**5. Reactive oxygen species (ROS)**

Superoxide anion (O2<sup>−</sup>), hydrogen peroxide (H2O2), hypochlorite (OHCl), and hydroxyl radical (OH) are highly reactive oxygen species (ROS) and their production occurs during normal metabolism of the cell. In semen ROS are produced mostly by the leukocytes and immature spermatozoa and are related to acrosome reaction, capacitation, mitochondrial stability, and fusion with oocyte. Imbalance between the formation of ROS and the inability of the antioxidants to neutralize the excessive production of ROS is defined as oxidative stress (OS). As seminal plasma contains antioxidants and has natural antioxidant capacity, it sustains the free radicals balance in the sperm; overproduction of ROS and OS results in lipid peroxidation, protein changes, DNA damage and sperm death, and this may affect male fertility [126, 127].

High concentrations of ROS as potential cause of male infertility have been studied since 1943 [128]. Potential internal yield of excess ROS could be consequence to damaged or abnormal spermatozoa, varicocele, cryptorchism, testicular torsion, infection, inflammation and aging. Some external factors such as exposure to toxins (toluene, methoxyethanol, sulfur dioxide), metals (cadmium) chemotherapy and ionizing radiation (cancer treatment) may also influence ROS levels and form OS.

Studies demonstrate association between elevated ROS levels and abnormal sperm concentration, motility, morphology, higher DNA damage and apoptosis. Comparison between infertile men and donors showed that excess ROS values had a sensitivity of 68.8% and specificity of 93.8% in correlation with poor semen parameters and could result in infertility [129, 130].

It is important to understand the physiological role of ROS as they are relevant to sperm capacitation, hyperactivation and sperm-egg fusion formation. ROS are involved in intracellular cyclic adenosine monophosphate (cAMP) increase followed by protein Kinase A activation and elevation of tyrosine phosphorylation. These changes lead to sperm capacitation and hyperactivation, sperm membrane becomes unstable and initiates acrosome reaction (releasing enzymes contained in the acrosome—nonzymogen acrosin, proacrosin, inhibitor-bound acrosin, hyaluronidase, acid phosphatase, beta-glucuronidase, beta-glucosidase, beta-Nacetylglucosaminidase, beta-galactosidase and beta-N-acetylgalactosaminidase) which allows the binding of sperm cell to oocytes zona pellucida (ZP) [131].

Imbalanced ROS levels could compromise semen quality and functions and keeping them in normal concentration is considered essential to fertility. Oxidative stress and nutritional status are of importance to every person as antioxidant deficiency and malnutrition may alter the health in general. ROS are also related to various respiratory and cardiovascular diseases, neurodegenerative, digestive disorders and even cancer. The clinical importance of OS in relation to fertility is thoroughly studied. The clinical awareness of nutritional balance in disease occurrence, progression and outcome is still limited, but the need of balanced diet nutrients and antioxidants is urged and necessary [132, 133].

### **6. Processing sperm samples** *in vitro*

The ability of a men to become biological father is not only a consequence of normal sperm count but is also linked to the normal function of the male reproductive

tract and sperm activity. Failure in sperm production or low sperm count and motility, poor morphology, disturbance in sperm movement and progressive passage through the cervical mucus, uterus, ampulla of the oviducts, capacitation and acrosome reaction, binding zona peluccida, etc. can result in male infertility.

To overcome male infertility in ART different protocols for sperm processing have been developed. There are still many debates on the exact influence of specific techniques used for sperm processing and their benefit to achieve pregnancy. Selecting a proper technique must be strongly individual according the couple's infertility history and ART treatment plan along with semen quality. Isolating an optimal fraction (higher count with progressive movement, morphologically normal rates) of spermatozoa gives the opportunity for selection and usage of the spermatozoa with a better fertilizability and higher chances to contribute for a viable fetus, for intrauterine insemination (IUI), IVF or ICSI.

Two of the most explored methods for sperm processing in ART—density gradient centrifugation (DGS) and swim-up (SU)—are investigated in details. Compared to fresh sample, the processed one has lower DNA fragmentation rates [134, 135] and lower concentration of ROS regardless which method was used [136, 137].

