Human Sperm Morphology as a Marker of Its Nuclear Quality and Epigenetic Pattern
Abstract
:1. Introduction
2. Materials and Methods
2.1. Patients
- -
- alpha (the type I error, i.e., the probability of wrongly rejecting H0 and detecting a statistically significant difference when the groups are not actually different) at 0.05 and
- -
- beta (the type II error, i.e., the probability of wrongly accepting H0 and not detecting a statistically significant difference when a specified difference between the groups in reality exists) at 0.1, the power of our analyses reached 90% (1-beta: 0.9) with 12 patients included. As we had already included 20 patients, we analyzed the results obtained in these 20 patients, thus exceeding the required number of patients.
2.2. Sperm Preparation
2.3. Sperm Selection under High-Magnification Microscopy (IMSI-like Methodology)
2.4. Selection of Epigenetic Marks of Interest in Male Fertility: Nuclear Proteins and PTMs
2.5. Fluorescent Immunocytochemistry and Two-Dimensional (2D) Microscopy
2.6. Three-Dimensional (3D) Microscopy
3. Results
3.1. Sperm Epigenetic Marks and Sperm Morphology under High-Magnification Microscopy
3.2. Anatomical Relationships between Vacuoles and Epigenetic Marks
3.2.1. 2D Microscopy
3.2.2. 3D- Deconvolution Microscopy
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Patients | Age | Etiology | Abstinence Time (Days) | Semen Volume (mL) | Sperm Concentration (106/mL) | Total Sperm Count (106/ Ejaculate) | Sperm Vitality (%) | Sperm Total Motility (%) | Sperm Progressive Motility (%) | Sperm Typical Forms 1 (%) |
---|---|---|---|---|---|---|---|---|---|---|
1 | 34 | Exclusively female | 2 | 3.2 | 34.3 | 109.8 | 78 | 60 | 40 | 25 |
2 | 42 | Idiopathic | 2 | 4.3 | 21.6 | 92.9 | 83 | 45 | 35 | 31 |
3 | 32 | Exclusively female | 3 | 2.6 | 42.7 | 111.0 | 65 | 50 | 40 | 24 |
4 | 38 | Idiopathic | 2 | 2.8 | 57.2 | 160.2 | 71 | 60 | 45 | 36 |
5 | 29 | Idiopathic | 4 | 5.9 | 19.9 | 117.4 | 81 | 60 | 50 | 39 |
6 | 26 | Exclusively female | 3 | 3.3 | 33.2 | 109.6 | 82 | 50 | 40 | 33 |
7 | 44 | Idiopathic | 5 | 4.1 | 48.3 | 198.0 | 76 | 60 | 45 | 27 |
8 | 48 | Idiopathic | 4 | 2.3 | 29.0 | 66.7 | 79 | 60 | 55 | 29 |
9 | 31 | Idiopathic | 3 | 2.6 | 28.3 | 73.6 | 64 | 45 | 35 | 32 |
10 | 32 | Exclusively female | 4 | 2.7 | 46.1 | 124.5 | 69 | 50 | 35 | 25 |
11 | 28 | Exclusively male | 2 | 6.4 | 2.2 | 14.1 | 64 | 30 | 20 | 13 |
12 | 34 | Combined male and female | 5 | 1.9 | 8.6 | 16.3 | 76 | 50 | 30 | 21 |
13 | 26 | Exclusively male | 2 | 5.8 | 1.9 | 11.0 | 45 | 20 | 15 | 5 |
14 | 47 | Exclusively male | 2 | 3.1 | 5.7 | 17.7 | 77 | 40 | 30 | 12 |
15 | 39 | Combined male and female | 3 | 4.2 | 6.3 | 26.5 | 71 | 40 | 30 | 17 |
16 | 44 | Combined male and female | 3 | 1.7 | 7.2 | 12.2 | 48 | 20 | 10 | 2 |
17 | 32 | Combined male and female | 2 | 2.5 | 10.3 | 25.8 | 52 | 30 | 15 | 13 |
18 | 38 | Exclusively male | 4 | 2.1 | 11.9 | 25.0 | 56 | 30 | 20 | 18 |
19 | 27 | Exclusively male | 2 | 3.0 | 9.4 | 28.2 | 82 | 50 | 30 | 20 |
20 | 33 | Combined male and female | 4 | 3.2 | 10.1 | 32.3 | 64 | 40 | 30 | 8 |
Nuclear Proteins (n = 7) | |
---|---|
Protamine (2) | protamine 1, protamine 2 |
Core histones (5) | H1, H2A, H2B, H3, H4 |
Protamine epigenetic marks (n = 3) | |
P1 (3) | ph1, ph27ac1, ph3 |
P2 (0) | |
