Failure of pregnancy after intracytoplasmic sperm injection with decapitated spermatozoa: Case report

J. Saïas-Magnan1,5, C. Metzler-Guillemain1,2, G. Mercier3, F. Carles-Marcorelles4, J.M. Grillo1,3 and M.R. Guichaoua1,2

1 Laboratoire de Biologie de la Reproduction, Pr Luciani, Hôpital de la Conception, 147 Boulevard Baille, 13385 Marseille, Cedex 05, 2 Laboratoire de Biologie du développement et de la Reproduction, Pr Luciani. Faculté de Médecine Nord, Boulevard P. Dramard, 13326 Marseille Cedex 15, 3 Laboratoire d'Histologie, Pr Grillo, Faculté de Médecine Timone, 27, Boulevard Jean Moulin, 13385 Marseille, Cedex 05 and 4 Centre de Procréations Médicalement Assistées, Pr Gamerre, Hôpital de la Conception, 147 Boulevard Baille, 13385 Marseille, Cedex 05, France


    Abstract
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
The case of a couple with a history of long standing primary infertility is reported in which the man presented with a decapitated sperm defect. The woman had a normal history and presented with normal clinical characteristics. The couple underwent one unsuccessful conventional in-vitro fertilization (IVF). Subsequently, embryos were obtained and transferred after assisted fertilization attempts: in all, three subzonal inseminations and four intracytoplasmic sperm injections. A total of 49 mature oocytes was injected in both studies, 25 embryos obtained and 20 embryos transferred, three of them after freezing and thawing. Despite the good embryo morphology, implantation was unsuccessful and no pregnancy occurred. The failure of implantation may have resulted from an arrest in early embryonic development related to the sperm anomaly. One hypothesis is that transferred embryos may carry a chromosomal imbalance that prevents them from progressing to the blastocyst stage. Nevertheless, we cannot exclude the possibility that the woman is responsible for the implantation failure. Co-culture associated with a further attempt could provide information regarding the ability of embryos to progress to the blastocyst stage and implant.

Key words: decapitated spermatozoa/ICSI


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
The presence of a structural abnormality which affects the whole sperm population, or a high proportion of it, is relatively uncommon in man. Among these abnormalities, only 10 cases of decapitated spermatozoa have already been described (Luders, 1976Go; Perotti et al., 1981Go; Holstein et al., 1986Go; Chemes et al., 1987Go; Bacceti et al., 1989; Toyama et al., 1995Go). The pathogenesis of this syndrome is not clear, but the most obvious ultrastructural anomaly is the absence of the implantation fossa and of the basal plate. This type of pathology causes the sperm neck to weaken and break, which in turn prevents the spermatozoon from progressing along the female genital tract or fertilizing eggs.

Here we report the case of an infertile couple in which the male presented with decapitated spermatozoa. This couple underwent one conventional in-vitro fertilization (IVF), three subzonal inseminations (SUZI) and four intracytoplasmic sperm injection (ICSI) attempts. Each ICSI attempt resulted in the transfer of embryos with good morphological characteristics, but no pregnancy occurred. The significance of these results is discussed here.


    Case report
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
At the time of consultation in our centre, the patient and his wife were 33 and 31 years old respectively; they had been unable to conceive over a period of 5 years. The woman had a normal clinical history and presented normal characteristics on examination, with regular menses, normal hysterosalpingography and hormonal assessment. The karyotypes of both individuals were normal. The man presented a normal male phenotype and no history of significant illness. Physical examination revealed no particular abnormality. Analysis of two semen samples collected by masturbation after 3 days of sexual abstinence showed sperm concentrations of 12.6 and 29.8x106 spermatozoa/ml. Direct light microscopic analysis showed numerous isolated motile tails and fewer isolated heads. In the two samples respectively, among intact spermatozoa, 60 and 80% were motile, with 5 and 20% showing rapid progressive motility. Most of the intact spermatozoa had a bent tail. Morphological examination of spermatozoa after Shorr staining confirmed a high rate of teratozoospermy (84 and 99%, according to the 1992 WHO criteria) with a predominance of isolated heads (46 and 84%) and bent tails (32 and 20%). Seemingly normal-shaped acrosomes were seen in the great majority of spermatozoa analysed (81 and 87%). Aniline blue staining revealed a normally condensed chromatin.

