The Hidden Factor of Azoospermia: Telomere Effect

Abstract

Nonobstructive azoospermia (NOA), which results from defective spermatogenesis, is the absence of spermatozoa in the semen. NOA is a complex and multigenetic disorder that is caused by genetic and environmental factors. For the process of spermiogenesis to be fully completed, the functions of telomeres and their length in reproduction are crucial. In recent years, many studies have been published on how leukocyte telomere length might play an important role in the pathophysiology of azoospermia. They show that shorter leucocyte telomere length (LTL) is strongly associated with NOA and defective spermatogenesis. Telomeres preserve human gametogenesis and fertility while preventing chromosomal ends from eroding. The length of the telomere significantly determines how it functions. The proteins are unable to attach to telomeric regions and cannot perform capping at chromosomal ends once telomere shortening rises above a crucial threshold. It is important to include LTL evaluations as a precursor test in the treatment planning that can be created for azoospermic patients.

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, 3, 4, 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, 11, 12] abnormal chromosomal numbers, autosomal mutations, abnormal karyotypes, or y chromosomal microdeletions [5, 13, 14, 15, 16, 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 follicle-stimulating 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, 24, 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, 26, 27, 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, 31, 32, 33, 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, 36, 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, 43, 44, 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, 48, 49, 50, 51, 52, 53, 54, 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, 62, 63, 64, 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].

2.2.2 Polymerase chain reaction-based techniques (qPCR, MMqPCR, and aTL qPCR)

This method was created in order to address the issue of requiring a significant amount of DNA in order to evaluate telomere lengths [50]. Quantitative PCR (qPCR), monochromatic multiplex quantitative PCR (MMqPCR), and absolute telomere length quantification are employed to carry out these processes. The DNA sequence is amplified by PCR using specially designed primers over the course of 20–40 cycles, with each run doubling the amount of the PCR product (the amplicon typically), a fluorophore is employed in qPCR to quantify the quantity of the target DNA sequence. After each turn, the amount of emitted fluorescence is evaluated [75]. Seven years later, the same study group improved this technique by completing the amplification of both the telomeric and single-copy DNA regions from the same tube. Monochrome multiplex quantitative PCR (MMqPCR) is the name of this novel method [76, 77]. The underlying qPCR-based method was further modified by the other research team [78]. This group referred to it as an absolute telomere length (aTL) qPCR method. Briefly, the procedure for using this approach is the same as for using the initial qPCR experiment.

Limitations of this method

• Differences between batches [79]

• Insufficient reference standards [80]

• Equipment for qPCR is required [81].

2.2.3 Single telomere length analysis (STELA)

These techniques do not offer information on individual telomere lengths; instead, they only offer evaluations of the specimen’s typical telomere length. Given that it has been proposed that one or a few short telomeres act as a marker that causes cellular senescence or death [82].

Advantages

• Makes it possible to find telomeres that are critically short.

• Requires no viable cells

• Without the need for specific equipment

Limitations

• Only it provides details for a limited number of certain chromosomal ends

• Provides no mean telomere data

• Does not recognize ends lacking a telomere

• Not accurately detecting extended telomeres

• Intense in work

2.2.4 Quantitative fluorescence in situ hybridization (Q-FISH)

Quantitative fluorescence in situ hybridization (Q-FISH) of telomeric repeats is performed by evaluating metaphase or interphase nuclei after hybridization or labeling with a fluorescent (CCCTAA) three probe [83]. The substrate for Q-FISH, in contrast to the TRF and PCR-based tests, is cells. When used for Q-FISH, cells can be fresh, frozen, formalin-fixed, paraffin-embedded, or permeabilized. The Q-FISH approach can be used for telomeres of any size, not just average or little ones. Additionally, only by this method of evaluation will “telomere-free” endings be identified [84, 85]. For understanding how telomere length varies between chromosomes and how frequently telomere-free ends are associated with chromosomal instability, metaphase Q-FISH experiments have proved essential [86]. It is believed to be particularly helpful for figuring out the telomere length in unusual cells [87]. To correct some of its shortcomings, the use of interphase cells rather than metaphase chromosomes in these modifications is one of the Q-FISH advancements [88, 89].

Advantages

• Single telomere alterations can be found.

• Telomere lengths in particular cell types can be measured.

• When applied to metaphase chromosomes, it can assess the mean telomere length, signal signal-free ends, and identify individual telomeres.

