Investigation of genetic causes in non-obstructive azoospermic patients

1. Introduction

Infertility is defined as the failure to achieve a pregnancy by a sexually active couple after one year of regular unprotected sexual intercourse [1]. It is a public health problem that affects approximately 15% of all couples in the world [2]. In recent years, there has been increasing concern about a worldwide decline in male fertility, and the male factor is thought to be responsible for 40–50% of all cases of infertility in the world [3].

Abnormal semen parameters such as oligozoospermia, teratozoospermia or azoospermia are observed in only 50% of cases with male factor infertility [4]. Azoospermia is the most severe form of male factor infertility and is seen in approximately 10–20% of infertile couples [5]. Azoospermia, which means the absence of sperm in the seminal fluid, is caused by obstructive or non-obstructive etiologies [6]. Obstructive azoospermia (OA) is the absence of both spermatozoa and spermatogenetic cells in the semen and post ejaculate urine due to bilateral obstruction of the epididymis, seminal vesicles o rejaculatory ducts [4]. Non-obstructive azoospermia (NOA) is the absence of any sperm in these men analysis, without any evidence of obstruction in these pathways [7, 8].

Approximately 7% of infertile men have structural or numerical chromosomal abnormalities. The prevalence of karyotype anomalies in azoospermia is 10–15%, 5% in oligozoospermia and less than 1% in patients with normal sperm counts [9]. However, many other genetic abnormalities leading to infertility are still unknown.

Genetic research of human populations has made significant advances in recent years thanks to the development and availability of NGS (next generation sequencing) platforms. In contrast to the laborious process of single-gene mutation screening by exon-exon amplification and Sanger sequencing, NGS enables interrogation of large gene panels in a single experiment at a reasonable cost [10]. In recent years, genetic mutations in many diseases with established clinical diagnoses can be revealed thanks to the whole exon sequencing with NGS. At the same time, it has become possible to classify, and reveal the etiologies of different subtypes of the same disease.

In this study, we have aimed to evaluate the genetic analysis results of patients who applied with complaints of infertility and underwent micro-TESE operation with the diagnosis of NOA, using the NGS method. Our main aim in the study will be to evaluate the effect of genetic defects that impairor prevent spermatogenesis in male infertility based on micro-TESE results.

2. Material and methods

This study included 26 azoospermic NOA patients aged 26–44 who underwent TESE at İnönü University Faculty of Medicine, Turgut Özal Medical Center, Departmentof Urology, between 2014 and 2020. Detailed anamnesis of all participants was taken and physical examinations were performed. Patients with a history of pituitary and thyroid pathology, systemic disorders (such as diabetes, hypertension), those who had received chemotherapy or exogenous testosterone supplements were excluded.

Lymphocyte cell cultures were performed in all cases for karyotype analysis using heparinized peripheral venous blood samples. Chromosome analysis was performed on the metaphase plates stained using the high-resolution (450–550) G banding technique according to the standard protocol.

Semen analyses were performed with samples collected from male partners who masturbated after an ejaculatory abstinence period of at least 48 hours. Candidates with low semen volume (<1.5 mL) to be evaluated later for retrograde ejaculation and other obstructive pathologies were not included in the study. After the samples were kept in the incubator at 37 °C for 30 minutes, sperm concentrations were determined using a Makler counting chamber under a microscope at 200 times magnification. The results were evaluated based on WHO (World Health Organization) 2010 semen analysis reference values. When evaluating azoospermia and oligozoospermia, the results of at least 2 semen analyses performed 2–4 weeks apart were taken into account.

Venous blood samples collected from the participants early in the morning after an overnight fast were analyzed for testosterone, prolactin, follicle stimulating hormone (FSH) and luteinizing hormone (LH). Testosterone was analyzed using the chemiluminescent immunoassay method on a (Siemens Immulite 2000 immunoassay system, Siemens Healthineers, Erlangen, BY, Germany), while for the analyses of other hormonal parameters the Siemens Advia Centaur XPT immunoassay system, Siemens Healthineers, Erlangen, BY, Germany) was used.

