Download Genetics of male subfertility: consequences for the clinical work-up

Human Reproduction, Vol. 14, (Suppl. 1), pp. 24-37, 1999
Genetics of male subfertility: consequences for
the clinical work-up
Wolfgang Kiipker1'5, Eberhard Schwinger2, Olaf Hiort3,
Michael Ludwig1, Nikos Nikolettos1, Peter N.Schlegel4
and Klaus Diedrich1
department of Obstetrics and Gynecology, institute of Human Genetics,
3
Department of Pediatrics, Medical University Liibeck, Ratzeburger
Allee 160, 23538 Liibeck, Germany and 4James Buchanan Brady
Foundation, Department of Urology, New York Hospital - Cornell
Medical Center, New York, USA
5
To whom correspondence should be addressed
Major principles of genetic failures, chromosomal alterations and the most
common syndromes associated with male subfertility should be taken into
account before medical therapy and sophisticated techniques of assisted
fertilization are applied to help a couple conceive. This review addresses the
most common genetic reasons for male subfertility or infertility with special
regard to the importance for the clinical work-up in daily routine and the
potential risks for the conceptus.
Key words: androgen receptor/chromosome disorders/genetics/male subfertility/
Y deletions
Introduction
Severe male subfertility is assumed to be the reason for infertility in up to 50%
of all childless couples. In about 30% of these cases genetic disorders are
suspected to be the basis. These disorders present with a large variety of clinical
features, but mostly severe oligoasthenoteratozoospermia or azoospermia is the
predominant symptom.
The human genome probably contains up to 100 000 genes. Alteration in even
one base pair of a gene can result in an absent or abnormal protein with
potentially serious phenotypic consequences. More than 1000 genes are known
to regulate the human fertility status. A mutation in a single gene can cause
fertility problems, but on the other hand it has to be taken into account that germ
line development, male gonad development and male somatic development are
under control of a complex genetic network. Genetic disorders currently known
to occur during human spermatogenesis include chromosomal aneuploidy and
mutations in spermatogenesis genes. These disorders are inherited or result from
new mutations. A screening for genetic failures and chromosomal alterations
24
© European Society for Human Reproduction and Embryology
Clinical work-up for male subfertility
associated with male subfertility should be carried out before medical therapy
and sophisticated techniques of assisted fertilization are applied to help a couple
conceive.
Numerical and structural chromosome disorders
Chromosome disorders are numerical or structural. Polyploidy is defined as a
chromosome number that is an exact multiple of 23. Triploidy or tetraploidy are
most unlikely to be compatible with life, and are often seen in spontaneous
abortions. Structural aberrations result from chromosome breakage. Different
types of chromosomal rearrangements have to be distinguished as there are losses
of genomic material resulting from deletions, duplications and inversions. Fusion
of both ends of a chromosome results in the formation of a ring chromosome. If
breakage involves more than one chromosome, translocations occur. In reciprocal
translocations genetic material distal to the breakage points is exchanged between
homologous and non-homologous chromosomes. If no genetic material has been
lost, the individual is phenotypically normal-appearing and healthy — a balanced
translocation carrier.
As early as 1957, Ferguson-Smith et al. suspected the impact of chromosomal
aberrations as the underlying basis for the infertility of their 91 azoo- and
oligozoospermic patients, where they were able to detect a Barr body in 10 cases
(Ferguson-Smith et al., 1957). Two years later it was demonstrated that those
men could be karyotyped as 47,XXY, the Klinefelter syndrome (Jacobs and
Strong, 1959). Another study reviewed the results of 20 studies concerning the
karyotypes of severely subfertile or infertile males (De Braekaleer and Dao,
1991). The most frequent aberration was Klinefelter syndrome, in almost 5% of
cases, which is a 44-fold higher prevalence compared to a normal fertile
population. A total of 1.8% of patients presenting with severe oligoasthenoteratozoospermia and 0.1% with azoospermia were found to have Robertsonian
translocations, a fusion of two acrocentric autosomes. In most of those cases a
13; 14 translocation was detected. Reciprocal translocations and pericentric
inversions have been seen in a 7-13-fold higher percentage among subfertiles.
