒Biedl syndrome (BBS), Joubert syndrome (JBTS), and nephronophthisis (NPHP), on the right. (B) Causal genes, listed by protein function, for ciliopathy‐associated asthenoteratozoospermia in humans, namely, the PCD‐related multiple morphological abnormalities of flagella (MMAF) phenotype (black), other PCD‐related sperm morphological defects (blue) and MMAF phenotype associated to other ciliopathies (violet).

As described in Chapters 2.2 and 2.3, the recent advances in the genetics of male infertility contributed to the identification of numerous gene mutations, which are associated with asthenozoospermia because of morphological defects of the sperm flagellum (Figure 2B). Importantly, several of these gene mutations were shown to induce asthenozoospermia (in particular MMAF phenotype) in association with PCD (Figure 4B and Table S3). Most of these genes code for axonemal components ( ARMC4 , CCDC39 , CCDC40 , CCDC65 , CCDC103 , CFAP43 , CFAP44 , CFAP45 , CFAP47 , CFAP74 , DNAH1 , DNAH2 , DNAH6 , DNAH8 , DNAH10, DNAJB13 , DNALI1 , DRC1 , DRC4 , GAS8 , HYDIN , RSPH1 , RSPH3 , RSPH9 , SPAG6 , and WDR63 ) and proteins involved in IFT and axoneme assembly ( ARMC2 , CFAP69 , CFAP300 , DNAAF3 , DNAAF4 , DNAAF6 , LRRC6 , and SPEF2 ) (Figure 4B). Few additionally identified genes code for centriolar elements ( CEP78 and CEP135 ) or enzymes ( AK7 and QRICH2 ). Some genes still lack a precise functional characterization ( ARL2BP , BRWD1 , CCDC34 , CFAP52 , MNS1 , TCC12 , and WDR66 ) but their mutations are clearly associated with both asthenozoospermia and a PCD phenotype (Figure 4B). Besides, a limited number of genes, whose mutations induce other morphological defects of the sperm flagellum than MMAF, have been demonstrated to induce asthenozoospermia and PCD ( CCDC103 , DNALI1 , RSPH1 , RSPH9 , CFAP300 , DNAAF3 , DNAAF6 , and LRRC6 ) (Figure 4B).

Using the above established list of causal genes (Figure 4B), physicians can now precisely inform the infertile patients about their PCD‐associated phenotype and the risk of transmission to the offspring when using ART. Conversely, when the causal genes identified are known to be solely expressed or functional in the sperm cells but not in ciliated cells, the physicians can inform the infertile patients of the absence of potential cilia‐related symptoms.

4.3. Diagnosis of asthenozoospermia associated with other ciliopathies

The remaining ciliopathies mainly result from the dysfunction of non‐motile cilia (9 + 0 pattern) and their phenotypical spectrum ranges from airways dysfunction, retinitis pigmentosa, polydactyly, brain malformations, skeletal malformations, kidney cysts, obesity, hepatobiliary disease to less frequent phenotypes, such as laterality defect, hyposmia/anosmia, mental retardation, and male microgenitalism (Figure 4A). To date, up to 35 distinct ciliopathies involving non‐motile cilia, have been reported and can be classified according to their predominant clinical features: renal, skeletal, cerebral, and retinal ciliopathies. ¹⁶⁷ Each ciliopathy is characterized by a core set of predominant features, as illustrated here for the Bardet‒Biedl syndrome (BBS) and nephronophthisis (NPHP) (Figure 4A). The genetic etiology of the ciliopathies has been the topic of several reviews in the field during the last decades ¹⁷¹ and will not be discussed here. We will rather discuss particular cases of causal genes whose loss of function are well established to induce a severe phenotype of ciliopathy but whose mutations were recently identified in rare cases of male infertility with none or very weak ciliary defects. In those cases, the different types of mutation are likely to induce a variability of the clinical manifestations, thus contributing to the phenotypical spectrum of ciliopathies.

