1 Introduction

Dysplasia of the Fibrous Sheath (DFS) is a severe form of flagellar pathology, which causes sperm immotility and shows familial incidence^[1-3]. A new variant of the Immotile Cilia Syndrome in which a DFS was associated with classical dynein arms deficiency in sperm flagella and respiratory cilia has been reported^[4-6].

Sterile males showing rudimentary sperm tails have been described in several species including man^[7,8]. Particularly in man, the Stump tail Syndrome has been repeatedly cited in the literature^[9,10]. The term Dysplasia of the Fibrous Sheath has been coined to describe in a more comprehensive way this anomaly, identifying the main underlying defect^[4]. DFS affects cytoskeletal constituents of the sperm tail, and modifications of the fibrous sheath are the key component of this pathology. Patients with DFS have primary infertility, they do not respond to clinical therapies, and classical in vitro fertilization treatments consistently fail to achieve fertilization or pregnancies. Thus, these patients are good candidates for high complexity microfertilization techniques.

2 Materials and methods

2.1 Patients

Six adult male patients (age: 30.84.4 years old, means), with normal sexual function, complaining of primary infertility were studied. One of them had a brother (not twin) with clinical and ultrastructure features of DFS determined by electron microscopy. According to the prevalence of tail abnormalities two groups of patients could be discerned. In some of them all spermatozoa were affected, while in some others the number of abnormal tails was about 70-80% with 20-30% in the normal configuration^[6]. These two groups have distinct characteristics and correspond to the complete and incomplete form of the DFS. Four patients suffered from the complete form of DFS and two from the incomplete form.

Past histories provided no information on clinical problems other than respiratory symptoms (rhino-sinusitis, bronchitis and or bronchiesctasis) dating back to early childhood. In one of the patients with the incomplete form of DFS, no translocations of thoracic or abdominal viscerae were detected. According to previous studies this patient can be considered a mosaic variant of the immotile cilia syndrome^[5]. All patients were subjected to a complete andrological investigation, which revealed total sperm immotility or extreme asthenozoospermia. No other andrological conditions were found.

One patient was treated empirically in other Center for several months with human Menopausal Gonadotropin  (Pergonal, Serono Laboratories, Mexico), containing 75 IU Follicle Stimulating Hormone (FSH) and 75 IU Luteinizing Hormone (LH), with no improvement noted in several followup sperm analysis. Patients signed an informed Consent for ICSI procedure.

2.2 Transmission and scanning electron microscopy

A fresh semen sample was processed in each patient during the work-up diagnosis for electron microscopy examination within 30 min of ejaculation, according to methods previously described^[6]. In brief, the spermatozoa were washed with phosphate buffer (0.1 mol/L, pH 7.4), pelleted by centrifugation and fixed in 3% glutaraldehyde followed by 1.3% osmium tetroxide. The pellets were embedded in Epon Araldite (Polysciences Inc., Warrington, PA, USA) and thin sections were examined and photographed in a Zeiss 109 Electron microscope (Zeiss Oberkochen, Germany) after double staining with uranyl acetate and lead citrate. For quantification of axonemal anomalies with the transmission electron microscope, at least 100 flagella were counted. For studies with the scanning electron microscope, the same fixatives were used. The spermatozoa were fixed in suspension with buffer washes between and after both fixatives. Sperm cells were subsequently sedimented on poly-L-lysine coated slide fragments, to assure sperm adherence to the glass, dehydrated in a graded series of ethanol followed by absolute acetone, dried in a Balzers CDP 030 critical point drying apparatus (Balzers Union Ltd, Balzers, Lichtenstein), using CO₂ as transition fluid, coated with gold-palladium in a Balzers Union SCD 040, and observed in a Philips 515 scanning electron microscope (Philips Netherland BV, Eindhoven, The Netherlands).

For quantification of axonemal anomalies with the transmission electron microscope, at least 100 flagella were counted. In one patient a second semen sample was processed and studied by Scanning Electron microscopy.

In all cases a small aliquot of fresh semen was studied under phase contrast microscopy, and motility, viability and light microscopy morphology were studied according to standard methods^[11].

