Human Reproduction Vol.18, No.3 pp. 562±567, 2003
DOI: 10.1093/humrep/deg121
Pregnancies after ICSI using sperm with abnormal
head±tail junction from two brothers: Case report
G.Porcu1, G.Mercier2,3, P.Boyer2, V.Achard3, J.Banet1, M.Vasserot3, C.Melone2,
J.Saias-Magnan4, C.D'Ercole5, C.Chau5 and M.R.Guichaoua4,6,7
1
Centre de ProcreÂation MeÂdicalement AssisteÂe, AP-HM, HoÃpital de la Conception, 147 Boulevard Baille, 13385 Marseille cedex 05,
Laboratoire de ProcreÂation MeÂdicalement AssisteÂe, HoÃpital Saint-Joseph, 26 Bd de Louvain, 13285 Marseille cedex 08,
3
Laboratoire d'Histologie et de Biologie de la Reproduction, Faculte de MeÂdecine Timone, 27 Boulevard Jean Moulin, 13385
Marseille cedex 05, 4Laboratoire de Biologie de la Reproduction, HoÃpital de la Conception, 147 Boulevard Baille, 13385 Marseille
cedex 05, 5Service de GyneÂcologie ObsteÂtrique, AP-HM, HoÃpital Nord, Chemin des Bourrellys, 13915 Marseille cedex 20 and
6
Laboratoire de BiogeÂnotoxicologie et MutageneÁse Environnementale (EA1784), IFR PMSE112, Faculte de meÂdecine 27, Boulevard
Jean Moulin, 13385 Marseille cedex 5 France
2
7
To whom correspondence should be addressed at: Laboratoire de Biologie de la Reproduction, HoÃpital de la Conception, 147,
Bd Baille, 13385 Marseille cedex 5, France. E-mail: [email protected]
We report ICSI pregnancies in two couples with a history of long standing primary infertility in which the sperm of
the male partner were either acephalic or had abnormal head±midpiece attachments. The two couples, in which the
men are brothers, underwent ICSI. Sperm were analysed by transmission electron microscopy and immunocytochemistry with an anti-MPM2 monoclonal antibody. The ®rst couple underwent two ICSI cycles, each consisting of the injection of two mature oocytes and the transfer of two embryos. A successful pregnancy occurred after
the second transfer and led to the birth to a healthy girl. The second couple underwent three ICSI cycles, each consisting of the injection of 18 oocytes and the transfer of two embryos; the last of these led to a triple ongoing
pregnancy which included two identical twins. Caesarean section led to the birth of three fetal-growth restricted
children. This case report demonstrates that ongoing pregnancies can be achieved in cases of abnormal development
of the head±neck attachment. The genetic origin of this syndrome is generally accepted, but the phenotypic heterogeneity observed by light and electron microscopy among published cases suggests that there are a variety of genetic
causes of this syndrome.
Key words: decapitated sperm defect/ICSI/male infertility/pregnancy
Introduction
The decapitated and decaudated sperm defect is a rare
syndrome in humans which involves the absence of the
implantation fossa and the basal plate. The morphological
aspects of this syndrome are well documented in humans,
and electron microscopy has clari®ed the ultrastructural
defects of these sperm with an abnormally high fragility
of the head±tail junction (Perotti et al. 1981; Baccetti et al.
1984, 1989; Holstein et al., 1986; Chemes et al., 1987, 1999;
Toyama et al., 1995, 2000; SaõÈas-Magnan et al., 1999).
Nevertheless, the pathogenesis of the syndrome is not clear;
at present, no explanation has been found as to why the
proximal centriole fails to connect to the nucleus. It has
been suggested that the proximal centriole/centrosome,
which induces the formation of the basal plate and the
implantation fossa, is also required to anchor the ¯agellum to
the nucleus. The decapitated sperm may therefore result from
the abnormal behaviour of the proximal centriole/centrosome,
562
which may be unable to induce the development of the
structures that anchor the ¯agellum. Spontaneous fertilization
cannot occur with these sperm, as the tail easily separates from
the head because of the neck's fragility, and ICSI is the only
way in which embryos can be obtained. To our knowledge, no
evolutive pregnancy has previously been reported (Chemes
et al., 1999; SaõÈas-Magnan et al., 1999). We report here the
birth of a healthy baby and a triple pregnancy achieved by ICSI
in two infertile couples in which the men are brothers who
produce sperm that are either acephalic or have abnormal
head±midpiece attachments.
