Recent decline in sperm motility among donor candidates at a sperm bank in Denmark.

STUDY QUESTION: Has there been variation in semen quality among men applying to be sperm donors (i.e. donor candidates) in Denmark in recent years (2017-2022)? SUMMARY ANSWER: The motile sperm concentration and total motile sperm count (TMSC) in ejaculates-both measures of sperm quality-declined by as much as 22% from 2019 to 2022. WHAT IS KNOWN ALREADY: Questions remain about whether human semen quality has declined in recent years. Whilst some studies provide evidence for a decline in human semen quality, these findings have been disputed owing to potential biases in the populations studied or in the methods used to measure semen quality. Resolution of this issue has important implications for human fertility, as well as for those involved in the recruitment of sperm donors for use in medically assisted reproduction. STUDY DESIGN, SIZE, DURATION: We obtained data on the semen quality of ejaculates previously collected from 2017 to 2022 at sperm bank locations in four cities in Denmark: Aarhus, Aalborg, Copenhagen, and Odense. Our study focuses on the single semen samples provided by 6758 donor candidates aged between 18 and 45 years old to determine whether their sperm quality met a minimum criterion for them to be accepted as sperm donors. PARTICIPANTS/MATERIALS, SETTING, METHODS: All ejaculates were analyzed within 1 hour of production. Semen volume (ml) was estimated by weight and both the concentration (106/ml) of sperm as well as the concentration of motile sperm (World Health Organization grades a and b) were measured using the same protocols and computer-assisted semen analysis system across all years at each site. Statistical analyses of the semen variables were controlled for age and donation site, as well as the average monthly high temperature when the ejaculate was produced. MAIN RESULTS AND THE ROLE OF CHANCE: From 2017 to 2019, semen volume, sperm concentration, and total sperm count in the ejaculates of donor candidates increased by 2-12%. Then, from 2019 to 2022, sperm concentration and total sperm count changed by 0.1-5% from year to year, but none of those changes were statistically significant. In contrast, both motile sperm concentration and TMSC declined significantly, by 16% and 22%, respectively, between 2019 and 2022. Thus, the concentration of motile sperm in donor candidates declined from 18.4 [95% CL: 17.0, 20.0] million/ml in 2019 to 15.5 [14.4, 16.7] million/ml in 2022, and TMSC declined from 61.4 [55.8, 67.5] million per ejaculate in 2019 to 48.1 [44.1, 52.4] million in 2022. LIMITATIONS, REASONS FOR CAUTION: We cannot determine from the available data the causes of the decline in semen quality of donor candidates from 2019 to 2022. However, as this period coincides with lockdowns and changes in work patterns during the coronavirus disease 2019 pandemic, it is possible that changes in motile sperm concentration and TMSC were the result of changes in the lifestyles of the men whose semen was analyzed. WIDER IMPLICATIONS OF THE FINDINGS: Men providing initial semen samples at sperm banks, when applying to be sperm donors, are a useful population in which to monitor changes in human semen quality over time. Our results have implications for human fertility and the recruitment of sperm donors for medically assisted reproduction, where motile sperm concentration is an essential selection criterion because it influences fertility. We suggest that gathering health and lifestyle data on donor candidates at sperm banks might help to identify causal factors for the decline of sperm quality that could be addressed and intervention, if desired, could be personalized for each accepted donor. STUDY FUNDING/COMPETING INTEREST(S): No external funding was obtained for this study. E.L. and A.-B.S. are employees of Cryos International. AP reports paid consultancy for Cryos International, Cytoswim Ltd, Exceed Health, and Merck Serono in the last 2 years of this study, but all monies were paid to the University of Sheffield (former employer). AP is also an unpaid trustee of the Progress Educational Trust (Charity Number 1139856). RM declares support from Cryos International to present results of this research at ESHRE 2023. None of the authors were directly involved in the collection or physical analysis of semen samples. TRIAL REGISTRATION NUMBER: N/A.

DNA mismatch and damage patterns revealed by single-molecule sequencing.

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases1,2. Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired3,4. However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing.

Single duplex DNA sequencing with CODEC detects mutations with high sensitivity.

Detecting mutations from single DNA molecules is crucial in many fields but challenging. Next-generation sequencing (NGS) affords tremendous throughput but cannot directly sequence double-stranded DNA molecules ('single duplexes') to discern the true mutations on both strands. Here we present Concatenating Original Duplex for Error Correction (CODEC), which confers single duplex resolution to NGS. CODEC affords 1,000-fold higher accuracy than NGS, using up to 100-fold fewer reads than duplex sequencing. CODEC revealed mutation frequencies of 2.72 × 10-8 in sperm of a 39-year-old individual, and somatic mutations acquired with age in blood cells. CODEC detected genome-wide, clonal hematopoiesis mutations from single DNA molecules, single mutated duplexes from tumor genomes and liquid biopsies, microsatellite instability with 10-fold greater sensitivity and mutational signatures, and specific tumor mutations with up to 100-fold fewer reads. CODEC enables more precise genetic testing and reveals biologically significant mutations, which are commonly obscured by NGS errors.

Single-strand mismatch and damage patterns revealed by single-molecule DNA sequencing.

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other genetic diseases1-4. Almost all of these mosaic mutations begin as nucleotide mismatches or damage in only one of the two strands of the DNA prior to becoming double-strand mutations if unrepaired or misrepaired5. However, current DNA sequencing technologies cannot resolve these initial single-strand events. Here, we developed a single-molecule, long-read sequencing method that achieves single-molecule fidelity for single-base substitutions when present in either one or both strands of the DNA. It also detects single-strand cytosine deamination events, a common type of DNA damage. We profiled 110 samples from diverse tissues, including from individuals with cancer-predisposition syndromes, and define the first single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumors deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples deficient in only polymerase proofreading. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. Since the double-strand DNA mutations interrogated by prior studies are only the endpoint of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable new studies of how mutations arise in a variety of contexts, especially in cancer and aging.