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Published in Human Reproduction,
Volume 13, Supplement 1, 1998
There no longer seem to be any categories of male factor infertility that
cannot be treated with intracytoplasmic sperm injection
(ICSI). Even for men with azoospermia caused either by obstruction
or by germinal failure, ICSI may be performed successfully. The only failures
will be in azoospermic men who have neither spermatozoa nor spermatids
retrievable from the testis, but these men comprise a small percentage
of the cases with severe male factor. The source of the spermatozoa and
the cause of the sperm defect appear to have no effect on the success
of the procedure, whether the spermatozoon is epididymal, fresh or frozen,
testicular, ejaculated, or from the testicles of men with severe defects
in spermatogenesis. Maturation arrest, Sertoli cell-only, cryptorchidism,
chemotherapy and mumps do not appear to have a major impact on the pregnancy
rate. Of all the factors studied in couples where the male is severely
infertile or azoospermic, the only factor that seems to matter (as long
as spermatozoa are retrieved) is the age of the wife and, to a considerably
lesser extent, her ovarian reserve. Extensive genetic and paediatric follow-
up studies of ICSI pregnancies have revealed no increased risk of congenital
malformation (2.6%), no increased risk of de-novo autosomal abnormalities,
and a 1.0% risk of sex chromosomal abnormalities. These results are very
reassuring, but point to the need for careful counseling of couples with
male infertility.
Progress of ICSI since 1992
Since the publication of the first papers on the use of intracytoplasmic
sperm injection (ICSI) for oligozoospermia in 1992 and 1993, an intense
flurry of scientific effort has been dedicated to extending its application
to virtually every type of male infertility (Palermo et al., 1992; Van
Steirteghem et al, 1993; Silber, 1995; Silber et al, 1995a). The first
extension came when Nagy et al. (1995a) confirmed that the most severe
cases of oligoasthenoteratozoospermia produced the same pregnancy rates
as mild cases of male factor infertility, which were no different from
those of men with normal spermatozoa undergoing conventional in-vitro
fertilization (IVF). Liu et al. (1994a) then demonstrated that the
way in which the spermatozoa are pre-treated prior to ICSI is immaterial,
and that any method for aspirating the spermatozoa into an injection pipette
and transferring them into the oocytes is adequate. Liu et al. (1995)
also reported that fertilization failure was always related either to
poor egg quality or to sperm non-viability. It appeared that neither the
most severe morphological defect, nor the most severe motility defect,
nor the tiniest number of spermatozoa in the ejaculate ('pseudoazoospermia'),
had any negative effect on the pregnancy rate with ICSI (see
Tables I and II). Only absolute immotility of ejaculated or epididymal
spermatozoa lowered the fertilization rate, and this was found to be not
due to the immotility of the spermatozoon, but rather to its non-viability.
Completely nonmotile spermatozoa which were viable were still capable
of normal fertilization and pregnancy rates (see
Figure 1).
ICSI then took another leap forward with the development of sperm aspiration
and extraction techniques which allowed couples in whom the male was absolutely
azoospermic to have pregnancy rates no different from those in whom the
male had a normal sperm count (Nagy et al, 1995b; Silber et al, 1995b).
The first successful attempts at sperm aspiration combined with ICSI were
reported by Silber et al. and Tournaye et al. in 1994. Conventional IVF
with aspirated epididymal spermatozoa yielded a pregnancy rate of only
9% and a delivery rate of only 4.5%, whereas ICSI with aspirated epididymal
spermatozoa in men with congenital absence of the vas deferens (CAVD)
yielded a pregnancy rate of 47% and a delivery rate of 33%. Furthermore,
there was no difference in pregnancy rate with epididymal spermatozoa
retrieved for any cause of obstruction, whether it was failed vasoepididymostomy,
CAVD, or simply irreparable obstruction (Silber et al, 1995c).
