An additional marker for sperm DNA quality evaluation in spermatozoa of male partners of couples undergoing assisted reproduction technique (IVF/ICSI): Protamine ratio

Abstract

The aim of this study was to evaluate the relationship between the protamine ratio (P1/P2), DNA fragmentation of spermatozoa and protamine deficiency. Patients were grouped into fertile (G1; n = 151) and sub‐fertile (G2; n = 121). DNA fragmentation in spermatozoa was analysed by a TUNEL assay (terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labelling), and the protamination was determined by CMA3 staining, while Western blot was used to measure protamine P1 and P2. While sperm DNA fragmentation (SDF) and protamine ratio were significantly elevated in G2 compared with G1 (12.31 ± 7.01% vs. 17.5 ± 9.5%; p = .001) and (0.91 ± 0.43 vs. 0.75 ± 0.42; p = .003); respectively, the CMA3 positive showed no difference at all between G1 and G2. In G1, the CMA3 positive correlated negatively with the P1/P2 ratio and SDF (r = −.586, r = −.297; p = .001 respectively). In contrast, the protamine ratio correlated positively with SDF (r = .356; p = .001). In G2, no correlation was observed between CMA3 positive, SDF and the P1/P2 ratio but the P1/P2 ratio showed a positive correlation with SDF (r = .479; p = .001). In conclusion, the spermatozoa DNA deterioration was closely associated with abnormal protamination but showed an association with the protamine ratio, more than with CMA3 positive. Therefore, for the evaluation of DNA damage in spermatozoa, the P1/P2 ratio might act as an additional biomarker.

Could high DNA stainability (HDS) be a valuable indicator of sperm nuclear integrity?

“…It is not the case. Consistent with our observations, others have reported that there is no correlation between HDS, TUNEL, DFI, CMA3, AB and TB [ 58 , 59 ]. However, some positive correlations have also been reported [ 60 – 62 ].…”

Tobacco smoking and its impact on the expression level of sperm nuclear protein genes: H2BFWT , TNP1 , TNP2 , PRM1 and PRM2

“…The relative proportion of protamine quantities P1 and P2 has been proposed in many studies as a biomarker of the maturity and integrity of the sperm chromatin (Amor et al., 2019; Hammadeh et al., 2010).…”

“…From each slide, 500 spermatozoa were evaluated, using a fluorescence microscope (Olympus, Japan). Spermatozoa were classified as follows: bright green spermatozoon (CMA3 positive) had low protamination, and dull green spermatozoa (CMA3 negative) had normal protamination (Amor et al., 2019).…”

“…500 spermatozoa were evaluated from each slide using a fluorescence microscope (Olympus, Japan). Spermatozoa were classified as follows: green stained spermatozoa (TUNEL‐positive), and blue stained spermatozoa (TUNEL‐negative; Amor et al., 2019).…”

Semiquantitative promoter methylation ofMLH1andMSH2genes and their impact on sperm DNA fragmentation and chromatin condensation in infertile men

“…In a follow-up study of the same group, a cut-off of 16.8% for DFI has been calculated with high specificity and high positive predictive value (Sharma, Ahmad, Esteves, & Agarwal, 2016 (Yang et al, 2019). In another study, among 272 infertile men who sought for treatment SDF evaluated by TUNEL was found to be negatively correlated with sperm concentration, progressive motility but not correlated with normal morphology (Amor, Shelko, Hamad, Zeyad, & Hammadeh, 2019). Also some studies showed a strong relationship between vitality and SDF (Aghazarian, Huf, Pfluger, & Klatte, 2019.…”