Azoospermia is the complete absence of spermatozoa in the ejaculate in two or more semen analyses after centrifugation. Nonobstructive azoospermia (NOA) represents the most severe form of male factor infertility accounting for 10%-15% of cases and stems from an impairment to spermatogenesis. Understanding of the hypothalamic-pituitary-testicular axis has allowed NOA to be subcategorized by anatomic and/or pathophysiologic level. The etiologies of NOA, and therefore, the differential diagnoses when considering NOA as a cause of male factor infertility, can be subcategorized and condensed into several distinct classifications. Etiologies of NOA include primary hypogonadism, secondary hypogonadism, defects in androgen synthesis and/or response, defective spermatogenesis and sperm maturation, or a mixed picture thereof. This review includes up-to-date clinical, diagnostic, cellular, and histologic features pertaining to the multitude of NOA etiologies. This in turn will provide a framework by which physicians practicing infertility can augment their clinical decision-making, patient counseling, thereby improving upon the management of men with NOA.
Humans ; Azoospermia/diagnosis* ; Male ; Spermatogenesis/physiology* ; Hypogonadism/complications* ; Infertility, Male/etiology* ; Testis/pathology*

Multiple morphological abnormalities of the flagella (MMAF) represent a severe form of sperm defects leading to asthenozoospermia and male infertility. In this study, we identified a novel homozygous splicing mutation (c.871-4 ACA>A) in the adenylate kinase 7 (AK7) gene by whole-exome sequencing in infertile individuals. Spermatozoa from affected individuals exhibited typical MMAF characteristics, including coiled, bent, short, absent, and irregular flagella. Transmission electron microscopy analysis showed disorganized axonemal structure and abnormal mitochondrial sheets in sperm flagella. Immunofluorescence staining confirmed the absence of AK7 protein from the patients' spermatozoa, validating the pathogenic nature of the mutation. This study provides direct evidence linking the AK7 gene to MMAF-associated asthenozoospermia in humans, expanding the mutational spectrum of AK7 and enhancing our understanding of the genetic basis of male infertility.
Humans ; Male ; Sperm Tail/ultrastructure* ; Homozygote ; Consanguinity ; Asthenozoospermia/pathology* ; Infertility, Male/genetics* ; Mutation ; Pakistan ; Adenylate Kinase/genetics* ; Adult ; Pedigree ; RNA Splicing ; Exome Sequencing ; Spermatozoa

Male infertility can result from impaired sperm motility caused by multiple morphological abnormalities of the flagella (MMAF). Distinct projections encircling the central microtubules of the spermatozoal axoneme play pivotal roles in flagellar bending and spermatozoal movement. Mammalian sperm-associated antigen 17 ( SPAG17 ) encodes a conserved axonemal protein of cilia and flagella, forming part of the C1a projection of the central apparatus, with functions related to ciliary/flagellar motility, skeletal growth, and male fertility. This study investigated two novel homozygous SPAG17 mutations (M1: NM_206996.2, c.829+1G>T, p.Asp212_Glu276del; and M2: c.2120del, p.Leu707*) identified in four infertile patients from two consanguineous Pakistani families. These patients displayed the MMAF phenotype confirmed by Papanicolaou staining and scanning electron microscopy assays of spermatozoa. Quantitative real-time polymerase chain reaction (PCR) of patients' spermatozoa also revealed a significant decrease in SPAG17 mRNA expression, and immunofluorescence staining showed the absence of SPAG17 protein signals along the flagella. However, no apparent ciliary-related symptoms or skeletal malformations were observed in the chest X-rays of any of the patients. Transmission electron microscopy of axoneme cross-sections from the patients showed incomplete C1a projection and a higher frequency of missing microtubule doublets 1 and 9 compared with those from fertile controls. Immunofluorescence staining and Western blot analyses of spermatogenesis-associated protein 17 (SPATA17), a component of the C1a projection, and sperm-associated antigen 6 (SPAG6), a marker of the spring layer, revealed disrupted expression of both proteins in the patients' spermatozoa. Altogether, these findings demonstrated that SPAG17 maintains the integrity of spermatozoal flagellar axoneme, expanding the phenotypic spectrum of SPAG17 mutations in humans.
Humans ; Male ; Infertility, Male/pathology* ; Sperm Tail/ultrastructure* ; Homozygote ; Microtubule-Associated Proteins/genetics* ; Axoneme/genetics* ; Spermatozoa/ultrastructure* ; Adult ; Mutation ; Sperm Motility/genetics* ; Pedigree ; Microtubules ; Microtubule Proteins/genetics*