However, this breakthrough for men with CAVD brought with it a serious
problem. It was soon discovered that CAVD is caused by mutations on the
cystic fibrosis transmembrane conductance regular gene (CFTR) located
on chromosome 7. Although now this is taken for granted, in 1992 it was
a startling discovery (Dumur et al., 1990; Silber et al, 1991; Anguiano
et al., 1992). This discovery meant that all patients and their wives
undergoing sperm aspiration with ICSI for CAVD required careful genetic
screening for cystic fibrosis, and if the wife was a carrier (4% risk
of carrier status in the general population), then the embryos should
undergo preimplantation genetic diagnosis using polymerase chain reaction,
so that only healthy embryos would be replaced. The first case of successful
preimplantation embryo biopsy for cystic fibrosis, on the embryo of a
man who had undergone microsurgical epididymal sperm aspiration (MESA)
and ICSI for CAVD, was reported by Liu et a'. (1994b) using the techniques
pioneered by Handyside et al (1993). The use of MESA and ICSI for CAVD
led to intense molecular study of the genetic mystery of how the condition
of CAVD is transmitted via defects in the cystic fibrosis gene (Chillon
et al., 1995; Silber et al, 1995c).
Soon after the MESA-ICSI procedure was developed in 1994, it was discovered
that testicular spermatozoa could fertilize as efficiently as ejaculated
spermatozoa and also result in normal pregnancies (Schoysman et al, 1993;
Devroey et al., 1995; Silber et al., 1995d). This procedure was coined
testicular sperm extraction (TESE). TESE truly
revolutionized the treatment of infertile couples with azoospermia. The
development of TESE meant that even patients with zero motility of the
epididymal spermatozoa or of ejaculated spermatozoa, or even men with
no epididymis could still have their own genetic child, so long as there
was normal spermatogenesis. It also meant that surgeons with limited raicrosurgical
skill could easily perform a testicle biopsy, enabling the retrieved spermatozoa
to be used for ICSI without the need for the microsurgical expertise required
to perform a conventional MESA procedure.
It was also demonstrated that epididymal spermatozoa, despite fairly weak
motility, could be frozen and, after thawing, yield pregnancy rates no
different from those obtained with freshly retrieved epididymal spermatozoa
(Devroey et al., 1994). This meant that men with obstructive azoospermia
could undergo a microsurgical reconstruction, without the need to have
the wife prepared for simultaneous IVF. Spermatozoa retrieved from the
epididymis at the time of the vasoepididymostomy could be frozen and stored.
If the vasoepididymostomy proved to be unsuccessful (10% of cases), the
frozen stored epididymal spermatozoa could serve as back-up to be used
for any number of future ICSI procedures without the husband having to
undergo further invasive surgery or aspirations (Silber, 1989a,b). Because
of the remarkable success with the freezing of very poor quality epididymal
spermatozoa for subsequent ICSI, couples did not have to time the MESA
exactly with the wife's egg retrieval, and in fact, the wife did not have
to go through any egg retrieval procedures unless the vasovasostomy or
vasoepididymostomy procedure proved to have failed. Then, at any later
date, at the couple's convenience, they could undergo ICSI with the frozen
stored spermatozoa. This also meant that men about to undergo chemotherapy
and/or radiotherapy for cancer could have a single ejaculate frozen, causing
no delay in treatment of the cancer, and this one ejaculate would be sufficient
for almost an infinite number of IVF-ICSI cycles.
Finally, in the majority of cases of patients with testicular failure
(caused either by maturation arrest, Sertoli cell-only syndrome, cryptorchid
testicular atrophy, post-chemotherapy azoospermia, or even Klinefelter's
syndrome), a very tiny number of spermatozoa or spermatids can usually
be extracted from extensive biopsies of the testicle and utilized for
ICSI (Devroey et al, 1995; Silber, 1995; Silber et al, 1995a,d, 1996).
