**1. Introduction**

#### **1.1 Background of nonobstructive azoospermia**

The World Health Organization (WHO) identifies infertility as a couple's failure to conceive despite having regular, unprotected sexual activity for a year. According to the most recent data, more than 50 million couples worldwide (about 15%) suffer from infertility [1, 2]. Male fertility problems affect about 50% of infertile couples [2–5], and 10–15% of this group are azoospermic patients [6]. A total of 40% of azoospermic patients have obstructive azoospermia (OA), and 60% have nonobstructive azoospermia. Normal spermatogenesis and endocrinological functions are present in OA patients; however, the male excurrent duct system is physiologically obstructed [7]. The NOA group is significantly more difficult and suffers from numerous genetically inherited issues. Chromosome abnormalities may involve autosomes or the sex chromosome. Several sex-specific chromosomal anomalies are more common in men who are azoospermic. Depending on the etiology, environmental factors might also

be before or after testicular development [8, 9]. IVF laboratories have been developed to provide a variety of treatment choices, although it was previously unable to treat the azoospermic portion of the population. However, it is crucial that the causes and contributing aspects of this issue can be identified with great clarity. Varicocele, prior febrile illness, cryptorchidism, orchitis, chemotherapy medicines, radiation, and hypogonadotropic hypogonadism are a few of the reasons for azoospermia that have been identified [10–12] abnormal chromosomal numbers, autosomal mutations, abnormal karyotypes, or y chromosomal microdeletions [5, 13–17]. ICSI patients who are male are chromosomally defective in approximately four percent of cases [18]. The two most common chromosomal diseases, Robertsonian translocations and Klinefelter syndrome (KFS), impact between ten and 20% of azoospermic men. Y chromosomal microdeletions, the most typical cause of azoospermia, are seen in between 5 and 10% of infertile males. The azoospermic factor region (AZF) on the Y chromosome long arm (Yq) is crucial for germ cell growth and differentiation [19]. The AZF region includes the AZFa, AZFb, and AZFc gene locations [20]. Compared to AZFa 5%, AZFb 16%, or the combined 14% deletions, AZFc is the most frequently identified 60% deletion. This is due in part to AZFc's length being four times that of AZFa [21, 22]. NOA is a result of the most typical sex chromosomal aneuploidy cause, Klinefelter syndrome (KFS). A total of 40% of azoospermic males have KFS [19, 20]. The 47, XXY chromosomal structure, smaller testicles, high levels of folliclestimulating hormone (FSH), low levels of testosterone (T), and high levels of luteinizing hormone (LH) are characteristics of KFS. The rare aneuploidy known as the 46, XX male disease affects 1 in 20,000 live births. A total of 90% of these males have an autosome, which is often recognizable there, or a translocation of the sex-determining region (SRY) from the Y chromosome to the X chromosome. The most common structural chromosomal defect is a Robertsonian translocation (RT). A total 1 in 1000 live newborns contains them. Infertile men experience reciprocal RTs nine times more frequently than healthy males [18]. On the other hand, there are still between 40% and 60% of people who are considered to be idiopathic [23–25]. Both the definition of this group's symptoms and the problem's origin are still unknown. The condition is characterized as idiopathic, and sperm maturation is impossible. Lifestyle and environmental variables can affect genetic makeup, but it is first essential to identify the mechanism by which the issue causes damage [8, 25–28]. These genetic developments relate to the identification of newly discovered genetic variations that cause spermatogenic failure and are used to diagnose male factor infertility [29]. The growing use of whole exome sequencing (WES) in infertility has sped studies on the telomere effect in recent years [30–34].

#### **1.2 The role of telomeric length**

Recent research findings suggest that some idiopathic reasons for male infertility and NOA may be related to telomeric structure [35–37]. The specialized DNA microsatellites known as telomeres are the linear eukaryotic chromosome ends that contain hexameric tandem repeats [38]. Terminologically, the terms "end" (telos) and "part" (meros) in Latin represent telomere. While working with Drosophila, Herman Muller discovered telomeres 80 years ago. The terminals of eukaryotic chromosomes include particular non-coding nucleoprotein structures. The functions of telomeres and their length in reproduction have been studied [39]**.** In humans, they develop from TTAGGG repeats and are connected to particular proteins. Telomeres serve as chromosomal stability structures and are complicated ribonucleoprotein structures

with repetitive DNA sequences (5′-TTAGG-3′) [40, 41]. The functions of telomeres and their length in reproduction have previously been studied [42–45]. Females have longer telomeres than men, and telomere length varies widely between species [46]. Studies have revealed that telomere shortening through cell divisions causes early cell degeneration, which is connected to the pathogenesis of a number of illnesses, including male infertility, especially in NOA patients [47–55].

