1 Department of Urology, Kobe University School of Medicine, 751 Kusunoki-cho, Chuo-ku, Kobe 6500017, and 2 Advanced Fertility Center, Fuchu Hospital, Izumifuchu, Japan
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Abstract |
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Key words: ART/flagellar ultrastructure/immotile spermatozoa/polycystic kidney
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Introduction |
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The present study described characteristics and outcome of ART in four men with totally immotile spermatozoa due to axonemal 9+0 defects. The association of such flagellar defects with ADPKD is also probed.
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Materials and methods |
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Each patient was asked to complete a questionnaire regarding his relevant medical and family histories, including problems with upper respiratory tract infections, any infertile siblings, duration of unprotected intercourse, previous treatment, and the gynaecological status of his wife. Physical examination included a check for gynaecomastia and varicocele, and the status of the vas deferens and epididymis was evaluated. Testicular volume was measured by an orchidometer, and size of the prostate was measured with a transrectal ultrasonogram.
Semen collection and assessment of sperm function
Semen samples were produced by masturbation, collected into sterile containers, and immediately transported to the laboratory. Semen analyses were performed after complete liquefaction. A conventional semen profile was obtained for each sample using the procedures described by the World Health Organization (WHO, 1992). A 5 µl aliquot of semen was placed on a Makler chamber to determine the sperm count. In addition, 5 µl aliquot of the sample was placed in a disposable counting chamber 20 µm deep (µ-Cell, Fertility Technologies, Notick, MA, USA) and analysed in a computer assisted analyzer (HTM 2030, Hamilton Thorne Research, Inc, Danvers, MA, USA) to measure the sperm motion parameters at 37°C (Okada et al., 1997). The latter included an evaluation of sperm motility, linearity, lateral head displacement, and beat cross frequency as previously described (Mortimer et al., 1988
). Each sample was assayed in triplicate. Eosin Y staining was also used to differentiate living from dead spermatozoa. Papanicolaou staining was used for morphological analysis. The zona-free hamster ova sperm penetration test was performed essentially as described by Yanagimachi et al. (1976) except for the use of cryopreserved hamster ova as described elsewhere (Nakagawa et al., 1997
). Briefly, cryopreserved zona-free hamster ova were pre-incubated in microdroplets (500 ml each) of BiggersWhittenWhittingham (BWW) medium (Biggers et al., 1971) under mineral oil for 3 h at 37°C in 4% CO2 in air. Next, a volume of 50 µl of sperm suspension (10x106/ml) was introduced into the droplet and co-cultured for 4 h under mineral oil at 37°C in 4% CO2 in air. The penetration rate was determined from observations made with an inverted phase-contrast microscope (Olympus IMT2, Olympus Co. Ltd., Tokyo, Japan), by one investigator (H.O.). At least 20 ova were used for each assay.
Laboratory tests
Blood chemistry was evaluated in each case, and included serum levels of creatinine, blood urea nitrogen, sodium, chloride, potassium, luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), testosterone, and oestradiol. Chromosomal analyses were done by G-banding and Q-banding as required. Total acrosin activity was measured by the procedures previously described (Nakagawa et al., 1997).
Mucociliary clearance and pulmonary function tests
In order to rule out the presence of impaired ciliary transport seen in Kartagener's syndrome, we assessed the saccharin transit time (Torikata et al., 1991). In addition, to evaluate the respiratory function, pulmonary function tests including vital capacity, forced expiratory volume in 1 s, functional residual capacity, forced vital capacity, residual volume, total lung capacity and maximal mid-expiratory flow rate, were performed.
Roentogenographic and ultrasonographic examination
Chest X-ray and abdominal computerized tomography (CT) scans were performed in each patient. Transrectal ultrasonography was done to check the prostate and seminal vesicles, and abdominal ultrasonography was done to evaluate the kidneys and liver.
Ultrastructural examination of sperm tail
Spermatozoa were washed twice with phosphate buffer, fixed with Karnovsky's fixative, washed with 0.1 M cacodylate buffer (pH 7.2), post-fixed with 1% osmium tetroxide, dried with ethanol and routinely embedded. The ultrastructure of sperm flagellum was evaluated by transmission electron microscopy. At least 20 slices of the different sperm tails were evaluated by one of us (H.F.) who were blind as to the source of the specimen.
