It is now common knowledge that classical sperm parameters: number, mobility and morphology give only partial information on their developmental capacity. With the advent of intracytoplasmic sperm injection (ICSI), the evaluation of nuclear integrity is now an unavoidable parameter in studying human infertility, irrespective of whether fertilization and blastocyst formation are obtained/observed after ART procedures. Increased DNA fragmentation (SDF/DFI, DNA fragmentation index) and an increase in the decondensation index (SDI), also called high DNA stainability (HDS), in the sperm chromatin structure assay, SCSA, are the two major negative parameters of sperm developmental capacity. These two markers do not affect fertilization but decrease the developmental capacity of the embryo obtained leading to a dramatic increase to the time to pregnancy (Buck Louis et al., Reference Buck Louis, Sundaram, Schisterman, Sweeney, Lynch, Kim, Maisog, Gore-Langton, Eisenberg and Chen2014).

The negative effect of sperm DNA fragmentation is obvious: as the SDF is more or less linked to oxidative damages; this leads to DNA strand breaks (Ménézo et al., Reference Ménézo, Entezami, Lichtblau, Belloc, Cohen and Dale2014a) or adduct products (Badouard et al. Reference Badouard, Ménézo, Panteix, Ravanat, Douki, Cadet and Favier2008), oxidized bases and abasic sites formation. All these insults have to be repaired at the time and immediately after fertilization and the problem is equally shared between the male and the female genomes (Lopes et al., Reference Lopes, Jurisicova and Casper1998), not specifically protected during the quiescent time before maturation. The DNA repair process is mandatory to avoid transmission of mutations to the next generation. The oocyte’s capacity to repair decay is important, redundant, but finite and decreases with maternal age (Ménézo et al., Reference Ménézo, Dale and Cohen2010); overwhelming this defence leads normally to embryo apoptosis. SDF affects IVF outcomes (Evenson et al., Reference Evenson, Jost, Marshall, Zinaman, Clegg, Purvis, de Angelis and Claussen1999) and increases with age (Belloc et al., Reference Belloc, Benkhalifa, Junca, Dumont, Bacrie and Ménézo2009; Deenadayal Mettler et al., Reference Deenadayal Mettler, Govindarajan, Srinivas, Mithraprabhu, Evenson and Mahendran2019) in relation to a decreased capacity to combat oxidative stress.

Three main processes are important in nucleus condensation, i.e. nucleus tertiary structure, measured by SDI/HDS. The replacement of histones by protamines, their padlocking and the final biochemical modification: deacetylation, methylation etc., lead to a spatial and biochemical nucleus conformation required for rapid access to the paternal genome after nucleus swelling (Ward et al., Reference Ward, Rizos, Corridan, Quinn, Boland and Lonergan2001; Ward, Reference Ward2010). This is mandatory for quick activation of the S-phase and the major methylation/epigenetic modifications affecting the male genome, which will be rapidly demethylated and then quickly remethylated (Park et al., Reference Park, Jeong, Shin, Lee and Kang2007). This aspect has been well documented in the bovine, in which fertility of one bull can be estimated from several thousands of inseminations (Eid et al., Reference Eid, Lorton and Parrish1994; Rahman et al., Reference Rahman, Schellander, Luceño and Van Soom2018; Kutchy et al., Reference Kutchy, Menezes, Ugur, Ul Husna, ElDebaky, Evans, Beaty, Santos, Tan, Wills, Topper, Kaya, Moura and Memili2019). HDS/SDI elevated values are related to reduced protamination or a failed padlocking of them, leading to nucleus structural loosening. Tertiary structure anomalies finally reduce the size of both pronuclei (Rahman et al., Reference Rahman, Schellander, Luceño and Van Soom2018). Sperm decondensation is highly significantly directly related to defective protamination in low fertility bulls (Kutchy et al., Reference Kutchy, Menezes, Ugur, Ul Husna, ElDebaky, Evans, Beaty, Santos, Tan, Wills, Topper, Kaya, Moura and Memili2019). The oocyte is poorly equipped to repair and manage a defective tertiary structure (Ménézo et al., Reference Ménézo, Russo, Tosti, El Mouatassim and Benkhalifa2007, Reference Ménézo, Entezami, Lichtblau, Belloc, Cohen and Dale2014a, Reference Ménézo, Evenson, Cohen and Dale2014b).

