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Pregnancies established through intracytoplasmic sperm injection (ICSI) using spermatozoa with dysplasia of  fibrous sheath

Santiago Brugo Olmedo, Vanesa Y. Rawe, Florencia N. Nodar, Germn D. Galaverna, Anbal A. Acosta, Hctor E. Chemes1

Centro de Estudios en Ginecologa y Reproduccin, CEGyR, Buenos Aires, Argentina
1Laboratory of Testicular Physiology and Pathology, Endocrinology Division, Children's Hospital, Buenos Aires, Argentina

Asian J Androl  2000 Jun; 2: 125-130

Keywords: spermatozoa; dysplasia; ciliary motility disorders; intracytoplasmic sperm injection
Aim: Dysplasia of the fibrous sheath (DFS) is an anomaly found in asthenozoospermic patients with extremely low or absent motility. In order to determine the efficacy of ICSI in these patients, a retrospective analysis of ICSI results in DFS patients has been done. Methods: Ten ICSI attempts were performed in 6 patients with diagnosis of Dysplasia of the Fibrous Sheath studied by transmission and scanning electron microscopy. Results: In the cases studied, sperm concentration was (29.6218.05)106/mL, total motility was 1.141.31%. Progressive motility was 0% except for one case with 0.1%. One hundred and three preovulatory oocytes were obtained and 94 metaphase II oocytes were injected. Sixty-nine of them showed two pronuclei (fertilization rate: 73.4%). Forty-nine embryos were obtained and 34 were transferred (mean: 3.4 embryos per transfer). Five pregnancies were diagnosed by -hCG plasma level determinations that resulted to be one preclinical abortion, one clinical abortion and three deliveries. Another pregnancy (ongoing) was achieved from a cryopreserved embryo transfer. Conclusion: These results showed that ICSI provides a suitable solution for patients suffering from irreversible sperm defects such as DFS. Nevertheless, it is mandatory to inform couples of possible transmission risks to offspring, which are unknown at present. Only when the etiology of this problem is disclosed, it will be possible to assess the real genetic risk.

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.

We report here the results of treatment using Intracytoplasmic Sperm Injection in six patients suffering from DFS with completely immotile spermatozoa (in one cycle) or spermatozoa with nonprogressive motility (in nine cycles).

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

Transcervical embryo transfer was performed between 48 and 72 h after oocyte retrieval, using a Frydman cannula (Prodimed, Neuilly, France). -hCG plasma level determinations were done two weeks later; and ultrasound was performed  four weeks after embryo transfer (6 weeks gestational age). Luteal phase was supplemented using 800 mg/day intravaginal micronized progesterone (UTROGESTAN NR, Rontag Laboratories, France) starting the day following oocyte retrieval until the 12th week of gestational age if confirmation of pregnancy was achieved.

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.

Patient number


















Volume (mL)











Sperm concentration**











Total motility (%)











Progressive motility (%)











*In these patients a positive -hCG level was detected. Patient 2 and 4 delivered two healthy girls. Patient 1 suffered from a preclinical abortion in the second attempt and a multiple delivery resulted (triplets) from the third attempt. Patient 3 suffered from an abortion during the first trimester and a pregnancy is ongoing after the transfer of cryopreserved embryos. **(n106 spermatozoa/mL). 

3.3 ICSI procedure

Estradiol level (means) on the day of hCG was 1539900 pg/mL.

The results obtained after ICSI are summarized in Table 2. Briefly, 103 preovulatory oocytes were obtained and a total of ninety-four oocytes were injected. In one cycle immotile spermatozoa were used and in nine cycles non-progressive motile spermatozoa were injected. No clinical differences were found in terms of fertilization rate and embryo quality when either motile or non-motile spermatozoa were used. In fact, one of the  pregnancies was achieved after injection of an immotile spermatozoon. Sixty-nine oocytes showed two pronuclei at the time of pronuclear visualization (fertilization rate: 73.4%). Thirty-four embryos were transferred (mean: 3.4 per transfer). Twenty zygotes and 15 cleaved embryos were cryopreserved. Five pregnancies were diagnosed by -hCG plasma levels determinations twelve days after embryo transfer. Another pregnancy was achieved after a cryopreserved embryo transfer in a subsequent artificially prepared cycle. From two different patients, two healthy girls were born on June 1995 and July 1998. The third pregnancy was a pre-clinical abortion, the fourth resulted to be a clinical abortion during the first trimester and the fifth pregnancy was multiple (triplets). Last patient delivered three healthy infants at the time of writing (two males and one female) by cesarean section at 33 week of gestation.

Table 2. ICSI and pregnancy results from ten cycles in six patients with DFS.




Preovulatory oocytes



Injected oocytes



Normal fertilization



Total zygotes obtained



Total embryos obtained



-zygotes cryopreserved



-embryos cryopreserved



-embryos transferred


88% good quality embryos

Positive -hCG plasma levels



Clinical abortion



Pre-clinical abortion






*One more pregnancy was achieved from a transfer of cryopreserved embryos.

4 Discussion

One of the most severe abnormalities of sperm structure is the Dysplasia of the Fibrous Sheath. As previously described, this condition affects various cytoskeletal components of the sperm tail which appears short and thick[6,7]. As a consequence, asthenozoospermia or total sperm immotility caused by serious disturbances in the organization of the sperm fibrous sheath are present. In a recent publication, in patients suffering from DFS we have shown that clinical treatments or classical in vitro fertilization were not successful in solving the infertility problem[6]. The results presented here confirm our previous observation that ICSI is a suitable procedure to overcome sperm immotility in DFS[12]. Similar results have been previously reported in a patient with severe sperm tail abnormalities and fertilizations have been obtained from patients with extreme asthenozoospermia or total sperm immotility[13-15]. A genetic origin of the syndrome has been suggested[6,9,16]. The familial incidence reported in all these series prompted the speculation about the genetic origin of this phenotype[17]. We have recently reported 5 pairs of brothers (10 patients) in a large series of 42 men with DFS[6]. Three of the patients here reported have brothers also affected by DFS. Recently, a gene, which encodes for a major fibrous sheath protein was identified and localized to the X chromosome, but to date there are no reports describing gene anomalies in patients with DFS[18]. Whether DFS is the consequence of the mutation/deletion of gene (s) coding for structural proteins of the flagellum, or depends on the failure of a regulatory system controlling proper flagellar assembly, will certainly be a matter of study in the future. For as long as the genetic transmission of the problem is unknown, it is necessary to inform these couples about the theoretical implications of the presence of this type of sperm pathology. Our results indicate that ICSI seems to be a successful technique to obtain an excellent fertilization and pregnancy rate. The possible transmission of infertility to the male offspring and of respiratory disease is a matter of concern. To date, respiratory manifestations in our DFS patients have not lead to serious respiratory insufficiency. Final decision should rest on the couples adequately informed of the risks involved.


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Correspondence to Santiago Brugo Olmedo, M.D., Viamonte 1438 (1055), Capital Federal, Buenos Aires,  ARGENTINA.
Tel: +54-11-4372 8289  Fax: +54-11-4371 7275

E-mail: sbo@impsat1.com.ar
Received 2000-04-10   Accepted 2000-05-24