DNA was extracted from the patients’ peripheral venous blood samples using a commercial kit (11796828001, High Pure PCR Template Preparation Kit, Roche, NRW, Germany) in accordance with the protocol specified below.

1. 200 μL blood sample of each patient was placed in 1.5 mL Eppendorf tubes containing 200 μL binding buffer and 40 μL proteinase K.

2. Blood samples were incubated at 70 °C for 10 minutes.

3. 100 μL of isopropanol was added into each tube and the mixture was transferred to filtered tubes and centrifuged at 8000g for 1 minute.

4. After centrifugation, the bottom of the tube was gently emptied and 500 μL inhibitor removal buffer was added into each tube and incubated at 8000g for 1 minute.

5. Following this process, the remnants at the bottom of the tube were emptied and each tube containing 500 μL wash buffer was centrifuged at 8000g for 1 minute.

6. The procedure number 5 was repeated once more.

7. The bottom of the tube was evacuated and centrifuged at 13,000g for 10 seconds.

8. After the filtered section was transferred into new tubes, 200 μL elution buffer was added and DNA was extracted by centrifuging at 8000g for 1 minute.

The amount and purity of the isolated DNA samples were determined by measuring them on the NanoDrop ND-2000c spectrophotometer.

From the DNA samples extracted, 44 infertility-associated genes in Table 1 were examined using the next generation sequencing (NGS) method [11].

3. Results

The patients participating in the study are numbered from 1 to 26 and the findings are stated in Table 2 to get her with the corresponding patient number. The INSL3 (insulin-like peptide 3) mutation in case number 14 was classified as Class 1, and all other mutations as Class 3 (Table 2).

Genetic analys is of all patients was performed and 47XXY Klinefelter syndrome was observed in 6. AZF (Azoospermia Factor) mutation in 2. FSHR variant change in 17, CFTR (Cystic fibrosis transmembrane conductance regulator) in 5, CATSPER1 (Cation Channel Sperm Associated 1) and TEX101 (Testis Expressed 101) in 1, INSL3 mutation in 1, LHCGR (Luteinizing Hormone/Choriogonadotropin Receptor) in 1, ZMYND15 (Zinc Finger MYND-Type Containing 15) in 1, DNAH5 (Dynein Axonemal Heavy Chain 5) in 2 and DNAH11 (Dynein Axonemal Heavy Chain 11) change in 1 patient, respectively.

4. Discussion

Among 44 genes we investigated, the FSHR (Follicle Stimulating Hormone Receptor) gene whose polymorphism was found in 17 (65.38%) patients, encodes the receptor that enables FSH to bind to the cell during initiation of spermatogenesis. It induces spermatogenesis in Sertoli cells in the testicles. In the absence of this receptor, FSH cannot bind to the cell and spermatogenesis cannot be initiated. The FSHR gene on chromosome 2p21 consists of 9 introns and 10 exons and a promoter region. Mutation screening has identified several single nucleotide polymorphisms (SNPs) in the FSHR gene [54]. Among these, linked SNPs at positions 307 and 680 in exon 10 of the FSHR gene have attracted wide attention. However, many studies investigating possible associations between Thr307Ala and Asn680Ser polymorphisms in the FSHR gene and male infertility, could not yield clear-cut results. Despite this uncertainty, FSHR polymorphisms presumably play an important role in male infertility. To further investigate the association between Thr307Ala and Asn680Ser polymorphisms in the FSHR gene and male infertility, a meta-analysis performed with a case-control study with 212 infertile and 164 fertile men from Northern China was published [55]. It has been shown that the FSHR gene polymorphisms found in this study have a higher frequency in infertile patients than in the control group [55].

In our study, FSHR polymorphism with “unclear pathologenic effect” was found in 17 patients. FSH levels were found to be higher than normal in 11 of these 17 patients (64.7%). In addition, FSHR polymorphisms were detected in 5 of our 6 patients with Klinefelter syndrome and FSH levels were observed to be higher than normal in 6 of them.