Multiple morphological abnormalities of sperm flagella (MMAF) is a severe form of asthenoteratozoospermia, characterized by morphological abnormalities and reduced motility of sperm, causing male infertility. Although approximately 60% of MMAF cases can be explained genetically, the etiology of the remaining cases is unclear. Here, we identified two novel compound heterozygous variants in the gene, dynein axonemal heavy chain 10 ( DNAH10 ), in three patients from two unrelated Pakistani families using whole-exome sequencing (WES), including one compound heterozygous mutation ( DNAH10 : c.9409C>A [p.P3137T]; c.12946G>C [p.D4316H]) in family 1 and another compound heterozygous mutation ( DNAH10 : c.8849G>A [p.G2950D]; c.11509C>T [p.R3687W]) in family 2. All the identified variants are absent or rare in public genome databases and are predicted to have deleterious effects according to multiple bioinformatic tools. Sanger sequencing revealed that these variants follow an autosomal recessive mode of inheritance. Hematoxylin and eosin (H&E) staining revealed MMAF, including sperm head abnormalities, in the patients. In addition, immunofluorescence staining revealed loss of DNAH10 protein signals along sperm flagella. These findings broaden the spectrum of DNAH10 variants and expand understanding of the genetic basis of male infertility associated with the MMAF phenotype.
Adult ; Humans ; Male ; Alleles ; Asthenozoospermia/pathology* ; Axonemal Dyneins/genetics* ; Dyneins/genetics* ; Exome Sequencing ; Infertility, Male/pathology* ; Mutation ; Pakistan ; Pedigree ; Sperm Tail/pathology*

This review focuses on the diagnostic algorithm for nonobstructive azoospermia (NOA), a significant male factor contributing to infertility. NOA, characterized by the absence of sperm in the ejaculate, requires a systematic diagnostic approach to identify reversible conditions, genetic factors, and prognosis for achieving pregnancy. The diagnostic pathway involves semen analysis and a comprehensive evaluation for hormonal deficiencies, anatomical abnormalities, and genetic factors. The importance of medical history, physical examination, endocrine evaluation, imaging, and genetic testing is emphasized. This review highlights the significance of differentiating NOA from obstructive azoospermia (OA) and outlines key considerations for effective management, including surgical sperm retrieval and assisted reproductive techniques. Testicular biopsy is discussed as a definitive method to distinguish obstructive cases from nonobstructive cases, providing valuable prognostic information. Overall, a thorough and systematic diagnostic approach is essential for the effective management of men suspected with NOA, offering insights into potential treatment options and reproductive outcomes.
Humans ; Azoospermia/therapy* ; Male ; Algorithms ; Semen Analysis ; Testis/pathology* ; Sperm Retrieval ; Biopsy ; Infertility, Male/etiology*

