Männliche Infertilität - biomed

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wissenschaft & praxis
tainty remains about reasonable selection of genetic analysis for the affected patients. Reviewing referrals and results of 515 own patients
we will try to give some recommendations for
selecting patients for Y-chromosomal screening
with regard to experiences of literature (2, 4, 5).
Männliche Infertilität
Das vorläufige Ergebnis einer Studie über männliche
Infertilität wurde als Poster beim 26. IFBLS-Weltkongress präsentiert. Wir freuen uns, den Inhalt dieses
Posters in der „mta austria“ wiedergeben zu dürfen.
q
wissenschaft
& praxis
Material
Blood samples from 515 infertile men affected with azoospermia, oligospermia, kryptozoospermia and oligoasthenoteratozoospermia
(OAT-syndrome). DNA extracted from fertile
females was used as a negative control and from
fertile males as a positive control. De-novo nature of the deleted region was proven by investigation of the patients father,
when available.
Genetic Analysis in Male Infertility: Screening Experience from
515 Patients and Recommendations for Selection Criteria
Methods
Gabriela Kronberger, Gabriele Sander and Olaf Rittinger;
Clinical Genetics, Children’s Hospital, Salzburger Landeskliniken, Austria
1
2
3
4
1
2
3
4
Introduction
Chromosome abnormalities have long been known as a
substantial cause of male sterility, appearing in approximately 5 – 15 % of male patients admitted to infertility clinics.
Amongst these, Klinefelter syndrome was shown to be, by far,
the most common aberration. Similarly, microdeletions of the
Y also represent an important cause of male infertility with incidence varying between 4 and 18 %, respectively, dependent
on the selection criteria. With the introduction of intracytoplasmatic sperm injection (ICSI) as a treatment for infertility,
genetic analysis of causative factors have gained increasing attention, aiming both to explain infertility and to prevent passing a genetic handicap on to the offspring. However, uncer-
Sch. J.
S. S.
H. A.
F. W.
R. H.
W. H.-J.
F. V.
H. J.
P. S.
E. M.
K. R.
N. A.
St. H.
W. O.
H. R.
M. A.
Sch. F.
S. K.
F. E.
Sch. P.
G. H.
P. K.
E. F.
F. H.
A. I.
B.K.
47,XX,del(Y)
46,XY
46,XY
46,X,del(Y)
45,X/46,X,del(Y)
45,X/46,X,del(Y)
46,XY
46,XY
46,XY
46,XY
46,XY
46,XX(46,X,del(Y)/47,XX,del(Y)
46,XY
46,X,del(Y)
46,X,del(Y)
46,XY
46,XY
46,XY
46,XY
46,XY
46,X,del(Y)
46,XY
46,XY
46,XY
46,XY
46,XY
Table 2: Patients showing a deletion in the Y-PCR-screening
sY160
sY159
sY158
sY157
sY255
AZFc
sY254
sY152
sY143
sY134
sY127
AZFb
sY114
sY135
sY88
sY87
sY83
sY84
sY82
AZFa
sY86
total
azoospermia
patient
515
242
pathological karyotype
76
14.76 %
56
23.14 %
47,XXY, 47,XXY-mosaics
42
8.16 %
39
16.12 %
other mosaics
12
2.33 %
5
2.07 %
translocation
10
1.94 %
3
1.14 %
46,XX male
4
0.78 %
4
1.65 %
Y-chromosomal deletions
26
5.05 %
17
7.02 %
Table 1: Pathological findings by cytogenetic and molecular (Y-PCR-screening) investigation in patients with impaired spermatogenesis
ZFY(X)
sY254
sY86
sY127
ZFY(X)
SRY
sY84
sY134
sY255
Figure 1: multiplex-PCR (1a Mix A, 1b Mix B):
1: 100 bp-ladder; 2: patient H.J.; 3: male control; 4: female control
Chromosome analysis: carried out on peripheral lymphocytes using standard techniques (GTG-banding).
Fluorescence in situ hybridisation (FISH) was done by
using Y-specific probes (alpha-satellite, classical satellite,
Yp11.2 [ONCOR] and Yp11.3 [Vysis]), according to manufacturer’s recommendations.
Molecular analysis was performed by polymerase chain
reaction (PCR) of selected regions of the Y-chromosome. We
were using two multiplex-mixes, according to Simoni et al.
