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Clinical and genetic particularities in male infertility

Autor: Stela Racoviţă Svetlana Capcelea Kiril Boiciuc Veaceslav Moșin Ninel Revenco Svetlana Hadjiu1 Mariana Sprincean
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Infertility is a worldwide problem affecting 15% of couples of reproductive age which had unprotected intercourse. In half of these cases, a male causal factor can be responsible of infertility [1,2]. A great proportion of these patients, approximately 15%, are diagnosed as azoospermic and genetic factors explain 21-29% of them [3,4]. Chromosomal abnormalities are one of the major causes of azoospermia, in about 15% of azoospermic men, these rates being much higher than the frequency in the general population which is approximately 0.6%. The most common chromosomal abnormality in men with azoospermia is Klinefelter syndrome with prevalence of 10% [5]. Several thousands of genes are involved in normal sexual development, testis determination, and spermatogenesis. Nowadays, only a few have routine clinical importance, but their number is rising with the new generation of sequencing. There is special interest in the genes of the long arm of the Y chromosome since they play an essential role in spermatogenesis and testes development. Most of these genes are located in a specific region known as the azoospermia factor region (AZF). There are three genetic domains in AZF (AZFa, AZFb, AZFc) on the Y chromosome, and gene microdeletions in these regions have been shown to be associated with azoospermia or severe oligozoospermia [6]. Microdeletions on the Y chromosome are the most frequent known molecular-genetic cause of male infertility. The frequency of Y chromosomal microdeletions in nonobstructive azoospermia is about 10% and in severe oligozoospermia is 5% [7].

In men with obstructive azoospermia it is also proven the practical importance of the CFTR (cystic fibrosis transmembrane conductance regulator) gene, which mutations cause cystic fibrosis and absence of vas deferens. Men with azoospermia caused by some of these genetic abnormalities can undergo sperm retrieval techniques and potentially father their own children. Using assisted reproductive technologies such as intracytoplasmic sperm injection (ICSI) and testicular sperm extraction (TESE) can bypass the natural selection, may result in transmission of genetic disorders to the offspring [8].
To determine chromosomal variations, Ychromosome microdeletion of the AZF and cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in infertile men, before assisted reproductive techniques (ART).
In this study we investigated 45 infertile men during genetic counseling among infertile couples referred for ART treatment, prior to intracytoplasmic sperm injection (ICSI). All patients signed an informed consent, approved by the local Ethics Committee and the study was performed according to the Helsinki declaration II. The 45 infertile men were azoospermic according to semen analyses following a 2-7 days’ period of sexual abstinence, according to the World Health Organization (WHO, 2010) guidelines for semen analysis. Criteria for including patients were fulfilled if they presented with azoospermia, raised or normal levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone. Karyotype analysis was performed on peripheral lymphocyte cultures using standard cytogenetic methods- G-banding. After 72 hours culturing, the cells were harvested, hypotonised and fixed using methanol. The metaphases were spread on slides. At least 10 metaphases were analyzed per individual and in cases of suspected of abnormalities the number of metaphases was increased. Chromosomal abnormalities were reported according to the recommendations of the International System for Chromosome Nomenclature (ISCN 2016). Genomic DNA was isolated according to standard procedure from peripheral blood samples and used to analyze AZF microdeletions and CFTR gene by PCR. The sequence-tagged sites used were sY84 and sY86 (AZFa), sY127 and sY134 (AZFb), sY254 and sY255 (AZFc), and SRY and ZFX/ZFY (controls). Specific primers used for multiplex mix and the expected PCR product lengths are shown in Table I. Two common mutations of the CFTR gene were tested ΔF508 and G542X.

Of 45 cases of infertile men with azoospermia, 34 had normal karyotype (46, XY). In 11 cases chromosomal abnormalities have been found, including 9 cases of the sexual chromosomes and 2 cases of the autosomal chromosomes abnormalities.

The most common abnormality of the sexual chromosomes was Klinefelter syndrome in 7 cases (figure 1, table 2) with classical form 47,XXY. As well we detected a rare case of sexual inversion in the man with karyotype 46,XX (figure 5), 1 patient with balanced autosomal rearrangement 46,XY,der(5),t(9;5)(9pter::5q23.3→ qter) (figure 2), and 2 persons with chromosomal polymorphism 46,XYqh+ (figure 3), 46,XY,14ps+. Most patients with Klinefelter syndrome were significantly taller than patients with normal karyotypes. As well they presented small, firm testes, hypogonadism and gynecomastia. The average age of patients with Klinefelter syndrome was 33.8, the main reason for consulting was infertility (Table II). Deletions of Y chromosome were seen in the AZFc regions of 2 patients (figure 4) with features of normal karyotype, deleted markers were sY254 and sY255. Both had history of infertility more than 5 years, slight increase in follicle stimulating hormone (FSH) and serum testosterone concentration was redused.