Ultrastructural investigations revealed the presence of numerous headless tails, which in all cases carried the proximal centriole and the segmented columns (Figure 1aGo); the break always occurred between the capitulum and the posterior nuclear pole. The rare intact spermatozoa all had a bent tail (Figure 1bGo). Longitudinal sections through the heads showed a normal shaped nucleus, with the exception of the caudal pole, which revealed an absence of the implantation fossa and basal plate. Longitudinal and transverse sections of the tails appeared to be normal.



View larger version (121K):
[in this window]
[in a new window]
 
Figure 1. (a) Longitudinal section of a headless tail in which the capitulum (large arrow), the proximal centriole sectioned longitudinally (small arrow) and the segmented columns (arrowhead) are clearly seen. (b) Longitudinal section of a rare intact spermatozoon with a bent tail showing segmented columns (small arrow) and middle piece. The basal plate and the implantation fossa are absent from the caudal pole of the nucleus (large arrow). The presence of a cross-section of a principal piece (arrowhead), near the neck region, surrounded by the same plasmalemma as the bent tail is difficult to interpret. It could be either multiple tails or a coiled tail. Scale bar = 1 µm (a and b).

 
The couple had previously undergone five IVF attempts in an other centre, including one classic IVF attempt where 10 mature oocytes were retrieved but no embryo was obtained. The other IVF attempts comprised three SUZI and one ICSI. In data pooled from these four attempts, 26 oocytes were retrieved, 24 of which were mature and therefore injected. Seven embryos were in this way obtained and transferred, but no pregnancy was achieved. The quality of the transferred embryos is not known.

Three ICSI operations were performed in our reproduction centre (Table IGo). After pituitary desensitization with leuproreline (Enantone 3.75 mg; Takeda, Puteaux, France), the patient's wife was stimulated using follicle stimulating hormone (FSH) (Metrodine HP, 48 ampoules; Serono, France). Oestradiol plasma levels and follicle growth were monitored every 2 days and human chorionic gonadotrophin (HCG) (Organon, St Denis, France) was administered after 12 days of stimulation. Oocyte retrieval was performed 36 h after HCG injection. In all, 27 oocytes were retrieved (Table IGo). Spermatozoa were separated from the seminal plasma after a mini swim-up procedure, which consisted of two washes of the spermatozoa in Eppendorf tubes with 1 ml of M1 medium (Bicef, l'Aigle, France), followed by a 15 min semi-horizontal migration in 20 µl of B2 medium (Bio Merieux, Montalieu, France). The suspension contained a few complete spermatozoa with bent tails, but numerous isolated tails with progressive motility. The excessive weakness of the sperm neck frequently caused the spermatozoon to break in the microinjection pipette. With gentle motions, however, we successfully injected an entire spermatozoon into each of 20 oocytes. Five oocytes were injected with a spermatozoon whose head and tail separated in the microinjection pipette. In the three attempts conducted in our centre, 18 embryos were obtained, 10 were transferred and three were frozen at the seven- to eight-cell stage, after 72 h. These three embryos were thawed and transferred in one induction cycle later, using clomifene (Clomid 500 mg; Marion Merrel, Puteaux, France) and FSH (Metrodine, three ampoules; Serono). At transfer, the embryos were graded according to morphological criteria previously described (Saïas-Magnan et al., 1993Go). All embryos transferred were of a regular size and shape, with 0–10% blastomeric fragmentation, the number of blastomeres ranging from four to eight. After each embryo transfer, the HCG plasma concentration was always negative.


View this table:
[in this window]
[in a new window]
 
Table I. Details of the three ICSI attempts accomplished in our IVF centre
 

    Discussion
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
The present case is the first report of ICSI using spermatozoa from a man presenting with the decapitated spermatozoa syndrome. This pathology is responsible for male infertility, and embryos cannot be obtained without assisted fertilization. In the three ICSI attempts performed in our laboratory, only head and tail from the same spermatozoon were injected, thus giving a cleavage rate similar to that of our other ICSI cases (75%). No pregnancy occurred, despite the transfer of good quality embryos. The reasons for these failures are unclear, but are probably related to the sperm anomaly. Indeed, this pathology partially confirms a previous experiment (Palermo et al., 1997Go), in which the authors assessed the ability of oocytes injected with a physically separated sperm segment (head only, head and tail separated, or tail only) to undergo normal embryonic development. In one study (Palermo et al., 1997Go), oocytes were monitored for cleavage for up to 72 h, and embryos were analysed by fluorescent in-situ hybridization (FISH). All of the embryos obtained after injection of a separated head and tail showed chromosome mosaicism.