Limitations

• Time-consuming

• Requires a great level of ability to assess chromosomes.

• Requires microscope (typically fluorescent)

• Unit of relative fluorescence for length

• When applied to metaphase chromosomes, it has the ability to recognize individual telomeres, measure end-to-end telomere length, and detect free.

Flow-FISH is another variation of the Q-FISH technique. This method combines flow cytometry with the hybridization of a pan-telomeric (binds to all telomeres) probe to cells in suspension rather than cells attached to slides, as is the case for metaphase and interphase. Cells in solution pass a laser one by one with the use of the technology known as flow cytometry. This method divides populations of cells based on their fluorescence emission or signals [90, 91, 92].

Advantages

• Using this technique, one can ascertain the mean “length” of a particular population of cells.

• When combined with antibodies, can offer details on particular cell types.

• Possibility of automation

Limitations

• Time-consuming

• Demands a high level of skill

• Requires flow sorting apparatus

• Relative fluorescence units are used to express length.

Primed in place telomeres can be labeled for the Q-FISH techniques using a primed in situ (PRINS) application. The telomeric sequences on metaphase chromosomes or interphase nuclei are labeled by PRINS using fluorescently-tagged nucleotides and PCR methods with telomeric primers [93, 94].

Advantages

• We can identify individual telomere modifications.

• Telomere lengths in particular cell types can be measured.

• When applied to metaphase chromosomes, it can assess the mean telomere length, signal signal-free ends, and identify individual telomeres.

Limitations

• Time-consuming

• Requires a great level of ability to assess chromosomes

• Needs a microscope, usually one that is fluorescent.

• Length as a unit of relative fluorescence

• Variability may be impacted by PCR efficiency.

• Requires only cells in metaphase to be mitotically active

2.2.5 Whole genome sequence

Next-generation sequencing has provided the opportunity to get genetic analysis for measuring telomere length measurement [95, 96].

Advantages

• More uniformity and breadth of coverage, including in the exome

• Commenting on clinically the results from genome-wide sequencing

• Data from whole genome sequencing is precise and detailed for identification.

Limitations

• The procedure produces a lot of data.

3. The role of TL and prognosis of NOA

In order to maintain genome integrity, recombination, and mitotic division, telomere length is crucial [97]. Telomere shortening has been linked to a variety of illnesses [98]. Numerous studies on male infertility with azoospermia have shown that the DNA of sperm and leucocytes from azoospermic person had shorter telomere lengths than that of males with viable sperm, indicating a potential role for telomere length in some unidentified male infertilities and azoospermia [96, 99, 100, 101]. The telomere size was evaluated in the blood leukocytes of azoospermic males [102]. In a study by Ferlin [103] on male infertility, LTL and SLT were found to be substantially associated [104, 105, 106].

4. Conclusion

The absence of mature spermatozoa in the pellet examination of concentrated seminal plasma after consecutive sampling at different times is called azoospermia. The azoospermia group without obstruction or duct problems after biochemical, genetic, and physical examinations is called nonobstructive azoospermia, and it has been determined that 1% of the population and ten percent of infertile men are affected by this problem [107]. Numerous infertile males have NOA that has been linked to a variety of genetic abnormalities, such as Y chromosomal microdeletions, karyotype disorders, and missense mutations in genes important in reproductive function. However, these alterations only explain around 25% of azoospermic cases, and around 75% of cases with severe spermatogenic impairment have an idiopathic origin [108, 109]. Recent researches strongly indicate that the pathogenesis of idiopathic NOA is complex and polygenic inheritance, which may affect spermatogenic function. Whereas the molecular mechanisms leading to NOA are still far from being understood, recent developments in genetic analysis have made it possible for our knowledge to increase significantly.

Although studies on this subject continue, telomere length and telomerase activity tests to be performed between indicated NOA patients and idiopathic patients will create an important database for azoospermia without any reason. That is important to fully clarify this possibility in the NOA patient group in order to decide the diagnosis of cryptozoospermia, which is seen in approximately 25–30% of azoospermic patient group patients. More clearly and most importantly, to inform the patient about their treatment.

5. Future trends

If it is possible to identify a target group with this type of telomere shortness for patients in the idiopathic NOA group, it may be possible to predict the rate of sperm finding in the posttreatment ejaculate or post-testicular operation procedure from these patient groups. However, there will be a need for studies to be carried out on this subject and the determination of target groups.