J.WJersson et al
SUber, S J., Nagy, Z., Uu, i. et al (1995) The use of epididymaJ and testicular
sperm injection: the genetic implications for male infertility. Hum. Reprod.,
10,2031-2043.
Sweet, CJL (1995) Intracytoplasmic sperm injection - 'pushing the envelope'
[letter]. FertiL Steril., 64, 223-224.
Toumaye, H., Liu, J., Nagy, Z. et al (1995) Intracytoplasmic sperm injection
(1CSI): the Brussels experience. Reprod. Fertil. Dev., 7, 269-279.
Yoshida, A., Tamayama, T., Nagao, K. et al (1995) A cytogenetic survey of
1,007 infertile males. Contracept. Fertil Sex., 23 (Suppl. no 9), S23.
The risk of chromosomal abnormalities
following ICSI
Ren6e H.Martin
Department of Medical Genetics, University of Calgary,
Alberta, Canada
During the past year, a number of studies have sounded a
warning about the safety of intracytoplasmic sperm injection
(ICSI). The Brussels group reported on prenatal diagnosis
results of 491 fetuses, detailing five sex chromosomal aneuploidies (47XXX; 47XYY; 47XXY (2); 46XX/47XXY);
(Bonduelle et al, 1995; Tournaye et al, 1995). This frequency
of ~ 1 % sex chromosomal aneuploidies after ICSI is higher
than expected since the incidences of 47XXX; 47XYY and
47XXY are about one in 1000 births. The recent report of In't
Veld et al. (1995) further highlights these concerns as five of
15 fetuses from 12 ICSI pregnancies had a sex chromosomal
abnormality.
Persson et al (1996) suggest that the cause of this high
frequency of sex chromosomal abnormalities may be that a
significant proportion of infertile males are in fact mosaic for
Klinefelter syndrome (46XY/47XXY). This genetic constitution could theoretically predispose to 24XX and 24XY spermatozoa. However, it is also possible that XXY germ cells, if
they were capable of completing meiosis, would produce only
23X and 23Y spermatozoa since it has been demonstrated
by both sperm karyotyping (Benet and Martin, 1988) and
fluorescence in-situ hybridization (FISH) analysis (Han et al,
1994) that 47XYY males produce chromosomally normal
spermatozoa. The extra chromosome appears to be eliminated
during spermatogenesis.
Three years ago we began a study of 10 infertile patients
to determine if these men, who were prime candidates for
ICSI, had an increased risk of chromosomal abnormalities in
their spermatozoa. The infertile men with oligo-, astheno-, or
teratozoospermia all had a normal lymphocyte karyotype,
normal concentrations of follicle stimulating hormone (FSH),
luteinizing hormone (LH) and testosterone and no known
exposure to radiation, drugs or environmental exposures.
Sperm chromosome karyotyping using the human spermatozoa/
hamster oocyte fusion system was performed on 518 sperm
chromosome complements from five men (Moosani et al,
1995). Sperm karyotyping is extremely labour intensive but it
does provide precise information on structural and numerical
abnormalities for all chromosomes. We found a significantly
increased frequency of numerical chromosomal abnormalities
924
in the infertile men (3.1%) compared with fertile donors
(0.84%). In order to increase our sample size, we used
multicolour FISH analysis with chromosome specific DNA
probes to study >200 000 spermatozoa from these five men
plus an additional five infertile men. A minimum of 10 000
sperm nuclei were scored per male per DNA probe for each
of chromosomes 1, 12, X and Y. There was a significant
increase in the frequency of disomy for chromosome 1 and
XY disomy in infertile men compared with normal men,
corroborating our results from sperm karyotypes (Moosani
et al, 1995, plus unpublished results). Pang et al (1995)
studied sperm aneuploidy by FISH analysis of chromosomes
7, 11, 12, 18, X and Y in nine oligoasthenoteratozoospermic
(OAT) patients and also found an increased frequency of
numerical chromosomal abnormalities, including sex chromosomal aneuploidy, in the infertile men. Miharu et al. (1994)
found no significant difference between the frequency of
aneuploidy as assessed by one-colour FISH in spermatozoa
from 12 infertile compared to normal donors; however, a
relatively small number of sperm nuclei were studied per male
and XY aneuploidy could not be evaluated since they did not
employ multicolour FISH.
