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 Procréation Médicalement Assistée, AP-HM, Hôpital de la Conception, 147 Boulevard Baille, 13385 Marseille cedex 05, 2 Laboratoire de Procréation Médicalement Assistée, Hôpital Saint-Joseph, 26 Bd de Louvain, 13285 Marseille cedex 08, 3 Laboratoire d’Histologie et de Biologie de la Reproduction, Faculté de Médecine Timone, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, 4 Laboratoire de Biologie de la Reproduction, Hôpital de la Conception, 147 Boulevard Baille, 13385 Marseille cedex 05, 5 Service de Gynécologie Obstétrique, AP-HM, Hôpital Nord, Chemin des Bourrellys, 13915 Marseille cedex 20 and 6 Laboratoire de Biogénotoxicologie et Mutagenèse Environnementale (EA1784), IFR PMSE112, Faculté de médecine 27, Boulevard Jean Moulin, 13385 Marseille cedex 5 France

7 To whom correspondence should be addressed at: Laboratoire de Biologie de la Reproduction, Hôpital de la Conception, 147, Bd Baille, 13385 Marseille cedex 5, France. e-mail: mguichaoua{at}ap-hm.fr


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 first 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 clarified the ultrastructural defects of these sperm with an abnormally high fragility of the head–tail junction (Perotti et al. 1981Go; Baccetti et al. 1984Go, 1989; Holstein et al., 1986Go; Chemes et al., 1987Go, 1999; Toyama et al., 1995Go, 2000; Saïas-Magnan et al., 1999Go). 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 flagellum to the nucleus. The decapitated sperm may therefore result from the abnormal behaviour of the proximal centriole/centrosome, which may be unable to induce the development of the structures that anchor the flagellum. 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., 1999Go; Saïas-Magnan et al., 1999Go). 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The two brothers consulted our centre for primary infertility. They came from a family of nine children, and the father and mother are first cousins. So far, the three younger brothers of the patients have expressed no wish to have children and refused to undergo semen examination.

Patient 1
The first 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 first union for both of them. Both had a normal phenotype, no history of significant 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, 1999Go). Direct light microscopy analysis of the two semen samples revealed severe oligozoospermia with 1.7 x 106 and 1.0 x 106/ml sperm respectively (the few tail-less heads observed in fresh semen were counted as sperm), and numerous acephalic sperm (7.9 x 106 and 16.8 x 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 first 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 x 106/ml) (Chemes et al., 1999Go). 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 Procréation Médicalement Assistée (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 were then transferred. The transferred embryos were graded according to previously described morphological criteria (Saïas-Magnan et al., 1993Go). 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 first union for both of them. Both had a normal phenotype, no history of significant 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 x 106 intact sperm/ml and 0.5 x 106 tail-less heads/ml. The concentrations of isolated motile tails were 34 x 106 and 4.5 x 106/ml respectively. Altogether, 20 and 10% respectively of intact sperm 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 first patient’s wife was used. During the first 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 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.



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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.

 
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 fixed at 4°C with 4% glutaraldehyde in 0.1 mol/l phosphate buffer, pH 7.2 containing 1.5% sucrose for 1 h, and postfixed 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 µl 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 fixed with 2% paraformaldehyde for immunocytology, according to the technique described by Colombero et al. (1999Go). 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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, 1999Go), 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 deficient, 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 fibres (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, 1981Go) but disappears in the sperm.



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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 fibrous sheath of the principal piece is normal. Scale bar = 1 µm. (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 fibrous sheath is enlarged, in the proximal region of the main piece, by a granular material surrounding the axoneme. Scale bar = 1 µm. (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 µm. (D) High magnification of a headless tail proximal end showing longitudinal section of a centriolar adjunct (arrow). Scale bar = 200µm.

 
Immunocytochemistry
With the anti-MPM-2 antibody, maximal signal intensity was confined to the proximal region of the flagellum, 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 fluorescent spots were located on the tail, the first 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).



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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 fluorescent spots are located at the proximal end of the tail. (C) A bend is observed at the more distal spot.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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., 1996Go; Van Blerkom, 1996Go). Simerly et al. (1995Go) 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., 1996Go). 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., 2002Go).

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 to obtain embryos. To our knowledge, ICSI has been reported in two such couples. In the first study (Chemes et al., 1999Go), four mature oocytes were injected in one attempt, but cleavage and syngamy did not occur. The second couple (Saïas-Magnan et al., 1999Go) 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 (Saïas-Magnan et al., 1999Go) 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. (2001Go) suggested that most genetic sperm defects seem to be due to recessive autosomal mutations; in the studies carried out by Baccetti et al. (1989Go) and Chemes et al. (1999Go), 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 identified 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, 1979Go; Perotti et al., 1981Go; Bacetti et al., 1984Go, 1989; Holstein et al., 1986Go; Chemes et al., 1987Go, 1999; Toyama et al., 1995Go, 2000; Saïas-Magnan et al., 1999Go). A variant of this syndrome has been described by Kamal et al. (1999Go); 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., 1981Go; Baccetti, 1984Go; Chemes et al., 1987Go, 1999; Toyama et al., 1995Go; Saïas-Magnan et al., 1999Go). Other kinds of decapitation have also been described. In the study carried out by Holstein et al. (1986Go), 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. (1989Go) 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. (1999Go); only one patient had absence of the basal plate in all sperm, and the three others analysed by electron 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., 1999Go; Saïas-Magnan et al., 1999Go), but no evolutive pregnancy has been reported until the present study. According to the study of Palermo et al. (1997Go) 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 significant 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 first 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 flagellum 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., (1999Go). 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.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 8, 2002; resubmitted on October 19, 2002; accepted on November 20, 2002.





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