Hypo-osmotic swelling test (HOST)
HOST was performed according to the procedures described by Jeyendran et al. (1984), with minor modification. Briefly, spermatozoa were washed with Earle's balanced salt solution and sperm suspension was prepared. Sperm suspension (100 µl) was incubated with 1 ml of hypo-osmotic solution (150 mOsm) at 37°C for 10 min. A Makler chamber filled with this sperm mixture was observed under phase-contrast microscopy (Olympus BHS-2) at x200 magnification without staining. At least 200 spermatozoa were observed and classified morphologically into seven categories according to the system described by Jeyendran et al. (1984). The spermatozoa presenting morphological change type G, which was the typical tail deformation pattern characterizing the reaction of living spermatozoa to the hypo-osmotic environment, were used for ICSI.
Treatment
Each couple provided written informed consent prior to undergoing assisted reproduction technology. Intrauterine insemination (IUI) had been performed using Percoll treated spermatozoa with monitoring of the maturation of the ova by ultrasonography and LH surge. Each insemination was done in the high intracervical area using 0.5 ml of the sperm suspension (120x106/ml) that was prepared by separation on Percoll density gradient.
In-vitro fertilization (IVF) was performed conventionally by the following procedures. Briefly, oocytes were retrieved after ovarian stimulation described below and inseminated in B2 medium (Menezo, Paris, France). Spermatozoa were also prepared by separation on Percoll density gradient. Enbryo transfer was performed at the 48-cell stage. Establishment of a pregnancy was determined by assay of ß-HCG and monitoring the fetal heart beat.
ICSI was performed according to the following procedures. Briefly, ovarian stimulation was accomplished by a long period protocol with FSH, HMG and HCG. 150 IU/day of FSH (Fertinorm; Serono Japan, Tokyo, Japan) treatment was followed by 150 IU/day administration of HMG (Pergogreen, Serono Japan). The diameter of the leading follicle was monitored with an ultrasonography until it became 18 mm. Then 5000 IU of HCG (Profasi; Serono Japan) was injected. Oocytes were retrieved by puncture 37 h after HCG administration as guided by vaginal ultrasonography. Metaphase II oocytes were injected with a single spermatozoon that had been pre-incubated in Earl's balanced salt medium at 37°C in an incubator with an atmosphere of 5% CO2, 5% O2, and 90% N2. A single spermatozoon was selected according to the morphology of its heads, and was injected through an injection needle (7 µm outer diameter, 5 µm inner diameter). In patient 1, HOST was performed and type G spermatozoa were utilized for ICSI. In the other patients oocytes were injected with randomly selected spermatozoa. Embryo transfer was performed at the 48-cell stage. Establishment of pregnancy was confirmed by the assay of urine ß-HCG 14 days after oocytes retrieval. It was re-confirmed by monitoring of the gestation sac by ultrasonography.
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Results |
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Patient 1 and patient 2 had undergone 10 cycles and 14 cycles of IUI in other institutions before referral respectively. But no pregnancy was achieved. These patients also underwent conventional IVF/embryo transfer for two cycles and one cycle respectively. In all attempts the fertilization rate was reportedly to be 0%. All four patients were eventually enrolled in the ICSI program. ICSI was then performed 15 times. Between five and 12 metaphase II oocytes were retrieved each ICSI cycle, and between one and 12 oocytes were injected with a spermatozoon. In three patients (nos. 13) a totally immotile spermatozoon was injected into an oocyte. In patient 4, immotile spermatozoa were used in the first attempt at ICSI, while motile spermatozoa that were found in the semen were used in the subsequent ICSI cycle, a pregnancy was achieved by ICSI with motile spermatozoa in this case (Table II). Totally 92 metaphase II oocytes were recovered and 70 were injected with a spermatozoa and 27 of them developed in two-pronuclear embryos (overall fertilization rate 38.6%). Finally, 15 cleaving embryos were obtained and transferred (overall cleavage rate 55.5%), and only one pregnancy was achieved.
