J Assist Reprod Genet (2012) 29:343–346

DOI 10.1007/s10815-012-9720-3

GENETICS

Case report: elevated sperm aneuploidy levels in an infertile

Robertsonian translocation t(21;21) carrier with possible
interchromosomal effect
N. Rogenhofer & S. Dürl & R. Ochsenkühn & M. Neusser &
E. Aichinger & C. J. Thaler & S. Müller

Received: 8 November 2011 / Accepted: 26 January 2012 / Published online: 9 February 2012
# Springer Science+Business Media, LLC 2012

Introduction translocations (reciprocal exchange between two chromo-

somes), inversions, Y-chromosome micro-deletions and bal-
Somatic chromosomal abnormalities are frequently found in anced Robertsonian translocations (fusion of two acrocentric
infertile men, particularly in those with low sperm count chromosomes, e.g. 13, 14, 15, 21, 22). Carriers of a balanced
[1–5]. Studies in infertile men have demonstrated that 2– Robertsonian translocation show impaired gametogenesis to a
14% carry constitutional chromosomal abnormalities [1] Re- variable degree and elevated levels of nullisomic or disomic
search in this area has become more relevant in the past few sperm (3–40%, mean 15%) [1–5]. The fertilization with an
years with the advent of assisted reproductive therapies aneuploid sperm results in monosomy or trisomy in the fetus
(ART), especially intracytoplasmatic sperm injection (ICSI). [7, 8]. Furthermore fetal aneuploidies are a major cause of
ICSI has been extremely successful for the treatment of male pregnancy loss and fetal malformations [9].
infertility, but transmission of cytogenetic defects to offspring Here we report on a sperm aneuploidy screening of all
is a major concern and demonstrated to be reality [1], therefore chromosomes (1–22,X,Y) in an infertile patient showing
it is recommended to investigate this. The incidence of chro- low-frequency mosaicism of a balanced Robertsonian trans-
mosomal aberrations in infertile men depends on the defini- location involving both chromosomes 21.
tion of “infertility”, and is estimated to be approximately 5%
in oligozoospermic men and 14% in azoospermic men [6].
Abnormalities mostly consist of numerical sex chromosome Case report
abnormalities (e.g. 47,XXY), balanced reciprocal
Patient
Capsule Sperm aneuploidy and Robertsonian translocation with
possible interchromosomal effect. The 36 years old man presented in our infertility clinic with
N. Rogenhofer : R. Ochsenkühn : C. J. Thaler sterility lasting for 2 years. His medical history showed no
Division of Gynecological Endocrinology and Reproductive evidence of relevant diseases, medication or nicotin abuse
Medicine, Department of Gynecology and Obstetrics, Großhadern, and BMI (body mass index) was unremarkable. Hormone
Ludwig-Maximilians University,
Munich, Germany
parameters of testosterone, follicule stimulating hormone
(FSH), luteinising hormone (LH), prolactine and thyroid
S. Dürl : M. Neusser : E. Aichinger : S. Müller parameters were in the normal range. Serological testing
Institute of Human Genetics, Ludwig-Maximilians-University, (hepatitis B/C, HIV, mumps) showed inconspicuous results.
Munich, Germany
The sperm count revealed an oligo-astheno-teratozoospermia
N. Rogenhofer (*) (Sperm concentration: 14,3 mio sperm per ml, sperm motility:
Division of Gynecological Endocrinology and Reproductive WHO a (rapid progressive motility): 9%, WHO b (moderate
Medicine, Department of Gynecology and Obstetrics, Ludwig- progressive motility): 10%, WHO c (no progressive motility):
Maximilians University Munich—Großhadern,
Marchioninistrasse 15,
31%, WHO d (immotile): 50% and 98% abnormal sperm
81377 Munich, Germany morphology). Somatic chromosome analysis from a blood
e-mail: [email protected] sample revealed a low-frequency (4%) mosaic of a balanced
344 J Assist Reprod Genet (2012) 29:343–346

