9.3.5 Cytogenetics and molecular genetics

Genetic aberrations are important causes of spermatogenic and endocrine testicular failure. Often, clinical skills are insufficient to demonstrate the primary genetic nature of a gonadal disorder, and cytogenetic and molecular tests should be considered for the diagnostic process (Table 9.5.3.1) (1–7). They are helpful, not only for establishing the basic aetiology of certain types of male endocrine disturbances, but also in that karyotyping and some DNA tests have attained a pivotal role in genetic risk counselling for severely infertile couples. Also, the diagnosis of a chromosomal abnormality or single gene mutation in an infertile man can have repercussions for other members of his family. They may carry the same type of genetic aberration, and thus be at increased risk for inadvertent reproductive outcomes.

The most time-honoured method in male endocrinology is the analysis of banded metaphase chromosome preparations from blood lymphocytes, which remains of undiminished practical importance (8, 9). This technique allows for the direct visualization of the complete set of chromosomes in a somatic cell lineage and provides information on both chromosome number and structure. However, a regular karyotype in somatic cells, such as lymphocytes, does not necessarily translate into normal meiotic pairing and segregation of the chromosomes in the germ cell lineage. Meiotic cell preparations and ejaculated spermatozoa may thus be included in the diagnostic work-up of an infertile man. The place of these techniques is more in the realm of research than of daily clinical practice, as discussed below. In contrast, several molecular genetic tests are firmly established as valuable diagnostic tools. Details concerning the two most important tests, mutation analysis of the CFTR gene and screening for Y-chromosomal microdeletions, are given below.

In human somatic cells, only one X chromosome is genetically active, while any further X chromosome undergoes inactivation. After staining with fuchsin or other dyes, the inactivated X chromosome is visible as a Barr body (also referred to as sex or X chromatin) at the rim of interphase cell nuclei. In a similar fashion, so called Y bodies can be demonstrated with quinacrine staining in the nuclei of smeared oral mucosa or other cells. They indicate the presence of a Y chromosome. Both the Barr and the Y body test had a limited role as a fast and cheap means to obtain information about the sex chromosome complement of a cell. By now, both are obsolete and have been supplanted by methods discussed below.

For most clinical purposes the analysis of banded lymphocyte chromosomes (8, 9) is the sole necessary cytogenetic test. Box 9.3.5.1 summarizes the most important indications for karyotyping in male endocrinology. At least 2 ml of blood are needed. Anticoagulation with heparin is best, as the metaphase preparations tend to be of poorer quality if EDTA-coated containers are used. The white blood cells are subjected to a short-term culture, and the lymphocytes induced to undergo mitosis by adding a phytohaemagglutinin. They are then arrested in metaphase by adding colcemide, and after several further preparative steps are fixed onto a microscopic slide. To obtain the characteristic and diagnostically important banding pattern, the chromosomes need to be stained. For routine purposes, the authors use a combination of GTG (Giemsa) and QFQ (quinacrine) banding, but some laboratories prefer other techniques such as R (reverse-staining Giemsa method) bands. All these methods yield a characteristic pattern of alternating dark and bright bands along the entire length of all chromosomes.

The analysis of the mitoses may be done directly at the microscope or on the computer screen with digitized images of metaphase spreads. The chromosome number is determined in 10–20 cells and 5–10 cells are fully karyotyped, meaning that the banding pattern is evaluated in detail. Traditionally, a banding resolution of 400 per haploid genome was considered as satisfactory. There has been a move towards higher banding resolutions in the order of 500–550 per haploid genome, as some laboratories now routinely obtain them (Fig. 9.3.5.1). Prometaphase preparations may even allow 850 or more bands to be distinguished, but the analysis of such mitoses is tedious. The higher the banding resolution, the more likely is the detection of very small structural rearrangements. It has not been demonstrated that, in the field of male endocrinology, high-resolution banding yields more clinically relevant information than an ordinary banding level of 400–550. The results of karyotyping should be reported in accordance with the International System for Human Cytogenetic Nomenclature (10). Its formulae allow any possible numerical or structural chromosomal aberration to be described in an unequivocal fashion.

Occasionally chromosomes display morphological features that cannot be classified, with one of the standard banding techniques, as either a clinically innocuous polymorphism or a truly pathological trait (11). In such cases it may be helpful to employ staining methods that specifically highlight certain parts of the chromosomes, such as centromeric and non-centromeric heterochromatin (C banding), or the nucleolus organizing regions of chromosomes 13, 14, 15, 21, and 22 (NOR staining). The most common normal variants in the human karyotype are an enlarged heterochromatic region on the proximal long arm of chromosome 9 (9qh+), a small pericentric inversion of chromosome 9, heterochromatic variants of the long arm of the Y chromosome, and very short or exceptionally large short arms of the acrocentric chromosomes 13, 14, 15, 21, and 22.