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Discussion |
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Since the flagella and cilia of the spermatozoa possess the same ultrastructure, there are three possible combination of the ultrastructural defects: defects in both flagella and cilia, defects in the flagella only, and defects in the cilia only. We did not investigate the ultrastructure of the respiratory cilia in the present study. None of the four patients had a history of upper respiratory tract disorders, and demonstrated no respiratory dysfunction nor any delay in nasociliary clearance. These results suggested that ultrastructural defects were restricted to the sperm flagella, and that the majority of the cilia functioned normally.
Three of four patients showed a total immotility of the viable spermatozoa, while one patient exhibited a few motile spermatozoa on separate occasions. The absence of the central microtubules was observed in the sperm flagella of 3 patients; that defect was present in 80% of the spermatozoa of the fourth patient. In a previous study, ~1.1% of the men attending a male infertility clinic had ultrastructural flagellar defects (Okada et al., 1993). Unpublished data from that clinic showed that eight out of 20 patients (40%) with totally immotile spermatozoa had axonemal 9+0 defects (Okada, unpublished data).
Several procedures of ART were tried in these four patients. All had failed to father a child by IUI and conventional IVF-ET. Although the acrosin activity of the spermatozoa was within normal limits, they failed to penetrate the oolemma of the hamster ova. This observation suggests that the failure of ICSI in these cases can be attributed to the inability of the spermatozoa to penetrate the oolemma.
While all four patients underwent ICSI, those in whom spermatozoa were completely lacking in the microtubules achieved successful fertilization, but even after successful embryo transfer, no pregnancy was achieved. However, patient 4, 80% of whose spermatozoa lacked central microtubules with the remaining 20% exhibiting a normal 9+2 axonemal structure, fathered a male infant. In our series of four subjects, only one patient demonstrated this phenomenon. It is noteworthy that in this patient at the first ICSI cycle, when immotile spermatozoa (probably 9+0 spermatozoa) were injected, no pregnancy had been achieved, whereas at the second attempt, all the oocytes were injected with a motile spermatozoon (possibly 9+2 spermatozoa). Pregnancy was then achieved. Vandervorst et al. (1997) has reported that patients with ultrastructural flagellar defects had no motile spermatozoa on subsequent attempts. The reason for this discrepancy cannot be explained from the present observation.
These results also suggest that a patient with PKD and totally immotile spermatozoa with an axonemal 9+0 defect can fertilize an ovum, but it may fail to reach the fetal stage. This differs from the observation by Vandervorst et al. (1997), that a patient with the 9+0 axonemal defects did not achieve fertilization. Our results indicate that the central microtubules play a role in fetal development.
Recently, Barros et al. (1997) described a new method to select suitable spermatozoa for ICSI among totally immotile spermatozoa. By using their modified HOST they reported a fertilization rate of 41.9% and two clinical pregnancies. Although the aetiology of immotility of spermatozoa was not investigated, their method seemed to be worth trying in patients like present cases.
Tournaye et al. (1998) has reported that in five couples whose spermatozoa were immotile because of ultrastructural defects no pregnancy was obtained. For their series eight cycles of ICSI were performed and overall fertilization rate was 23.2%. The overall cleavage rate was 40%. In our series overall fertilization rate was 38.6% and overall cleavage rate was 55.5%. The difference of axonemal defects in these two reports may explain the difference in the outcome.
Interestingly the four patients we describe all had ADPKD. Three different loci are known to cause this disorder: PKD1 in chromosome region 16p13.3 (Reeders et al., 1985); PKD2 at 4q1323 (Kimberling et al, 1993
) and PKD3 at an unmapped locus (Daoust et al, 1995
). There has been no genetic study of the defect in the central microtubules in infertile men; thus, the genetic linkage between ADPKD and this defect in the sperm flagellum remains to be determined. It is strongly suggested that the gene responsible for constructing the central microtubules of the flagella may be located near the aforementioned genes.
Only one of four men had been previously diagnosed with PKD. We recommend that abdominal CT scans or ultrasonograms be performed to explore this unusual combination of disorders. The genetic linkage of ADPKD and the axonemal 9+0 defect should be investigated by molecular biological techniques to allow us to improve counselling of such patients who wish to undergo ICSI.