Histone variants (n = 23) | |
H1 (3) | H1.4, H1.t, H1.t2 |
H2A (11) | H2A.1a, H2A.2b, H2A.2c, H2A.3, H2A.J, H2A.V, H2A.X, H2A-bbd 2/3, H2A.Z, macroH2A.1, macroH2A.2 |
H2B (6) | tH2B., H2B.1b, H2B.1c/e/f, H2B.1d, H2B.1 l, H2B.2f |
H3 (3) | H3, H3.3, H3.1t |
H4 (0) | |
Histones PTMs (n = 104) | |
H1 (4) | K43ac, R50me1, K62me1, K63ac |
H1t (13) | K112me1, K113ac, K122ac, K124me1, K170ac, K173me1, K183ac, K183me3, R185me1, S180ph, S187ph, K188me1, K190ac |
H2A (3) | K5ac, R11me2, R29me1 |
H2AV/Z (21) | K4ac, K4me2/3, K7ac, K7cr, K7me1/2/3, S9ph, K11cr, K11me1/2/3, K13cr, K13me2/3, K15ac, R19me1, K27ac, K37cr, K37me1 |
H2B (2) | K16me1, K20me3 |
tH2B (15) | K6ac, T9ph, K12ac, K12me1/3, K13me1/3, K16me1/3, K17me1, K28ac, K29ac, R30me2, K86ac, R87me1 |
H3 (31) | T3ph, K4me1/2/3, K9ac, K9me1/2/3, K14ac, K18ac, K18me1, K23ac, K23me1/3, R26me1/2 K27me1/2/3, K36cr, K36me1/2/3, K37ac, K37me2/3, R53me1, K56ac, K79me1/2, K12Ox |
H4 (15) | S1ph, R3me1, K5ac, K8ac, K12ac, K16ac, K9ac, K20me1/2/3, K31ac, R35me1, M84Ox, K91ac, R92me1 |
Pairing | Rabbit Antibody Cy3 (Red) | Mouse Antibody FITC (Green) |
---|---|---|
1 | Pan H3 Millipore 06-755 | Protamine 2 Briar Patch Biosciences Hup-2B |
2 | H3K27me3 Millipore 07-449 | H3K9me2 Active motif 39683 clone MABI 0307 |
3 | H3K9me1 Millipore ABE101 | H3K4me2 Millipore 05-1338 clone CMA303 |
4 | H3K4me3 Abcam ab8580 | H4K20me2 GeneTex GTX630545 clone GT1851 |
5 | H3K9me3 Active motif 39765 | H3K4me1 GeneTex GTX50902 clone MABI0302 |
Non-Vacuolated Sperm (%) | Vacuolated Sperm (%) | ||||||
---|---|---|---|---|---|---|---|
Nuclear Proteins and PTMs | Mean ± SEM | Median | Q3–Q1 | Mean ± SEM | Median | Q3–Q1 | p-Value |
H3 | 74.8 ± 4.8 | 80.9 | 62.5–90 | 88.1 ± 2.7 | 90 | 83.7–95 | 0.009 * |
P2 | 82.1 ± 4.4 | 88.8 | 67.8–100 | 50.2 ± 6.2 | 35 | 28.8–76.3 | <0.001 * |
H3K4me1 | 5.4 ± 0.8 | 5.0 | 3.1–8 | 6.2 ± 0.8 | 8 | 5–8.5 | 0.325 |
H3K4me2 | 6.4 ± 1.0 | 5.0 | 5.0–10 | 6.3 ± 0.9 | 6.5 | 5– 8.3 | 0.71 |
H3K4me3 | 49.1 ± 7.4 | 46.3 | 13.8–76.7 | 78.5 ± 5.2 | 88.2 | 73.3–95 | <0.001 * |
H3K9me1 | 31.1 ± 6.3 | 25 | 10–43.5 | 42.3 ± 4.8 | 35 | 33.3–68.2 | 0.165 |
H3K9me2 | 22.6 ± 6.5 | 7.5 | 0.0–32.5 | 15.4 ± 4.7 | 73 | 0.0–25.9 | 0.13 |
H3K9me3 | 61.8 ± 6.9 | 69.1 | 26.7–85.7 | 62.2 ± 4.9 | 65 | 47.7–71.2 | 0.911 |
H3K27me3 | 73.6 ± 5.1 | 85.7 | 58.8–90 | 63.9 ± 6.3 | 79.2 | 42.5–85.9 | 0.028 * |
H4K20me2 | 18.7 ± 2.6 | 17.4 | 9.9–27.3 | 24.7 ± 4.5 | 17.4 | 9.1–31 | 0.121 |
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Bendayan, M.; Caceres, L.; Saïs, E.; Swierkowski-Blanchard, N.; Alter, L.; Bonnet-Garnier, A.; Boitrelle, F. Human Sperm Morphology as a Marker of Its Nuclear Quality and Epigenetic Pattern. Cells 2022, 11, 1788. https://doi.org/10.3390/cells11111788
Bendayan M, Caceres L, Saïs E, Swierkowski-Blanchard N, Alter L, Bonnet-Garnier A, Boitrelle F. Human Sperm Morphology as a Marker of Its Nuclear Quality and Epigenetic Pattern. Cells. 2022; 11(11):1788. https://doi.org/10.3390/cells11111788
Chicago/Turabian StyleBendayan, Marion, Liliana Caceres, Emine Saïs, Nelly Swierkowski-Blanchard, Laura Alter, Amélie Bonnet-Garnier, and Florence Boitrelle. 2022. "Human Sperm Morphology as a Marker of Its Nuclear Quality and Epigenetic Pattern" Cells 11, no. 11: 1788. https://doi.org/10.3390/cells11111788