The syndrome of multiple morphological abnormalities of the sperm flagella (MMAF) is one of the most serious kinds of sperm defects, leading to asthenoteratozoospermia and male infertility. In this study, we use whole-exome sequencing to identify genetic factors that account for male infertility in a patient born from a consanguineous Pakistani couple. A homozygous frameshift mutation (c.1399_1402del; p.Gln468ArgfsTer2) in axonemal dynein light chain domain containing 1 ( AXDND1 ) was identified in the patient. Sanger sequencing data showed that the mutation was cosegregated recessively with male infertility in this family. Papanicolaou staining and scanning electron microscopy analysis of the sperm revealed severely abnormal flagellar morphology in the patient. Immunofluorescence and western blot showed undetectable AXDND1 expression in the sperm of the patient. Transmission electron microscopy analysis showed disorganized sperm axonemal structure in the patient, particularly missing the central pair of microtubules. Immunofluorescence staining showed the absence of sperm-associated antigen 6 (SPAG6) and dynein axonemal light intermediate chain 1 (DNALI1) signals in the sperm flagella of the patient. These findings indicate that AXDND1 is essential for the organization of flagellar axoneme and provide direct evidence that AXDND1 is a MMAF gene in humans, thus expanding the phenotypic spectrum of AXDND1 frameshift mutations.
Humans ; Male ; Sperm Tail/ultrastructure* ; Frameshift Mutation ; Infertility, Male/pathology* ; Pakistan ; Pedigree ; Consanguinity ; Axonemal Dyneins/genetics* ; Adult ; Spermatozoa ; Exome Sequencing

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by impaired motility of cilia and flagella. Mutations in cilia- and flagella-associated protein 300 ( CFAP300 ) are associated with human PCD and male infertility; however, the underlying pathogenic mechanisms remain poorly understood. In a consanguineous Chinese family, we identified a homozygous CFAP300 loss-of-function variant (c.304delC) in a proband presenting with classical PCD symptoms and severe sperm abnormalities, including dynein arm deficiency and acrosomal malformation, as confirmed by transmission electron microscopy (TEM). Histological analysis revealed multiple morphological abnormalities of the sperm flagella in CFAP300 -mutant individual, whereas immunofluorescence demonstrated markedly reduced CFAP300 expression in the spermatozoa of the proband. Furthermore, tandem mass tag (TMT)-based quantitative proteomics showed that the CFAP300 mutation reduced key spermatogenesis proteins (e.g., sperm flagellar 2 [SPEF2], solute carrier family 25 member 31 [SLC25A31], and A-kinase anchoring protein 3 [AKAP3]) and mitochondrial ATP synthesis factors (e.g., SLC25A31, cation channel sperm-associated 3 [CATSPER3]). It also triggered abnormal increases in autophagy-related proteins and signaling mediator phosphorylation. These molecular alterations are likely to contribute to progressive deterioration of sperm ultrastructure and function. Notably, successful pregnancy was achieved via intracytoplasmic sperm injection (ICSI) using the proband's sperm. Overall, this study expands the known CFAP300 mutational spectrum and offers novel mechanistic insights into its role in spermatogenesis.
Humans ; Male ; Infertility, Male/pathology* ; Acrosome/pathology* ; Sperm Tail/pathology* ; Pedigree ; Spermatozoa ; Adult ; Loss of Function Mutation ; Ciliary Motility Disorders/genetics* ; Spermatogenesis/genetics* ; Female

OBJECTIVE: The aim of this study is to analyze the clinical features and genetic etiology of a patient with multiple morphological abnormalities of the sperm flagella (MMAF) retrospectively. METHODS: A severely oligospermic patient from the Reproductive Center of the First Affiliated Hospital of Ningbo University was selected as the study subject. Clinical data and examination results were collected. High-throughput sequencing and bioinformatics were used to analyze the genetic etiology. And Sanger sequencing was employed to validate findings in the family. Transmission electron microscopy (TEM) was used to observe the sperm ultrastructure, and immunofluorescence analysis was performed to examine the localization of FSIP2 protein in the sperm. RESULTS: The patient presented with severe oligospermia, and sperm morphology displayed MMAF. TEM revealed fibrous sheath and 9+2 microtubule structural disruptions in the sperm. Sequencing identified compound heterozygous variants in the FSIP2 gene (c.17798C > T, c.5927T > G), inherited from the father and mother, respectively. According to the guidelines of the American College of Medical Genetics and Genomics, the variants were classified as pathogenic. The patient's spouse underwent intracytoplasmic single sperm injection, resulting in one embryo, but no clinical pregnancy occurred after embryo transfer. CONCLUSION This study reported the mutation of FSIP2 gene c.17798C > T, c.5927T > G in a patient with MMAF. These findings expand the mutational spectrum of the FSIP2 gene and provide insights for genetic and assisted reproductive counseling for patients with MMAF.
Humans ; Male ; Sperm Tail/pathology* ; Heterozygote ; Oligospermia/genetics* ; Spermatozoa ; Mutation ; Infertility, Male/genetics* ; Adult ; Pedigree ; Retrospective Studies ; Sperm Injections, Intracytoplasmic