Mutations in the CFTR gene cause cystic fibrosis (CF) [56]. This disease is characterized by progressively worsening lung disease, pancreatic dysfunction, elevated sweat electrolytes, and male infertility. Given the fact that almost all male patients with CF are infertile due to congenital bilateral absence of the vas deferens (CBAVD), the question has arisen as to whether CFTR is also involved in infertility due to CBAVD alone. A small-scale French study reported that patients with CF had ongenital aplasia of the epididymis and vas deferens seven cases out of 17 azoospermic men (41%), while only 2.8% of individuals in the general population were reportedly heterozygous for the F508del mutation [57]. In another study, CFTR gene mutation was found in 9 of 750 unselected oligozoospermic patients [58]. Similar results have recently been described in a German population study. A heterozygous CFTR gene mutation was found in 5 (19.23%) of 26 non-obstructive azoospermic patients. Each of the mutations was located in different regions of genes.

The CATSPER1 gene encodes a calcium channel that is involved in the acquisition of motility of mature sperm in the final stage of spermatogenesis. It is predicted that mutations in this gene will disrupt the motility of the sperm flagellum and cause infertility [59]. In our study, we found CATSPER1 mutation in only one of our patients (number 19). This patient who had also TEX101 mutation underwent 47 XXY chromosome analysis.

TEX101 is a glycosylphosphatidyl inositol (GPI)-anchored glycoprotein that has been identified as a molecular marker of germ cells. Although there are indications that malfunction in TEX101 may affect male fertility, little is known about its exact physiological function and underlying molecular mechanisms. Recently, a study has showed that in the absence of the TEX101 gene, sperms were unable to pass through the uterotubal junction or bind to the ZP (zona pellucida), leading to male infertility [60]. Additionally, a study found that a group of mice with the TEX101 mutation had a uterotubal migration defect and that sperms had no ability to adhere to the surface of the female genital tract [61]. In our study, TEX101 mutation was found in patient number 19 with a diagnosis of Klinefelter syndrome who also had a CATSPER1 mutation.

It has been shown that INSL3 and its receptor LGR8 (leucine-rich repeat-containing G-protein-coupled receptor 8), the LH/-LHCGR system are effective in the descent of the testicles from the inguinal canal into the scrotal sac [62]. The INSL3 gene localized on chromosome 19, close to the Janus kinase 3 (JAK3) gene contains two exons with an intron interrupting the C-peptide coding domain [63]. INSL3, produced by prenatal Leydig cells, is required for the transabdominal descent of testicles through stimulation of growth hormone and differentiation of the gubernaculum, as shown in rodents [64]. Since several mutations leading to amino acid substitution have been found incases with INSL3 and LGR8 mutations, it has been suggested that they may have a role in human cryptorchidism [65]. A review of the literature has shown that these mutations are prevalent in 4–5% of men with a past or present history of cryptorchidism [66]. In addition to its role in testicular descent and cryptorchidism, INSL3 hormone has possible important but undefined endocrine and paracrine effects in adults. Deficiency of this hormone may represent an important manifestation of functional hypogonadism [66]. In a recent study, LGR8 expression in germ cells showed that INSL3 is linked to LGR8. Suppression of apoptosis by binding suggested that INSL3 plays an active role in the survival of germ cells [67]. A study comparing serum INSL3 levels in 135 fertile men and 85 male patients with testicular disorders showed that serum INSL3 levels were within the normal range in fertile men, but decreased in patients with testicular disorders [68]. In our study, INSL3 mutation and FSHR gene polymorphism were detected in patient number 14. The patient’s bilateral testicles are located scrotally and their sizes are smaller than normal.

It is well known that LH and its receptor play a role in testicular descent. Patients with hypogonadotropic hypogonadism or inactivating mutations of the LHCGR gene have undescended testes due to androgen deficiency [69].

A study conducted on 278 patients with abnormally located testicles, 277 infertile men, and 271 men with normal spermatogenesis showed that patients with testicular disorders had a high LH level to maintain normal testosterone levels but this LH elevation was not associated with any LHCGR genotype.

It has been determined that the presence of LHCGR mutation is associated with spermatogenic damage rather than testicular dysfunction [70]. In our study, homozygous LHCGR mutation was observed in patient number 12.