(3). In the case of a deletion of one or more PCR fragments,
Im Rahmen des 26. Weltkongresses der IFBLS (International
Federation of Biomedical Laboratory Science), der vom 13. bis
18. Juni 2004 in Stockholm/Schweden stattfand, hatte ich
zum einen die Möglichkeit, als Delegierte des Österreichischen Berufsverbandes der MTA aktiv am berufspolitischen
Teil dieser Veranstaltung teilzunehmen und damit Österreich
gemeinsam mit weiteren sechs Kolleginnen (Helene Breitschopf, Christina Mayerl, Klaudia Pigl, Anna Schlögl, Christine
Seebacher und Eva Weisz) zu vertreten. Zum anderen war es
mir eine große Freude, dass der von mir eingereichte Poster
„Genetic Analysis in Male Infertility: Screening Experience
from 515 Patients and Recommendations for Selection Criteria“ zur Präsentation angenommen worden und bei der Veranstaltung sehr gut angekommen ist.
Details zu diesem Poster, welches das vorläufige Ergebnis einer Studie ist, die seit 1997 an der Klinischen Genetik in Salzburg läuft, entnehmen Sie bitte nachfolgendem Beitrag.
Gabriela Kronberger, Dipl. MTA
[email protected]
wissenschaft & praxis
led „azoospermic region“ (Yq11.2 ) were detected in a total of
26 patients (5.05 %) (table 2, figure 1a,b). Most microdeletions
were found in patients with azoospermia and extreme oligospermia. One patient showed a deletion in the AZFa-c, three in
AZFb, four in AZFb-c, fifteen in AZFc and three only in the heterochromatic region. In eight cases the deletion was also cytogenetically visible, all of them including the heterochromatic region (figure 3b).
Recommendations / Conclusion
All couples referred for ICSI should be offered traditional
chromosome analysis. Y-chromosome DNA analysis, however, should be reserved for men with azoospermia or at least
extreme oligospermia (less than 106 spermatozoa / ml), be-
Figure 2: Karyogram of a patient with OAT-syndrome:
46,XY,t(5;13)(q33;p11)
the deletion was confirmed by analysing the corresponding
genomic patient’s DNA, in single primer pair PCR controls,
and was extended to STS loci known to cross the proximal
and the distal borderlines of each AZF region.
3a
3b
CEP X
Results
76 patients showed an abnormal karyotype (14.76 %), but
was higher than expected among men with azoospermia, at
23.14 % (table 1). Klinefelter’s syndrome, including mosaics,
were shown to be the most common cytogenetic anomaly
(8.16 %). Other findings consisted in low-level gonosomal mosaics (2.33 %) and translocations (1.94 %) (figure 2). In four patients with XX-maleness (0.78 %) we identified some material of the Y-chromosome containing at least the SRY region on
the short arm of one X-chromosome, which was proven by
FISH (figure 3a). By means of PCR, microdeletions of the so-cal-
patient
pathological karyotype
47,XXY, 47,XXY-mosaics
other mosaics
translocation
46,XX male
Y-chromosomal deletions
total
Azoosp.
515
76
42
12
10
4
26
242
56
39
5
3
4
17
< 1 mio
sperms
99
8
1
4
3
0
5
SRY
CEP X
46,XX-male
46,X,del(Yq)
Figure 3: FISH-investigation
3a: LSI SRY SpectrumOrange / CEP X SpectrumGreen (Vysis) hybridized to a specimen obtained from an XX-male
3b: Quint Essential Y-specific DNA probe (Appligene Oncor) hybridized
to the AMELY gene on the short arm of the Y-chromosome (Yp11.2)
1-5 mio
sperms
100
6
1
3
2
0
2
>5 mio
sperms
30
0
0
0
0
0
0
unkown
44
5
3
0
2
0
2
Table 3: Correlation between the number of sperms and the occurrence of pathological karyotype and
Y-chromosomal deletions.
References
1.Tiepolo L., Zuffardi O: Localization of factor controlling spermatogenesis in the non-fluorescent portion of the human Y
chromosome long arm. Hum Genet 1976; 24: 119-124
2.Chandley AC: Chromosome anomalies and Y chromosome
microdeletion as causal factors in male infertility. Hum Reprod
1998; 13: 45-50
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■
3.Simoni M. et al: Laboratory guidelines for molecular diagnosis
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