**1.6 Smoking and molecular alteration: epigenetics, miRNA/noncoding RNA of spermatozoa**

Different lifestyles and environmental factors alter epigenetic profiles: chromatin modifications, DNA methylation, and noncoding RNAs, thereby altering chromatin structure and changing gene expression [63].

The protamines are fundamental in the sperm chromatin condensation and the protection of the paternal genomic DNA from alterations [56, 64, 65]. It has also been proposed that the deficiency in protamine may lead to the accumulation of lesions at the level of the spermatic DNA [66], morphological abnormalities, and the triggering of apoptotic pathways, the inactivation of mitochondria, and consequently, the decrease in the sperm motility [67].

The alterations of protamine ratio (P1/P2) at the level of the interval (0.8–1.2) in the semen have been clearly associated with the male infertility [68].

Hammadeh et al. also investigated the association between smoking and protamine deficiency of sperm chromatin and demonstrated that the high P1/P2 ratios in smoker are due to an underrepresentation of P2. This suggests that ROS production in smokers deteriorates chromatin condensation and change protamine 1 to protamine 2 ratio of spermatozoa [9].

Smoking is probably behind the underexpression of protamine ending with high levels of histone to protamines ratios [69]. Overall, the alteration from the normal P1/P2 ratio seems to be important in male fertility, although the precise manner by which this happens can differ from person to person.

Abnormalities during the arrangement of chromatin may also cause infertility [70, 71], affecting embryo development [72, 73].

Epigenetic modifications change the gene expression without altering the DNA sequence and can be transferred to next generation through both meiotic and mitotic cell divisions [74].

Different studies have investigated the relationship between the effects of smoking on epigenetic profiles such as chromatin modifications and DNA methylation and genes transcription [75, 76].

Cigarette smoking adversely affects DNA methylation patterns [77–79]. A previous study from our laboratory showed that smoking may lead to biochemical changes in many regions of the sperm DNA that are related to MAPK8IP and TKR gene. And that has negative effects on semen parameters [80].

Moreover, benzo[a]pyrene and nicotine induce alterations in sperm chromatin during histone-protamine transition, which may alter the methylation pattern of CpG in the promoter regions of DNA in the offspring of heavy smokers [81].

Furthermore, many studies took in considerations the interaction between the gene and the environment. They studied the association between tobacco smoking and genetic polymorphisms, involving DNA repair genes and genes involved in carcinogen metabolism [82, 83].

Over 100 miRNAs were found in spermatozoa. Twenty eight of them were differentially expressed between nonsmokers and smokers. In infertile men, the expression of has-miR-146b-5p, has-miR-509-5p, has-miR-146d, and has-miR-652 was altered [83]. These four miRNAs are involved in different pathways such as cell proliferation, differentiation, and apoptosis in spermatozoa as well as early embryogenesis [83].

Altered spermatozoal mRNA profiles and miRNA changes have been shown in smokers [84, 85].

An increased risk of idiopathic male infertility was reported in male smokers, while nonsmokers did not show an increased risk of infertility. These men carried 462Ile/Val genotype of the CYP1A1 gene [86].

Moreover, a significant relationship was observed between smoking and the GSTM1+/GSTT1 del genotypes and the GST gene GSTP1 105IV/GSTT1 polymorphisms in infertile men. The GSTP1, GSTM1, and GSTT1 genes are engaged in the development of idiopathic male infertility [87].

Amor et al. demonstrated that *H2BFWT*, *TNP1*, *TNP2*, *PRM1*, and *PRM2* genes were differentially expressed (p < 0.01), and these genes were downregulated in the spermatozoa of heavy smokers [7].

### **1.7 Male smoking and assisted reproductive treatment (ART)**

Almost 50 million couples worldwide are facing infertility issue [88, 89]. Infertility is described as a disease characterized by a failure to conceive after regular unprotected intercourse of 1 year and is used interchangeably with the term "subfertility" [90].

ART technique was the solution for such couples to solve their infertility issues and to achieve pregnancy. Different lifestyles and environmental factors showed to have an adverse effect on a male and female fertility and consequently conceiving. Tobacco smoking is one of the lifestyle factors that was associated with infertility.

A smoking habit in males also has an adverse effect on pregnancy outcomes among in vitro fertilization (IVF) intracytoplasmic sperm injection (ICSI) patients [8, 91]. An association between cigarette smoking and altered ICSI and IVF outcomes was reported [41]. In a study by Klonoff-Cohen et al., the number of retrieved oocytes decreased by almost 46% in smokers. The males were active smokers, and the females were passive smokers [92]. In addition, a decrease in live birth rates was noticed in 166 couples seeking pregnancy using ART [93].

Although spermatozoa with damaged DNA is still capable of fertilization, but its effect is prominent in the later stages such as apoptosis, poor fertilization rate, high frequency of miscarriage, and morbidity of off springs [94, 95].

Because of the faulty transition histones-protamines, sperm DNA breaks increased, and this may cause poor embryo morphology at early cleavage stages. An abnormal protamine ratio was associated with poor preimplantation [56].

However, other studies have reported that there is no significant relationship between smoking and fertility outcomes in humans [96].