This surprising ability of men who appear to produce no spermatozoa whatsoever
to have their own genetic child developed from application of basic studies
in quantitative analysis of testicle biopsy begun by Steinberger and Zuckerman
and continued by Silber and Rodriguez-Rigau (Steinberger and Tjioe, 1968;
Zuckerman et al, 1978; Silber and Rodriguez-Rigau, 1981). These early
studies of the kinetics of spermatogenesis in the testicle demonstrated
that often a tiny amount of spermatogenesis was present if one examined
quantitatively and carefully the testicle biopsy of men who were azoospermic
from non-obstructive testicular failure. However, the significance of
this 'threshold' phenomenon was not appreciated until the era of ICSI,
when it was realized that these spermatids could be harvested, and normal
pregnancy rates achieved, in the ~60% of such patients who possessed this
minuscule degree of spermatogenesis in otherwise completely deficient
testicles.
There has been some excitement generated over the possibility of using
'round cells' derived from testicular tissue (or even from the ejaculate),
which are presumably early spermatids, for ICSI in the absence of elongated
spermatozoa. Infertility clinics around the world are now endeavoring
to use this technique, which may have unfortunate implications.
The landmark study of Ogura and Yanagimachi (1993; Ogura et al, 1993,
1994) demonstrated in mice that fertilization and occasional liveborn
offspring could arise from the use of early round spermatids. However,
these mice had normal spermatogenesis, resulting in the availability of
many mature spermatozoa, which would give much higher fertilization and
pregnancy rates than the round spermatids. This work was followed by a
host of clinical efforts attempting to fertilize human eggs with ICSI
using 'round cells' (Sofikitis et al, 1994; Fishel et al, 1995; Tesarik
and Mendoza, 1996; Tesarik et al, 1996).
The problems with this approach are: (i) in human spermatogenesis, it
is widely known that maturation arrest' is a problem associated with meiosis,
and not with sperm maturation. Wherever there are early round spermatids,
there will also be elongated mature spermatids with a tail (Silber et
al, 1996; W.Schulze, personal communication, 1996); and (ii) clinics not
aware of this reality are tempted to inject 'round cells' when they cannot
retrieve spermatozoa. The truth is, that if those round cells were genuinely
spermatids, then a better search would have revealed the presence of mature
spermatozoa. On the other hand, it is extremely difficult, using Hoffman
optics, to distinguish with certainty a round spermatid from a Sertoli
cell nucleus with its prominent nucleolus, or even from some spermatocytes.
Where there are truly no spermatozoa, there may be many 'round cells'
either with Sertoli cell only, or with maturation arrest, that are not
round spermatids.
The current ability of most men to father a child, regardless of the quality
of the sperm count, even if there are apparently no mature spermatozoa
at all, is dramatic. It appears that the results of ICSI are related neither
to the source of the spermatozoa (whether ejaculated, testicular or epididymal),
nor to the quality of the spermatozoa (morphology or motility). ICSI results
are not influenced by whether the spermatozoon has been frozen or is fresh,
whether it is retrieved with ease (as in the cases of normal spermatogenesis
or with ejaculated spermatozoa), or whether the spermatozoon was extracted
directly from the Sertoli cell after hours of painstaking searching of
a testis sample (see
Table III). With regard to the infertile or azoospermic male and ICSI,
none of these factors has had any significant influence on fertilization,
cleavage or pregnancy rate. In fact, the only significant factor in the
success of ICSI appears to be the age of the female partner (Silber et
al, 1995c). Regardless of the source of the spermatozoa, their quality
or the diagnosis in the male, the success rate appears to be determined
only by the age of the wife (see
Table IV).
(top)
Are the babies normal?: the
genetics of ICSI
If the most important refinement of ICSI in 1994 and 1995 was
the development of TESE-ICSI for cases of non-obstructive azoospermia,
the major development in 1996 was the detailed follow-up study of ICSI
babies and the information it provided about the genetics of infertility.