Microdeletions in each region of AZFa-sY84, sY86; AZFb-sY124, sY134; AZFc-sY254, sY255, and presence of gene SRY and ZFY were detected in the patient with XX testicular disorder of sex development (figure 6). The results of endocrine markers of the patient were as follows: slight increase in FSH of 11.5 mlU/ml (normal range of 2.0-10.0 mlU/ml); serum testosterone concentration 1.3 ng/ ml (normal range 2.0-6.9 ng/ml); while luteinizing hormone (LH) was in the normal range of 5.5 mlU/ ml (normal range 2.0-12.0 mlU/ml). The phenotype and psychological identity of the patient were male, height and weight being within the norm limits, genital organs characteristic of male sex with hypogonadism. The result of spermograme presented azoospermia.

Two men have been found as carriers of a CFTR gene mutation ΔF508 (figure 7); for calculating the risk of recurrence in offspring were also investigated their spouses, who were homozygous.

The frequency of chromosomal aberration in our patients are significantly higher – 24,4% (11/45), than the one quoted in other bibliographic sources. The results of our study are, of course, due to the fact that we investigated men with azoospermia. It is well known that chromosomal abnormalities increase with decreasing sperm counts [9]. Sex chromosomal anomalies were predominant with 9 cases than autosomal chromosome anomalies in 2 cases. The most common abnormality of the sexual chromosomes was 47, XXY – 15,5% (7/45), (figure 1, table II). This high incidence of Klinefelter’s syndrome among azoospermic men is also reported in bibliographic sources [10]. The median patients age was 33.8 (range 24 to 47) years, and their median duration of infertility was 7.8 (range 5 to 15) years (table 2). These cases reflected reality that a large number of cases are detected after puberty among patients with azoospermia presenting to infertility clinics. The large majority, more than 90% of nonmosaic 47, XXY males, are azoospermic and would not be able to naturally conceive [7,11]. However, nowadays patients with Klinefelter’s syndrome, including the non-mosaic type, need no longer be considered irrevocably infertile. ICSI offers the opportunity for reproduction even when spermatozoa are not present in the ejaculate, but they risk producing offspring with chromosomal abnormalities [12,13]. In the current study, one patient was diagnosed with XX karyotype (figure 5). The two categories of this syndrome are described in the literature, SRY positive in 80% of patients and 20% in SRY negative. The case presented also refers to the SRY positive category, with the SRY gene being detected by the multiplex PCR molecular examination (figure 6). This rare condition occurs in approximately 1/20,000 and was initially named – XX male syndrome, in 2005 was revised its current nomenclature of 46,XX testicular disorder of sex development (DSD) [14]. Literature data describe sporadic cases of this syndrome, although family cases have also been reported. In this case, it was also considered sporadic on the basis of family history. Early diagnosis of XX in male is often missed due to the relatively normal development of sexual organs and the phenotype. After puberty majority of them presented hypogonadism, azoospermia and infertility. Our case was diagnosed at the age of 31 due to the same cause mentioned above azoospermia and infertility. With 46,XX DSD, the fertility possibility are restricted.
The following options can be artificial insemination with donor semen (AID) or adoption. Balanced autosomal translocations has been reported in bibliographic sources among oligozoospermic and azoospermic men [15]. In the present study reciprocal translocations 46,XY,der(5),t(9;5)(9pter::5q23.3→qter) (figure 2) were seen in one azoospermic men, the frequency being 2.2% (1/45 patients). The patients that are carriers of balanced translocations have normal phenotype but may produce unbalanced gametes with the potential for this unbalanced phenotype to be inherited by an ICSI-derived child [16]. Male infertility is associated in more and more studies in recent years with chromosomal polymorphisms.
In our study they were present in two participants 46,XYqh+ (figure 3), 46,XY,14ps+. The role of chromosomal polymorphisms in infertility has been studied previously for many authors and despite of being overrepresented in infertile couples, this subject continues to be an intriguing question, without having a fully elucidated clinical relevance [17]. In the current study, AZF microdeletions was detected in 2 (4.4%) of 45 azoospermic cases. Both patients presented the loss of sY254, and sY255, that corresponded with DAZ gene from AZFc region (Figure 4). AZFa and AZFb deletions have not been detected in either patient. Both cases with deletions had normal karyotype (46,XY). The DAZ (Deleted in Azoospermia) gene family is reported to be the most frequently deleted AZF candidate gene with the frequency of 13% in azoospermic men, in good agreement with the 5% to 20% rate of microdeletions [18, 19, 20, 21]. The DAZ gene belongs to a gene family, that encodes RNA-binding proteins that are exclusively expressed in the germ cells and control spermatogenesis. Deletions of the AZFc locus cause spermatogenic defects varying in severity, ranging from azoospermia due to Sertoli cell-only to oligozoospermia with different testicular phenotype [22]. Cystic fibrosis (CF) is an autosomal-recessive disorder caused by mutations in the CFTR gene. In the present study have been found two men as carriers of a CFTR gene mutation- ΔF508 (figure 7). The ΔF508 mutation is the most common mutation of CFTR gene with frequency in about 50– 80% [23]. For men with CFTR mutations, female partner testing and genetic counseling is important prior to ART. For calculating the risk of recurrence in offspring were also investigated their spouses, who were homozygous.
This study highlights for all couples with the diagnosis of male infertility with azoospermia the need of genetic testing and counseling prior to employment of assisted reproduction techniques. This is important for providing a firm diagnosis and fertility treatment to couples with infertility.

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