4.3.1. Bardet‒Biedl syndrome, Joubert syndrome, and asthenozoospermia

BBS is a rare and severe developmental genetic disorder resulting from the dysfunction of primary cilia. It is characterized by retinal dystrophy, obesity, polydactyly, intellectual disability, renal dysfunction, urogenital malformations and male hypogonadism ¹⁷² , ¹⁷³ (Figure 4A). To date, more than 20 causal genes were identified and encoded components of the BBSome complex located at the ciliary basal body together with chaperone and microtubule associated proteins. ¹⁷⁴ , ¹⁷⁵ Biallelic loss of function mutations in IFT74 were previously reported to induce BBS with a clear phenotype of obesity, polydactyly, retinal dystrophy, and intellectual disability. IFT74 mutations were also shown to induce Joubert syndrome (JBTS), a closely‐related ciliopathy (Figure 4A). ¹⁷⁶ Surprisingly, Lorès et al. recently identified two unrelated asthenozoospermic MMAF patients, carrying homozygous missense mutations in IFT74 (c.256G>A; p.Gly86Ser), which resulted in the production of mutant proteins with abnormal subcellular localization along the sperm flagella. ¹⁷⁷ The two MMAF individuals with the IFT74 c.256G>A mutation had neither clinical features indicative of BBS nor other ciliopathy, only weight excess was reported for one individual. ¹⁷⁷ This study suggested that the presence of both normal and mutant IFT74 proteins in the MMAF individuals allowed to maintain systemic ciliary function but not sperm flagella function, thus resulting in a phenotype of isolated male infertility. Overall, this indicates that the degree of severity of the mutations in IFT74 (complete loss of function vs. point mutation) will translate into variable phenotypes of ciliopathies, ranging from nearly isolated male infertility to more severe symptomatic pictures.

In the same line, retinal degeneration is a clinical entity associated with several ciliopathies ¹⁷⁸ and mutations in POC1B , encoding a protein involved in the function of photoreceptor primary cilia, are well known to be associated with JBTS ¹⁷⁸ and cone‐rod dystrophy (CORD). ¹⁷⁹ Recently, Hua et al. identified the c.151delG variant in POC1B, in two brothers with CORD and oligoasthenozoospermia. ¹⁸⁰ Importantly, they showed that mutation of Poc1b in the mice induced CORD and sperm flagellum defects, confirming that POC1B is essential for both retina and sperm flagella. ¹⁸⁰ This study also highlights the phenotypical continuum between flagellar defects and ciliopathies.

4.3.2. Nephronophthisis‐related ciliopathies and asthenozoospermia

NPHP is a renal ciliopathy resulting from the dysfunction of cilia from the polarized epithelial cells that line the renal tubules. ¹⁸¹ NPHP is mainly characterized by the presence of cystic kidneys, renal fibrosis, and interstitial inflammation resulting in the inability of the kidneys to concentrate solutes ¹⁸¹ (Figure 4A). NPHP is manifested by polyuria, impaired sodium reabsorption that causes hypovolemia and hyponatremia, anemia, and growth delay. The genetic etiology of NPHP is relatively well established ¹⁷¹ and NPHP4 has long been considered as a causal NPHP gene, accounting for a substantial proportion of NPHP cases. ¹⁸² Interestingly, NPHP4 mutations were also associated with the cerebello‐oculo‐renal syndrome, another ciliopathy, and male infertility. ¹⁸³ Mutation of Nphp4 in the mice also induced male infertility because of sperm count and motility defects. ¹⁸⁴ Very recently, Ali et al. identified the homozygous missense variant c.1490C>G (p.P497R) in NPHP4, which segregates with male infertility because of MMAF in two affected brothers from a consanguineous Pakistani family. ¹⁸⁵ While Ali et al. did not confirm the deleterious impact of the identified variant on NPHP4 transcript and proteins in semen samples from the probands, they performed in silico analyses, which strongly supported the pathogenicity of the c.1490C>G (p.P497R) variant. Similar to the case of IFT74 mutations reported above, this highlights the variable phenotypical translation of different types of mutations in ciliopathy causal genes.

As a final note of this chapter, it is important to stress that we have focused in this review on asthenozoospermia but that ciliopathies can include other phenotypes of male infertility. First, at a general level, the dysfunctions of cilia during embryonic development and at the adult stage can induce both male and female infertility because of genito‐urinary malformations and hormonal deregulation (hypogonadotropic hypogonadism), for instance. In addition, in regard to PCD, motile cilia are also present within the male genital tract, at the surface of the efferent duct epithelium. ¹⁸⁶ In consistence with this, some PCD patients were described with an infertility phenotype of azoospermia, defined by the absence of spermatozoa in their ejaculate. This point is supported by the identification of mutations in the PCD causal genes CCNO and MCIDAS, which induce azoospermia both in mice and in humans by affecting the multiciliated cells of the efferent ducts. ¹⁸⁷

5. CLINICAL PERSPECTIVES

5.1. Development of therapeutical strategies for functional asthenozoospermia

Several studies were performed in vitro on human and animal spermatozoa to search for potential therapeutical strategies. In vivo studies were also performed in mouse models and empiric clinical studies were performed on asthenozoospermic men in 1970–1980s. In this chapter, we will provide an update of the different strategies and compounds that were tested in vitro and in vivo, in animals and humans.