The prevalence of chromosomal anomalies can be expected to be significantly
increased in a range of 2.1% up to 8.6% in a non-selected group of males with
subfertility as reported by several authors (Chandley, 1979; Abrahamsson et al.,
1982; Zuffardi and Tiepolo, 1982; Yoshida et al, 1995) as compared with the
general male population (0.7-1%). Severity of spermatogenic impairment and
prevalence of chromosomal anomalies seem to be positively correlated. Studies
on male and female in-vitro fertilization (IVF) patients demonstrated an increase
of translocations and chromosomal mosaics. Our own studies concerning chromosomal abnormalities among 440 subfertile males designated for an intracytoplasmic sperm injection (ICSI) treatment revealed a prevalence of 4.3% (19/
440). All patients presented with a sperm concentration below 20xl0 6 /ml. Thus,
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W.Kiipker et al.
treatment options such as ICSI exclusively involve a male population at a
particular higher risk for chromosomal abnormalities.
The most common numerical chromosome anomaly in infertile men is 47,XXY;
90% of affected males display the classic condition of 47,XXY, and mosaic
forms 46,XY,47,XXY also exist. The classic Klinefelter syndrome results from
meiotic non-disjunction of the X chromosome. Mosaic forms are caused by
mitotic non-disjunction of the X chromosome after fertilization. Patients with
Klinefelter syndrome mostly present with a small testes volume and azoospermia.
Plasma testosterone concentrations are decreased whereas oestradiol concentrations tend to be elevated. The mosaic variant is not as severe as the classic form.
Testes size may be normal, azoospermia is less common. Focal spermatogenesis
can be detected (Ferguson-Smith et al., 1957; Gordon et al., 1972). The karyotype
XXY is encountered in -1:700-1000 of newborns. Few cases have been reported
for men with Klinefelter syndrome having very few motile spermatozoa in the
ejaculate (Foss and Lewis, 1971) It is not known why this chromosome
constitution causes infertility, but the balance of sex chromosomes seems to play
an important role. It was previously reported that testicular sperm extraction
yielded motile spermatozoa in four out of nine patients, but pregnancies were
not established (Tournaye et al., 1996). It is interesting that spermatozoa from
an XXY male have been found to be gonosomal haploid at a very high percentage
(Martini et al., 1996).
Men with azoospermia and a 46,XX karyotype are found less frequently (1:20
000 newborns). Male development in these patients is due to a chromosomal
translocation of the short arm of the Y chromosome on the short arm of the X
chromosome. The phenotype of those men depends on the breakpoint of the
translocated Y material. Men with azoospermia and the karyotype 45,X are rare.
They have an unbalanced karyotype because the distal part of the long arm of
the Y chromosome is lost. The other part of the Y chromosome including the
SRY locus is translocated to mostly acrocentric autosomes.
In order to evaluate risk factors for inheritance of chromosomal abnormalities,
analysis of meiotic segregation patterns can be applied.
Meiotic segregation patterns
With the introduction of fluorescent in-situ hybridization (FISH) and its application
in human spermatozoa, a great number of sperm nuclei can be investigated
within a short time. The segregation mode for most of the translocations can be
studied to determine the ratio of chromosomally balanced, unbalanced and normal
spermatozoa. But the frequency of chromosomally unbalanced spermatozoa
varies and is dependent on the underlying aberration. Even in different patients
with the same anomaly, the rate of unbalanced spermatozoa can vary greatly.
Most investigators who looked for meiotic segregation patterns (Balkan and
Martin, 1983; Pellestor et al., 1987; Martin et al, 1992; Syme and Martin, 1992)
reported a tendency to a much higher rate of normal and balanced gametes than
26
Clinical work-up for male subfertility
unbalanced. However, the segregation pattern and the percentage of normal
haploid or balanced spermatozoa in the ejaculate of a chromosomal aberrant
patient is important for evaluating the risk of inheritance when techniques of
assisted fertilization such as ICSI are applied.
Azoospermia factor (AZF) and deletions of the Y chromosome
Recently, there have been several studies devoted to defining key regions on the
Y chromosome which are suspected to contain important genes responsible for
spermatogenesis. These regions are located on the long arm of the Y chromosome
(Yqll). A variety of spermatogenic defects have been associated with microdeletions of these regions. The corresponding gene locus is known as azoospermia
factor (AZF). In the first report of the presence of such a locus (Tiepolo and
Zuffardi, 1976) 1170 subfertile men were karyotyped, of whom six azoospermic
males were found to have microscopic deletions on the long arm of the Y
chromosome. Thus, the existence of the AZF was claimed.
