This study compares the relative effects of advancing male age on multiple genomic defects in human sperm [DNA fragmentation index (DFI), chromatin integrity, gene mutations, and numerical chromosomal abnormalities], characterizes the relationships among these defects and with semen quality, and estimates the incidence of susceptible individuals for a well characterized nonclinical nonsmoking group of 97 men (22–80 years). Adjusting for confounders, we found major associations between age and the frequencies of sperm with DFI and fibroblast growth factor receptor 3 gene (FGFR3) mutations associated with achondroplasia (P< 0.01) with no evidence for age thresholds. However, we found no associations between age and the frequencies of sperm with immature chromatin, aneuploidies/diploidies,FGFR2mutations (Apert syndrome), or sex ratio in this cohort. There were also no consistent correlations among genomic and semen-quality endpoints, except between DFI and sperm motility (r= −0.65,P< 0.001). These findings suggest there are multiple spermatogenic targets for genomically defective sperm with substantially variable susceptibilities to age. Our findings predict that as healthy males age, they have decreased pregnancy success with trends beginning in their early reproductive years, increased risk for producing offspring with achondroplasia mutations, and risk of fathering offspring with Apert syndrome that may vary across cohorts, but with no increased risk for fathering aneuploid offspring (Down, Klinefelter, Turner, triple X, and XYY syndromes) or triploid embryos. Our findings also suggest that the burden of genomic damage in sperm cannot be inferred from semen quality, and that a small fraction of men are at increased risk for transmitting multiple genetic and chromosomal defects.

Significance: We present experimental evidence showing that meiosis is an important source of germline mutations. Because sites of meiotic recombination experience recurrent double-strand breaks at hotspots, recombination has been previously suspected to be mutagenic. Yet inferences made from sequence comparisons have not found strong evidence for a mutagenic effect of recombination. Here, we directly sequenced a large number of single sperm DNA molecules and found more new mutations in molecules with a crossover than in molecules without a recombination event. We also observed that GC alleles are transmitted more often than AT alleles at polymorphic sites. Our data demonstrate that both mutagenesis and biased transmission occur during crossing over in meiosis and are important modifiers of the sequence content at recombination hotspots.

The lifelong spermatogonial stem cell divisions unique to male germ cell production are thought to contribute to a higher mutation frequency in males. The fact that certain de novo human genetic conditions (e.g., achondroplasia) increase in incidence with the age of the father is consistent with this idea. Although it is assumed that the paternal age effect is the result of an increasing frequency of mutant sperm as a man grows older, no direct molecular measurement of the germ-line mutation frequency has been made to confirm this hypothesis. Using sperm DNA from donors of different ages, we determined the frequency of the nucleotide substitution in the fibroblast growth factor receptor 3 ( FGFR3 ) gene that causes achondroplasia. Surprisingly, the magnitude of the increase in mutation frequency with age appears insufficient to explain why older fathers have a greater chance of having a child with this condition. A number of alternatives may explain this discrepancy, including selection for sperm that carry the mutation or an age-dependent increase in premutagenic lesions that remain unrepaired in sperm and are inefficiently detected by the PCR assay.

Our understanding of the details of mammalian meiotic recombination has recently advanced significantly. Sperm typing technologies, linkage studies, and computational inferences from population genetic data have together provided information in unprecedented detail about the location and activity of the sites of crossing-over in mice and humans. The results show that the vast majority of meiotic recombination events are localized to narrow DNA regions (hot spots) that constitute only a small fraction of the genome. The data also suggest that the molecular basis of hot spot activity is unlikely to be strictly determined by specific DNA sequence motifs in cis. Further molecular studies are needed to understand how hot spots originate, function and evolve.

