Assisted reproduction for infertile patients with 9+0 immotile spermatozoa associated with autosomal dominant polycystic kidney disease

Hiroshi Okada1,3, Hitoshi Fujioka1, Noboru Tatsumi1, Masato Fujisawa1, Kazuo Gohji1, Soichi Arakawa1, Hiroshi Kato2, Shin-Ichiro Kobayashi2, Shinzo Isojima2 and Sadao Kamidono1

1 Department of Urology, Kobe University School of Medicine, 7–5–1 Kusunoki-cho, Chuo-ku, Kobe 650–0017, and 2 Advanced Fertility Center, Fuchu Hospital, Izumifuchu, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We investigated the clinical feature of patients with totally immotile spermatozoa due to 9+0 ultrastructural flagellar defects and polycystic kidney disease. We also tried to establish the feasibility of applying modern assisted reproduction technology (ART) in these patients. During 6-year interval a total of 1956 Japanese men were referred to the male infertility clinic. Of them, 16 were diagnosed to have immotile spermatozoa and four of them exhibited axonemal 9+0 defects in the sperm flagella. These four also had autosomal dominant polycystic kidney disease (ADPKD). Intrauterine insemination (IUI) and conventional in-vitro fertilization and embryo transfer failed to achieve fertilization. Intracytoplasmic sperm injection (ICSI) with 100% immotile spermatozoa was performed in all four cases. Two-pronuclear fertilization was obtained in 27 of the 70 (38.6%) of the successfully injected oocytes, but no pregnancy resulted. In one case, a few motile spermatozoa were present at the second cycle of ICSI, a pregnancy was successfully achieved using these spermatozoa. While immotile spermatozoa from patients with the axonemal 9+0 defect achieved fertilization by ICSI, the embryos failed to develop. Our results indicate that the central microtubules may play a role in fetal development. Since the 4 patients with 9+0 defects also had ADPKD, the genetic linkage between these two conditions should be studied by molecular biological methods so as to aid our ability to counsel such patients.

Key words: ART/flagellar ultrastructure/immotile spermatozoa/polycystic kidney


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Intracytoplasmic sperm injection (ICSI) is a powerful tool for overcoming the problem of male infertility in such conditions as oligozoospermia or astheonozoospermia. However, the use of totally immotile spermatozoa reportedly has a negative effect on fertilization and pregnancy rates (Nagy et al., 1995Go). Afzelius (1976) first reported that the ultrastructural defects of flagella or cilia may cause some diseases. We also described that some patients have demonstrated defects in the sperm flagella on ultrastructural evaluation (Okada et al., 1993Go). There are two representative causes of immotility in spermatozoa; one is an ultrastructural defect of sperm flagella. In this case, the spermatozoa are alive but cannot move. The other is the necrozoospermia. In this case spermatozoa in the ejaculate are dead because of a partial obstruction of ejaculatory ducts. In the former condition, ICSI with spermatozoa from ejaculate is a treatment of choice, however, in the latter situation spermatozoa retrieved from testes were successfully used for ICSI (Tournaye et al., 1998Go).

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
A total of 1956 Japanese men were referred to the male infertility clinic of Kobe University Hospital between January 1991 and December 1996. Of that number, 21 men were found to have totally immotile spermatozoa. Those 21 patients were further evaluated to try to determine the aetiology of the immotility of the spermatozoa.

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., 1997Go). The latter included an evaluation of sperm motility, linearity, lateral head displacement, and beat cross frequency as previously described (Mortimer et al., 1988Go). 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., 1997Go). Briefly, cryopreserved zona-free hamster ova were pre-incubated in microdroplets (500 ml each) of Biggers–Whitten–Whittingham (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., 1997Go).