ZMYND15 gene has been shown to act as a histone deacetylase-dependent transcriptional repressor which regulates the normal transient expression of haploid cell genes during spermatogenesis. Inactivation of ZMYND15 results in premature activation of the transcription of several important haploid genes, including Prm1 (Protamine 1), Tnp1 (Transition Protein 1), Spem1 (Spermatid Maturation 1) and Catpser3, causing late spermatid exhaustion and male infertility. ZMYND15 is the first transcriptional repressor gene identified as essential for sperm production and male fertility [51]. In a study, ZMYND15 mutation was found in 3 patients in Sanger sequencing of 219 patients with severe oligozoospermia [71]. In our study, ZMYND15 mutation and FSHR gene polymorphism were observed in patient number 11 with Klinefelter syndrome.

Kartagener syndrome, which is a type of Primary Ciliary Dyskinesia (PCD), has three components: chronic bronchitis and bronchiectasis, infertility with immotile sperm or low motility, and situs inversus. The diagnosis can be confirmed by electron microscopic examination of ciliary axonemal structures [72]. Most of the mutations identified to date have occurred in the DNAH5 (dynein axonemal heavy chain 5) and DNAI1 (dynein axonemal intermediate chain 1) genes, and a very small number of mutations involve other genes. A study conducted with 90 asthenozoospermic and 200 normospermic individuals, in which DNAH5, DNAI1 and DNAH11 mutations were investigated, reported that mutations were found in 7 patients in the asthenozoospermic group [73]. In our study, we found DNAH11 variant in patient number 6 and DNAH5 class 3 variant in patients number 7 and 9. Additionally, patients number 7 and 9 had FSHR gene polymorphisms.

The reason why pathogenic and possible pathogenic mutations were not detected in most of the patients in our study is that there may be other genes associated with infertility. In addition, many changes classified as “variants of uncertain clinical significance” were detected in our study according to the ACMG 2015 criteria and in-silico generated databases. However, these changes are not directly associated with infertility today; some of them are classified as polymorphisms and some as variants of unknown significance. These criteria may change as databases are constantly updated and new data is added.

Our study has some limitations. Most importantly our study population consisted of small number of patients. Therefore, mutations that might turn out to be pathogenic in a higher number of patients may have been evaluated as mutations with unknown pathogenic effects in this study. In addition, due to the small number of patients in our study population, rarely seen gene mutations might not be detected. Secondly, due to the lack of a control group in the study, the mutations detected could not be compared with those found in fertile male individuals. In fact, the high cost of commercial kits has restricted our ability to study infertility-associated mutations in greater number of patients. Nevertheless, we believe that our further study on the genetic evaluation of patients with non-obstructive azoospermia will contribute to the literature and lead to conduction of more comprehensive studies.

5. Conclusions

As a result, although many mutations and polymorphisms we found in our study were evaluated as “variants of uncertain clinical significance”, they may be considered “pathogenic” variants in the future. We think that more research is needed to understand whether these changes cause infertility, and that next-generation sequencing can be used as a routine diagnostic test to determine mutations and polymorphisms related to FSHR, DNAH5 and DNAH11 genes in infertility cases of unknown etiology.

Availability of data and materials

The data presented in this study are available on reasonable request from the corresponding author.

Author contributions

BD and IG—designed the research study. BD and AK—performed the research. CE and FO—provided help and advice on collection of samples. EA—analyzed the data. BD and EA—wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

This study was conducted with the approval of the ethics committee of Inonu University Faculty of Medicine, Turgut Ozal Medical Center (ethics committee approval no: 2020/105). The patients provided informed consent and agreed to publication of the details of this research.

Acknowledgment

We would like to thank Gürkan Kazancı, who is a member (#345) of The International Federation of Translators (FédérationI nternationaledes Traducteurs: FIT, 57 rued’Amsterdam, 75008 Paris, France: www.fit-ift.org) for proof reading of the manuscript.

Funding

This research received no external funding.

Conflict of interest

The authors declare no conflict of interest.