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Original Articles: Hormones and Reproductive Health |
Immunogénétique Humaine (C.K., L.Q.-M., K.M.), Institut Pasteur, 75724 Paris Cedex 15 France; Unità di Andrologia (C.K.), Dipartimento di Fisiopatologia Clinica, Viale Pieraccini, 6, 50139 Firenze, Italy; and University Department of Growth and Reproduction (E.R.-D.M., L.F.-L., N.E.S.), Rigshospitalet, 9, Blegdamsvej, DK-2100 Copenhagen, Denmark
Address all correspondence and requests for reprints to: Ken McElreavey, Immunogénétique Humaine, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. E-mail: kenmce{at}pasteur.fr
Abstract
Y chromosome microdeletions have been reported as a possible genetic factor of male infertility. Despite a large number of studies in this subject, there is still considerable debate and confusion surrounding the role of Y chromosome microdeletions in male infertility. This has been further compounded by observations of Y microdeletions in fertile males. The aim of the present study was to evaluate: 1) the incidence of Y microdeletions in control male population and infertile males, where complete semen and hormonal analysis was available to define whether Y microdeletions are specific for spermatogenic failure or if they can be found also in normospermic men; and 2) whether the suboptimal semen quality reported in Denmark is associated with a higher incidence of Y microdeletions in respect to other populations. Double-blind molecular study of deletions was performed in 138 consecutive patients seeking intracytoplasmic sperm injection treatment, 100 men of known fertility, and 107 young military conscripts from the general Danish population. Microdeletions or gene-specific deletions were not detected in normospermic subjects or in subfertile men with a sperm count of more than 1 x 106/mL. Deletions of the Azoospermia factor (AZF)c region were detected in 17% of individuals with idiopathic azoo/cryptozoospermia and in 7% of individuals with nonidiopathic azoo/cryptozoospermia. The data indicate that: 1) the composition of the study population is the major factor in determining deletion frequency; 2) Y chromosome microdeletions are specifically associated with severe spermatogenic failure; therefore, the protocol described here is reliable for the routine clinical workup of severe male factor infertility; and 3) the frequency of Yq microdeletions in the Danish population is similar to that from other countries and argues against the involvement of microdeletions in the relatively low sperm count of the Danish population.
RECENTLY, CONSIDERABLE attention has focused on the role of genetic factors in spermatogenic failure (1), with an emphasis on the Y chromosome. Deletions of three nonoverlapping regions of the Y chromosome long arm have been described, associated with male infertility. Deletions of the AZFa region are mainly associated with Sertoli cell only syndrome (SCOS), deletions of the AZFb region are mainly associated with spermatogenic arrest, and a range of phenotypes from azoospermia to oligozoospermia are associated with the absence of the AZFc region (2).
Individuals defined as idiopathic severe oligo- or azoospermic have the highest frequency of Y microdeletions, particularly involving the AZFc region. However, there remains considerable debate concerning the frequency, the position, and the phenotypes associated with Y chromosome microdeletions. For example, deletion frequency has been reported between 1–55% in different studies (3). It is not clear whether these wide variations, particularly in deletion frequency, are attributable to differences in the study design, the geographic or ethnic origins of the study population, or to experimental error. In addition, the contribution of Y microdeletions to male subfertility in the general population has not been evaluated; and the incidence of Y microdeletions in control males, where complete semen and hormonal analysis is available, has not been investigated. A complete investigation of the latter group is necessary because Y microdeletions have been repeatedly reported in fertile men (2, 4, 5, 6), which suggests that microdeletions may not be confined to infertile subjects. Until these questions have been definitively resolved, the utility of Y chromosome microdeletion screens in a clinical context remains controversial.