The Y chromosome consists of three different structural and functional parts,
the euchromatic part on the short arm (Ypll), the euchromatic part on the long
arm (Yqll) and the heterochromatin on the long arm (Yql2). G-banding exhibits
12 different parts of the heterochromatin, which consists almost completely of
repetitive DNA sequences. The entire Y chromosome comprises 59 megabases
of genetic information in the euchromatic regions on both arms (Vogt et al.,
1997). To date, three deletion maps of the Y chromosome have been constructed
(Affara et al., 1996). The commonly used map separates the euchromatic region
into seven intervals and several subintervals (Vollrath et al., 1992). Ma et al.
(1992) identified distinct deletions in two karyotypically normal azoospermic
males in interval 6 of Yqll (JOLAR, in subinterval 1 and KLARD, between
subintervals 12 and 14), on which basis 1 year later the same authors characterized
two genes as being responsible for azoospermia, YRRM1 and YRRM2 (Y
chromosome RNA Recognition Motif) which are now known to be RBM (RNAbinding motif) (Ma et al., 1993). Another study described the presence of another
gene within the AZF region which they called DAZ (Deleted in Azoospermia)
(Reijo et al., 1995). They studied 89 azoospermic men using a polymerase chain
reaction (PCR)-based analysis with 84 sequence-tagged sites (STS). Of these
men, 12 (13%) were found to have overlapping deletions on Yq. Subsequent
analysis of severely oligozoospermic males revealed that DAZ deletions may
also be present in men with sufficient spermatogenesis to have spermatozoa in
the ejaculate (Reijo et al., 1996). Vogt et al. (1996) published a study on 370
azoospermic and severely oligozoospermic men in which 12 of them were found
to have submicroscopic deletions of intervals 5 and 6. They claimed the existence
of three genetically active regions, AZFa, AZFb and AZFc. It is postulated that
these three regions on the Y-chromosome correspond to different histological
patterns of spermatogenic failure. Whereas patients with deletions in AZFa
display a complete absence of germ cells, men with deletions in AZFb exhibit a
27
W.Kupker et al.
maturation arrest of spermatogenesis before or at meiosis. On the other hand,
deletions in AZFc which correspond to the DAZ region seem not to be associated
with a specific interruption phase of spermatogenesis and can result either in
azoospermia or in oligozoospermia with few mature spermatozoa in the ejaculate.
Since the first reports of Y chromosome-specific deletions many studies have
been performed (Table I, Figure 1) to look for deletions in azoospermic and
oligozoospermic patients referred to assisted fertilization programmes. There is
strong evidence that Y deletions are actually involved in the development of
spermatogenesis failure, but the apparently diverse findings concerning the
prevalence are confusing for the scientific community. It remains to be clarified
if these differences are due to patient selection, number of analysed STS or
technical problems of PCR application. Reijo et al. (1996) analysed 84 STS in
Yqll of patients with non obstructive azoospermia and detected deletions in
13.5%. The same authors (Reijo et al., 1996) found 5.3% in severely oligozoospermic patients using 110 STS. The overall results of Vogt et al. (1997) indicate
a significantly fewer (4.6%) after the screening of 700 patients. They applied 27
STS in their PCR assay comprising the relevant Y regions of AZFa, AZFb and
AZFc (DAZ). Our own data obtained after screening 245 men for deletions in
the DAZ region (six STS in single PCR technique) appear rather disappointing.
Out of 92 non-obstructive azoospermic males only one patient displayed a large
deletion comprising the complete DAZ region (Ludwig et al., 1998). All blood
samples of patients with non-obstructive azoospermia were additionally analysed
in a reference laboratory (P.N.Schlegel, New York) using single and multiplex
PCR of 35 STS with identical results. On the other hand, using the same 35 STS
Schlegel detected a prevalence of 6.3% among his subfertile patients (Girardi
et al, 1997).