One explanation for the absence of pregnancy in the present couple is that the embryos transferred may carry a chromosomal imbalance and are therefore incapable of progressing to the blastocyst stage. The mechanism that causes this chromosomal mosaicism among the blastomeres is unclear. The most likely explanation is that the spindle becomes defective as a result of the abnormal behaviour of the centrosome, and cannot therefore ensure normal segregation of the chromosomes at each mitotic division of the embryo. Indeed, this study (Palermo et al., 1994Go) provides evidence that the human sperm centrosome controls the first mitotic divisions after fertilization. The interaction of the centrosome and nucleus appears to be important during spermatogenesis and fertilization, where the centriole often remains in close contact with the decondensing sperm nucleus. This structural association has obvious functional implications for cell polarity, as well as for cell divisions (Omura and Fukui, 1985Go). The fact that fertilization of oocytes with physically separated sperm segments leads to mosaic embryos (Palermo et al., 1997Go) suggests that physical disruption of the sperm neck in decapitated spermatozoa compromises the ability of the centrosome to function normally in the zygote.

The origin of this syndrome is very probably genetic, since a case of two brothers bearing the same pathology has been reported (Baccetti et al., 1989Go). The same defect has also been reported in cattle (Blom and Birch-Andersen, 1970Go) and pigs (Toyema and Itoh, 1996). The mechanism which gives rise to this anomaly is discussed. It has been suggested (Holstein et al., 1988Go) that the proximal centriole, which induces the development of the basal plate and the implantation fossa, is responsible for its own anchorage to the nucleus. An overproduction of vesicles arising from the Golgi complex was described (Bacetti et al., 1984) in a human with decapitated spermatozoa, and it was proposed that this had interfered with the attachment of the centriole to the nucleus. This overproduction of vesicles was not observed in the present case, and thus the sperm defect observed may be due to the dysfunction of the sperm centrosome (Van Blerkom and Henry, 1991Go). Our observation support previous conclusions (Perotti and Gioria, 1981Go; Perotti et al., 1981Go; Chemes et al., 1987Go) that the proximal centriole may be unable to induce development of the basal plate and the implantation fossa, either because it is prevented from moving to the caudal pole of the nucleus, or because of a functional anomaly.

Thus, the arrest of embryonic development could be related either to the presence of a functionally abnormal centriole, or to the loss of interaction between the nucleus and the proximal centriole during spermatogenesis and fertilization. In the first hypothesis, infertility may result from a defect in centrosome reconstitution following fertilization: the centrosome may be able to bind {gamma}-tubulin and other centrosome-associated proteins for some, but not all, division cycles (Simerly et al., 1995Go). In the second hypothesis, the developmental arrest may result from the disturbance of the sperm aster function, which is essential for the physical union of the male and female pronuclei. Abnormal distribution of chromosomes between blastomeres could result from this abnormal spindle and would explain the precocious degeneration of the embryos before implantation.

The most likely explanation for the absence of pregnancy in this couple is that the embryos' chromosomal imbalance prevents their progression to the blastocyst stage. A high incidence of mosaicism in humans has been reported (Palermo et al., 1998Go) in other conditions. FISH analysis of the embryos derived from the reactivation of unfertilized oocytes by injection of sperm cytosolic factor showed an abnormal chromosomal complement of the blastomeres. Nevertheless, it is impossible to exclude the possibility that the wife is responsible for the implantation failure. Indeed, a case has been reported where pregnancy and delivery of a healthy baby resulted after two ICSI cycles using only sperm heads (Tucker et al., 1996Go), but these contained the centrosome. Despite seven unsuccessful assisted fertilization attempts, the patient and his wife remain highly motivated, and an ICSI with co-culture is underway. This will enable us to evaluate the ability of the embryos to progress to the blastocyst stage. Further reports of similar ICSI cases using decapitated spermatozoa will be required, in order to elucidate the relationship between the sperm defect and the reproductive failure of these patients. In the large majority of cases, severe sperm defects have no predictive value with regard to the success of ICSI and do not impair the fertilization process (Küpker et al., 1998Go). Nevertheless, the present defect is perhaps an example of one of the rare limits to the ICSI process.

