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 dHistologie 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: decapitated sperm defect/ICSI/male infertility/pregnancy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 1999). 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., 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 Procréation Médicalement Assistée (CPMA) of the La Conception Hospital, Marseilles. Following pituitary desensitization with leuproreline (Enantone 3.75 mg; Takeda, Puteaux, France), the patients 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 headtail misalignment and two embryos were then transferred. The transferred embryos were graded according to previously described morphological criteria (Saïas-Magnan et al., 1993). They were all of regular size and shape, with 010% blastomeric fragmentation and the number of blastomeres ranged from 34 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 headtail 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 patients 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 headtail 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.
|
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. (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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transmission electron microscopy
Ultrastructural investigations revealed that both brothers had a mixture of sperm with abnormally aligned headmidpieces, loose heads and acephalic sperm (Figure 2AD). 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, headtail 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, 1981) but disappears in the sperm.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 headtail 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., 1999), four mature oocytes were injected in one attempt, but cleavage and syngamy did not occur. The second couple (Saïas-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 headtail 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., 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 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, 1979
; Perotti et al., 1981
; Bacetti et al., 1984
, 1989; Holstein et al., 1986
; Chemes et al., 1987
, 1999; Toyama et al., 1995
, 2000; Saïas-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. Headtail separation usually occurs at the headneck 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
; Saïas-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 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; Saïas-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 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 Downs 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., (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 |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baccetti, B., Burrini, A.G., Collodel, G., Magnano, A.R., Piomboni, P., Renieri, T. and Sensini, C. (1989) Morphogenesis of the decapitated and decaudated sperm defect in two brothers. Gamete research, 23, 181188.[ISI][Medline]
Baccetti, B, Capitani, S., Collodel, G., Di Cairano, G., Gambera, L., Moretti, E. and Piomboni, P. (2001) Genetic sperm defects and consanguinity. Hum. Reprod., 16, 13651371.
Chemes, H.E., Carizza, C., Scarinci, F., Brugo, S., Neuspiller, N. and Schwarsztein, L. (1987) Lack of a head in human spermatozoa from sterile patients: a syndrome associated with impaired fertilization. Fertil. Steril., 47, 310316.[ISI][Medline]
Chemes, H.E., Puigdomenech, E.T., Carizza, C., Olmedo Brugo, S., Zanchetti, F. and Hermes, R. (1999) Acephalic spermatozoa and abnormal development of the head-neck attachment: a human syndrome of genetic origin. Hum. Reprod., 14, 18111818.
Colombero, L.T., Takeuchi, T., Sills, E.S., Breed, W.G., Rosenwaks, Z. and Palermo, G.D. (1999) A comparison of human spermatozoa immunolabeling features using xenogenic reagents for centrosomal proteins. Clin. Exp. Obst. Gyn., 26, 141146.
Holstein, A.F. and Roosen-Runge, E.C. (1981) Atlas of Human Spermatogenesis. Grosse Verlag Berlin (eds), pp. 133143.
Holstein, A.F., Schill, W.B. and Breucker, H. (1986) Dissociated centriole development as a cause of spermatid malformation in man. J. Reprod. Fertil., 78, 719725.[Abstract]
Kamal, A., Mansour, R., Fahmy, I., Serour, G., Rhodes, C. and Aboulghar, M. (1999) Easily decapitated spermatozoa defect: a possible cause of unexplained infertility. Hum. Reprod., 14, 27912795.
Le Lannou, D. (1979) Teratospermie consistant en labsence de tête spermatique par défaut de connexion tête col chez lhomme. J. Gynecol. Obstet. Biol. Reprod. Paris, 8, 4345.[Medline]
Palermo, G.D., Colombero, L.T. and Rosenwaks, Z. (1997) The human sperm centrosome is responsible for normal syngamy and early embryonic development. Rev. Reprod., 2, 1927.
Perotti, M.E., Giarola, A. and Giora, M. (1981) Ultrastructural study of the decapitated sperm defect in an infertile man. J. Reprod. Fert., 63, 543549.[Abstract]
Rawe, V.Y., Terada, Y., Nakamura, S., Chillik, C.F., Brugo Olmedo, S. and Chemes, H.E. (2002) A pathology of the sperm centriole responsible for defective sperm aster formation, syngamy and cleavage. Hum. Reprod., 17, 23442349.
Saïas-Magnan, J., Zarka, V., Dumont, M.C., Carles, F., Melone, C., Guichaoua, M.R., Erny R. and Luciani J.M. (1993) Qualité des embryons dans les stérilités inexpliquées. Contracept. Fertil. Sex., 21, 501504.[ISI][Medline]
Saïas-Magnan, J., Metzler-Guillemain, C., Mercier, G., Carles-Marcorelles, F., Grillo J.M. and Guichaoua, M.R. (1999) Failure of pregnancy after intracytoplasmic sperm injection with decapitated spermatozoa. Hum. Reprod., 14, 19891992.
Sathananthan, A.H., Ratnam, S.S., Ng, S.C., Tarin, J.J., Gianaroli, L. and Trounson, A. (1996) The sperm centriole: its inheritance, replication and perpetuation in early human embryos. Hum. Reprod., 11, 345356.[Abstract]
Simerly, C., Wu, G.J., Zoran, S., Ord, T., Rawlins, R., Jones, J., Navara, C., Gerrity, M., Rinehart, J., Binor, Z. et al. (1995) The paternal inheritance of the centrosome, the cells microtubule-organizing center in humans and the implication for fertility. Nat. Med., 1, 4752.[ISI][Medline]
Toyama, Y., Kasama, T., Fuse, K.H. and Katayama, T. (1995) A case of decapitated spermatozoa in an infertile man. Andrologia, 27, 165170.[ISI][Medline]
Toyama, Y., Iwamoto, T., Yajima, M., Baba, K. and Yuasa, S. (2000) Decapitated and decaudated spermatozoa in man, and pathogenesis based on the ultrastructure. Int. J. Androl., 23, 10915.[ISI][Medline]
Tucker, M.J., Morton, P.C., Wright, G., Sweitzer, C.L., Ingargiola, P.E. and Chan, S.Y. (1996) Paternal influence on embryogenesis and pregnancy in assisted human reproduction. JBFS1(2). Hum. Reprod., 11 (Natl. Suppl.), 9095.
Van Blerkom, J. (1996) Sperm centrosome dysfunction: a possible new class of male factor infertility in the human. Mol. Hum. Reprod., 2, 349354.[Abstract]
World Health Organization (1999) World Health Organization Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction. Cambridge University Press, Cambridge.
Submitted on July 8, 2002; resubmitted on October 19, 2002; accepted on November 20, 2002.