Genetics of Male Infertility: Evolution of the X and Y Chromosome and Transmission of Male Infertility to Future Generations

The purpose of this chapter is to put in perspective the accumulating molecular data on Y chromosomal X chromosomal, and autosomal spermatogenesis genes, and their transmission to ICSI offspring. The current gene search on the Y chromosome is just a starting point for locating many other spermatogenesis genes that are widespread throughout the genome. Now that the Y has been sequenced, many more genes are being discovered that impact spermatogenesis. The presence on the Y chromosome of testis specific genes, which arrive from autosomal homologues, or from persistence of ancestral X genes which eventually acquire male specific function, is a recurrent theme in the evolution of spermatogenesis of all animals with sex determining chromosomes. A summary of the evolutionary history of our X and Y chromosome explains why the Y chromosome was a good place to start in the molecular search for spermatogenesis genes. However, it is clear that numerous genes on the X chromosome as well, and on autosomes, also impinge on spermatogenesis and may thus be transmitted to ICSI offspring. The presence of Y deletions does not prevent fertilization or pregnancy for azoospermic and severely oligospermic (<2x10) men either with ICSI, or occasionally with no treatment at all. The Y deletion (and presumably infertility) is transmitted to the male offspring. However, there are many spermatogenesis genes involved in male infertility, and we have barely scratched the surface with what have been (up until very recently) very gross mapping techniques. Whether or not these currently detectable gross "microdeletions" are found in an infertile male patient does not obviate the likelihood of there being a genetic cause for his azoospermia or severe oligospermia. If a defective gene (or genes) is located on his Y chromosome, then all of the male offspring will inherit his problem. However, if genes on the X chromosome are responsible for the infertility, then daughters will be carriers and grandsons may inherit the defect. If autosomal dominant genes are the cause of the infertility, then only half of the male offspring will be infertile, and half of the daughters will be carriers. There is no way of knowing what effect, if any at all, the carrier state for male infertility will have on the daughter. All cases of male infertility need to be considered genetic until proven otherwise, and patients so counseled. A negative Y deletion assay as currently widely practiced, and a normal 46 XY karyotype (because these techniques have such low resolution) does not in any way rule out that the infertility is genetically transmissable. With sequence-based techniques we are now identifying many genes that in a polygenic fashion determine the sperm count. The current enthusiasm for STS mapping of Y deletions is just a very crude beginning, and is only identifying huge deletions. Smaller mutations and point deletions are certain to be more common causes of male infertility. THE USE OF ICSI IN AZOOSPERMIC AND OLIGOSPERMIC MEN: INTRODUCTION TO THE PROBLEM Since the introduction in 1992 of intracytoplasmic sperm injection, there has been a revolution in our thinking about male infertility (Palermo et al., 1992; Van Steirteghem et al, 1993). The most severe cases of male infertility, even with apparently 100% abnormal morphology, and even just rare sperm in the ejaculate, could now have pregnancy and delivery rates apparently no different from conventional IVF with normal sperm (Nagy et al, 1995; Liu et al., 1994; Liu et al., 1995). In 1993, testicular sperm extraction (TESE) and microsurgical epididymal sperm aspiration (MESA) in conjunction with ICSI was introduced for the treatment of obstructive azoospermia (Schoysman et al., 1993; Devroey et all, 1994; Silber et al., 1994, 1995a; Tournaye et al., 1994; Devroey et al., 1995a). Eventually this technique was also used for “non-obstructive” azoospermia (Devroey et al., 1995b; Silber et al., 1995b, 1996, 1998a). Many azoospermic men have a minute amount of sperm production in the testis that is not quantitatively sufficient to “spill over” into the ejaculate, but is adequate for ICSI (Silber et al., 1995b, 1995c, 1997a, 1997b; Silber and Rodriguez-Rigau, 1981; Steinberger & Tjioe, 1968; Zukerman et al., 1978). It is with these cases of non-obstructive azoospermia and severe oligospermia that the greatest concern has been registered for the well-being of offspring generated by ICSI. Thus, if severe oligospermia or azoospermia is of genetic origin, in many cases, ICSI creates a potential problem of proliferation of male infertility (Silber 1998b; Faddy et al., 2001). The purpose of this chapter is firstly to explain the accumulating molecular data on Y chromosomal spermatogenesis genes, and their transmission to ICSI offspring. The second purpose is to outline the reasons for concentrating on the evolution of the Y chromosome, and the light it sheds on the existence of many more spermatogenesis genes that are widespread throughout the genome, and that may also be responsible for transmitting