Here, for the first time, we describe a Yq microdeletion screen of infertile, subfertile, and normospermic men from the Danish population, where complete semen analysis and hormonal profile was available. The DNA samples were analyzed in a double-blind manner using 14 anonymous and 6 gene-specific Y chromosome markers. The choice of Danish subjects for this study was not completely coincidental. A recent study demonstrated that a relatively large fraction of the otherwise healthy Danish male population seems to have suboptimal semen quality (7). Thus, an additional aim of this study was to investigate a possible contribution of Y microdeletions to this worrying phenomenon. We show that Y chromosome microdeletions are specific for spermatogenic failure, that the variation in deletion frequency reported in other studies is probable attributable to study design, and that Y microdeletions do not seem to contribute to the relatively low sperm count of the general Danish population.
Materials and Methods
The study population has been recruited and studied through a comprehensive andrological examination, including semen analysis and hormonal analysis by the Department of Growth and Reproduction (Rigshospitalet, Copenhagen, Denmark). Samples were analyzed in a double-blind manner. DNA samples were sent from the Department of Growth and Reproduction in a coded way to the laboratory of Immunogénétique Humain (Institut Pasteur, Paris, France), where Y chromosome screening was performed. No exchange of information concerning clinical or molecular data took place until the completion of the study.
Subjects
A total of 345 subjects were studied. These consisted of 138
consecutive patients seeking intracytoplasmic sperm injection (ICSI)
treatment, who were referred to the Rigshospitalet for a full
andrological workup; 100 men of known fertility recruited from a Danish
obstetric clinic who recently made their partners pregnant; and 107
young military conscripts from the general Danish population. All
subjects gave an informed consent for molecular analysis of their blood
samples, and the study was approved by a local ethical committee. A
subdivision of the whole study population, on the basis of the sperm
count, is shown in Table 1
. Among
patients seeking ICSI treatment, 62 were affected by idiopathic
infertility; whereas, in 61 cases, abnormal andrological findings
(including cryptorchidism and >2 degree varicocele) were reported; 12
patients were normozoospermic; and for 3 patients, no clinical
information, other than infertility possibly due to a male factor, was
available (Table 2).
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Semen analysis was performed accordingly to the WHO 1992
guidelines (8). Serum concentrations of FSH, LH, and sex
hormone binding globulin were measured using time-resolved
immunofluorometric assays from DELFIA, Wallac, Inc.,
Turku, FIN. Testosterone and estradiol were measured by RIA
(Coat-a-Count, Diagnostic Products, Los Angeles, CA) and
Immuno, Diagostic Systems Laboratories, Inc. (Boldon, UK),
respectively. Serum inhibin B was measured in duplicate in a
double-antibody enzyme immunometric assay using a monoclonal antibody
raised against the inhibin ßB-subunit in combination with a labeled
antibody raised against the inhibin 
-subunit, as previously
described.
Molecular analysis
PCR analysis of genomic DNA, extracted from lymphocytes, was
performed. A total of 14 anonymous STS markers and 6 Y-specific genes,
spanning the 3 AZF regions, were screened [in AZFa: sY82, sY86, sY87,
and the gene DBY; in AZFb: sY114, sY116, sY125, sY133, and the genes
eIF-1AY, XKRY, and CDY; in AZFc: sY145, sY147,
sY152, sY158, and the genes BPY2 and DAZ
(sY254)]. sY98 and sY100 (between AZFa and AZFb) and sY160 (distal to
AZFc in the heterochromatin) were also included (Fig. 1). The PCR
primers and conditions were described previously, with modifications
(9, 10). Duplex amplifications were performed using the
following combinations: Mix A sY254, sY125; Mix 2 sY98 and sY100; Mix 3
sY160 and sY145; mix 4 sY87 and sY152. For each duplex reaction, the
PCR conditions were 95 C for 5 min, followed by 35 cycles of 95 C for 1
min, 60 C for 1 min, and 72 C for 1 min.
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Results
Microdeletions were detected in nine infertile subjects affected
by azoospermia and cryptozoospermia (<1 x
106/mL; six and three patients, respectively).
