Normozoospermic infertile men possess subpopulations of sperm varying in DNA accessibility, relating to differing reproductive outcomes

Abstract

Study question: Can a reliable assay be developed to quantify DNA accessibility in human sperm to help with the assessment of pre-implantation development affected by dense packaging of mammalian sperm's genetic material?

Summary answer: We adapted NicE-view, an assay that directly labels accessible DNA, for use in human sperm and applied it to examine spermatozoa from infertile individuals with distinct reproductive outcomes.

What is known already: Existing data suggest a connection between sperm chromatin compaction and reproductive outcomes. The assays used to generate this data, however, measure chromatin compaction indirectly and thus understanding their meaning is challenging.

Study design, size, duration: Between April 2020 to December 2023, 60 normozoospermic infertile men were invited to participate in an experimental study and asked to provide a semen sample.

Participants/materials, setting, methods: Among the 60 individuals forty had undergone at least one treatment with ART. Among these ART-treated participants, 20 were included in the study because after fertilization only one or no embryos developed during embryo culture (low blastocyst growth rate, LBGR). The other 20 men were included as at least 50% of cultured embryos developed to the blastocyst stage (high blastocyst growth rate). Additionally, 20 previously infertile individuals obtained a pregnancy naturally (NATP) and were included as well. Washed spermatozoa obtained from seminal plasma or prepared by swim-up procedure were processed for NicE-view to determine DNA accessibility as a marker of chromatin condensation using confocal microscopy. Images of more than 3 million spermatozoa were acquired. Computer-assisted image segmentation was used to identify individual sperm heads and DNA accessibility levels were then quantified in each. We also compared NicE-view to chromomycin A3 (CMA3), a conventional marker of chromatin de-condensation, and ATAC-see, an alternative assay for measuring DNA accessibility.

Main results and the role of chance: Both semen and swim-up samples of participants contained two well-delineated subpopulations of spermatozoa with distinct DNA accessibility levels, the frequencies of which varied among individuals. Interestingly, individuals with high frequencies of highly accessible sperm DNA, as measured in semen, possessed decreased sperm concentrations. Moreover, participants with high frequency of highly accessible sperm DNA were more common in the LBGR sub-group. Surprisingly, selection of motile sperm by swim-up enriched for sperm with high DNA accessibility in participants from all three sub-groups. Chromatin accessibility measurements by Nice-view were distinct from DNA staining with the fluorescent CMA3 dye, and NicE-view allowed much clearer separation of sperm subpopulations than ATAC-see.

Limitations, reasons for caution: This was a single-centre study with a cohort of 60 individuals. Sperm samples containing very high frequencies of sperm with increased DNA accessibility were more common in the LBGR sub-group. The number of individuals with this pattern was, however, limited, even within this category.

Wider implications of the findings: High DNA accessibility is associated with poor pre-implantation embryonic development in vitro and NicE-view may be used for the prediction of abnormal embryonic development in ART. Further studies examining samples from larger cohorts of participants and the localization of accessible regions within the sperm genome are needed to fully evaluate the utility of this method.

Study funding/competing interest(s): Swiss National Science Foundation (Grant No. 189264), Swiss Center for Applied Human Toxicology (SCAHT) research (Grant No. 1 'male reproductive toxicity') (both to C.D.G.), and the Novartis Research Foundation (to A.H.F.M.P.). M.E.G., C.D.G., and A.H.F.M.P. are authors on a patent application (EP23210754.0) on the use of NicE-view for the assessment of sperm.

Trial registration number: ClinicalTrials.gov ID NCT04256668.

Keywords: ART; ICSI; IVF; chromatin accessibility; human reproduction; pre-implantation embryo; sperm.

Figure 1.