2.3 Ovarian stimulation

Ovarian stimulation of patient's wives was performed using a combination of FSH (METRODINE NR, Serono Laboratories, Mexico) and hMG (PERGONAL NR, Serono Laboratories, Mexico, HUMEGON NR, Organon Laboratories, Buenos Aires, Argentina and hMG, Massone Laboratories, Buenos Aires, Argentina), under Leuprolide Acetate (LUPRON NR, Abbot Laboratories, Buenos Aires, Argentina) suppression starting on day 21 of the previous menstrual cycle in two different protocols: 1 mg daily subcutaneous dose reduced to 0.5 mg after ovarian suppression was confirmed (quiescent ovaries at ultrasound and serum Estradiol levels  30 pg/mL) and continued until hCG administration; and a depot preparation (LUPRON DEPOT NR 3.75 mg Abbott, Buenos Aires, Argentina) in one intramuscular dose applied on day 21 of the previous menstrual cycle. Estradiol plasma levels and ovarian follicular size were monitored daily. Ten thousand IU of hCG (PROFASI NR, Serono Laboratories, Mexico) was administered intramuscularly when 2 or more follicles 17 mm were present at ultrasound. Oocyte retrieval was performed 35 h after hCG administration by ultrasonically guided follicular puncture.

2.4 ICSI procedure

Oocyte-cumulus complexes were retrieved and placed in four well dishes with 0.5 mL of Human Tubal Fluid (HTF, Irvine Scientific Laboratories, Santa Ana, CA, USA) supplemented with 15% Synthetic Serum Substitute (SSS, Irvine Scientific, Santa Ana, California, USA). After approximately two hours, oocyte-cumulus complexes were treated for 30 s with hyaluronidase (Sigma Laboratories, St. Louis, MO, USA) at a concentration of 80 IU/mL, and immediately washed in Hepes buffer-Human Tubal Fluid (H-HTF, Irvine Scientific Laboratories, Santa Ana, CA, USA) supplemented with 1% Bovine Serum Albumin (BSA, Sigma Laboratories, St. Louis, MO, USA) to remove the granulosa cells from the corona radiata. The maturation state of the oocytes was checked after stripping off the granulosa cells, and the oocytes were left to stabilize in HTF+15%   SSS until the time of injection.

Ejaculated sperm were processed using discontinuous Percoll gradients  (50-95%). In one cycle the 95% layer was washed in Earle's medium and centrifuged at 1800g for 5 min. In the other nine cycles, pellets were washed by centrifugation at 1800g for 5 min, and resuspended in H-HTF+1% BSA.

Metaphase II oocytes were placed in 5 L drops of H-HTF+1% BSA under embryo tested-light mineral oil (Sigma Laboratories, St. Louis, MO, USA), and the sperm injection was performed using an inverted microscope (Nikon Diaphot, Nikon Corporation, Tokyo, Japan) with a heated plate and Narishige micromanipulators (Nikon Corporation, Tokyo, Japan). In all attempts except one, motile spermatozoa were injected. All injected spermatozoa had the characteristic stump tail morphology and their tails were broken with the micropipette before injection in order to activate the spermatozoon.

When ICSI procedure was finished, between 4- 6 injected oocytes were placed in a 40 L drop of HTF+15% SSS under mineral oil, and incubated at 37 in 5% CO₂, until the moment of pronuclear visualization (between 12-18 h post injection). Twenty four hours after pronuclear visualization, embryo quality and cleavage was checked: embryo quality was determined using the following criteria: good quality embryos were those with almost no fragments present, uniform blastomeres and normal cleavage rate, and bad quality embryos were those with more than fifty percent of their surface covered with fragments, nonuniform blastomeres and/or low cleavage rate present.

Supernumerary zygotes and cleaved embryos not selected for transfer were cryopreserved.

2.5 Embryo transfer

3 Results

Ten ICSI cycles in six patients having spermatozoa with Dysplasia of the Fibrous Sheath were performed between November 1994 and November 1999. In four patients all spermatozoa were affected, while in two patients the number of abnormal tails was about 70-80% with 20-30% in the normal configuration. These two groups have distinct characteristics and correspond to the complete and incomplete form of the DFS.

3.1 Electron microscopy of the sperm

Under transmission electron microscopy most flagella were grossly abnormal showing marked hypertrophy and hyperplasia of the Fibrous Sheath as well as variable axonemal disruption and partial defects of the inner dynein arms of microtubular doublets.

In some spermatozoa, the 9+2 axonemal structure was completely distorted while in some others it was preserved in the center of dense rings of hyperplastic fibrous sheaths (Figure 1). Besides the serious distortion of the Fibrous Sheath, midpiece was not formed and mitochondria were poorly assembled or absent. The scanning electron microscopy study disclosed thick and distorted tails and absence of a middle piece (Figure 1).

Figure 1. Electron microscopy of spermatozoa with Dysplasia of the Fibrous Sheath. (A): Two spermatozoa showing short, thick and irregular tails typical of the DFS. The length of the tail is approximately 11 m (normal length 60 m). Magnification: 7 000. Scale bar: 3 m. (B): A transversal section of a sperm flagellum with marked hyperplasia and disorganization of the fibrous sheath around the axoneme. Scale bar: 0.5 m. Magnification: 44 000.