Some rare structural aberrations, such as marker chromosomes, require a more extensive work-up, for instance chromosomal microdissection and analysis with appropriately selected fluorescence in situ hybridization (FISH) probes. In clinical dysmorphology and paediatric genetics, the high-resolution scanning of the whole genome for submicroscopic deletions and duplications with microarray-based assays has attained a prominent role (12). It is too early to judge the possible impact of these new techniques in the field of male endocrinology.

The finding of two or more different karyotypes in a blood or other cell sample is referred to as chromosomal mosaicism. In male endocrinology, the suspicion of a mosaic state arises in the rare non-azoospermic individual with Klinefelter’s syndrome (see Chapter 9.4.3). Such patients may carry a cell line with a normal set of chromosomes. If mosaicism is suspected, the number of cells analysed for their chromosomal complement must be increased above the standard level of 10–20, usually to a total count of 50–100. Abnormal chromosomal complements confined to single cells are biologically insignificant. To diagnose true mosaicism, one needs to demonstrate at least two cells with the same karyotype in each of the cell lines. When the phenotype strongly suggests a karyotypic anomaly, but none is apparent upon analysis of a blood cell sample, one may consider studying another tissue. Fibroblasts from a skin biopsy are suitable for this purpose. More conveniently, one may use epithelial cells brushed from the oral mucosa. Because they are in the interphase, only the FISH technique is suitable to test them for aneuploidy, e.g. of the sex chromosomes, in suspected 47,XXY/46,XY or 45,X/46,XY mosaicism.

The analysis of meiotic chromosomes is performed by only few specialized laboratories. This diagnostic technique is not part of the routine workup of the infertile male. It has been used for two main purposes: (1) to demonstrate directly the effect of known constitutional chromosome anomalies on meiosis; and (2) to search among infertile men with normal somatic karyotypes for abnormalities of meiotic chromosome pairing that upon karyotyping of blood cells would not be apparent. The most commonly used material for such studies are testicular biopsies, but ejaculated immature germ cells are also suitable (13, 14). In the biopsied cases up to 50% of the patients were reported to display abnormalities of meiotic chromosome pairing. The clinical significance of these findings is controversial.

Sperm chromatin is highly compacted. It requires special efforts to investigate the chromosomal contents of ejaculated male germ cells (15, 16). One way is to let them decondense in the cytoplasm of hamster oocytes, with which they can fuse spontaneously after co-incubation. This procedure allows for the analysis of the complete chromosomal complement of a spermatozoon, but has the disadvantages of being very laborious and not permitting the study of more than small numbers of sperm.

FISH is an alternative approach to studying the genetics of ejaculated spermatozoa. Sperm are incubated with one or more chromosome-specific probes that bind to selected target DNA sequences, and can subsequently be visualized under the fluorescence microscope. This procedure allows for the analysis of vast numbers of germ cells. A drawback compared with the hamster ovum technique is that without special arrangements, information is obtained only about chromosome number, but not structure. In addition, a single FISH assay can at best target three to four of the 23 sperm chromosomes at a time. For a complete overview encompassing all chromosomes, multiple parallel assays would be required. As this vastly increases the resources needed for a FISH study, in practice the analysis is usually limited to a few chromosomes, and the results extrapolated to the complete genome.

Sperm chromosome analysis has applications in andrology (16), but its role in routine clinical practice has remained marginal. One use is to analyse the spermatozoa of carriers of constitutional chromosome abnormalities, for example translocations or mosaics for numerical aberrations. It is reasonable to assume that a man who produces many genomically unbalanced sperm may have a particularly increased risk for fathering a child with a chromosomal disorder. Should his partner conceive, the recommendation for invasive prenatal diagnosis would be made with particular emphasis. However, a low rate of unbalanced spermatozoa in the FISH or hamster ovum test would still not obviate the need to recommend a prenatal chromosome test in any ensuing pregnancy.

Not only translocation carriers, but also infertile men with a normal somatic karyotype produce on average more chromosomally unbalanced sperm than their fertile peers. The aneuploidy rates in this heterogeneous group of patients vary from normal to grossly increased. Children conceived via intracytoplasmic sperm injection (ICSI) techniques have a risk of carrying a chromosomal aberration that moderately exceeds the population baseline. In part, this is a consequence of the increased aneuploidy rate in the germ cells of their fathers.

Compared to cytogenetic methods, most diagnostic DNA tests provide highly focused information that relates not to the complete genome or an entire chromosome, but to a single gene or parts of it. For the gene under study, the analysis can reach the one base pair level of resolution. The narrow focus of this type of diagnostic procedures implies that the clinical indications for ordering them are more specific than for karyotyping. One notable exception to this rule is the DNA test for Y-chromosomal microdeletions, now used widely as a screening procedure in unexplained male infertility with azoospermia or severe oligozoospermia.