Until now little has been known about the origins and mechanisms of the
molecular rearrangements in Yqll resulting in deletions and loss of Y-specific
genetic material. Y chromosomal rearrangements and possibly the loss of genetic
material are expected to occur during meiosis associated with sister chromatid
exchange, spermatid development or in the spermatozoa of the germ line of the
patients' fathers or de novo during early embryogenesis (Edwards and Bishop,
1997). A different mechanism leading to Y deletions could relate to the specific
patterns of DNA condensation and packing that occur during spermiogenesis. It
seems unlikely that very small deletions of, for example, one STS cause severe
spermatogenic impairment (Pryor et al., 1997): this has to be ruled out if these
deletions are definitely polymorphisms or rather represent inefficient amplification
due to technical problems of PCR procedure. Most studies could demonstrate
that subfertile men typically present with deletions of at least 0.5-1 X106 base
pairs of DNA in length. If there is any relation between the extent of deletion
and its region with regard to the testis, histology has to be studied in more detail
within the near future.
Since Y chromosome inheritance is of paternal origin, men with somatic Y
deletions are likely to have male offspring with similar genetic defects after
assisted fertilization. Moreover, there is evidence that mosaicism in the paternal
28
Clinical work-up for male subfertility
Figure 1. Microdeletions of the Y chromosome as reported in the literature.
germ cell line is possible in subfertile males when Y-specific deletions are not
detectable in the somatic cell line. These patients could transmit deletions to
their male offspring (Kent-First et al., 1996). However, to enable a detailed
analysis of the origin of AZF deletions, single cell analysis of spermatids and
29
W.Kupker et al.
Table I. Results of Yqll microdeletions in azoo- and oligozoospermic men (according to Vogt,
1998)
First author
n
Azoo
Nagafuchi (1993)
Kobayashi (1994)
Reijo (1995)
Kent-First (1996)
Najmabadi (1996)
Nakahori (1996)
Qureshi (1996)
Reijo (1996)
Stuppia (1996)
Vogt(1996)
Brown (1997)
Foresta (1997)
Girardi (1997)
Kremer (1997)
McElreavey (1997)
Simoni (1997)
Stuppia (1997)
Van der Ven (1997)
Vogt(1997)
50
63
89
32
60
153
100
35
33
370
345
38
160
164
100
168
50
204
700
50
59
89
13
50
135
53
0
19
Oligo + AZF
0
4
0
19
10
18
4
35
14
370
228
16
118
53
64
74
0
44
117
22
42
111
36
94
50
158
700
STS
AZF
26
16
84
64
23
23
22
110
14
76
82
15
35
8
35
4
27
27
27
6
8
12
4
13
20
8
0
6
13
39
11
10
7
9
5
0
2
32
Oligo
0
2
0
3
1
3
2
2
1
6
9
3
. 3
3
1
2
6
1
12
%a
12.0
16.0
13.5
9.4
18.0
13.0
8.0
5.3
18.0
5.0
11.3
18.4
6.3
4.3
14.0
3.0
12.0
1.0
4.6
Percentage of patients with AZF deletions.
n = number of patients; Azoo = patients with azoospermia; Oligo + AZF — patients with
oligozoospermia; STS = sequence tagged sites; AZF — patients with deletions in AZFa, b and c;
Oligo = patients with oligozoospermia and AZF deletions.
spermatozoa might be essential. If we assume that about one in 1000 men is
azoospermic and that up to 15% of them will have Y deletions, then the frequency
of this genetic defect would be about one in 10 000 births if techniques of
assisted fertilization are applied in those infertile couples.
Each Y gene, such as RBM and DAZ which are expressed in the testis, is
located in Yqll in a position overlapping one of the AZF regions. RBM and
DAZ encode an mRNA binding protein whose definite function is not yet known.
SPGY (SPermatogenesis Gene locus on the Y; described by Vogt et al., 1996)
belongs to the same gene family as DAZ (now DAZ1) and was recently renamed
as DAZ2. Moreover, DAZ has one additional gene copy on the short arm of
chromosome 3 and is called DAZH (for DAZ Homologue) or DAZL1 (for DAZlike 1). Most recently, 12 new Y genes having multiple copies on the Y
chromosome or homologues on the X chromosome have been described (Lahn
and Page, 1997). It is up to further research to reveal the secrets of function of
these new genes and how they are involved in spermatogenesis.