5.1.1. In vitro studies

Supplementation with epididymal peptides—in vitro treatment

Epididymal peptides are additional promising candidates for in vitro sperm maturation and motility rescue. In particular, DEFB126 ( β‐defensin 126) was observed to increase the motility of co‐cultured human spermatozoa. ¹⁸⁸ The C‐type Natriuretic Peptide, which is normally secreted by the epididymal epithelium and promotes sperm capacitation by activating the cGMP/PKG pathway, ¹⁸⁹ was shown to improve the motility in vitro of spermatozoa from asthenozoospermic patients, and in vivo, when injected to cyclophosphamide (CTX)‐induced asthenozoospermic mice. ¹⁹⁰

Mechanotherapy or photo‐biomodulation

Alternative and innovative strategies have also been proposed and rely on mechanotherapy or photo‐biomodulation. In the first case, bull and human spermatozoa were exposed to high‐frequency ultrasound for 20 s and showed an improvement of sperm motility without affecting sperm viability or inducing DNA fragmentation; the authors propose this method to improve IVF procedures. ¹⁹¹ In a second trial, researchers observed a slight but significant improvement of progressive motility after sperm irradiation with different LED wavelengths within the range of 470–850 nm. ¹⁹²

Supplementation with antioxidants

Vitamin B8, also known as Inositol, acts on mitochondrial membrane potential and can increase in vitro the motility of spermatozoa collected from asthenozoospermic patients. ¹⁹³ Curcumin also showed beneficial effects in vitro on sperm motility, possibly by regulating ROS production and improving mitochondrial function. ¹⁹⁴

5.1.2. In vivo studies with mouse models

Supplementation with miRNAs—in vivo treatment in mouse models

Additional potential targets for innovative therapeutic strategies are miRNAs, many of which are being detected as differentially expressed in semen from asthenozoospermic patients. ¹⁹⁵ Hence, miR‐24‐3p, which affects the stability of glycogen synthase kinase 3 beta (GSK3β) mRNA and alters the sperm energy balance, was shown to be upregulated in spermatozoa from asthenozoospermic patients. A recent trial succeeded in improving in vivo sperm motility of gossypol‐induced asthenozoospermic mice by supplementation of an inhibitor of miR‐24‐3p to germ cells, using Sertoli cell‐derived extracellular vesicles. ¹⁹⁶

Supplementation with ATP—in vivo treatment in mouse model

Other researchers proposed a similar experimental approach by administration of nanocages enriched in ATP through a tail vein injection in the gossypol‐induced asthenozoospermia mouse model. ¹⁹⁷ In this case, the human H‐ferritin nanocarrier loaded with ATP, significantly rescued the sperm motility defects, when delivered in the testis (spermatid cells).

5.1.3. In vivo studies with asthenozoospermic patients

In the first studies with asthenozoospermic patients, researchers tested empiric administration of different molecules such as the protease kallikrein, the vasoactive agent pentoxifylline, hormones (testosterone, human chorionic gonadotropic hormone) and anti‐inflammatory agents (Ketotifen), and observed, in vivo, an improvement of sperm motility. ¹⁹⁸ , ¹⁹⁹ , ²⁰⁰ , ²⁰¹ , ²⁰² Since these pioneer studies, the variety of the tested compounds has increased and antioxidants stand out as the most tested and used compound with the aim of reducing the established deleterious impact of reactive oxygen species (ROS) (see reviews ²⁰³ , ²⁰⁴ ).

