Researchers identify a rare cause of male infertility and discover a potential cure

By Hugo Francisco de SouzaReviewed by Sophia CoveneySep 18 2023

In a recent study published in the eBioMedicine journal, researchers investigated the genetic underpinning of asthenozoospermia, the leading cause of male fertility.

Their multidisciplinary examinations were able to identify adenylate kinase 9 (AK9), an enzyme involved in sperm energy metabolism and cellular nucleotide homeostasis, as essential to fertilization by enabling sperm to swim toward the ovum even in sugar-free media.

Mutations in the AK9 encoding gene were found to cause male infertility, both in murine models and human study participants. While these genetic mutations are life-long, the team discovered that intracytoplasmic sperm injections (ICSI) were able to rescue afflicted patients from infertility, thereby curing the condition.

Male infertility and asthenozoospermia

Infertility, the inability to conceive even after a year of frequent unprotected sex, is a common condition affecting approximately 15% of all couples of childbearing age.

Research suggests that infertility can be caused by numerous factors, including genetics, diet, mental well-being, and, especially in women, age. Almost half of all cases of infertility can be attributed to men, with asthenozoospermia being the leading cause of sterility.

Asthenozoospermia, sometimes called asthenospermia, is a condition wherein sperm motility is severely impaired, making otherwise fertile sperm incapable of successfully reaching the female ovum for conception.

Previous studies have identified genetic contributors to asthenozoospermia, including the A-kinase anchoring protein (AKAP), human tRNAGlu (TTC), the dynein axonemal heavy chain (DNAH), and the cilia and flagella associated (CFAP) gene families. Unfortunately, the genetic etiology and molecular pathogenesis underpinning asthenozoospermia remain poorly understood.

Sperm motility is entirely due to the beating of sperm flagella, an energy-driven process. Adenosine triphosphate (ATP) is the primary source of this energy, produced by the flagellum via glycolysis and by the sperm mitochondria via oxidative phosphorylation.

Investigations in male mice models have shown that alterations to any of the aforementioned gene families in sperm result in severe impairment to energy generation and, in turn, sperm motility, resulting in infertility and the asthenozoospermia phenotype.

Despite being found in both invertebrates and vertebrates, mammalian sperm is special because it remains motile even in the presence of glycolysis inhibitors, suggesting that in addition to glycolysis and oxidative phosphorylation, mammalian sperm motility is regulated by other poorly understood energy metabolisms.

Adenylate kinases (Aks) have been suggested to fulfill this role by transferring phosphate groups to adenosine diphosphate (ADP), thereby producing ATP for flagellar use. Nine Aks have been hitherto identified (AK1-AK9), all of which have some role in sperm motility but do not otherwise affect fertility.

Notably, AK9 is highly expressed in the human testis and is involved in maintaining the homeostasis of cellular nucleotides by [catalyzing] the interconversion of nucleoside phosphates. However, because of the lack of selective AK inhibitors, the physiological effect of AK9 in sperm and its role in the nucleotide homeostasis and energy metabolism has not been fully uncovered.

Sha et al. (2023)

About the study

In the present study, researchers used a multidisciplinary approach to identify the genetic etiology and molecular pathogenesis of asthenozoospermia, with a focus on the effects of mutations in the AK9 gene and its encoded AKD2 protein. They used evidence from human asthenozoospermia patients and knockout AK9 (Ak9 KO) mice to identify the role of the gene in affecting sperm motility.

Human recruitment for this study was conducted at the Women and Children's Hospital of Xiamen University. One hundred and sixty-five Chinese men presenting idiopathic asthenozoospermia (cases) and 200 men with normal fertility (controls) were enrolled. Preliminary tests revealed that in all physical and semen parameters except sperm motility, case and control cohorts were clinically identical. Case cohorts presented reduced sperm motility ranging from 0 to 32%.

To elucidate genetic mutations involved with the AK9 gene and their impacts on motility, whole-exome sequencing (WES) of participant DNA was carried out. Sanger sequencing of identified mutant genotypes was used for fine-scale data generation.

Wild-type (WT) and mutant AK9 protein structures were predicted and visualized using the AlphaFold database and UCSF Chimera tool, respectively. Semen characteristics analyses of sperm from both cohorts were undertaken as prescribed by the World Health Organization Laboratory Manual for the Examination and Processing of Human Semen (5^th edition).