3.2 Semen analysis

Results of the 6 patients' semen analysis on the day of ICSI procedure in the 10 cycles are summarized in Table 1. In one cycle only immotile spermatozoa were available and in nine cycles non progressive motile spermatozoa were found. Under light microscopy, spermatozoa had short, rigid and thick flagella, frequently displaying irregular contours and/or coiled tails.

Table 1. Seminal parameters on the day of ICSI procedure in six patients with DFS.

References

[1] Eliasson R, Mossberg B, Cammer P, Afzelius BA. The immotile cilia syndrome: a congenital ciliary abnormality as an etiology factor in chronic airway infections and male sterility. N Engl J Med 1977; 297: 1-6.
[2] Antonelli M, Modesti A, De Angelis M, Marcolini P, Lucarelli N, Crifo S. Immotile cilia syndrome: radial spokes deficiency in a patient with Kartagener' s triad. Acta Paediatr Scand 1981; 70: 571-3.
[3] Torikata C, Kawai T, Nogawa S, Ikeda K, Shimizu K, Kijimoto C. Nine Japanese patients with immotile-dyskinetic cilia syndrome: an ultrastructural study using tannic acid-containnig fixation. Hum Pathol 1991; 22: 830-6.
[4] Chemes HE, Brugo Olmedo S, Zanchetti F, Carrere C, Lavieri JC.  Dysplasia of the fibrous sheath: an ultrastructural defect of human spermatozoa associated with sperm immotility and primary sterility. Fertil Steril 1987; 48: 664-9.
[5] Chemes HE, Morero JL, Lavieri JC. Extreme asthenozoospermia and chronic respiratory disease.  A new variant of the immotile cilia syndrome. Int J Androl 1990; 13: 216-22.
[6] Chemes HE, Brugo Olmedo S, Carrere C, Oses R, Carrizza C, Leisner M, et al. Ultrastructural pathology of the sperm flagellum: Correlation between flagellar pathology and fertility prognosis in severely asthenozoospermic men. Hum Reprod 1998; 9: 101-6.
[7] Maqsood M. An abnormality of mammalian spermatozoa. Experientia 1951; 7: 304.
[8] Blom E. A sterilizing tail stump sperm defect in a Holstein-Friesian bull. Nord Vet Med 1976; 28: 295-8.
[9] Baccetti B, Burrini AG, Capitani G, Collodel G, Moretti E, Piombini P, et al. Notulae seminologicae. 2. The short tail and stump defect in human spermatozoa. Andrologia 1993; 25: 331-5.
[10] Barth AD, Oko RJ. Defects of the sperm tail. In: Abnormal Morphology of bovine Spermatozoa. Ames: Iowa State University Press; 1989. p 214.
[11] World Health Organization Laboratory manual for the examination of human semen and semen cervical mucus interaction. Cambridge: Cambridge University Press; 1987. 
[12] Brugo Olmedo S, Nodar F, Chillik C, Chemes HE. Successful intracytoplasmic sperm injection with spermatozoa from a patient with dysplasia of the fibrous sheath and chronic respiratory disease. Hum Reprod 1997; 7: 1497-9.
[13] Stalf T, Sanchez R, Kohn FM, Schallesu U, Kleinstein J, Hinz V, et al. Pregnancy and birth after intracytoplasmic sperm injection with spermatozoa from a patient with stump tail syndrome. Hum Reprod 1995; 10: 2112-4.
[14] Bongso T A, Sathananthan AH, Wong PC, Ratnam SS, Ng Sc, Anandakumar C, et al. Human fertilization by microinjection of immotile spermatozoa. Hum Reprod 1989; 4: 175-9.
[15] Terriou P, Giorgetti C, Hans C, Spach JL, Salzmann J, Carlon N, et al. Subzonal sperm insemination and total or extreme asthenozoospermia: an effective technique for an uncommon cause of male infertility. Fertil Steril 1993; 60: 1057-61.
[16] Bisson JP, Leonard C, David G. Caractere Familial de certaines perturbations morphologiques des spermatozoides. Arch Anat Cytol Path 1979; 27: 230-3.
[17] Kastury K, Taylor WE, Arver S, Fisher CE, Gutierez M, Bhasin S, et al. cDNA cloninig, chromosomal mapping and tissue distribution of the human axonemal dynein light chain gene for the immotile cilia syndrome. J Androl 1997; Jan/Feb (Suppl): 56.
[18] Moss SB, Vanscoy H, Gerton GL. Mapping of a haploid transcribed and translated sperm-specific gene to the mouse X chromosome. Mammalian Genome 1997; 8: 37-8.