The frequency of the most common sporadic Apert syndrome mutation (C755G) in the human fibroblast growth factor receptor 2 gene (FGFR2) is 100–1,000 times higher than expected from average nucleotide substitution rates based on evolutionary studies and the incidence of human genetic diseases. To determine if this increased frequency was due to the nucleotide site having the properties of a mutation hot spot, or some other explanation, we developed a new experimental approach. We examined the spatial distribution of the frequency of the C755G mutation in the germline by dividing four testes from two normal individuals each into several hundred pieces, and, using a highly sensitive PCR assay, we measured the mutation frequency of each piece. We discovered that each testis was characterized by rare foci with mutation frequencies 10³ to >10⁴ times higher than the rest of the testis regions. Using a model based on what is known about human germline development forced us to reject (p < 10⁻⁶) the idea that the C755G mutation arises more frequently because this nucleotide simply has a higher than average mutation rate (hot spot model). This is true regardless of whether mutation is dependent or independent of cell division. An alternate model was examined where positive selection acts on adult self-renewing Ap spermatogonial cells (SrAp) carrying this mutation such that, instead of only replacing themselves, they occasionally produce two SrAp cells. This model could not be rejected given our observed data. Unlike the disease site, similar analysis of C-to-G mutations at a control nucleotide site in one testis pair failed to find any foci with high mutation frequencies. The rejection of the hot spot model and lack of rejection of a selection model for the C755G mutation, along with other data, provides strong support for the proposal that positive selection in the testis can act to increase the frequency of premeiotic germ cells carrying a mutation deleterious to an offspring, thereby unfavorably altering the mutational load in humans. Studying the anatomical distribution of germline mutations can provide new insights into genetic disease and evolutionary change. Some human disease mutations occur 100–1,000 times more frequently than would be predicted from genome wide studies of mutation in different species. In Apert syndrome, for example, two-thirds of all new causal mutations occur at only one base in the affected gene. This unusually high frequency suggests that something about that DNA base or its local surroundings makes it highly susceptible to mutation. We studied this hypothesis by examining the location of cells containing this mutation in the testes of normal men. We found that mutant cells were not uniformly distributed throughout the testes, as would be expected for random mutations. Instead, we found 95% of the mutants in small clusters containing only a few percent of the total testes cells. A higher-than-average mutation rate could not explain the data. We propose that these mutations arise at the expected rate, but that mutated cells gain a selective advantage that allows them to increase their frequency compared to nonmutant cells. Our results—which argue against the mutational hot spot model in favor of a selection model—suggest how germline selection in animals can alter the mutational load of a species.

For decades, classical crossover studies and linkage disequilibrium (LD) analysis of genomic regions suggested that human meiotic crossovers may not be randomly distributed along chromosomes but are focused instead in “hot spots.” Recent sperm typing studies provided data at very high resolution and accuracy that defined the physical limits of a number of hot spots. The data were also used to test whether patterns of LD can predict hot spot locations. These sperm typing studies focused on several small regions of the genome already known or suspected of containing a hot spot based on the presence of LD breakdown or previous experimental evidence of hot spot activity. Comparable data on target regions not specifically chosen using these two criteria is lacking but is needed to make an unbiased test of whether LD data alone can accurately predict active hot spots. We used sperm typing to estimate recombination in 17 almost contiguous ~5 kb intervals spanning 103 kb of human Chromosome 21. We found two intervals that contained new hot spots. The comparison of our data with recombination rates predicted by statistical analyses of LD showed that, overall, the two datasets corresponded well, except for one predicted hot spot that showed little crossing over. This study doubles the experimental data on recombination in men at the highest resolution and accuracy and supports the emerging genome-wide picture that recombination is localized in small regions separated by cold areas. Detailed study of one of the new hot spots revealed a sperm donor with a decrease in recombination intensity at the canonical recombination site but an increase in crossover activity nearby. This unique finding suggests that the position and intensity of hot spots may evolve by means of a concerted mechanism that maintains the overall recombination intensity in the region. Meiotic crossover events are not randomly distributed across the human genome, but are concentrated in many small regions of a few kb with high recombination rates compared to surrounding regions. How the distribution of recombination events affects the association of different alleles along the chromosome (linkage disequilibrium, or LD) was recently addressed using sperm typing in regions already known or suspected to contain unusually high recombination intensities. In the current paper, the authors used sperm typing to examine recombination in a region not known or suspected of containing recombination hot spots. They first established the crossover distribution pattern within a 103-kb region of human Chromosome 21. Then, they compared their data to predictions of crossover distributions estimated by statistical analyses of polymorphism in the region. They found a good concordance between the two, although it was not perfect. To the authors' knowledge, this work is the first to compare LD-based estimates of recombination to sperm-typing data from regions not previously known or suspected of containing recombination hot spots. In addition, one of the studied hot spots revealed an example of a decrease in recombination intensity with a concurrent increase at a nearby site. This unique observation suggests that the activity of hot spots may evolve in a concerted fashion such that the overall recombination activity of the region is maintained.