Many genes assumed to be involved in spermatogenesis are not located on
the Y chromosome. It is not known if the Y chromosome contains a specific set
of spermatogenesis genes serving as primary controls or only as fine tuners in
the process of spermatogenesis (Burgoyne, 1991). The genetic control of human
spermatogenesis is not only based on the function of single genes but also on
regulatory and interactive gene networks.
30
Clinical work-up for male subfertility
Obstructive azoospermia and mutations in the CFTR gene
Cystic fibrosis (CF) is the most common autosomal recessive disorder with an
incidence of 1:2.500 live births and a carrier frequency of 1:20. The cystic
fibrosis gene was identified in 1989 and encodes the cyclic adenosine monophosphate-regulated chloride channel found in secretory epithelian cells (Kerem et al.,
1989). The gene is called cystic fibrosis transmembrane conductance regulator
gene (CFTR). More than 800 gene mutations have been detected in the meantime.
Clinical features of classic CF are chronic pulmonary obstruction and infections,
insufficiency of the exocrine pancreas, neonatal meconium ileus, elevated sweat
electrolytes and male infertility. The loss or reduction of chloride channel function
as well as defective acidification of intracellular compartments in CF epithelia
likely account for the majority of the classical clinical symptoms in CF. Infertility
results from congenital bilateral absence or atrophy of the vas deferens (CBAVD).
The body of the epididymis may also be affected but the testicular efferent ducts
tend to be spared and some may be dilated. Semen analysis reveals azoospermia
in almost all adults with CF. But CBAVD can also be detected as an isolated
anomaly of the Wolffian duct structures. It has been shown that anomalies of the
Wolffian duct structures seem to be associated with mutations of the CFTR gene
without any other clinical manifestations of CF. Recently, it could be demonstrated
that men who are affected by CBAVD have a striking increase (60%) of
heterozygous mutations in the CFTR gene including compound heterozygotes,
which means different mutations in each gene copy (Patrizio and Asch, 1994).
A majority of these heterozygotes could be shown to have a DNA variant in a
non-coding sequence of the CFTR gene, the 5 T (thymidines) allele in the 3'
splicing region of intron 8 (Chillon et al., 1995). Seven T or nine T are normally
found in this region, whereas the reduction of T (5 T) results in the reduction of
efficiency of splicing exon 9 to code for transcription of CFTR mRNA (Figure
2). In the presence of 5 T of two alleles with only 10-40% of CFTR mRNA is
produced in its complete form, resulting in the reduction of CFTR protein or the
expression of non-functional proteins, although the amount of CFTR protein may
be sufficient to prevent CF symptoms in lung and pancreas. In this way,
obstructive azoospermia can be an important marker for mild features of CF in
phenotypically healthy males. Moreover, there are reports of occasional fertile men
with CF mutations and with minimal evidence of disease (Barreto et al., 1991).
Another condition associated with obstructive azoospermia and chronic sinopulmonary infections is Young's syndrome. Although an increased frequency of
CFTR gene mutations has been observed in males with Young's syndrome, a
clear association between the syndrome and CF could not be established
(LeLannou et al., 1995).
Androgen receptor gene mutations in male subfertility
Spermatogenesis is dependent on functioning androgen metabolism, mainly on
testosterone. Testosterone potentiates the prevention of Sertoli cell apoptosis
31
W.Kiipker et al.
5T
7T
9T
-GTGTG(T) n AACAG CFTR gene
Exon 10
Exon9
Exon8
Intron
Intron
CFTRmRNA
Exon 8 Exon 9 Exon 10
CFTRmRNA
Functional CFTR
Translation
CFTRmRNA (no exon 9)
Inactive CFTR
Figure 2. Impact of different numbers of T (thymidines) on CFTR gene expression.
synergistically with follicle stimulating hormone to maintain sperm maturation
(Tesarik et al., 1998). A form of minimal androgen resistance was believed to
play a biochemical role in some cases of male infertility (Aiman et al., 1979);
however, its importance is controversial (Aiman et al., 1982; Bouchard et al.,
1986). Testosterone and dihydrotestosterone act via the androgen receptor (AR)
to regulate transcription of target genes in these cells and thus the AR gene can
be a candidate gene for male infertility.