As there are studies exploring telomere length in reproductive cells (oocytes and spermatozoa) and their connection to infertility, shorter telomeres in spermatozoa might be assumed as a factor causing idiopathic infertility [138]. Truncated telomeres and altered DNA integrity in sperm could negatively influence fertilization, pronuclei formation, embryo morphology and quality and thus could compromise blastocyst formation and implantation. Spermatozoa obtained by either DGC or SU have longer telomeres compared sperm cells in the raw semen [139].

Some substances such as pentoxifylline (methylxanthine derivate primarily used in intermittent claudication and other vascular disorders treatment) might enhance the motility and quantity of motile sperm after processing. By using pentoxifylline primarily on samples with poor quality increased sperm viability in infertile men with oligoasthenozoospermia, was observed. Samples obtained by PESA or TESE could also be improved by implementing this xanthine derivative in cultural media and thus improve sperm motility [140].

Sperm preparation methods along with technical advantages of MSOME allow the selection of sperm cells, with best predictive values, for ART treatments. There are some limitations related to each method used and that is now an open field to research and establish new noninvasive protocols for sperm selection in the routine practice.

### **7. Conclusion**

Spermatogenesis is a complexed process of division and formation of male reproductive cells. It is highly sensitive to various internal (hormonal regulation, transmitters, growth factors) and external (nutritive substances, therapeutics, drugs, hormones and their metabolites, different toxic substances or X-radiation, increased temperature) factors [141]. Given that the time frame for formation of every new generation of spermatozoa takes approximately 3 months, it should be considered that unfavorable effects purge would be the consequence to time consuming treatment or lifestyle changes.

Modern day society—environment, lifestyle and diet are suspected to be harmful to different processes in the organism such as spermatogenesis and could negatively affect the quality and quantity of life through human lifespan including the ability to reproduce. Considering that sex formation takes place during early fetal development attention to mother's nocuous habits, lifestyle, and environmental specifics should be advert. Events during pregnancy could also influence male

**11**

*The Sperm: Parameters and Evaluation DOI: http://dx.doi.org/10.5772/intechopen.90677*

urological or fertility problems.

**Acknowledgements**

**Conflict of interest**

**used through the report**

АМА advanced maternal age

BMI body mass index

ART assisted reproduction technology

CASA computer assisted sperm analysis cAMP cyclic adenosine monophosphate

DGC density gradient centrifugation ICSI intracytoplasmic sperm injection

IUI intrauterine insemination IVF in vitro fertilization

OA oligoasthenozoospermia

ROS reactive oxygen species SAT sperm aneuploidy test

OS oxidative stress

TESE testicular sperm TEX 11 testis expressed gene WHO World Health Organization

ZP zona pellucida H2O2 hydrogen peroxide O2<sup>−</sup> superoxide anion OHCl hypochlorite OH hydroxyl radical

SU swim up

CBAVD congenital bilateral absence of vas deferens

MESA microsurgical epididymal sperm aspiration MSOME motile sperm organelle morphology examination

PESA percutaneous epididymal sperm aspiration

PGS preimplantation genetic screening extraction

PGD preimplantation genetic diagnosis

TAILS tail axoneme intra-lumenal spital

NOA nonobstructive oligoasthenozoospermia

fertility later in life [142]. In some specific cases, when there was a long exposure to high dosage of toxins, chemotherapy or radiotherapy, spermatogenesis regeneration would most probably take years or may never be restored. Healthy life style along with regular medical check and tests could indicate on time and even prevent

The authors wish to thank their IVF unit colleagues at Ob/Gyn Hospital "Dr. Shterev" for most helpful discussions while summarizing this chapter.

The authors report no financial or commercial conflicts of interest.

**Appendix: this appendix presents the definitions of terms/notations** 

CFTR cystic fibrosis transmembrane conductance regulator gene

IMSI intracytoplasmic morphologically selected sperm injection

*The Sperm: Parameters and Evaluation DOI: http://dx.doi.org/10.5772/intechopen.90677*

fertility later in life [142]. In some specific cases, when there was a long exposure to high dosage of toxins, chemotherapy or radiotherapy, spermatogenesis regeneration would most probably take years or may never be restored. Healthy life style along with regular medical check and tests could indicate on time and even prevent urological or fertility problems.