male infertility to future generations. A third, and simpler, goal is to review the more routinely appreciated cytogenetic aspects of male infertility, and its impact on ICSI offspring. EARLY GENETIC STUDIES OF AZOOSPERMIC AND SEVERELY OLIGOSPERMIC MEN For several decades, it had been speculated that there was a genetic etiology to many cases of male infertility (Silber et al., 1995b; Silber 1989). This suspicion originally arose from cytogenetic evidence reported over 25 years ago in a very small percentage (0.2%) of azoospermic men who were otherwise phenotypically normal, but who had grossly obvious terminal Y chromosome deletions (Fig. 1a & 1b) (Tiepolo & Zuffardi, 1976). Figure 1a and 1b. Karyotype of the azoospermic male with cytogenetically visible Yq deletion compared to karyotype of an azoospermic male with a normal Y chromosome. Simple karyotyping of infertile men also raised the possibility of infertility being associated with autosomal translocations (Van Assche et al., 1996; Bonduelle et al., 1995, 1996, 1998a, 1998b, 1999; Egozcue et al., 2000). A massive summary of karyotyping results in newborn populations, reviewed by Van Assche, revealed an incidence of balanced autosomal translocations in a normal newborn population of 0.25% but an incidence of 1.3%, in infertile men (Table Ia) (Van Assche et al., 1996). In fact, karyotyping of oligospermic males (i.e. less than 20 million per cc) reveal a 3% incidence of some type of autosomal chromosome anomaly, either balanced Robertsonian translocations, balanced reciprocal translocations, balanced inversions, or extra markers. These translocations could conceivably be transmitted to offspring if ICSI allowed them to conceive. However, because of the limitations of the resolution of cytogenetics, and the very small percentage of these readily discernable karyotypic abnormalities found in infertile men, until recently it had been a convoluted struggle to study the genetic causes of male infertility, and the possible transmission of these genetic errors to the offspring of couples with male infertility (Egozcue et al., 2000). The possibility that many more cases of male infertility might be genetic was bolstered by the failure of most clinical therapies to correct deficient spermatogenesis (Devroey et al., 1998; Baker et al., 1981; Baker et al., 1984, 1985; Baker & Kovacs, 1985; Baker 1986; Nieschlag et al., 1995, 1998; Nilsson et al., 1979; Rodriguez-Rigau et al., 1978; Schoysman 1983; Silber et al., 1995b; Silber 1989). The heritability of sperm count demonstrated in the wild (O'Brien et al., 1986, 1987; Short 1995), classic studies of naturally occurring pure sterile Y deletions in Drosophila, and very early molecular investigations of the Y chromosome in humans led to what has now become an intense search for genes which control spermatogenesis and which may be defective in many or most infertile males (Johnson et al., 1989; Ma et al., 1992, 1993; Eberhart et al., 1996; Hockstein et al., 1995). However, only recently has the frequent genetic etiology of male infertility related to defects in spermatogenesis (not to mention obstruction) become widely acknowledged via molecular methodology (Kent-First et al., 1996; Kremer et al., 1997, 1998; Krausz and McElreavey, 2001; Silber et al., 1995b; Vogt 1996, 1997; Reijo et al., 1995; Chillon et al., 1995; Shin et al., 1997; Anguiano et al., 1992). If male infertility is of genetic origin, its possible transmission to offspring of successfully treated infertile men is a serious social concern (Page et al., 1999; Mulhall et al., 1998; Silber 1998b; Faddy et al., 2001). Y CHROMOSOME MAPPING OF INFERTILE MEN AND ICSI With simple karyotyping, it has been known that a very small number of azoospermic men (0.2%) have large defects visible in the long arm of the Y chromosome that are not present in their fertile fathers. This implied the existence of an azoospermic factor somewhere on Yq. (Tiepolo & Zuffardi, 1976). However, smaller defects (i.e. "microdeletions") could not be discerned with those limited early cytogenetic methods (Fig. 1a,1b). Therefore, these defects in Yq were considered to be rare even in azoospermic men. In 1992, comprehensive Y chromosomal maps were constructed using yeast artificial chromosomes (YACS) and sequenced tagged sites (STS), and this created the possibility for more detailed study of the Y chromosome in infertile men (Foote et al., 1992; Vollrath et al., 1992). Using polymerase chain reaction (PCR), a more refined search for Y chromosome deletions could be pursued by testing for as many as 52 DNA landmarks (STSs, or sequence tagged sites) across the entirety of the Y chromosome. All Y-DNA markers employed were placed on a physical map of the chromosome, the markers representing all gene families that were then known in the nonrecombining region of the Y chromosome (Vogt et al., 1997; Foote et al., 1992; Vollrath et al., 1992; Lahn & Page, 1997). Using these molecular mapping techniques, which have much greater resolution than cytogenetics, a large series of severely infertile men with clearly identified phenotypes revealed deletions in 13% of azoospermic males (Reijo et al., 1995) (Fig. 2).