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Notes |
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References |
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Afzelius, B.A., Dallai, R., Lanzavecchia, S. et al. (1995) Flagellar structure in normal human spermatozoa and in spermatozoa that lack dynein arms. Tissue Cell., 27, 241247.[ISI][Medline]
Barros, A., Sausa, M., Angelopoulos, T. and Tesarik, J. (1997) Efficient modification of intracytoplasmic sperm injection technique for cases with total lack of sperm movement. Hum. Reprod., 12, 12271229.[ISI][Medline]
Biggers, J.D., Whitten, W.K. and Whittingham, D.G. (1976) The culture of mouse embryos in vitro. In Naniel, J.C. Jr. (ed.), Methods in Mammalian Embryology. Freeman, San Francisco, USA, pp. 86116.
Daoust, M.C., Reynolds, D.M., Bichet, D.G. et al. (1995) Evidence for third genetic locus for autosomal dominant polycystic kidney disease. Genomics, 10, 733736.
Escalier, D, and David G. (1982) Pathology of the cytoskelton of the human sperm flagellum. Axonemal and pre-axonemal anomalies. Biol. Cell, 50, 3752.
Jeyendran,E.S., Van der Ven, H.H., Perez-Pelaez,M. et al. (1984) Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Fertil., 70, 219228.[Abstract]
Kimberling, W.J., Kumar, S., Gabow, P.A. et al. (1993) Autosomal dominant polycystic kidney disease: localization of the second gene to chromosome 4q13-q23. Genomics, 18, 467472.[ISI][Medline]
Mortimer, D., Serres, C., Mortimer, S.T. et al. (1988) Influence of image sampling frequency on the perceived movement characteristics of progressively motile human spermatozoa. Gamete Res., 20, 313327.[ISI][Medline]
Nagy, Z., Liu, J., Joris, H. et al. (1995) The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. Hum. Reprod., 10, 11231129.[Abstract]
Nakagawa, H., Okada, H., Fujisawa, M. et al. (1997) Relationship of acrosin to sperm function tests. Andrologia, 29, 103108.[ISI][Medline]
Okada, H., Hayashi, A., Tanaka, H. et al. (1993) Ultrastracture of immotile spermatozoa obtained from infertile male patients. Jpn. J. Urol., 84, 18791882.
Okada, H., Tatsumi, N., Kanzaki, M. et al. (1997) Formation of reactive oxygen species by spermatozoa from asthenospermic patients: response to treatment with pentoxifylline. J. Urol., 157, 21402146.[ISI][Medline]
Reeders, S.T., Breuning, M.H., Davis, K.E. et al. (1985) A highly polymorphic DNA marker linked to adult polycystic kidney disease on chromosome 16. Nature, 317, 542544.[ISI][Medline]
Tokikata, C., kawai, T, Nogawa, S. et al. (1991) Nine Japanese patients with immotile-dyskinetic cilia syndrome; an ultrastructural study using tannic acid-containing fixation. Hum. Pathol., 22, 830836.[ISI][Medline]
Tournaye, H., Joris, H., Vandervorst, et al. (1998) Intracytoplasmic sperm injection with ultrastructurally abnormal spermatozoa. In Ombelet, W., Bosmans, E, Vandeput, H. et al. (eds), Modern ART in the 2000s. Andrology in the nineties. Proceeding of an International Symposium on Male Infertility and Assisted Reproduction. Parthenon Publishing Group, New York, London, pp. 157162.
Vandervorst, M., Tournaye, H., Camus, M. et al. (1997) Patients with absolutely immotile spermatozoa and intracytoplasmic sperm injection. Hum. Reprod., 12, 24292433.[Abstract]
Yanagimachi, R., Yanagimachi, H., Rogers, B.J. (1976) The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. Biol. Reprod., 15, 471476.[ISI][Medline]
World Health Organization. (1992) WHO Laboratory Manual for the Examination of Human Semen and SemenCervical Mucus Interaction. Cambridge University Press, Cambridge, UK.
Submitted on March 20, 1998; accepted on October 9, 1998.