Our results from both FISH analysis and sperm karyotypes
demonstrated a significantly increased frequency of aneuploidy
in spermatozoa from infertile men, particularly for the sex
chromosomes. These results were most striking in oligozoospermic men. Quantitative problems, including oligozoospermia and azoospermia, have been associated with pairing
abnormalities within both the autosomes and the sex chromosomes. Egozcue et al (1983) reported that in infertile males,
there is an increased frequency of pairing disruptions resulting
in meiotic arrest. It is possible that a pairing abnormality in
these infertile males could lead to meiotic arrest in some cells
causing oligozoospermia and aneuploidy in other cells capable
of completing spermatogenesis. The sex chromosome bivalent
is particularly susceptible to pairing abnormalities since there
is generally only one crossover in the pseudoautosomal region.
Speed and Chandley (1990) studied infertile men with a normal
somatic karyotype and found reduced XY synapsis compared
with normal men. Furthermore, Hassold et al. (1991) have
determined that there is a reduction in recombination for the
XY bivalent in meiosis leading to a 47XXY karyotype.
Thus, it is quite plausible that infertile men have decreased
recombination and pairing leading to both meiotic arrest
(oligozoospermia) and non-disjunction of the sex chromosomes^
Persson et al (1996) have advocated karyotyping infertile
men before ICSI to 'divide infertile males into euploid 'low
risk' or aneuploid 'high risk' groups.' This is based on their
hypothesis that the cause of the increased frequency of sex
chromosomal aneuploidy in these men is due to mosaicism.
However, according to our hypothesis of a pairing abnormality
responsible for both the oligozoospermia and non-disjunction,
karyotyping would not distinguish a high or low risk male. In
fact, all of our infertile patients had a normal karyotype
and yet they still had an elevated frequency of aneuploid
spermatozoa. This suggests that it would be prudent to offer
Genetic consequences of ICSI
prenatal diagnosis for all couples contemplating infertility
treatment by ICSI.
References
Benet, J. and Martin, R. (1988) Sperm chromosome complements in a 47.XYY
man. Hum. Genet., 78, 313-315.
Bonduelle, M , Legein, J., Derde, M.-P. et aL (1995) Comparative follow-up
study of 130 children bom after intracytoplasmic sperm injection and 130
children bom after in-vitro fertilization. Hum. Reprod., 10, 3227-3331.
Egozcue, J., Templado, C , Vidal, F. et aL (1983). Meiotic studies in a series
of 1100 infertile and sterile males. Hum. Genet., 65, 185-188.
Han, X, Ford, J., Flaherty, S. et al. (1994) A fluorescent in situ hybridisation
analysis of the chromosome constitution of ejaculated sperm in a 47, XYY
male. Clin. Genet., 45, 67-70.
Hassold, T., Sherman, S., Pettay, D. et aL 0991). XY chromosome
nondisjunction in man is associated with diminished recombination in the
pseudoautosomal region. Am. J. Hum. Genet., 49, 253-260.
In't Veld, P., Brandeburg, H., Verhoeff, A. et aL (1995) Sex chromosomal
abnormalities and intracytoplasmic sperm injection [letter]. Lancet, 346,773.
Miharu, N., Best, R, and Young, S. (1994) Numerical chromosome
abnormalities in spermatozoa of fertile and infertile men detected by
fluorescence in situ hybridization. Hum. Genet., 93, 502-506.
Moosani, N., Pattinson, H., Carter, M. et al. (1995) Chromosomal analysis of
sperm from men with idiopathic infertility using sperm karyotyping in situ
hybridisation. FertiL Steril., 64, 811-817.
Pang, M., Zackowski, J., Hoegerman, S. etal. (1995) Detection by fluorescence
in situ hybridisation of chromosome 7, 11, 12, 18, X and Y abnormalities
in sperm from oligoasthenospermic patients of an in vitro fertilisation
progam. J. Assist. Reprod Genet., 12, 53S.
Persson, J., Peters, G. and Saunders, D. (19%) Is ICSI associated with risks
of genetic disease? Implications for counselling, practice and research.
Hum. Reprod., 11, 921-924.
Speed, R. and Chandley, A. (1990) Prophase of mciosis in human spermatocytes
analyzed by EM microspreading in infertile men and their controls and
comparisons with human oocytes. Hum. Genet., 84, 547-554.