The deletions removed the entire AZFc region (sY145, sY147, sY152, and
sY158, including the genes DAZ and BPY2) in each
case. No difference in the extent of the deletion was detected. The
clinical details of each patient with an AZFc deletion are described in
Table 3. Microdeletions were not detected
in the group of oligozoospermic (>1 x
106/mL) or normospermic subjects.
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). Microdeletions were only observed in the subgroup
of men seeking ICSI treatment. Within this subgroup, the incidence of
microdeletions in idiopathic azoo/cryptozoospermic men was 17% (Table 2). Microdeletions were also found in three cases of nonidiopathic
infertility (7%). These subjects presented with unilateral
cryptorchidism, varicocele grade 2, and hypoandrogenism with sexual
dysfunction. In the first two of these three cases, gonadal histology
was available, which revealed both spermatogenic arrest and SCOS. These
observations are consistent with the pattern of histology associated
with Y chromosome microdeletions. Isolated gene-specific deletions were
not detected. Although all the microdeletions seemed to have the same
molecular extent in each case, a genotype/phenotype correlation could
not be established on the basis of the semen analysis and testicular
histology. Sperm concentration ranged from 0–0.135 x
106/mL. Testicular histology varied from
spermatocytic arrest combined with Sertoli cell only (SCO) pattern to
bilateral SCO pattern combined with diffused Leydig cell hyperplasia.
The hormonal parameters showed a more homogeneous picture: serum
inhibin B levels were below the normal range in each patient, whereas
FSH levels were above the mean (3.94 U/L) in all cases, with abnormally
high values in seven of nine patients. Discussion
The data presented here clearly show that the composition of the
study population is a major factor influencing deletion frequency.
Patients affected by idiopathic infertility and severe oligo- or
azoospermia have a greater probability of harboring microdeletions,
compared with the nonidiopathic group or moderate oligospermic men,
respectively. We found that the highest deletion frequency in the group
defined as idiopathic azoospermic/cryptozoospermic was 17% (Table 2).
A subdivision of the study population, on the basis of sperm number,
indicates a deletion frequency of 11.5% in the group of
azoospermic/cryptozoospermic patients (this includes both idiopathic
and nonidiopathic patients). The incidence progressively decreases with
the inclusion of less severe phenotypes, to 8.9% (oligospermic with
<5 x 106 sperm per mL) and 7.1%
(oligospermic with <20 x 106 sperm per mL)
(Table 1). These figures are consistent with two other studies of the
French and Italian populations, where uniformly defined clinical groups
were studied using a similar study protocol (9, 10 ; Fig. 2). This
suggests that variation in deletion frequency is mainly attributable to
the clinical composition of the study group; and it also indicates
that, at least in Europe, there is no evidence to support a variation
in deletion frequency between different populations/ethnic groups or
different geographic regions.
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All deletions detected in the current study involved the AZFc region, and the associated phenotype ranged from azoospermia to cryptozoospermia. This supports previous observations that AZFc deletions are the most frequent, and they are associated with a variable phenotype (3, 11). Because serum inhibin B concentrations were uniformly very low in the deleted patients, it is recommended that abnormally low levels of inhibin B should be included among the indications for a Y-chromosome-microdeletion screen. Gene-specific deletions were not detected, indicating that such events, which have been recently reported (12), are either rare or associated with a well-defined clinical subgroup of patients. The use of a PCR-based deletion detection method, such as that used in this study, does not formally exclude other rearrangements of the Y chromosome, such as reduction in gene copy numbers or other structural rearrangements of the Y chromosome (inversions, duplications, and others) that may lead to male subfertility.
In this study, three men defined as nonidiopathic infertile (presenting cryptorchidism, varicocele, and hypoandrogenism) were found to harbor microdeletions of the AZFc region. The extent of these three deletions was identical to that observed in the idiopathic infertile men. Though it is possible that other factors could have contributed to reduced sperm production, the gonad histology in two of these cases was consistent with previous histological findings in cases where Y microdeletions have been detected. Microdeletions have occasionally been reported together with cryptorchidism, varicocele, obstructive azoospermia, and hypogonadotrophic hypogonadism (10, 11, 13) and are considered to be chance association. This finding indicates that Y microdeletion screening has to be extended to all patients affected by severe oligozoospermia, regardless of the presence of abnormal andrological findings.