Robertsonian translocation 45,XY,t(21;21)(q10;q10) in a nor- The investigated sperm cells of the mosaic t (21;21)
mal 46,XY male background. patient showed a significantly elevated level of disomy 21
(1,08% of all cell nuclei analyzed). However, also for chro-
mosomes 2 (0,38%), 4 (0,29%), 6 (0,19%), 9 (0,29%), 15
(0,29%) and most notably for chromosome 16 (0,87%)
Materials and methods
significantly increased disomy frequencies, compared to
baseline frequencies from the literature, were observed
We screened a sperm sample of this patient for elevated
(see Fig. 1). The remaining chromosomes showed no evi-
disomy levels of all chromosomes (1–22, X and Y) and for
dence of elevated disomy rates. The overall diploidy rate
increased diploidy rates by multicolour interphase fluores-
was also within the normal range.
cence in situ hybridisation (sperm-FISH). Patient sperm cells
were prepared for FISH according to standard procedures
[10]. In brief, cells were fixed with methanol/acetic acid (3/
1 v/v) and then dropped onto glass slides. Subsequently, the
Discussion
sperm cell chromatin was decondensed by 10 mM LIS (Lith-
ium-diiodosalicylat)/10 mM DTT (Dithiothreitol) treatment
It is well established that patients with Robertsonian trans-
and finally the cells were permeabilized by pepsinization.
locations may have higher aneuploidy rates in sperm cells.
FISH to sperm cell nuclei with chromosome-specific centro-
This can be associated with infertility, leading to an in-
mere (CEP, kindly provided by M. Rocchi, www. biologia.
creased risk for miscarriages and fetal malformations [1,
uniba.it/rmc) or BAC (LSI, obtained from http://bacpac.chori.
13, 14]. In this case report we were able to extend this view
org or Abbott Molecular) probes was performed for all human
by demonstrating that even low-frequency mosaic translo-
chromosomes (1–22, X and Y) as described in Williams 1993
cation carriers may be affected by increased germ cell an-
[11] with minor modifications). Subsequently, the number of
euploidy rates and the resulting secondary symptoms.
FISH-signals in each nucleus was determined by microscopic
Moreover, these aneuploidies may also involve other
analysis according to standardized criteria [10].
chromosomes than the translocation products. The occur-
At least 1000 nuclei per chromosome probe (>24.000 cell
rence and impact of this so-called interchromosomal effect
nuclei) were scored. Statistical analyses were performed
(ICE) is controversially discussed, and some authors ap-
with the Statistical Package for Social Sciences (SPSS for
proved the effect [3, 15–19], while others did not [20–22].
Windows 16.0, SPSS Inc., Chicago IL, USA). An increase
It can be assumed that the ICE is the consequence of a
of the aneuploidy rate in the patient by over two standard
general disturbance of meiotic processes caused by the
deviations compared to the mean baseline level in healthy
translocation product, mainly the synapsis and disjunction
controls described in the literature (see Results section) was
of other chromosome pairs.
considered statistically significant (P<05%).
Chen et al. investigated meiotic segregation behaviour in
spermatozoa from six males with Robertsonian translocations
(t(13;14), t(14;22), t(13;21)) by FISH [23]. Furtheron, they
Results studied chromosome 18, X and Y. They found a higher
aneuploidy frequency for sex chromosomes in three patients
In order to obtain an updated overview about baseline aneu- and increased rates of diploidy in two t(13;14) carriers. Anton
ploidy rates in human sperm, we performed a meta-analysis of et al. also analysed the segregation of chromosomes 13 and 14
the current literature on sperm FISH aneuploidy analysis in in 7 male t(13;14) carriers as well as chromosomes 18, 21, 22,
healthy control individuals revealed 64 publications reporting X and Y [13]. They report a significant increase in the disomy
on 620 individuals (complete reference list available upon rates for the sex chromosomes in two carriers and considered
request). It is of note that not all 24 chromosomes were these findings correlated with an ICE.
analyzed in each individual, because most studies focussed However all previous studies investigated only a limited
on the clinically relevant chromosomes 13, 18, 21, X and Y. set of chromosomes. Most studies included only chromo-
Therefore, the reliability of the presently available data may somes 21, X and Y [13]. Less frequently, disomy of chro-
still vary considerably between chromosomes and for some mosomes 13 and 18 have been investigated. Some authors
chromosomes statistically meaningful data is still missing (e. have also looked for aneuploidy of other chromosomal
g. chr. 3, 5, 11, 19 and 20). An average diploidy rate of 0.2% pairs, such as pairs 1, 4, 9, and 22. Others investigated
and a mean disomy frequency of 0.18% were described for chromosomes 7, 9, 13, 18, 21, X and Y in transolcation
healthy individuals. However, chromosome-specific differen- carriers [2]. Overall, only 10 or less individuals were ana-
ces are evident [12], for example when comparing chromo- lysed for chromosomes 3, 11 and 20. Moreover, there is no
some 200.08% and chromosome 2200.42%) (Fig. 1). data available so far for chromosomes 5 and 19.
J Assist Reprod Genet (2012) 29:343–346 345

Fig. 1 Summary of sperm

aneuploidy analysis in healthy
control individuals and in the
mos t(21;21) patient. The
investigated sperm cells of the
t(21;21) patient showed
significantly elevated level of
disomy 21 (arrow). However,
also other chromosomes
showed significantly increased
frequencies (arrows)

Here, we performed–for the first time- screening of a sperm possible cause for male infertility. We conclude that analysis
sample from a low frequent t(21;21) patient for elevated dis- of sperm aneuploidy by FISH may be helpful technique in a
omy levels of all chromosomes (1–22, X and Y) by multi- comprehensive clinical work-up and may increase the accu-
colour sperm-FISH. In this case, we see a clear indication for racy of a prognostic prediction compared to standard karyo-
the presence of an ICE. We found significantly increased levels type analysis on somatic cells alone. Sperm-FISH screening of
of chromosome 21 disomic sperm as well as elevated level of all 24 chromosome types is labour intensive, but the procedure
disomy for chromosomes 2, 4, 6, 9, 15 and 16. Therefore in our can be automated. It can be used to clarify the significance of
t(21;21) patient, an ICE may play a role and further increases the so far elusive ICE, and the results obtained may direct
the overall frequency of aneuploid sperm cells. further chromosome analysis during in vitro fertilization.
It is not known, if the increase of disomy rates in Therefore, cytogenetic studies on spermatozoa could be inte-
translocation-carriers is truly the result of ICE or if it is grated in the genetic exploration of the aetiology of infertility,
associated with other abnormal semen parameters. Indeed, and may give men with fertility problems a personalized
several studies have shown increased frequencies of aneuploi- assessment of the aneuploidy risk in their offspring.
dy in spermatozoa of males with severe oligozoospermia or
oligoasthenoteratozoospermia, despite having a normal leuco-
cytic karyotype [3, 15, 16, 24, 25]. In our study elevated
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