Toumaye, H., Liu, J., Nagy, Z. et aL (1995) Intracytoplasmic sperm injection
(ICSD: the Brussels experience. Reprod. FertiL Dev., 7, 269-279.
Micromanipulative assisted
fertilization—still clinical research
The-Hung Bui 13 and Hakan Wramsby 24
department of Clinical Genetics, and Reproductive
Medical Centre, Division of Obstetrics and Gynecology,
Department of Women and Child Health, Karolinska
Hospital, S-171 76 Stockholm, Sweden, and department
of Obstetrics and Gynecology, Division of Fetal and
Maternal Medicine, Huddinge University Hospital,
Huddinge, Sweden.
4
To whom correspondence should be addressed
Intracytoplasmic sperm injection (ICSI) is a much more
efficient micromanipulative technique than subzonal insemination (SUZ1) or partial zona dissection (PZD), resulting in
improved fertilization and pregnancy rates (Tarfn, 1995). Its
rapid implementation in in-vitro fertilization (TVF) centres is
dramatically changing the treatment of severe male-factor
infertility (Hamberger et al., 1995; Tournaye et al, 1995), and
has altered the outlook of repeated failed fertilization after
conventional IVF (Asch et al, 1995; Nagy et al, 1995).
Indeed, a recent survey of all 13 FVF centres in Sweden
revealed that the ICSI technique is used clinically or is being
established in 11 of these centres (T.-H.Bui and L.Hamberger,
unpublished data). However, there is an increased perception
of potential genetic and teratological risks for the offspring
conceived by these microinvasive techniques if used indiscriminately (Meschede et al, 1995; Patrizio, 1995; Persson
et al, 1996). Several issues of practical and theoretical
importance pertaining to ICSI and genetics are presented and
further discussed here.
The risk of chromosome defects
The use of seemingly 'unfertilized' but inseminated oocytes
for subsequent reinsemination or ICSI should-raise concerns
for polyploidy, since the fertilization process might have
arrested at several previously undetectable phases after sperm
penetration (Asch et al, 1995). In addition, microinjection of
sperm heads (Bourne et al., 1995) or immature sperm cells
might lead to an increased risk of mosaicism due to defective
or absent sperm centrosomes (Ogura and Yanagimachi, 1995;
Sofikitis et al, 1995). Thus, further genetic evaluation of the
effect of the centrosome on spindle formation is strongly
needed before widespread use of these methods (Asch et al,
1995; Simerly etal, 1995).
It has been argued that an increased risk for chromosome
defects in the offspring resulting from ICSI could originate
from the underlying chromosomal pathology within the male
population selected for treatment particularly when microepididymal sperm aspiration (MESA) or testicular sperm
extraction (TESE) are used (Persson et al, 1996). In our view,
there is enough evidence (Kjessler, 1974; De Braekeleer
and Dao, 1991; Persson et al, 19%) to recommend that a
chromosome analysis should be routinely performed on the
male when ICSI is to be used. Surprisingly, a karyotype of
the men treated with ICSI was not routinely carried out in as
many as eight of the eleven Swedish centres using ICSI
(T.-H.Bui and L.Hamberger, unpublished data). If this reflects
a common practice, there is an urgent need for evidence based
guidelines on the genetic workup of men with severe infertility.
Others have advocated that a chromosome analysis should
also be performed on the female partner merely as a general
measure of risk evaluation (e.g. Meschede et al, 1995),
although the rationale for such a routine analysis is not clear
(Chandley, 1990).
Invasive prenatal diagnosis is commonly offered because
of the potential increased aneuploidy risk for the offspring
conceived by ICSI and limited follow-up data presently available (Bonduelle et al, 1995; Persson et al, 1996). However,
in our experience about only one third of the couples would
have prenatal diagnosis performed, the majority would refuse
it mostly for fear of fetal loss. Although a major autosomal
chromosome aberration can be suspected at birth a sex chromosome defect in the offspring cannot be detected in most cases
without prenatal diagnosis. In such a situation, a chromosome
analysis may be obtained from a cord blood sample at delivery
taking into consideration that a sufficient numbeT of metaphases
should be analysed to allow the diagnosis of, at least, moderate
levels of mosaicism. Moreover, for research purposes, chromosome analysis of fetal tissue should be performed when an
ICSI pregnancy miscarries to substantiate whether there is an
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