The human Y chromosome is not only the dominant sex determinator, but is also enriched with genes exclusively expressed in the testis and supposedly involved in spermatogenesis. That microscopically visible deletions or other Y-chromosomal rearrangements (see Chapter 9.4.6) can impair or ablate male fertility has been known since the 1970s. More recently, distinct microdeletions, not detectable through microscopic chromosome analysis, have been discovered as cause of spermatogenic failure resulting in male infertility and were classically denominated azoospermia factor loci a, b and c (AZFa/b/c, respectively). As these deletions are found in frequencies of 2–10% (or even higher, depending on the study population), they are the second most frequent genetic cause of male infertility after Klinefelter’s syndrome.

The portion of the male-specific region of the Y chromosome (MSY) affected by deletions was completely sequenced in 2003, which allowed the molecular mechanism of microdeletions to be identified as homologous recombination between identical sequences in palindromes (17). The breakpoints of deletions are well characterized today, and five main microdeletion patterns have been identified, named AZFa, AZFb (P5-proximal P1), AZFbc (P5-distal P1 or P4-distal P1) and AZFc (b2/b4) with an overlap of AZFb and AZFc. It is well established that microdeletions of the Y chromosome occur in infertile but not in fertile men, establishing a clear cause-effect relationship. The frequency of deletions differs remarkably between countries, possibly depending on the selection criteria of the patients and on the ethnic background (18). The vast majority (about 80%) are deletions involving the AZFc region, while the other deletions are found much more rarely.

Complete deletions of AZFa, AZFb or AZFbc are always associated with azoospermia. Complete, bilateral Sertoli cell-only (SCO) syndrome is found in testicular biopsies of men with AZFa deletions, and a mixture of SCO and maturation arrest is observed in men with AZFb and AZFbc deletions. Patients with AZFc deletions have a slightly milder phenotype, with mixed atrophy and residual spermatogenesis in about 50% of the patients, although SCO is present in the majority of these patients. This is reflected by the semen parameters, which show azoospermia in about half of the patients with AZFc deletions, and only a few spermatozoa present in the ejaculate of the other half. In general, testicular sperm extraction (TESE) is possible in patients with AZFc deletions with a probability of about 50% of sperm recovery, but no sperm retrieval has so far been reported in patients with complete AZFa, AZFb or AZFbc deletions. Therefore, performing molecular genetic testing in these patients has a definite prognostic value for TESE. There are no clinical parameters beyond azoospermia or severe oligozoospermia which can be used to predict the occurrence of a microdeletion of the Y chromosome (18).

The AZFa region contains the two single copy genes (USP9Y and DBY), while the AZFb and AZFc regions together comprise 24 genes, most of which are present in multiple copies for a total of 46 copies. The complete AZFb deletion removes 32 copies of genes and transcription units, while the AZFc deletion removes 21 copies. One gene of the AZFc region is DAZ (deleted in azoospermia), which is present as four copies arranged in two complexes of two genes. Expression of DAZ mRNA has been demonstrated in the male germ cell lineage, but the exact cellular function of the protein product remains unknown. The function of the other genes is also subject of current research, but as yet no single gene has been demonstrated to cause the severe spermatogenic phenotype found in men with complete deletions.

The molecular diagnosis of Y-chromosomal microdeletions is relatively easy and cheap, justifying its popularity, which now makes it one of the most frequently performed diagnostic tests in molecular genetics. Since the publication of best practice guidelines for Y-chromosomal microdeletion screening, the analysis has been standardized and an external quality control scheme is also available (19). In short, a set of anonymous DNA markers (sequence tagged sites, STS) resident on the long arm of the Y chromosome is amplified by means of the polymerase chain reaction (PCR). According to the guidelines, two separate multiplex PCR reactions with one STS primer for each AZF region are performed, leading to a total of two primers for each AZF deletion. Each STS marker provides an amplification product that can be visualized on an electrophoresis gel as a distinct band. Lack of amplification of both primers of one region suggests the presence of a microdeletion, which can in close to 100% of cases be considered complete. It is estimated that this basic protocol for routine microdeletion screening is sufficient to detect over 95% of clinically relevant deletions, although very rare exceptions of partial deletions within the above-mentioned regions might occur. These partial deletions, however, are of unclear pathogenetic significance and their characterisation is still experimental. Commercial kits for routine diagnosis are available, but care should be used in the choice of the kit. Avoid those using too large a number of markers, which makes them prone to analytical errors without improving the diagnostic power.

