established through intracytoplasmic sperm injection (ICSI) using spermatozoa
with dysplasia of fibrous
Brugo Olmedo, Vanesa Y. Rawe, Florencia N. Nodar, Germn D. Galaverna,
Anbal A. Acosta, Hctor E. Chemes1
de Estudios en Ginecologa y Reproduccin, CEGyR, Buenos Aires, Argentina
Asian J Androl 2000 Jun; 2: 125-130
AbstractAim: 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.
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].
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
this anomaly, identifying the main underlying defect. 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
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.
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.
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. All patients were subjected
to a complete andrological investigation, which revealed total sperm immotility
or extreme asthenozoospermia. No other andrological conditions were found.
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.
Transmission and scanning electron microscopy
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. 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).
quantification of axonemal anomalies with the transmission electron microscope,
at least 100 flagella were counted. In one patient a second semen sample
and studied by Scanning Electron microscopy.
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.
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
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
SSS until the time of injection.
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
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.
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
zygotes and cleaved embryos not selected for transfer were cryopreserved.
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.
Electron microscopy of the sperm
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.
some spermatozoa, the 9+2 axonemal structure was completely distorted
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.
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.
1. Seminal parameters on the day of ICSI procedure in six patients with
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
Estradiol level (means) on the day of hCG was 1539900 pg/mL.
results obtained after ICSI are summarized in Table 2. Briefly, 103 preovulatory
oocytes were obtained and a total of ninety-four oocytes were injected.
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.
2. ICSI and pregnancy results from ten cycles in six patients with DFS.
more pregnancy was achieved from a transfer of cryopreserved embryos.
4 DiscussionOne 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. The results presented here confirm our previous observation that ICSI is a suitable procedure to overcome sperm immotility in DFS. 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. We have recently reported 5 pairs of brothers (10 patients) in a large series of 42 men with DFS. 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. 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.
Eliasson R, Mossberg B, Cammer P, Afzelius BA. The immotile cilia syndrome:
ciliary abnormality as an etiology factor in chronic airway infections
and male sterility. N Engl J Med 1977; 297: 1-6.
Brugo Olmedo, M.D., Viamonte 1438 (1055), Capital Federal, Buenos Aires,