The AR belongs to the large steroid receptor superfamily and consists of three
major functional domains (Figure 3). Transcriptional regulation is mediated by
sequences within the N-terminal end, while distinct DNA- and hormone-binding
regions are present. In clinical human disease, the AR has drawn attention
because mutations of the AR gene cause various virilization disorders in 46,XY
individuals with phenotypes ranging from unequivocally female through to
patients with ambiguous genitalia and to males with only slight inhibition
of masculinization (Hiort et al., 1996). Interestingly, patients with androgen
insensitivity all have a severe defect of spermatogenesis. The AR gene is located
on the X chromosome and mutations are inherited through healthy female
carriers. It comprises 8 exons encoding for a protein of 919 amino acids. Within
the large first exon, two polymorphic repeat regions are located, one CAG (polyglutamine) repeat and one GGN (poly-glycine) repeat. However, the exons 2 to
8 are highly conserved within the steroid receptor family.
With the cloning of the AR gene, over 300 patients with distinct mutations
have been described (Gottlieb et al., 1998). These mutations can be spread
throughout the gene, affecting all major domains of the AR protein. However,
32
Clinical work-up for male subfertility
X-Chromosome
q 11-12
2
2
3
3
4
4
5
5
6
6
7
7
8
3'
COOH
NH2
AR
+ 7*-. ++
Z n +++
Zn
Steroid binding site
Figure 3. Gene expression and structure of the androgen receptor.
even with the same mutation, the phenotype may be highly variable (Hiort et al,
1996; Wang et al, 1998a). To date, nine males have been described with a
distinct point mutation in the AR gene and a severe defect of spermatogenesis
without a virilization deficit (Akin et al, 1991; Tsukada et al., 1994; Yong et al.,
1994; Hiort et al, 1997; Wang et al, 1998a,b; Shkolny et al, 1999). These
males suffered from severe oligoteratozoospermia or even from azoospermia.
However, in three males successful pharmacological treatment with clomiphene,
tamoxifen, or high dose androgen treatment resulted in major improvement of
the sperm count and even in pregnancy (Gooren, 1989; Akin, 1993; Yong
et al, 1994).
The CAG-trinucleotide repeat within the first exon has drawn specific attention.
This polymorphism usually comprises 13-30 repeats. Elongation of this repeat
region encoding for more than 40 glutamines is associated with the development
of a spinal and bulbar muscular atrophy (Kennedy's disease), a neurodegenerative
disorder affecting adult males who also present with a slight virilization deficit.
It was recently demonstrated that infertile males have significantly longer CAG
repeats within the normal range than normal controls (Tut et al, 1997). This
elongation is associated with a reduced transactivational capability of the AR.
This concept, however, was challanged by Giwercman et al. (1998), who could
not demonstrate this finding in a large cohort of infertile males from Sweden.
In conclusion, the AR gene may play an important role as a genetic determinant
of male subfertility. Whether infertile or subfertile males with AR gene mutations
generally respond favourably to conservative treatment options still remains to
be investigated. However, further identification of molecular abnormalities of the
33
W.Kiipker et al.
AR gene in those males will give interesting insight into the mechanisms of
androgen action needed for normal spermatogenesis.
Conclusions
Male subfertility is based on genetic abnormalities in about 30% of total cases.
According to Engel and Schmidt, the prevalence of genetically linked subfertility
would increase from 1.8 to 2.3% within 600 years, if genetically linked subfertility
accounted for 100% of subfertility and conception was achieved exclusively by
using assisted reproductive techniques (Engel and Schmidt, 1995). Thus, according
to them assisted reproduction does not significantly influence the human genetic
pool. When carrying out assisted reproduction treatment for male and female
infertility practitioners are obliged to assure the transmission of the minimum of
genetic alterations. Therefore the assessment of the chromosomal state and their
inheritance is recommended. Thus in patients presenting with oligoasthenoteratozoospermia, chromosome analysis must be included in the diagnostic procedure.
The role of deletions of the Y chromosome and the androgen receptor remains
to be further investigated. Patients with obstructive azoospermia should be
investigated for mutations in the CFTR-gene. To analyse the segregation patterns
in spermatozoa and thus to assess the risk for inheritance, the FISH technique is
extremely useful. Moreover, besides the technical screening, the first and most
important step in undergoing infertility treatment is the physician's support and
guidance to enable couples to make individual decisions.
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