Supplementation with antioxidants—in vivo treatments

Supplementation of vitamin D3, which contributes to calcium and phosphorus homeostasis, was shown to increase sperm motility upon daily administration in men showing asthenozoospermia. ²⁰⁵ Oral treatment with vitamin E either alone or combined with vitamin C did not show any effects on semen parameters, ²⁰⁶ while it appeared to improve sperm motility in combination with l‐carnitine (LC). ²⁰⁷ Similarly, LC plus l‐acetyl carnitine (LAC) showed greater in vivo efficacy in increasing sperm motility than single therapy with LAC. ²⁰⁸ Oral treatment with Coenzyme Q10, involved in the mitochondrial oxidative phosphorylation chain, revealed a moderate but significant improvement in sperm motility of idiopathic asthenozoospermic patients. ²⁰⁹ , ²¹⁰ , ²¹¹

Overall, oral antioxidants supplementation constitutes the most prescribed treatment for infertile patients, including asthenozoospermic men. Based on recent meta‐analyses, there are evidence that they have favorable effect on semen parameters of infertile men but large randomized, well‐designed, placebo‐controlled trials are still needed to confirm the consistent benefit for the patients. ²¹² , ²¹³ Importantly, there is currently no clear consensus on the optimal composition for antioxidant supplementation.

5.2. Development of male contraceptive strategies

The phosphorylation of sperm proteins is established as a prerequisite and a hallmark for sperm motility and hyperactivation during the transit in the male and female genital tracts, respectively. Two recent studies indicate that targeting signaling enzymes regulating protein phosphorylation events in sperm cells, such as protein phosphatase 1 (PP1) and the sAC, is a relevant and attractive strategy to inhibit sperm motility and subsequently male fertility.

5.2.1. Contraceptive strategy targeting the phosphatase PP1

Among the phosphatases expressed in sperm cells, the spermatozoa‐specific PP1 isoform, called PP1γ2, plays a major role in sperm motility (for review, see ref. ²¹⁴ ). Inhibition of its catalytic activity is required for the acquisition of sperm motility within the epididymis and is mediated by its interaction with proteins named Regulatory Interactors of Protein Phosphatase One (RIPPOs). An attractive contraceptive strategy consists in restoring the activity of the phosphatase PP1 in order to inhibit sperm motility. With this objective, Silva et al. have designed synthetic peptides, termed bioportides, which were able to enter sperm cells and selectively disrupt the interaction between PP1γ2 and RIPPO proteins (i.e., the phosphatase inhibitor 2 and the A kinase anchor protein 4). They showed that, in vitro, these bioportides were efficiently delivered to human and bovine spermatozoa, and as expected, they significantly reduced the percentage of progressively motile spermatozoa while the proportion of immotile spermatozoa was increased. ²¹⁵ This strongly demonstrates that targeting the interface between PP1γ2 and PP1‐interacting proteins represents a relevant strategy to inhibit sperm motility. These results are major in the field of andrology and research efforts are currently ongoing to evaluate the use of bioportides as potential novel male contraceptives (Male Research Contractive Consortium, University of Wolverhampton).

5.2.2. Contraceptive strategy targeting the sAC

During capacitation, the cytoplasmic increase in bicarbonate and calcium levels in the sperm cells, directly activates the sAC, which induces a rise of cAMP and promotes the activity of the PKA, triggering downstream phosphorylation events. The sAC is a key enzyme in the signaling pathways regulating sperm motility and capacitation. As a result, sAC deficiency in mice was shown to result in male sterility because of sperm motility and capacitation defects, precluding sperm migration to the fertilization site (for reviews, see refs. ²¹⁶ , ²¹⁷ ). As described above, mutations in sAC were also identified in asthenozoospermic individuals, ¹⁰³ confirming an essential role of sAC in humans too.

Recently, Balbach et al. identified a selective and potent inhibitor of the sAC, called TDI‐11861, which, in vitro, can abrogate the bicarbonate‐induced cAMP accumulation in epididymal mouse and human ejaculated spermatozoa, at a nanomolar concentration range. ²¹⁸ This was accompanied by a marked decrease in flagellar beat frequency and the inability of sperm cells to undergo the acrosome reaction. ²¹⁸ Importantly, in vivo administration of a single‐dose of TDI‐11861 to male mice was shown to block the bicarbonate‐induced cAMP accumulation and protein phosphorylation in epididymal mouse sperm cells, resulting in the loss of motility approximately 15 min after the treatment; this inhibition was maintained in ejaculated spermatozoa retrieved from the uterus of female mice after copulation. Notably, this led to 2.5 h of complete loss of male fertility, which was fully restored 24 h post‐treatment. This study is the first one to provide an in vivo proof of concept for a contraceptive strategy targeting the sAC and the downstream sperm signaling pathway. Efforts are currently being made by the Sacyl Pharmaceuticals company to improve the potency of preclinical sAC inhibitors with the objective of providing an acute, non‐hormonal and on‐demand male contraceptive in the next 5 years (∼2030).