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 (1–20x106/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 4–8-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 4–8-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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 21 of the 1956 patients (1.1%) were found to have spermatozoa that were totally immotile. As spermatozoa from five of these 21 patients were dead according to eosin-Y staining, their diagnosis was necrozoospermia. Semen samples from the remaining 16 patients contained viable but immotile spermatozoa at a frequency ranging from 15 to 80%. Ultrastructural analyses of the sperm flagella of four of the 16 patients with totally immotile spermatozoa revealed a lack of central microtubules in 80–100% of the spermatozoa (Figure 1Go). These four men were also found to have polycystic kidney disease (PKD), as shown by abdominal ultrasonography and CT scans (Figure 2Go). Only one of the four patients (no. 1) had been previously diagnosed with PKD. Analysis of the pedigree showed that all four men had inherited PKD from the maternal side; the diagnosis was thus autosomal dominant polycystic kidney disease (ADPKD). Further detailed investigation of the spermatozoa was performed in these four patients. The sperm count in the four cases ranged from 3.5 to 61 x106/ml. Totally immotile spermatozoa were observed in three patients (Nos. 1–3). Patient 4 initially presented totally immotile spermatozoa, however, in the following semen analyses his semen contained a few motile spermatozoa (twice in six occasions). Only this patient showed this phenomenon. About 4% of the spermatozoa of patient 4 exhibited motility on the third and fourth semen analyses. From 10 to 50% of the spermatozoa were stained with eosin Y (Table I). The curvilinear velocity of the spermatozoa in patient no. 4 was as slow as 0.5–1 µm/s; thus they could not swim forward effectively. Repeated hamster testing revealed no penetration of hamster ova by the spermatozoa of any of the four patients. Total acrosin activity of the spermatozoa of four patients was within the normal range used at our institution (>26 µIU/106 spermatozoa) (Nakagawa et al., 1997Go).



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Figure 1. Transverse section of sperm flagellum of patient 1. The sperm tail lacks a pair of central tubules (original magnification x75 000). Bar = 200nm.

 


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Figure 2. Abdominal computerized tomography (CT) scan in patient 1 shows the presence of multiple cysts in both kidneys; typical of autosomal dominant polycystic kidney disease.

 
Concerning their medical history, three patients (nos. 1,2,4) had no history of recurrent bronchitis or sinusitis, whereas one patient (no. 3) had a history of recurrent bronchitis and sinusitis during childhood. No situs inversus or bronchiectasis was found in any of the four patients. Saccharin transit time was within the normal limit in all patients, as were pulmonary function tests. Palpation of the intrascrotal components showed no varicocele of abnormality in the epididymis and vas deferens. Transrectal ultrasonography showed a prostate of normal size in all four patients. All four men had a normal male chromosomal phenotype, and serum hormonal assay showed a normal endocrine profile in each case.

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 1–5 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. 1–3) 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.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Male infertility with the demonstration of completely immotile spermatozoa can be caused by an ultrastructural flagellar defect or by necrozoospermia. We have evaluated patients with the former condition with particular interest since 1991 (Okada et al., 1993Go). The axoneme of a cilium is composed of nine microtubular doublets equipped with an inner and outer dynein arm that is arranged in a circle around two central microtubules (Afzelius et al., 1984). The sperm flagellum demonstrates the same ultrastructure as a cilium. To date, several ultrastructural defects including a lack of central microtubules, lack of dynein arms and other abnormalities have been described in immotile spermatozoa (Escalier et al., 1982). In the present study, 16 patients demonstrated completely immotile spermatozoa due to various defects in the flagella observed on ultrastructural examination. Since 1993 we observed four patients whose spermatozoa lacked the central microtubules (the axonemal 9+0 defect) of the flagella. Our first patient with an axonemal 9+0 defect demonstrated numerous cysts in both kidneys and PKD was diagnosed. We become interested in this rare combination of disorders, and began to perform abdominal ultrasonography and CT scans in patients with completely immotile spermatozoa. We identified an additional three patients with completely immotile spermatozoa due to an axonemal 9+0 defect and PKD. The present study described the characteristics of these four patients and the outcome of treatment using ICSI.

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., 1993Go). 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., 1985Go); PKD2 at 4q13–23 (Kimberling et al, 1993Go) and PKD3 at an unmapped locus (Daoust et al, 1995Go). 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.


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on March 20, 1998; accepted on October 9, 1998.