NicE-view reveals variation between DNA accessibility in individual human sperm. (A) Cartoon showing different staining conditions used to measure presence of accessible DNA and nicked DNA in sperm samples. (B) Example images showing results of staining outlined in (A). The right panels show merged images from the two left panels (with DAPI signal in magenta and the DNA labelling signal in green). Brightness and contrast for Nick translation image were enhanced relative to the (T4) Nice-view images to make signal more visible. The corresponding unenhanced image is shown in Supplementary Fig. S1. Scale bars = 10 μm. (C) Density plot showing distributions of log2 transformed integrated intensities for sperm stained with conditions indicated in (A). Total sperm numbers per condition are indicated. (D) Correlation plots showing Spearman’s ρ for a set of selected features measured in all (total and swim-up) sperm samples.

Figure 2.

Frequency of sperm with differing levels of DNA accessibility varies between individuals and preparation methods. (A) Split violin plots showing the distribution of single sperm integrated intensity values from 57 individuals stained with Nick translation, NicE-view and T4 NicE-view. Black distributions (left) represent total sperm, while grey distributions (right) refer to sperm selected for motility via swim-up preparation. (B) Beeswarm plots showing distributions of frequencies of highly labelled sperm (for each assay) obtained from thresholding distributions seen in (A), coloured by participant category. For total sperm n = 60, for swim-up n = 57. (C) Correlation plot showing Spearman’s ρ for a set of participant-level features. Values are only displayed for correlations with an FDR-corrected P-value of <0.05.

Figure 3.

Sperm concentration is negatively correlated with the frequency of sperm with high levels of DNA accessibility. (A) Table showing breakdown of participant parameters following re-categorization based on NicE-view frequencies (from Fig. 2B). Data from 57 individuals possessing both total and swim-up samples are shown. Double low: <50% of sperm with high DNA accessibility in both total and swim-up sample. Double high: ≥50% of sperm with high accessibility in both total and swim-up sample. Swim-up high: ≥50% of sperm with high accessibility in swim-up sample, but not in total sample. For numerical variables, a Kruskal–Wallis rank sum test was used to compare groups, while for Original Category a Fisher’s exact test was used. Statistical comparisons of different parameters were corrected for multiple hypothesis testing using a false discovery rate calculation (listed as ‘qvalue’ in table); values in bold are statistically significant. (B) Scatter plots comparing Sperm concentration or the % Nick translation⁺ swim-up sperm to the % NicE-view^high sperm from total (left) or swim-up (right) sperm. Points are coloured based on NicE-view Categories as defined in (A). Values shown are Spearman’s ρ by category. (C) Heatmap showing relationship between spermatological parameters, DNA accessibility, and DNA nicking in all samples examined. Data are ordered by % NicE-view^high sperm in swim-up sample.

Figure 4.

NicE-view intensity barely correlates with chromomycin A3 (CMA3) and provides better separation of lowly and highly labelled sperm than ATAC-see. (A) Split violin plot showing the distribution of log₂ integrated T4 NicE-view intensities from individual sperm from samples fractionated by FACS according to CMA3 staining intensity from six individuals. Sperm with low CMA3 levels are shown in blue on left, while sperm with high CMA3 levels are shown in red on right. The first and third plots from the left are derived from infertile individuals with increased anogenital distance, while the others are from fertile males. (B) Split violin plot showing the distribution of log₂ integrated ATAC-see intensities from individual sperm from total and swim-up preparations from five individuals.

Figure 5.

DNA accessibility and DNA nicking correlate in human sperm samples. (A, B) Scatter plot showing relationship between % NicE-view^high and % T4 NicE-view^high (A) or % Nick translation⁺ (B) sperm in total (left) and swim-up (right) samples. Points are coloured by participant category. Values presented are Spearman’s ρ per category. (C) Cartoon describing double-labelling experiment to label DNA nicks and accessible regions in the same cell. (D) Scatter plot showing the results of double-labelling experiments. Red points indicate Nick translation followed by Nick translation, while blue points indicate Nick translation followed by NicE-view. For both samples, value on y-axis represents Nick translation level. For red points, value on x-axis represents incomplete DNA repair by T4 DNA ligase (likely from highly damaged sperm), while for blue points value on x-axis represents NicE-view signal. Inset shows subpopulations identifiable in scatter plot. Data presented represent a pool of samples from six individuals.