Materials and methods
The two brothers consulted our centre for primary infertility. They
came from a family of nine children, and the father and mother are ®rst
cousins. So far, the three younger brothers of the patients have
expressed no wish to have children and refused to undergo semen
examination.
ã European Society of Human Reproduction and Embryology
Pregnancies with abnormal head±tail junction sperm
Figure 1. (A) and (B) The two spermatozoa of patient 2 from whom the three evolutive embryos originated showed that tails are misaligned
with the heads as a consequence of the connecting piece defect.
Figure 3. Immunostaining of sperm with a monoclonal anti-MPM-2 antibody (recognizing entrosomal phosphoproteins) in a normal control
(A) and in patient 1 (B and C). (B) Two ¯uorescent spots are located at the proximal end of the tail. (C) A bend is observed at the more
distal spot.
Patient 1
The ®rst patient and his wife were 25 and 24 years old respectively.
They had been unable to conceive over a period of 4 years. It was the
®rst union for both of them. Both had a normal phenotype, no history
of signi®cant illness and a normal karyotype. The wife had irregular
menses, normal hysterosalpingography and hormonal assessment.
Two semen samples were collected by masturbation after 3 days of
sexual abstinence. Semen parameters were studied according to
standard methods (World Health Organization, 1999). Direct light
microscopy analysis of the two semen samples revealed severe
oligozoospermia with 1.7 3 106 and 1.0 3 106/ml sperm respectively
(the few tail-less heads observed in fresh semen were counted as
sperm), and numerous acephalic sperm (7.9 3 106 and 16.8 3 106/ml
respectively). Of the intact sperm, 15 and 30% respectively were
motile but no sperm showed progressive motility. Morphological
examination of intact and tail-less sperm after Shorr staining on the
®rst sample showed 100% teratozoospermy with a predominance of
tail-less heads (71%). The discrepancy between the isolated head
frequencies in fresh semen and in smears can be explained by the
mechanical decapitation of the sperm during the smear preparation
(sperm were concentrated by centrifugation at 500 g if the
concentration was <2 3 106/ml) (Chemes et al., 1999). In 18% of
the intact sperm, the tail was misaligned with the head. Apparently
normal-shaped acrosomes were observed in 62% of the sperm
analysed. Sperm vitality was normal (53%).
The couple underwent two ICSI cycles at the Centre de ProcreÂation
MeÂdicalement AssisteÂe (CPMA) of the La Conception Hospital,
Marseilles. Following pituitary desensitization with leuproreline
(Enantone 3.75 mg; Takeda, Puteaux, France), the patient's wife
was stimulated using FSH (Gonal-F, Serono, France). The estradiol
plasma level and follicle growth were monitored every 2 days, and
hCG (Organon, St Denis, France) was administered after 12 days of
stimulation. Oocyte retrieval was performed 36 h after the hCG
injection. For each ICSI procedure, two mature oocytes were
retrieved, both of which were successfully injected with intact
sperm lacking any apparent head±tail misalignment and two embryos
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G.Porcu et al.
Figure 2. (A) Longitudinal section of a misaligned head±tail spermatozoon showing separation of head and tail which are only connected by
an enlarged cytoplasmic droplet surrounding all the midpiece. The mitochondrial sheath is completely disorganized. The ®brous sheath of the
principal piece is normal. Scale bar = 1 mm. (B) Longitudinal section of an acephalic spermatozoon showing no discontinuity of the plasma
membrane which covers the proximal end of the neck. Mitochondria are rare and disorganized in the cytoplasmic droplet. The ®brous sheath
is enlarged, in the proximal region of the main piece, by a granular material surrounding the axoneme. Scale bar = 1 mm. (C) Longitudinal
section of a tail-less head. The karyoplasm is normally condensed with the presence of vacuoles. Note the absence of the implantation fossa
and the basal plate and the presence of an enlarged cytoplasmic droplet hanging against the head. The cross-section of a midpiece shows a
normally constituted axoneme (arrow). Scale bar = 500 mm. (D) High magni®cation of a headless tail proximal end showing longitudinal
section of a centriolar adjunct (arrow). Scale bar = 200mm.
were then transferred. The transferred embryos were graded according
to previously described morphological criteria (SaõÈas-Magnan et al.,
1993). They were all of regular size and shape, with 0±10%
blastomeric fragmentation and the number of blastomeres ranged
from 3±4 at 48 h. An ongoing pregnancy occurred after the second
transfer, leading to the birth of a healthy girl whose birth weight at
full-term was 2480 g and whose Apgar was 10.