In this volume, Liebaers and Bonduelle describe the extensive follow-up
studies of ICSI babies who underwent chromosomal evaluation at amniocentesis
or chorionic villus sampling (CVS), as well as a detailed 2 year paediatric
follow-up. In the first 877 consecutive ICSI babies born in the Brussels
Dutch-Speaking Free University program, thus far there has been no greater
incidence of major or minor congenital abnormalities than is seen in routine
screenings of normal large populations. The chromosomal studies of ICSI
pregnancies are similarly reassuring. Firstly, of the 877 ICSI babies
who underwent detailed paediatric follow-up by Bonduelle et al, 448 were
male and 429 were female, indicating no significant difference in sex
ratio. The overall incidence of major congenital malformations in these
877 children was 2.6%, which is no different from the incidence of similar
major malformations in many studies involving very large samples of normal
populations (from 2.1 to 3.6%; see
Tables V, VI and VII). In 1995, the ICSI task force also reported
18 major malformations in 763 children, which corresponds to the report
of Bonduelle et al of 2.6% [Office of Population Consensus and Surveys,
1987-1988; Congenital Malformation Statistics, 1979-1985, London. HMSO
(OPC Series MB3); and National Perinatal Statistics Unit and the Fertility
Society of Australia, 1992; IVF and GIFT Pregnancies,
Australia and New Zealand, 1990; Sydney National Perinatal Statistics
Unit (NPSU); and New York State Department of Health, 1990, Congenital
Malformations Registry Annual Report; Statistical Summary of Children
Born in 1986 and Diagnosed through 1988.
The results of chromosomal studies from amniocentesis and CVS of the first
486 fetuses undergoing prenatal testing, although reassuring, deserve
special consideration (see
Tables VIII, IX and X). Six of these 486 prenatal chromosomal evaluations
(1.2%) showed de-novo abnormalities not transmitted directly from the
father. Five of these abnormalities were sex chromosomal in nature, and
only one (0.2%) was an autosomal abnormality. This latter abnormality
was a Down'syndrome (47XY,T- 21) picked up on CVS in a 41 year old woman,
and this pregnancy was terminated. A 0.2% incidence of trisomy 21 in a
41 year old woman is to be expected in any population of women of that
age and certainly is in no way attributable to the ICSI procedure or to
the infertility of the husband.
However, the five de-novo sex chromosomal abnormalities (1.0%) represent
an incidence greater than the 0.2% incidence expected in a normal population
of newborns. Of these five anomalies, one was 47,XXX, one 46,XX/47,XXX
mosaic, two 47,XXY Kimefelter's, and one 47,XYY. Although this incidence
of sex chromosomal aneuploidies certainly exceeds the expected population
norm seen in newborns, most parents did not express serious concern about
this and elected to interrupt the pregnancy in only one case, a Kimefelter's.
These children with sex chromosomal abnormalities appear to be normal
in every other way.
The incidence of autosomal chromosomal abnormalities inherited from the
parents was 1.0%. All were paternally transmitted chromosomal aberrations
seen in the father prior to ICSI during the initial counseling. The transmission
of these abnormalities from the father to the child was not of serious
concern to most of the parents. These inherited structural chromosomal
anomalies included three inversions and two balanced translocations. Thus,
the results of detailed follow-up of ICSI pregnancies and delivered babies
are very reassuring, but suggest that there may be a very slightly higher
risk (1%) than normal of sex chromosomal abnormalities in these children.
Furthermore, attention must be given to the possibility of the occasional
balanced translocation in the fetus.