Since this pathology might have a genetic origin, patients must be informed of the transmission risk, but genetic counselling is difficult because the mutations involved are unknown. In order to allow further characterization of the molecular basis of this centriolar pathology, genomic DNA has been isolated from this patient.


    Notes
 
5 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
Baccetti, B., Selmi, M.G. and Soldani, P. (1984) Morphogenesis of `decapitated' spermatozoa in a man. J. Reprod. Fertil., 70, 395–397.[Abstract]

Baccetti, B., Burrini, A.G., Collodel, G. et al. (1989) Morphogenesis of decapitated and decaudated sperm defect in two brothers. Gamete Res., 23, 181–188.[ISI][Medline]

Blom, and Birch-Andersen, (1970) Ultrastructure of the `decapitated sperm defect' in Guernsey bulls. J. Reprod. Fertil., 23, 67–72.[Medline]

Chemes, H.E., Carizza, C., Scarinci, F. et al. (1987) Lack of head in human spermatozoa from sterile patients: a syndrome associated with impaired fertilisation. Fertil. Steril., 47, 310–316.[ISI][Medline]

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, 719–725.[Abstract]

Holstein, A.F, Roosen-Runge, E.C. and Schirren, C. (1988) Illustrated Pathology of Human Spermatogenesis. Grosse, Berlin.

Küpker, W., Schulze, W. and Diedrich, K. (1998) Ultrastructure of gametes and intracytoplasmic sperm injection: the significance of sperm morphology. Hum. Reprod., 13, 99–106.[Abstract]

Luders, G. (1976) A defect of the head-flagellum-connection of human spermatozoa. Andrologia, 8, 365–368.[ISI][Medline]

Omura, F and Fukui, Y. (1985) Dictyostelium MTOC: structure and linkage to the nucleus. Protoplasma, 127, 212–221.[ISI]

Palermo, G.D., Munné, S. and Cohen, J. (1994) The human zygote inherits its mitotic potential from the male gamete. Hum. Reprod., 9, 1220–1225.[Abstract]

Palermo, G.D., Colombero, L.T. and Rozenwaks, Z. (1997) The human sperm centrosome is responsible for normal syngamy and early embryonic development. Rev. Reprod., 2, 19–27.[Abstract/Free Full Text]

Palermo, G.D., Avrech, O.M., Colombero, L.T. et al. (1998) Human sperm cytosolic factor triggers Ca+ oscillations and overcomes activation failure of mammalian oocytes. Mol. Hum. Reprod., 3, 367–374.[Abstract]

Perotti, M.E. and Gioria, M. (1981) Fine structure and morphogenesis of headless human spermatozoa associated with infertility. Cell. Biol. Int. Rep., 5, 113.[ISI][Medline]

Perotti, M.E., Giarola, A. and Giora, M. (1981) Ultrastructural study of the decapitated sperm defect in an infertile man. J. Reprod. Fertil., 63, 543–549.[Abstract]

Saïas-Magnan, J., Zarka, V., Dumont, M.C. et al. (1993) Qualité des embryons dans les stérilités inexpliquées. Contracept. Fertil. Sex., 21, 501–504.[ISI][Medline]

Simerly, C., Wu, G.J., Zoran, S. et al. (1995) The paternal inheritance of the centrosome, the cell's microtubule-organizing center, in humans, and the implications for infertility. Nature Med., 1, 47–52.[ISI][Medline]

Toyama, Y. and Itoh, Y. (1996) Ultrastructural features and pathogenesis of decapitated spermatozoa in a boar. Andrologia, 28, 109–115.[ISI][Medline]

Toyama, Y., Kazama, T., Fuse, K.H. and Katayama, T. (1995) A case of decapitated spermatozoa in an infertile man. Andrologia, 27, 165–170.[ISI][Medline]

Tucker, M.J., Morton, P.C., Wright, G. et al. (1996) Paternal influence on embryogenesis and pregnancy in assisted human reproduction. Hum. Reprod., 11, 90–95.[Medline]

Van Blerkom, J. and Henry, G. (1991) Dispermic fertilisation of human oocytes. J. Electron. Microsc. Tech., 17, 437–449.[ISI][Medline]

World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction. Cambridge University Press, Cambridge.

Submitted on January 25, 1999; accepted on April 27, 1999.