Patient 2
The second patient and his wife were 36 and 31 years old respectively,
and they had been unable to conceive over a period of 8 years. It
was the ®rst union for both of them. Both had a normal phenotype,
no history of signi®cant illness and a normal karyotype. The wife
had regular menses, normal hysterosalpingography and hormonal
assessment. Analysis of two semen samples collected by masturbation
after 3 days of sexual abstinence showed a sperm concentration of
4.5 3 106 intact sperm/ml and 0.5 3 106 tail-less heads/ml. The
concentrations of isolated motile tails were 34 3 106 and 4.5 3 106/ml
respectively. Altogether, 20 and 10% respectively of intact sperm
564
were motile, but none showed a progressive motility. The
teratozoospermy was 100 and 98% respectively, with 45 and 83%
of isolated heads; 29 and 6% respectively of the entire sperm showed
head±tail misalignment. Sperm vitality was low (40%).
Two ICSI cycles were carried out in the CPMA at the La
Conception Hospital. The same stimulation protocol as for the ®rst
patient's wife was used. During the ®rst ICSI cycle, four mature
oocytes were retrieved and injected and two embryos were obtained
and transferred. A biochemical pregnancy occurred. The couple
underwent a second ICSI procedure: two mature oocytes were
retrieved and two embryos were obtained after microinjection and
were transferred, but no pregnancy occurred. The couple then
underwent a third ICSI cycle in another centre (the CPMA at the
Saint Joseph Hospital). The same stimulation protocol was used; 12
oocytes were retrieved, 11 of which were mature and were therefore
injected, resulting in 10 embryos of which two were transferred. At the
time of the third ICSI cycle, all the available sperm showed head±tail
misalignment; the two sperm which were injected are shown in
Figure 1A and B. A triple ongoing pregnancy was achieved, including
Pregnancies with abnormal head±tail junction sperm
identical twins. Caesarean section for fetal-growth restriction at 30
weeks gestation led to the birth of two boys and one girl, with no
malformations; birth weights were 900, 900 and 1000 g respectively.
Transmission electron microscopy
A semen sample was collected by masturbation after 3 days of sexual
abstinence. After 30 min of liquefaction, the sample was washed with
Ferticult (Fertipro N.V.) then ®xed at 4°C with 4% glutaraldehyde in
0.1 mol/l phosphate buffer, pH 7.2 containing 1.5% sucrose for 1 h,
and post®xed at 4°C with 2% osmium tetroxide in the same buffer
(without sucrose) for 15 min. The tissue was dehydrated in a graded
acetone series and embedded in Epon. Thin sections (100 nm) were
picked up upon 300-mesh grids and stained with uranyl acetate and
lead citrate.
Immunocytochemistry
This was carried out on the sperm of the two brothers and two controls
of known fertility. Semen samples were collected by masturbation
after 3 days of sexual abstinence. Following 30 min of liquefaction,
the samples were washed with Tris buffer 10 mmol/l pH 8 and
resuspended in 1X PBS; 200 ml of the suspension was spread onto a
cytoslide (Shandon) by cytocentrifugation at 40 g for 5 min with a
cytospin 3 (Shandon). After drying, the number of sperm and the
quality of the spread was evaluated with a phase-contrast phase
microscope and adjusted if necessary. Slides were ®xed with 2%
paraformaldehyde for immunocytology, according to the technique
described by Colombero et al. (1999). Immunostaining of the
centrosome with a murine anti-MPM-2 monoclonal antibody
(Mitotic Protein Monoclonal, Dako Corporation) was carried out for
the two patients and two normal controls; negative controls without
MPM-2 were carried out for both controls and patients. MPM-2 is an
antibody which recognizes phosphoproteins found at the centrosome.
Results
Connecting piece misalignment
Initial microscopic investigation on fresh semen and analysis
of smears after Schorr staining revealed abnormal head±
midpiece attachments; the heads were attached either to the tip
of the midpiece and were in misalignment with the sperm axis,
resulting in so called `bent neck' and `asymmetrical insertion'
forms (World Health Organization, 1999), or were attached to
the side of the midpiece. On the smears, some sperm showed a
gap between head and tail, with a cytoplasmic droplet
maintaining the connection between both structures.
Numerous headless tails were observed on the smears, 360
and 414% for patients 1 and 2 respectively. A cytoplasmic
droplet, located in the midpiece, was observed in 36 and 42%
of the sperm in patients 1 and 2 respectively. This defect was
either isolated or associated with one or more of the previous
anomalies.