6. CONCLUDING REMARKS

Infertility is a major public health problem and presents significant medical and financial challenges. Asthenozoospermia is the predominant cause of male infertility and to date, although clinical and genetic diagnosis of the condition have been much improved, the only possible treatment is assisted reproductive technology, and in particular, intracytoplasmic sperm injection. In addition to the financial cost, assisted reproductive technology involves substantial medical procedures for the partners of infertile men, as they require hormonal injections to induce superovulation, ovary puncture to collect the oocytes, followed by in vitro culture and embryo transfer in the uterus after intracytoplasmic sperm injection.

The establishment of a genetic diagnosis of the infertility can benefit to patients and affected couples, at different levels. First, the genetic diagnosis will permit to inform the patients of the risk of transmission to their offspring when using assisted reproductive technology procedures. Importantly, it will specify the potential association of some gene mutations with cilia‐related disorders such as primary ciliary dyskinesia and more severe ciliopathies. This is of importance as some clinical features of these ciliopathies could manifest later in the life of the affected patient at mild degrees and/or manifest in their offspring in the context of genetic and environmental susceptibility factors. Second, the knowledge obtained about the genetic causes associated with male infertility could be helpful to establish genotype/phenotype correlation and provide information regarding potential gene mutations which preclude proper intracytoplasmic sperm injection. This would ultimately help in providing a prognosis for assisted reproductive technology (intracytoplasmic sperm injection) outcome to the patients from the early stages of the procedures, and eventually suggest alternative solutions, such as sperm donation or adoption, if relevant. Last, based on the knowledge obtained through the analysis of the gene and molecular mechanisms associated with functional asthenozoospermia, it is now possible to develop potent strategies to stimulate sperm motility and fertilization potential. Such strategies are initiated in several laboratories and if successful, they could constitute at long‐term an alternative to intracytoplasmic sperm injection by directly treating infertile men; conversely, the development of negative modulators of sperm motility, could help in developing male contraceptives. These innovative strategies would provide men with the possibility to be more active in the control of their fertility, therefore being of great benefit in terms of both societal and economical aspects.

As a next step to further progress in this field, it will be essential to develop robust and large pipelines for genetic screening of functional asthenozoospermia, which remains poorly investigated in contrast to asthenozoospermia because of morphological defects of the sperm flagella. The establishment of well‐designed studies with comprehensive sperm analyses enabling proper categorization of each enrolled patient will be crucial for routine genetic analysis and the generation of solid genotype‒phenotype conclusions.

AUTHOR CONTRIBUTIONS

The authors have performed a review of the literature and wrote the following parts of the manuscript: Introduction (Joao C. Ribeiro), Background—structure of the sperm flagellum (Aminata Touré), Morphological defects of asthenozoospermia—MMAF (Aminata Touré), Morphological defects of asthenozoospermia—annulus and others (Marjorie Whitfield), Background—sperm functional maturation (Aminata Touré), Functional defects of asthenozoospermia—signaling pathways (Emma Cavarocchi), Functional defects of asthenozoospermia—energy metabolic pathways (Maëva Drouault), Functional defects of asthenozoospermia—other functional defects (Maëva Drouault), Diagnosis of male infertility and ICSI outcome (Aminata Touré), Diagnosis of asthenozoospermia associated with ciliary defects (Aminata Touré), Therapeutics strategies of asthenozoospermia (Emma Cavarocchi), Male contraceptive strategies (Violaine Simon), Concluding remarks (Aminata Touré), and Supplemental tables with mutation types and allele frequencies (Emma Cavarocchi). Emma Cavarocchi and Aminata Touré made the illustrations. Joao C. Ribeiro made the graphical abstract. Aminata Touré critically read and corrected the whole manuscript. All authors have read and approved the manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare they have no conflicts of interest.

Supporting information

Supporting Information

Cavarocchi E, Drouault M, Ribeiro JC, Simon V, Whitfield M, Touré A. Human asthenozoospermia: Update on genetic causes, patient management, and clinical strategies. Andrology. 2025;13:1044–1064. 10.1111/andr.13828