(top)
THE GENETICS OF INFERTILE MEN
ABOUT TO UNDERGO ICSI
Genetics of oligozoospermia and germinal failure
The increased incidence of paternally transmitted translocations and inversions
in infertile men has been deduced from detailed chromosomal evaluations
performed on 694 patients and their wives prior to undergoing the ICSI
procedure, as well as from a detailed review of the literature of chromosomal
evaluations in infertile men (see
Tables XI and XII). From 1975 until the present time, reports have
been published on 7876 infertile men who have undergone karyotyping. Of
these, 3.8% were found to have sex chromosomal abnormalities and 1.3%
were found to have autosomal chromosomal abnormalities, giving a total
of 5.1% chromosomal abnormalities in this large population. This compares
to the incidence in newborn infants of sex chromosomal abnormalities of
0.14%, autosomal chromosomal abnormalities of 0.25%, and of total chromosomal
abnormalities in the newborn population of 0.38% (Koulischer and Schoysman,
1974; Chandley, 1979; Abramsson et al, 1982; Zuffardi and Tiepolo, 1982;
Gardelle et al, 1983; Matsuda et al, 1989; Yoshida et al, 1995). The incidence
of sex chromosomal abnormalities in newborn infants (0.14%) is therefore
much less than that in azoospermic males. The average 3.8% incidence of
sex chromosomal abnormalities obtained from all these studies was different
from that shown in the study of Matsuda et al (1989), which found no sex
chromosomal abnormalities in azoospermic men but a 1.7% incidence of autosomal
abnormalities. The great majority of sex chromosomal abnormalities in
azoospermic men appear to be XXY Klinefelter's syndrome, with other sex
chromosomal abnormalities occurring in only 0.6% of azoospermic men.
Of the total of 1.3% of infertile men in these published reports who had
autosomal abnormalities, there was an approximately equal number of Robertsonian
translocations and reciprocal translocations, with a smaller number of
inversions and extra markers. The incidence of autosomal abnormalities
in newborn infants (0.25%) is one-fifth of that found in infertile men.
In azoospermic males, 1.1% have autosomal abnormalities. In the Brussels
series of severe oligozoospermic, asthenozoospermic and teratozoospermic
men, as well as men with oligoasthenoteratozoospermia, only 0.3% had sex
chromosomal abnormalities, while none of the men who failed to exhibit
any of the three defects had sex chromosomal abnormalities. On average,
2% of men with severe oligozoospermia or oligoasthenoteratozoospermia
exhibited chromosomal defects, a rate 5-6 times greater than that of a
normal population. These chromosomal defects result in a higher rate of
miscarriage and the transmission of paternal chromosomal defects to the
offspring. Therefore, in a very small percentage of infertile men (2%),
chromosomal abnormalities create meiotic difficulties that appear to interfere
with spermatogenesis. However, there are many more subtle genetic defects
that appear to be responsible for male factor infertility that will not
show up in routine chromosomal analysis.
In a study initiated by David Page and myself, 89 men with non-obstructive
azoospermia caused either by maturation arrest, Sertoli cell-only, or
a combination of these two histological defects, underwent detailed sequence-tagged
sites (STS) mapping of the Y chromosome This demonstrated microdeletions
in 13% of patients, located at the distal portion of the euchromatic region
of the Y, which is positioned approximately in the middle of the long
arm of the Y. These microdeletions can be detected with mapping signposts,
which currently have a sensitivity of only 20 000 base pairs. Thus, it
is very possible that many more, much smaller, mutations in the Y chromosome,
perhaps in the region termed DAZ, may be responsible for varying degrees
of azoospermia and oligozoospermia (Reijo et al, 1995; Silber, 1995; Silber
et al, 1995a,c). This region of the human genome is particularly difficult
to sequence accurately because of the presence of so many confusing 'Y-specific
repeats.' These repeats also explain why this region is so very prone
to spontaneous mutations and perhaps also why male infertility is very
common in the human.
It is easy to interpret incorrectly as deletions what are in truth polymorphisms,
because most of the Y chromosome does not undergo recombination and is
truly a degenerate chromosome. However, our study controlled for that
possibility by finding no such deletions in the fathers or brothers of
these azoospermic men, who incidentally were fertile. The questions can
be raised, why do some of these men have occasional spermatozoa recoverable
from the testes, and also what causes azoospermia in the other 87% of
azoospermic men with Sertoli cell only or maturation arrest? One new avenue
to explore is the finding that the Y deleted DAZ gene also occurs on autosome
3 in humans, thus introducing issues of recombination and dominance (Saxena
et al, 1996).