Transmission electron microscopy
Ultrastructural investigations revealed that both brothers had a
mixture of sperm with abnormally aligned head±midpieces,
loose heads and acephalic sperm (Figure 2A±D). No normal
forms were observed. Decapitation had occurred between the
nucleus and the centriolar region, so that the detached tails
contained the whole midpiece (Figure 2B and D). In the same
way, head±tail misalignment always occurred in this region; in
one spermatozoon, the head position was at a 180° angle to the
tail (Figure 2A). In all of the intact sperm, the proximal end of
the tail was separated from the head, and both were connected
by a cytoplasmic mass. The majority of the heads analysed had
abnormal shaped nuclei with granulous chromatin and abnormal acrosomes (Figure 2A) and longitudinal sections revealed
that the post nuclear region was de®cient, lacking the
implantation fossa and the basal plate (Figure 2C).
Longitudinal sections of the headless tails showed no discontinuity of the cell membrane covering the proximal end of the
neck and the normally organized connecting piece (Figure 2B
and D). The presence of an enlarged cytoplasmic droplet
around the neck and midpiece was observed on all the intact
and acephalic sperm analysed (Figure 2A, B and D); a
cytoplasmic droplet was also observed at the basal part of the
tail-less heads (Figure 2C). The mitochondria in the mitochondrial sheath were disorganized (Figure 2A, B and D).
Transverse sections of the tails showed a normal axoneme
and outer dense ®bres (Figure 2C). A centriolar adjunct was
present in some headless tails (Figure 2D). This structure
normally emerges from the distal end of the proximal centriole
in differentiating spermatids (Holstein, 1981) but disappears in
the sperm.
Immunocytochemistry
With the anti-MPM-2 antibody, maximal signal intensity was
con®ned to the proximal region of the ¯agellum, in all of the
control sperm (Figure 3A). In the two patients with the
decapitated and decaudated sperm defect, all of the intact
sperm and all of the headless tails presented the same
homogeneous labelling pattern with the anti-MPM-2 antibody:
two ¯uorescent spots were located on the tail, the ®rst at the
proximal end of the tail and the second at the distal end of the
midpiece (Figure 3B), a bend was often observed in this region
(Figure 3C).
Discussion
It is now well established that the sperm centrosome plays an
important role in the process of fertilization and cell division in
the fertilized oocyte, since it organizes the sperm aster (Tucker
et al., 1996; Van Blerkom, 1996). Simerly et al. (1995) suggest
that some human infertilities may result from defects in the
centrosome at fertilization. The sperm centrosome is composed
by the proximal sperm centriole and the surrounding
pericentriolar material; a defect of centrosomal function
could be attributed to either or both of these components
(Sathananthan et al., 1996). Proximal centriole/centrosomal
dysfunction may compromise embryo development by inducing irregular cleavage, or chromosomal aberration, leading to
retardation or arrest of embryo development (Rawe et al.,
2002).
Until now, no pregnancy has been reported in a couple in
which the man presents a primary infertlity resulting from a
defect in the connecting piece, this syndrome being in such
cases; the abnormally high fragile head±tail junction facilitates
separation of the head and tail, and spontaneous fertilization is
thus rendered impossible. Assisted fertilization is the only way
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G.Porcu et al.
to obtain embryos. To our knowledge, ICSI has been reported
in two such couples. In the ®rst study (Chemes et al., 1999),
four mature oocytes were injected in one attempt, but cleavage
and syngamy did not occur. The second couple (SaõÈas-Magnan
et al., 1999) underwent three subzonal inseminations (SUZI)
and four ICSI cycles (three of which were performed in our
reproduction centre); a total of 20 embryos was transferred,
three of them after freezing and thawing. Despite good embryo
morphology, implantation was unsuccessful and no pregnancy
occurred. The present study demonstrates that an evolutive
pregnancy can be achieved with sperm presenting a fragility of
the head±tail junction.
Several hypotheses may be proposed in order to explain the
discrepancies between the ICSI results in previous studies and
our present study. The failure to achieve pregnancy may have
been due either to an additional and unknown sperm or oocyte
defect (SaõÈas-Magnan et al., 1999) or to chance; indeed, a
reasonable possibility of conception remains after seven
cycles. Alternatively, it may be that there are distinct genetic
origins of this syndrome. Indeed, the genetic origin of this
syndrome is now accepted. Baccetti et al. (2001) suggested that
most genetic sperm defects seem to be due to recessive
autosomal mutations; in the studies carried out by Baccetti et al.