#### **1.3 Mechanism of the effect of telomere length on male infertility**

Telomeres play significant roles in meiosis by facilitating key development of gamete stages such as chromosomal pairing, synapsis, and crossing over [56]. Telomerase is a cell-based reverse transcriptase that keeps telomere length stable [57]. Telomeres in healthy somatic cells shorten with each mitotic division until they finally reach a threshold length that triggers aging, cell cycle arrest, and apoptosis [58]. Between somatic and spermatogenetic cells, there are three distinct telomere-specific changes that are known. First off, telomeres in sperm are not shortened with age, unlike those in somatic cells, ensuring that chromosomes are passed down through generations intact. Indeed, several studies have reported that increasing paternal age is actually associated with longer telomeres in spermatogenetic cells and in the leukocytes [59]. Second, numerous telomere-binding proteins have been uncovered in spermatogenetic cells [60]. Third, telomere connections are made as telomeres move toward the nuclear membrane during spermatogenesis [60].

A reverse transcriptase known as telomerase contains two structurally distinct subunits: the catalytic "telomerase reverse transcriptase" (TERT) and the human RNA matrix known as the "telomerase RNA component" (TERC). By lengthening the guanine-rich sequence and slowing the ongoing DNA loss during cell division, this ribonucleoprotein can reduce telomere shortening [61]. In contrast to stem, embryonic, and germ cells, somatic cells do not express telomerase [62, 63]. Telomere length is conserved and enhanced throughout spermatogenesis in male germ cells with active telomerase [39, 41, 45, 61–65]. Telomerase is present in testes from fetal to adulthood. The spermatogenetic stage (spermatogonia level compared to spermatocyte and spermatid stage) has increased telomerase activity**.** However, in spermatozoon, no telomerase activity was measured [46]. The role of sperm telomeres is not completely known, and recent many studies reported the relationship between male infertility and azoospermia [48, 55]. Some of these studies have been published on sperm telomere length in relation to spermatogenesis [55, 66, 67].

These studies show that sperm telomere length (STL) could affect male infertility and that it might be a potential new biomarker of sperm maturity. Many studies work on the relationship between telomere length (TL) in somatic cells, such as leukocyte telomere length (LTL) in azoospermia.

## **2. Methods for quantifying telomere length**

#### **2.1 Diagnosis of azoospermia**

Azoospermia was defined as the absence of spermatozoa in the centrifuged pellet of two or three sequential specimens. These patients were further identified as OA or NOA by evaluating their medical history, physical examination, serum hormone levels (FSH, LH, total testerone, PRL, E2, and progesterone), karyotyping, Y

chromosome microdeletion analysis, and imaging studies, evaluation of percutaneous epididymal sperm aspiration (PESA) or testicular sperm aspiration (TESA) results. Physical blockage of the male reproductive system was the definition of OA. These patients typically have normal hormone levels, indurated epididymides, and normal testicular volume. Spermatogenic dysfunction, which was used to define nonobstructive azoospermia, is characterized in these patients by aberrant hormone levels, tiny and soft testes [68].

For diagnosis of azoospermia, sequential semen samples (2 or 3) must be centrifugated after liquefication for 10 or 15 minutes at least 3.000 g, and after discarding of süpernatant, the remaining pellet (minimum 0,5 ml) must be evaluated under 400× magnification as wet preparation. At least two distinct day semen samples acquired more than 2 weeks apart should be analyzed, and evaluation should be carried out in accordance with 2010 World Health Organization standards [69, 70]. If even small amounts of sperm are found in the centrifuged material in cases where they have been labeled as cryptozoospermia, there is a chance of sperm cryopreservation for ICSI cycles. Numerous studies revealed that 35% of men who were assumed to have nonobstructive azoospermia actually had mature spermatozoa [71]. After several detailed evaluations of centrifugated sequential semen samples, if no mature sperm was found, this prognosis can be identified as azoospermia.

### **2.2 DNA isolation and methods for measurement of telomere length**

Genomic DNA was extracted from peripheral blood leucocytes by conventional kits. The length of telomere length (LTL) was then determined using some different techniques.

#### *2.2.1 Terminal restriction fragmentation (TRF)*

The first method for determining telomere length was terminal restriction fragment (TRF) analysis, which is referred to be the "gold standard" approach with a combination of frequently cutting restriction enzymes that are unable to cut telomeric DNA because genomic DNA is digested using this method as the telomeric and subtelomeric regions lack recognition sites. By a probe designed specifically for telomeric DNA, the telomeric component is then identified by southern blotting or in-gel hybridization. The undamaged telomeres from each chromosome are then separated by agarose gel electrophoresis into groups based on their size. Depending on their size and intensity, telomeres will imprint at varying lengths [72, 73]. This benefit is crucial for the implementation of this technology because it means that it does not need expensive or specialist equipment. The advantages of this method include the opportunity to compare results with those from other studies and the provision of a kilobase size evaluation for telomere length. The use of restriction enzymes results in the containment of subtelomeric DNA that is close to the telomere, which causes the genuine telomere length to be incorrectly estimated as a flaw in this approach. The polymorphisms in these subtelomeric and telomeric regions may potentially result in incorrect findings interpretation. The results may also differ according to the restriction enzymes that were used. Other limitations of the TRF test include the need for significant amounts of DNA (micrograms) and the preference for telomere length analysis in blood samples rather than other tissue types. Because very short telomeres might not be able to sufficiently bind the probe, this approach cannot detect short telomeres on a small number of chromosomes [74].