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) endonuclease (Cas9) (CRISPR-Cas9) technology was used to construct male Ak9 KO mice, murine representatives of the asthenozoospermia phenotype. Electron microscopy (both scanning [SEM] and transmission [TEM]) of human and murine sperm was undertaken for spermatozoa ultrastructure visualization.

Immunofluorescence and western blotting assays were employed to identify AK9/AKD2 protein concentrations in sperm samples. The sperm chromatin structure assay (SCSA) and flow cytometry were used for sperm DNA stainability and fragmentation detection. Excised Ak9 KO mice testicles were then stained using hematoxylin and eosin dyes to visualize the testes’ structure.

Liquid chromatography-mass spectrometry (LC/MS) was used to detect and characterize sperm adenosines and measure phosphotransfer rates. In tandem with flow cytometry, the mitochondrial membrane potential (MMP) assay kit detected the mitochondrial membrane potential of sperm samples.

Finally, for men identified as having AK9 mutations as their causes of infertility and asthenozoospermia, intracytoplasmic sperm injection (ICSI), a process via which sperm from a donor is extracted and directly inserted into an ovum, was carried out.

Study findings

Of the 165 case males presenting the idiopathic asthenozoospermia phenotype, genetic analysis revealed five with bi-allelic mutations in their AK9 gene. Of these, two presented homozygous mutations and were found to be from unrelated consanguineous families.

One had a homozygous frameshift insertion mutation, while the other had a different homozygous frameshift insertion mutation, a heterozygous non-frameshift deletion mutation, and a stop-loss mutation.

In silico analysis of the human AK9 transcript revealed that both the above-referenced mutations significantly altered the three-dimensional structure of the normal AK9/AKD2 protein. Cross-referencing these obtained structures against the ExAC, 1000 Genomes Project, gnomAD _exome (All), and GnomAD _exome (East Asian) databases revealed that these mutations were absent or rare across a sizeable global populace, suggesting that the AK9 gene is highly conserved.

Taken together, these findings imply that AK9 gene mutations are inherited from parental heterozygous carriers following Mendelian patterns, and the mutations are autosomally recessive.

Comparisons of physical, secondary sexual, and, surprisingly, sperm morphology characteristics from case and control cohorts for both humans and mice revealed that phenotypes were identical between cohorts. SEM and TEM images revealed that AK9 WT and mutants were indistinguishable from each other even at the ultrastructure level.

Targeted metabolomic analysis revealed, however, that functionally, AK9-deficient individuals showed significantly reduced AMP and ADP levels compared to their WT counterparts.

Due to the unique role of AK in-phosphotransfer, we evaluated the phosphoryl moiety in ATP. Surprisingly, O-labelled-ATP was significantly reduced, indicating insufficient AK-catalysed phosphotransfer in the sperm of patients with AK9 deficiency. These results suggest that bi-allelic mutations in AK9 disrupt glycolytic metabolic homeostasis and inhibit AK-catalysed phosphotransfer in human sperm.

Sha et al. (2023)

Mass spectrometry analyses of AK9 mutated sperm elucidated that 211 proteins were upregulated, and 195 proteins were downregulated in mutant sperm compared to WT. Gene ontology analyses of these differentially expressed proteins revealed that they are involved in energy production and conversion, carbohydrate transport and metabolism, secondary metabolite biosynthesis, signal transduction, and catabolism.

Finally, ICSI performed on both mice asthenozoospermia models and human patients showed successful results. Three of the five human patients with asthenozoospermia participated in the study, with all cases resulting in a successful pregnancy and the delivery of healthy babies.

This highlights that AK9 affects only sperm motility and energy modalities but does not alter the fertilization ability of the spermatozoa. ICSI can thus be used in future clinical trials as a rescue from male infertility for couples whose male partner has mutations in his AK9 gene.

Conclusions

In the present study, researchers investigated the genetic etiology and molecular pathogenesis of asthenozoospermia, the primary cause of male infertility.

Their results highlight that mutations in the AK9 gene severely reduce sperm motility and alter protein expression while leaving sperm structure and fertility unchanged. Their findings highlight the genetic underpinnings of the condition and present ICSI as a potential rescue for couples wherein the male partner suffers from asthenozoospermia.