(1989) and Chemes et al. (1999), and in the present study, the
defect is observed in two sterile brothers. Nevertheless, neither
the genes nor the mutations involved in this defect have been
identi®ed and several kinds of decapitated sperm defect have
been described. This syndrome can manifest a variety of
clinical aspects: in most studies, direct light microscopic
analysis shows numerous isolated motile tails, and either fewer
isolated sperm heads or no recognizable sperm head (Le
Lannou, 1979; Perotti et al., 1981; Bacetti et al., 1984, 1989;
Holstein et al., 1986; Chemes et al., 1987, 1999; Toyama et al.,
1995, 2000; SaõÈas-Magnan et al., 1999). A variant of this
syndrome has been described by Kamal et al. (1999); the
authors reported 16 cases of easily decapitated sperm defect in
which semen analysis was normal, but minimal micromanipulation for the purposes of ICSI resulted in decapitation of the
sperm during immobilization.
Variability in the location of the break was observed by
electron microscopy analysis of the tail anomalies. Head±tail
separation usually occurs at the head±neck interface, thus
preserving the integrity of the connecting piece; the absence of
basal plate and implantation fossa have been observed at the
caudal pole of the nucleus (Perotti et al., 1981; Baccetti, 1984;
Chemes et al., 1987, 1999; Toyama et al., 1995; SaõÈas-Magnan
et al., 1999). Other kinds of decapitation have also been
described. In the study carried out by Holstein et al. (1986), the
basal plate and the implantation fossa were normal, and the
decapitated sperm syndrome results from the dissociation of
the proximal and the distal centrioles. Baccetti et al. (1989)
have described a patient in which the break either occurred
between the nucleus and the centriolar region, between the
anterior and the posterior part of the midpiece or between the
midpiece and the principal piece. The same heterogeneity was
found in the easily decapitated sperm defect described by
Kamal et al. (1999); only one patient had absence of the basal
plate in all sperm, and the three others analysed by electron
566
microscopy showed heterogeneous defects such as degenerative basal plate and centriole, separation between the proximal
and distal centriole, absence of the proximal centriole,
separation of the nucleus and the basal plate and the presence
of a cytoplasmic droplet in some sperm.
Thus, it appears that several types of decapitated and
decaudated sperm defect may exist, but the most important
problem posed by this syndrome is that of embryo development
when the sperm are used for ICSI. Indeed, normal fertilization
rates have been obtained in couples undergoing ICSI (Chemes
et al., 1999; SaõÈas-Magnan et al., 1999), but no evolutive
pregnancy has been reported until the present study. According
to the study of Palermo et al. (1997) during which oocytes were
injected with separated heads and tails, the main risk for the
embryos is chromosome mosaicism due to the abnormal
distribution of the chromosomes between the blastomeres. A
signi®cant imbalance leads to precocious degeneration of the
embryos before implantation. In the case of evolutive pregnancies, the embryos are either chromosomally normal or are
affected by a chromosomal number abnormality that is
consistent with embryo development. In this study, prenatal
diagnosis was not performed for the ®rst couple, whose main
concern was that the pregnancy should proceed. In the case of
the second couple, the triple pregnancy made prenatal diagnosis impossible. In both cases, several ultrasound examinations
were carried out with the aim of detecting malformations of the
embryo resulting from chromosomal abnormalities, in particular Down's syndrome features.
The two cases presented here suggest that certain types of
defect in the connecting piece are compatible with the
formation of a normal sperm aster and normal embryo
development. Indeed, Figure 1 clearly shows the physical
disruption between the ¯agellum and the nucleus in the two
sperm from which the triple pregnancy originated. In both
brothers, the sperm defect is caused by a failure in the caudal
migration of the centrioles, but the behaviour of the proximal
centriole during fertilization was normal. A difference was
observed between the labelling patterns obtained with the
murine anti-MPM-2 monoclonal antibody in the patients and
the controls. Nevertheless, immunohistochemical staining of
intact sperm and decapitated tails from both patients was the
same as that obtained by Colombero et al., (1999). The
labelling pattern of the two patients' sperm may therefore be
considered as normal, and does not provide any clues as to the
mechanisms involved in the centriolar function failure in these
patients. Genetic counselling presents a problem as the genes
mutated in the decapitated sperm defect are presently
unknown, and little is understood about the nature of the
primary defect of this syndrome, but it is essential that several
ultrasound examinations be carried out during the pregnancy.
Acknowledgements
The authors would like to thank D.Rousseau, J.Pellegrin and
J.Paterniti for their technical assistance. This study was supported
by grants from the Assistance Publique of Marseille and the
Association pour la Recherche pour le Cancer.
Pregnancies with abnormal head±tail junction sperm
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