Genetic Architecture of Azoospermia-Time to Advance the Standard of Care.

BACKGROUND: Crypto- and azoospermia (very few/no sperm in the semen) are main contributors to male factor infertility. Genetic causes for spermatogenic failure (SPGF) include Klinefelter syndrome and Y-chromosomal azoospermia factor microdeletions, and CFTR mutations for obstructive azoospermia (OA). However, the majority of cases remain unexplained because monogenic causes are not analysed. OBJECTIVE: To elucidate the monogenic contribution to azoospermia by prospective exome sequencing and strict application of recent clinical guidelines. DESIGN, SETTING, AND PARTICIPANTS: Since January 2017, we studied crypto- and azoospermic men without chromosomal aberrations and Y-chromosomal microdeletions attending the Centre of Reproductive Medicine and Andrology, Münster. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We performed exome sequencing in 647 men, analysed 60 genes having at least previous limited clinical validity, and strictly assessed variants according to clinical guidelines. RESULTS AND LIMITATIONS: Overall, 55 patients (8.5%) with diagnostic genetic variants were identified. Of these patients, 20 (3.1%) carried mutations in CFTR or ADGRG2, and were diagnosed with OA. In 35 patients (5.4%) with SPGF, mutations in 20 different genes were identified. According to ClinGen criteria, 19 of the SPGF genes now reach at least moderate clinical validity. As limitations, only one transcript per gene was considered, and the list of genes is increasing rapidly so cannot be exhaustive. CONCLUSIONS: The number of diagnostic genes in crypto-/azoospermia was almost doubled to 21 using exome-based analyses and clinical guidelines. Application of this procedure in routine diagnostics will significantly improve the diagnostic yield and clinical workup as the results indicate the success rate of testicular sperm extraction. PATIENT SUMMARY: When no sperm are found in the semen, a man cannot conceive naturally. The causes are often unknown, but genetics play a major role. We searched for genetic variants in a large group of patients and found causal mutations for one in 12 men; these predict the chances for fatherhood.

Healthy ageing and spermatogenesis.

Delayed family planning and increased parental age increase the risk for infertility and impaired offspring health. While the impact of ageing on oogenesis is well studied, this is less understood on spermatogenesis. Assessing ageing effects on the male germline presents a challenge in differentiating between the effects of ageing-associated morbidities, infertility and 'pure' ageing. However, understanding the impact of ageing on male germ cells requires the separation of age from other factors. In this review, we therefore discuss the current knowledge on healthy ageing and spermatogenesis. Male ageing has been previously associated with declining sperm parameters, disrupted hormone secretion and increased time-to-pregnancy, among others. However, recent data show that healthy ageing does not deteriorate testicular function in terms of hormone production and spermatogenic output. In addition, intrinsic, age-dependent, highly specific processes occur in ageing germ cells that are clearly distinct from somatic ageing. Changes in spermatogonial stem cell populations indicate compensation for stem cell exhaustion. Alterations in the stem cell niche and molecular ageing signatures in sperm can be observed in ageing fertile men. DNA fragmentation rates as well as changes in DNA methylation patterns and increased telomere length are hallmarks of ageing sperm. Taken together, we propose a putative link between the re-activation of quiescent Adark spermatogonia and molecular changes in aged sperm descending from these activated spermatogonia. We suggest a baseline of 'pure' age effects in male germ cells which can be used for subsequent studies in which the impact of infertility or co-morbidities will be studied.

A germ cell-specific ageing pattern in otherwise healthy men.

Life-long sperm production leads to the assumption that male fecundity remains unchanged throughout life. However, recently it was shown that paternal age has profound consequences for male fertility and offspring health. Paternal age effects are caused by an accumulation of germ cell mutations over time, causing severe congenital diseases. Apart from these well-described cases, molecular patterns of ageing in germ cells and their impact on DNA integrity have not been studied in detail. In this study, we aimed to assess the effects of 'pure' ageing on male reproductive health and germ cell quality. We assembled a cohort of 198 healthy men (18-84 years) for which end points such as semen and hormone profiles, sexual health and well-being, and sperm DNA parameters were evaluated. Sperm production and hormonal profiles were maintained at physiological levels over a period of six decades. In contrast, we identified a germ cell-specific ageing pattern characterized by a steady increase of telomere length in sperm and a sharp increase in sperm DNA instability, particularly after the sixth decade. Importantly, we found sperm DNA methylation changes in 236 regions, mostly nearby genes associated with neuronal development. By in silico analysis, we found that 10 of these regions are located in loci which can potentially escape the first wave of genome-wide demethylation after fertilization. In conclusion, human male germ cells present a unique germline-specific ageing process, which likely results in diminished fecundity in elderly men and poorer health prognosis for their offspring.

Molecular Aging Markers in Patients with Klinefelter Syndrome.

Molecular aging markers provide the opportunity for biological age determination in humans and to study factors, such as genetic determinants, affecting the ageing process. In males with Klinefelter syndrome (KS, non-mosaic karyotype 47, XXY), which is the most common sex chromosome aneuploidy, age-related morbidity and mortality are increased, and a significantly reduced life span has been observed. The aim of this study was to investigate whether Klinefelter patients exhibit molecular signs of premature ageing. We studied, specifically, age-associated DNA methylation patterns (by pyrosequencing) and relative telomere length (TL; by quantitative polymerase chain reaction) in blood in a cohort of Klinefelter patients (n=178 and 266 for DNA methylation and TL, respectively) aged 18-71 years and compared them to the data of age-matched healthy male (n = 184 and 196 for DNA methylation and TL, respectively) and female controls (n = 50). Age-associated DNA methylation patterns were not indicative of accelerated ageing in Klinefelter men. Significantly longer telomeres were found in the young Klinefelter subjects aged 18-24 years (mean=1.51 vs. 1.09 and 1.26 in female and male controls, respectively). However, telomere length in subsequent age groups showed no difference to controls. Gonosomal aneuploidy in Klinefelter syndrome is associated with higher baseline TL at adolescent age, but comparable TL with progressive age in other age groups.