Several reports indicated that male sperm counts have declined during the last 40–50 yr (14, 15, 16). This decline is associated with an increase in the frequency of anomalies of the male reproductive system, including ambiguous genitalia and testicular cancer. These data and the association between male subfertility and subsequent high risk of testicular cancer are consistent with the hypothesis that male subfertility and testicular cancer share important aethiological factors (17). Recently, a high frequency of young men with suboptimal semen quality has been reported in Denmark (18). More than 40% of young adult Danish men have sperm counts below 40 x 106/mL, which, according to a recent study (7), is associated with decreased fertility. Although a number of factors (such as pesticides, exogenous estrogens, and heavy metals) may have a negative impact on spermatogenesis, none of these factors have been formally demonstrated to be responsible for the reported decline in sperm quality in some countries, including Denmark.
Although Y microdeletions of specific regions on the long arm are a cause of male infertility, the data in this report argues against the involvement of microdeletions in the recently observed high incidence of reproductive abnormalities, including relatively low sperm count of the Danish population. The role of nongenetic factors, such as environmental pollutants, needs to be further investigated. The data also shows that the marker set used in the study is specific for reduced sperm production and can therefore be used in the routine clinical workup of both idiopathic and nonidiopathic severe oligozoospermic and azoospermic men. The demonstration of a Y chromosome microdeletion in an infertile man is not only important in defining the etiology of the condition but is also of clinical prognostic value (19).
Acknowledgments
We thank Drs. Anne-Grethe Andersen, Elisabeth Carlsen, and Niels Joergensen for their clinical contribution; Jane Hinrichsen for her technical assistance; and the technical staff of the Rigshospitalet for hormone and semen analyses.
Footnotes
1 This study was supported, in part, by the Telethon, Italy (Grant n
281/b), Association pour la Rechèrche sur le Cancer, Institut
National sur le Rechèrche Médicale post-vert, and grants
from the European Union and from the Danish Medical Research
Council. 
Received September 21, 2000.
Revised February 15, 2001.
Accepted February 28, 2001.
References
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|
|
|
|
 |
|
 E. Rajpert-De Meyts Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects Hum. Reprod. Update, May 1, 2006; 12(3): 303 - 323. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
 G. Vinci, F. Raicu, L. Popa, O. Popa, R. Cocos, and K. McElreavey A deletion of a novel heat shock gene on the Y chromosome associated with azoospermia Mol. Hum. Reprod., April 1, 2005; 11(4): 295 - 298. [Abstract] [Full Text] [PDF]
|
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|
 |
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|
|
|
|
 |
|
 M. Joffe Infertility and environmental pollutants Br. Med. Bull., December 1, 2003; 68(1): 47 - 70. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
J. J. Kurinczuk Safety issues in assisted reproduction technology: From theory to reality--just what are the data telling us about ICSI offspring health and future fertility and should we be concerned? Hum. Reprod., May 1, 2003; 18(5): 925 - 931. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Frydelund-Larsen, C. Krausz, H. Leffers, A. M. Andersson, E. Carlsen, S. Bangsboell, K. Mcelreavey, N. E. Skakkebaek, and E. Rajpert-De Meyts Inhibin B: A Marker for the Functional State of the Seminiferous Epithelium in Patients with Azoospermia Factor c Microdeletions J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5618 - 5624. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Oates, S. Silber, L. G. Brown, and D. C. Page Clinical characterization of 42 oligospermic or azoospermic men with microdeletion of the AZFc region of the Y chromosome, and of 18 children conceived via ICSI Hum. Reprod., November 1, 2002; 17(11): 2813 - 2824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
 |
|
 L C Layman Human gene mutations causing infertility J. Med. Genet., March 1, 2002; 39(3): 153 - 161. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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