In human, poor sperm morphology is linked to high HDS/SDI (Zini et al., Reference Zini, Phillips, Courchesne, Boman, Baazeem, Bissonnette, Kadoch and San Gabriel2009). Pronuclear formation can be impaired, leading to a syndrome called ‘no fertilization post ICSI’, an unfortunate concept (Junca et al., Reference Junca, Gonzalez Marti, Tosti, Cohen, De la Fontaine, Benkhalifa and Ménézo2012). It may induce poor embryo development and lead to recurrent miscarriages (Booze et al., Reference Booze, Brannian, Von Wald, Hansen and Evenson2019; Jerre et al., Reference Jerre, Bungum, Evenson and Giwercman2019). The tertiary structure is of major importance to allow the correct timing in preimplantation embryo development. A three-phase first step, involving peroxidation of lipids by NADPH oxidase, followed by a reaction with oxidized glutathione, followed by glutathione peroxidase activity, is mandatory for this protamine padlocking (Ménézo et al., Reference Ménézo, Entezami, Lichtblau, Belloc, Cohen and Dale2014a). This is probably why sperm nucleus condensation increases with age and so HDS/DFI decrease: HDS/SDI and SDF/DFI are independent parameters.

Chromomycin

Chromomycin (CMA3) is presented here as a standard for nucleus decondensation testing and protamination quality. Chromomycin is a fluorescent dye specific for DNA G–C bonds, as is oligomycin. It is first of all a marker of DNA, but also binds to protamines. If co-incubation is performed with soluble salmon protamines, CMA3 binding decreases (Manicardi et al., Reference Manicardi, Bianchi, Pantano, Azzoni, Bizzaro, Bianchi and Sakkas1995). However, if CMA3 staining is performed prior to TUNEL, the sensitivity to nick translation is ‘drastically reduced’ (Manicardi et al., Reference Manicardi, Bianchi, Pantano, Azzoni, Bizzaro, Bianchi and Sakkas1995). Moreover, De Iuliis et al. (Reference De Iuliis, Thomson, Mitchell, Finnie, Koppers, Hedges, Nixon and Aitken2009) found first a strong overlap/correlation (r = 0.956, P < 0.001) between TUNEL and CMA3. This confirmed the strong affinity between DNA and CMA3. Finally, a very high correlation was observed by De Iuliis et al. (Reference De Iuliis, Thomson, Mitchell, Finnie, Koppers, Hedges, Nixon and Aitken2009) between 8-OHdG, the major product of DNA oxidation, and CMA3 (r = 0.610, P < 0.001) that was similar to the correlation observed, in the same laboratory, between DNA fragmentation (TUNEL) and 8-OHdG (r = 0.671, P < 0.001; Aitken et al. Reference Aitken, De Iuliis, Finnie, Hedges and McLachlan2010) (Table 1).

Table 1.

Correlation between, CMA3, TUNEL and 8-OHdG (de Luliis et al., Reference De Iuliis, Thomson, Mitchell, Finnie, Koppers, Hedges, Nixon and Aitken2009; Aitken et al., Reference Aitken, De Iuliis, Finnie, Hedges and McLachlan2010)

Therefore, CMA3 did not allow a dichotomy between condensation and DNA fragmentation, due to oxidative stress insults. Confirming this fact, the paternal age effect measured by CMA3 showed an increase in decondensation with age (Belloc et al., Reference Belloc, Benkhalifa, Junca, Dumont, Bacrie and Ménézo2009), which could be coherent with a decrease in protamination, but did not fit with the increase in sperm DNA fragmentation related to paternal age. CMA3 cannot be considered as the standard test for sperm decondensation. Manicardi et al. (Reference Manicardi, Bianchi, Pantano, Azzoni, Bizzaro, Bianchi and Sakkas1995) showed that the real measurement of condensation with CMA3 was the difference in staining between CMA3 staining minus TUNEL staining, if reduced by two-thirds. The real evaluation of condensation included protamination, padlocking and the final biochemical modifications of nuclear structure (methylation, phosphorylation, acetylation and their combinations).