For a patient with a positive Y microdeletion test it can be attempted to obtain DNA from his father or a fertile brother. Normal results of their STS marker analysis allow the diagnosis of a de novo microdeletion to be made with confidence for the index patient. Some case reports of natural transmission of an AZFc deletion exist, showing that this deletion can be compatible with fertility in rare cases. In any case, an AZFc deletion will be transmitted to sons of the patient (possibly through TESE/ICSI) and therefore genetic counselling is advised.

Given the palindromic nature of MSY, it cannot be ruled out that other deletion patterns exist, but according to current knowledge this situation can be considered extremely rare. One partial deletion of the AZFc region, the gr/gr deletion, has been extensively studied and confirmed as risk factor for reduced sperm counts and male infertility. However, since this deletion is also found in fertile men with normal spermatogenesis, no consequence of the procedure or indication for counselling can be derived. Screening for this deletion is therefore not currently advised in clinical routine.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene not only cause the full clinical picture of cystic fibrosis (CF), but also a distinct form of male infertility unaccompanied by lung and pancreatic disease. Congenital bilateral absence of the vas deferens (CBAVD) falls into this class of cystic fibrosis-related disorders (20). CBAVD leads to obstructive azoospermia with the pathognomonic clinical features of normal testicular volume and FSH in the presence of reduced seminal pH, semen volume, fructose and α-glucosidase content. The definitive diagnosis is achieved by testicular biopsy showing normal spermatogenesis. For any patient suspected to have or diagnosed with CBAVD, a CFTR gene mutation analysis should be entertained. CFTR mutations have also been detected in men with congenital unilateral absence of the vas deferens (CUAVD) and in patients with oligo-/azoospermia without clinical features of obstruction. In the CUAVD group, the CFTR mutation rate does exceed the population baseline, but to a lesser degree than in CBAVD. A more controversial issue is whether this also holds true for men with oligozoospermia or non-obstructive azoospermia. Although some studies have reported positive findings, the bulk of evidence is against an increased CFTR gene mutation rate in this patient group.

The CFTR gene spans approximately 250 000 base pairs of genomic DNA. Mutation detection is technically demanding, not only because of this large size, but also because of the heterogeneity of CFTR mutations. With a share of about 70% of the cystic fibrosis alleles, a three-base pair deletion termed F508del (formerly ΔF508) predominates in the Caucasian population. The remaining 30% represent rare or exceedingly rare mutations, some of which have been described only in single patients or families. For routine diagnosis, most laboratories test for a limited panel of the mutations that are most prevalent in the local population. Restriction analysis, allele specific amplification, heteroduplex analysis, or other techniques may be employed for this purpose. The large stretches of DNA between the targeted potential mutation sites are not covered. This limited approach may be supplemented by an unspecific mutation screening technique, such as single strand conformation polymorphism (SSCP) analysis or denaturing gradient gel electrophoresis (DGGE). These procedures allow whole exons, or larger parts of them, to be tested for deviations from the normal base sequence. PCR products showing abnormal patterns in the DGGE or SSCP analysis should be characterized through sequencing. It is important to note the principal limitations of the currently used laboratory techniques. In patients of German descent, testing for F508del and the 27 next common mutations will leave about 15% of cystic fibrosis alleles undetected.

CF and CBAVD both follow an autosomal recessive mode of inheritance. This implies that CF or one of the CF-related disorders will result only when a mutation is present in both CFTR alleles. A common problem in the routine analysis arises when only one mutated allele can be found in some patients. If the clinical presentation is typical, there is good reason to assume that a second mutation is present, but has been missed by the laboratory tests. The spectrum of mutations encountered among men with CBAVD is similar to, but distinct from, that of cystic fibrosis patients. It also varies significantly in accordance with the individual’s ethnic background. Ideally, the panel of mutations searched for should be tailored both to the clinical indications and to the ethnicity of the patient. In routine practice, most laboratories will test for the same set of mutations in any given patient, but will take this into consideration in genetic risk calculation. Current guidelines are available for molecular genetic analysis of cystic fibrosis, including CFTR-related disorders, and include extensive flow charts explaining stepwise testing procedures (20).

Detecting CFTR gene mutations in a patient with CF or CBAVD confirms the clinical diagnosis and provides a causal explanation for the disease, an important psychological benefit for the affected individual. Beyond this, mutation analysis is the basis for genetic risk analysis and counselling of patients desiring treatment for their fertility problem. In central European populations, clinically unapparent heterozygosity for CFTR gene mutations has a substantial prevalence of about 4%. Therefore, there is a real possibility that the female partner of a man with CBAVD will be a mutation carrier. Calculating the risk for cystic fibrosis in a patient’s offspring is oftentimes complex and should be left to a trained geneticist. Factors that need to be taken into account are: the results of mutation analysis in the patient and his partner, the family history on both sides, the clinical diagnosis, the ethnic background of the couple, and the type of laboratory tests that were employed.