Even Klinefelter's syndrome patients (XXY), who were originally thought
not to be able to father children, often have a minute amount of sperm
production that can be discovered in the testis. These patients can undergo
the TESE- ICSI procedure and produce normal embryos that presumably lead
to a pregnancy. The question arises as to whether the spermatozoa from
these Klinefelter's patients, which are presumably like the spermatozoa
from other severely infertile men, yielding offspring with a higher incidence
of sex chromosomal abnormalities, may be a heterogeneous population of
some that are disomic for XX, some that are disomic for XY, as well as
some that are normal haploid Y or normal haploid X. If this were the case,
it would indicate that an offspring would have a 50% chance of also having
a sex chromosomal abnormality. However, if the opposite theory is true,
that it is only the mosaically normal areas of the seminiferous tubules
that are producing spermatozoa, then presumably the ratio of X and Y spermatozoa
in the testes of these men would not differ from normal, and there would
be no increased risk of sex chromosomal abnormalities in the children.
To test this concept, routine preimplantation genetic diagnosis has been
performed on all Klinefelter's patients undergoing TESE-ICSI (Staessen
et al, 1996). Thus far, the replaceable embryos have not had sex chromosomal
abnormalities, with the exception of one which was a mosaic of XXY and
XY. Therefore, this matter still remains under question.
Thus, because the ICSI procedure allows us to produce fertilized eggs
using spermatozoa from almost any man, no matter how apparently sterile,
we have been better able to study and understand the genetic causes of
male infertility, and the possible transference of male infertility to
the male offspring generated by the ICSI procedure. Perhaps this will
lead to a greater understanding in the future, and to improved treatment
of these genetic causes of male infertility.
(top)
Cystic fibrosis
The genetics of cystic fibrosis
and congenital male obstructive infertility have been studied in great
detail as a result of the introduction of ICSI (Silber et al, 1991; Anguiano
et al, 1992; Chillon et al, 1995). Previously, there was no evidence to
suggest that congenital absence of the vas might be a genetic condition
transmitted via the cystic fibrosis gene. The only clue was the clinical
observation that all men with cystic fibrosis also have congenital absence
of the vas deferens. However, the majority of men visiting fertility clinics
because of azoospermia caused by congenital absence of the vas deferens
had normal sweat chloride tests and no clinical signs of cystic fibrosis.
Yet, genetic studies showed that 70% of these men had common cystic fibrosis
mutations on one allele, and 10% had common cystic fibrosis mutations
on both alleles. In those cases where both alleles were affected, one
of the mutations was always extremely mild. Today, men with frank cystic
fibrosis, with both alleles having strong mutations, are also presenting
at fertility clinics to attempt to achieve pregnancy with MESA-ICSI.
The big mystery has been why 30% of cases show no cystic fibrosis mutations,
while 60% have only one allele affected. Studies performed by the Cystic
Fibrosis Consortium in Europe in which the entire coding region of the
cystic fibrosis genes was scanned revealed absolutely no mutations in
these men, meaning that the problem was not one of a hidden, undiscovered
mutation in any of the exons or coding regions. However, a splicing error
in intron 8, called the T5 allele, was found to be on the opposite allele
of a patient who was heterozygous for cystic fibrosis, but in both alleles
of those who showed no mutations. This resulted in a defective production
of CFTR protein that was adequate to prevent cystic fibrosis but inadequate
to prevent congenital absence of the vas deferens.
Thus, use of ICSI for treatment of the most severe cases of male factor
infertility is leading to major molecular genetic discoveries that would
never have been anticipated in the era of classical andrology.
(top)
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See also:
Microsurgical TESE and the
distribution of spermatogenesis in non-obstructive azoospermia--By
Dr. Silber
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