This study assessed the feasibility of testis tissue cryopreservation (TTC) for fertility preservation in prepubescent boys with cryptorchidism. From January 2014 to December 2022, the University Hospital of Copenhagen (Rigshospitalet, Copenhagen, Denmark) implemented TTC for 56 boys with cryptorchidism to preserve their reproductive potential. Testis tissue samples were collected during orchiopexy (32 cases) or at subsequent follow-up procedures (24 cases), necessitated by an increased risk of infertility as indicated by hormonal assessments and/or findings from initial surgical biopsies. Testis samples were procured for TTC and pathological analysis. The cohort had an average age of 1.3 (range: 0.3-3.8) years at the time of orchiopexy, with 91.1% presenting bilateral cryptorchidism. The study revealed a median germ cell count of 0.39 (range: 0-2.88) per seminiferous tubule, with germ cells detected in 98.0% of the bilateral biopsies and 100% of the unilateral, indicating a substantial potential for fertility in these immature tissues. A dark spermatogonia (Ad) was detected in 37 out of 56 patients evaluated, with a median Ad spermatogonia count of 0.027 (range: 0.002-0.158) per seminiferous tubule. A total of 30.2% of the samples lacked Ad spermatogonia, indicative of potential gonadotrophin insufficiency. The median hormone levels measured were as follows: follicle-stimulating hormone (FSH) at 0.69 (range: 0.16-2.5) U l -1 , luteinizing hormone (LH) at 0.21 (range: 0.05-3.86) U l -1 , and inhibin B at 126 (range: 17-300) pg ml -1 . Despite early orchiopexy, 20%-25% of boys with cryptorchidism remain at risk for future infertility, substantiating the necessity of TTC as a precaution. The study highlights the need for refined predictive techniques to identify boys at higher risk of future infertility.
Humans ; Male ; Cryptorchidism/pathology* ; Cryopreservation ; Testis/pathology* ; Fertility Preservation/methods* ; Child, Preschool ; Infant ; Orchiopexy ; Spermatogonia/pathology* ; Infertility, Male/etiology*

Teratozoospermia is a rare disease associated with male infertility. Several recurrent genetic mutations have been reported to be associated with abnormal sperm morphology, but the genetic basis of tapered-head sperm is not well understood. In this study, whole-exome sequencing (WES) identified a homozygous WD repeat domain 12 (WDR12; p.Ser162Ala/c.484T>G) variant in an infertile patient with tapered-head spermatozoa from a consanguineous Chinese family. Bioinformatic analysis predicted this mutation to be a pathogenic variant. To verify the effect of this variant, we analyzed WDR12 protein expression in spermatozoa of the patient and a control individual, as well as in the 293T cell line, by Western blot analysis, and found that WDR12 expression was significantly downregulated. To understand the role of normal WDR12, we evaluated its mRNA and protein expression in mice at different ages. We observed that WDR12 expression was increased in pachytene spermatocytes, with intense staining visible in round spermatid nuclei. Based on these results, the data suggest that the rare biallelic pathogenic missense variant (p.Ser162Ala/c.484T>G) in the WDR12 gene is associated with tapered-head spermatozoa. In addition, after intracytoplasmic sperm injection (ICSI), a successful pregnancy was achieved. This finding indicates that infertility associated with this WDR12 homozygous mutation can be overcome by ICSI. The present results may provide novel insights into understanding the molecular mechanisms of male infertility.
Humans ; Pregnancy ; Female ; Male ; Animals ; Mice ; Teratozoospermia/pathology* ; Semen/metabolism* ; Infertility, Male/metabolism* ; Spermatozoa/metabolism* ; Mutation ; RNA-Binding Proteins/metabolism* ; Cell Cycle Proteins/genetics*