H3K4me2 accompanies chromatin immaturity in human spermatozoa: an epigenetic marker for sperm quality assessment

Figures & data

Table 1. Overview of sperm parameters according to semen quality.

	N (22)	A (63)	OA (14)	p-value
Age (years)	34 (31–37)^ab	36 (33–40)^a	32.5 (31–37)^b	0.037
Volume (ml)	2.70 (2.10–3.40)^a	3.15 (2.50–4.20)^a	3.50 (2.40–5.10)^a	0.139
Concentration (mil/ml)	84.00 (69.00–109.00)^a	49.00 (29.00–78.00)^b	7.00 (4.00–10.00)^c	<0.001
Total motility (%)	66.00 (64.00–73.00)^a	49.00 (37.00–56.00)^b	42.00 (36.00–56.00)^b	<0.001
Progressive motility (%)	37.50 (30.00–50.00)^a	8.00 (3.00–14.00)^b	0.00 (0.00–10.00)^b	<0.001
% HDS	6.87 (3.98–10.09) ^a	10.43 (6.63–18.32)^a	11.77 (5.52–30.38)^a	0.044
MitoTracker+/YO-PRO1- (%)	71.31 (63.65–84.33) ^a	53.94 (43.50–66.37) ^b	51.095 (38.95–57.94) ^b	0.001
MitoTracker-/YO-PRO1+ (%)	14.05 (9.76–18.41) ^a	22.78 (17.18–30.89) ^b	24,17 (20.59–30.47) ^b	0.004

Values are expressed as medians (quartiles). Different superscript letters within a row denote significant differences (p < 0.05). N, normozoospermic; A, asthenozoospermic; OA, oligoasthenozoospermic; % HDS, high DNA stainability index. Numbers of patients in N, A, and OA groups are indicated above each column.

Table 2. Overview of sperm parameters according to the HDS.

	%HDS ≤ 15 (69)	%HDS > 15 (30)	p-value
Age (years)	36 (32–39)	34.5 (31–39)	0.278
Volume (ml)	3.30 (2.40–4.50)	3.00 (2.50–3.20)	0.101
Concentration (mil/ml)	57.00 (29.00–86.00)	31.50 (18.00–70.00)	0.046
Total motility	52.00 (42.00–65.00)	49.00 (33.00–61.00)	0.041
Progressive motility	11.00 (5.00–24.00)	6.00 (0.00–16.00)	0.043
MitoTracker+/YO-PRO1- (%)	61.00 (49.50–71.23)	52.45 (39.50–66.92)	0.067
MitoTracker-/YO-PRO1+ (%)	19.29 (14.18–28.90)	23.33 (18.49–34.52)	0.180
%DFI	11.59 (7.35–18.41)	13.45 (11.03–19.31)	0.124

Values are expressed as medians (quartiles) and as significant are considered differences at p < 0.05. HDS, High DNA stainability; DFI, DNA fragmentation index. Numbers of patients in the group with low and high HDS are indicated above each column.

Table 3. Summary of Spearman’s rank correlations between %HDS and other sperm parameters.

	%HDS	p-value
Age (years)	−0.125	0.220
Volume (ml)	−0.072	0.480
Concentration (mil/ml)	−0.331	<0.001
Total motility (%)	−0.270	0.007
Progressive motility (%)	−0.310	0.002
MitoTracker+/YO-PRO1- (%)	−0.261	0.023
MitoTracker-/YO-PRO1+ (%)	0.171	0.140
%DFI	0.200	0.047

Correlations are considered significant at p < 0.05. HDS, high DNA stainability index; DFI, DNA fragmentation index.

Table 4. Summary of Spearman’s rank correlations between H3K4me2 and other sperm parameters.

	H3K4me2	p-value
Age (years)	−0.120	0.261
Volume (ml)	0.120	0.260
Concentration (mil/ml)	−0.570	<0.001
Total motility (%)	−0.093	0.374
Progressive motility (%)	−0.250	0.015
MitoTracker+/YO-PRO1- (%)	−0.175	0.140
MitoTracker-/YO-PRO1+ (%)	0.010	0.410
%DFI	0.162	0.120
%HDS	0.470	<0.001

Correlations are considered significant at p < 0.05. HDS, high DNA stainability index; DFI, DNA fragmentation index.

Figure 1. Representative images of the subcellular localization of H3K4me2 in sperm. A) Immunocytochemical localization of H3K4me2 and negative control. B) Difference in H3K4me2 abundance between high and low HDS samples, as determined by western blot densitometry, with an anti-alpha tubulin antibody as a loading control. The data are expressed as means, including min–max whiskers, and different superscripts indicate statistical significance (p < 0.05). HDS, high DNA stainability index.

Figure 2. Relationship between H3K4me2 labeling and semen quality or chromatin maturity. A) Representative images of H3K4me2 histograms generated by flow cytometry, including a normospermic sample with HDS ≤ 15; B) and a sample with pathological semen quality and HDS > 15. C) Comparison of H3K4me2 fluorescence intensity between normozoospermic (N), asthenozoospermic (A), and oligoasthenozoospermic (OA) samples; D) and between samples with HDS ≤ 15 and HDS > 15. The data are expressed as medians and appropriate quartiles, and different superscripts indicate statistical significance (p < 0.05). HDS, high DNA stainability index; DFI, DNA fragmentation index.

Figure 3. Relationship between semen quality and DNA fragmentation or chromatin maturity measured by SCSA. A) Representative histograms and scatter diagrams of the SCSA results for samples with HDS ≤ 15; B) and HDS > 15; C) Comparison of %DFI; D) and %HDS between normozoospermic (N), asthenozoospermic (A), and oligoasthenozoospermic (OA) samples. The data are expressed as medians and appropriate quartiles, and different superscripts indicate statistical significance (p < 0.05). HDS, high DNA stainability index; DFI, DNA fragmentation index.