**10. Oxidative DNA Damage**

Spermatozoa harbor the haploid paternal genome and also important epigenetic information with regulatory roles for early embryo development [186]. Recently, it has been reported that biotechnologies such as cryopreservation damage sperm genes with important roles in fertilization and early embryo development, even in the absence of detectable DNA fragmentation [187,188]. Cryopreservation can also damage the sperm epigenome [189]. Many assays have been developed to investigate DNA integrity in the spermatozoa [190,191]. It is considered that most of the DNA damage is caused by an oxidative mechanism. Oxidation of nucleotides can cause abasic pairs in DNA, increasing the risk of replication errors. Loss of a base in DNA, i.e., creation of an abasic site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously, or under the action of radiation or alkylating agents, or enzymatically as an intermediate in the repair of modified or abnormal bases. The abasic site lesion is mutagenic or lethal if not repaired. From a chemical view point, the abasic site is an alkali-labile residue that leads to strand breakage through beta- and delta- elimination [192,193]. More

recently, multiple consequences of the electrophilic nature of abasic lesions have been revealed [194], and oxidized abasic sites are nowadays considered irreparable, leading to the most deleterious form of DNA damage, inter-strand cross links and double strand breaks [195,196]. Detection of oxidized nucleotides in sperm with flow cytometry has been reported using a specific antibody against the oxidative derivative of guanosine, 8-hydroxyguanosine [109,197], and threshold values for fertility have recently been reported in humans [198]. Another newly developed flow-cytometry-based assay, for evaluation of oxidative stress in sperm DNA, is the γHA2AX assay [199]. Although most histones are replaced by protamines, a small fraction remain in the nucleosome (5-15% in humans). This fraction contains the H2AH histone that is phosphorylated in Ser139 when under oxidative stress. The detection of γHA2AX (the phosphorylated form of the histone) has proven to be more sensitive than the TUNEL assay to detect DNA fragmentation, and also to be better correlated with pregnancy outcome in humans [200].

### **11. Impact of Early Embryo Development (EED)**

Fecundation of the egg by spermatozoa with compromised redox regulation or experiencing non-lethal oxidative stress has important consequences with regard to embryo viability and the health and well-being of the offspring [201]. Assisted reproductive technologies such as in vitro fertilization and ICSI are associated with an increased incidence of birth defects in offspring [202]. Animal studies indicate that fecundation with spermatozoa experiencing oxidative stress may cause embryonic death [203], an effect that has been linked to oxidative damage in the spermatozoa [204]. Recent research from our laboratory has compared the effect of cryopreservation on the transcriptome of early equine embryos [205]. Using the same ejaculate, half processed as fresh sperm and the other half frozen and thawed, we obtained embryos from the same mare and stallion after artificial insemination with the aliquot of fresh sperm and, in the mare's next cycle using the frozen thawed semen aliquot. The transcriptional profile of embryos obtained with frozen thawed spermatozoa differed significantly from that of embryos obtained with the fresh sperm aliquot of the same ejaculate. Significant downregulation of genes involved in biological pathways related to the gene ontology (GO) terms *oxidative phosphorylation, DNA binding, DNA replication,* and *immune response*. Interestingly, many genes with reduced expression were orthologs of genes in which knockouts are embryonic lethal in mice [205]. While the exact mechanism behind these changes remains to be elucidated, redox deregulation and oxidative stress in the spermatozoa seem to be an important factor. The spermatozoa is known to carry proteins [201], and numerous ncRNAs [206] to the oocyte, with important functions in early embryogenesis. However, it has recently been reported that caput epidydimal mouse sperm, which has not ye<sup>t</sup> incorporated RNAs, can support full development [207]. The impact of redox deregulation on sperm proteins is well recognized and has recently been reviewed [51,208], so it is not unlikely that oxidized proteins can be incorporated by the embryo impacting its development. Recently, preimplantation proteins in the human embryo with potential sperm origin have been identified [201]. In particular, 93 different proteins have been proposed as related to zygote and early embryo development before implantation in humans, moreover up to 560 sperm proteins with known roles in the regulation of gene expression in other cells or tissues have also been identified [201]. Even though further investigation is needed in this field, oxidative damage to sperm proteins with important functions during early embryo development may occur. Further supporting this hypothesis is the fact that biological processes such as *DNA binding and replication,* and *Histone Acetylation* were downregulated in embryos obtained with cryopreserved spermatozoa [205], and many of the proteins mentioned above have roles in these processes [201].
