Successful in-vitro fertilization pregnancy with spermatozoa from a patient with Kartagener's syndrome: Case Report

V.J. Kay1,3 and D.S. Irvine2

1 Department of Obstetrics and Gynaecology, Royal Infirmary of Edinburgh, 1 Lauriston Place, Edinburgh EH3 9EF and 2 MRC Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh, UK


    Abstract
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
This paper reports on the successful treatment by in-vitro fertilization (IVF) of a couple in whom the male partner had Kartagener's syndrome. His spermatozoa were severely asthenozoospermic with deficient dynein arms and disordered microtubular configuration. On computer-assisted sperm analysis (CASA) motile spermatozoa displayed straight non-progressive motility with minimal amplitude of lateral head displacement and none were hyperactivated. This is the first case report in which spermatozoa with axonemal disruption in a man with immotile cilia syndrome (ICS) have been shown to be able to penetrate the zona pellucida and fertilize oocytes. IVF may be a suitable treatment for certain variants of ICS.

Key words: dynein arm deficiency/immotile cilia syndrome/immotile spermatozoa/Kartagener's syndrome


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
Immotile cilia syndrome (ICS) is an inherited condition in which dynein arms are absent in axonemal microtubules in ciliary and flagellar structures, resulting in the following clinical disorders: infertility, bronchiectesis and sinusitis. When situs invertus is present the disorder is referred to as Kartagener's syndrome (Kartagener, 1933Go).

Until recently there was no treatment available for male infertility due to ICS. The development of assisted reproductive techniques has allowed rational treatment for these patients and to date there have been reported pregnancies using subzonal insemination (Bongso et al., 1989Go; Wolf et al., 1993Go; Nijs et al., 1996Go) and intracytoplasmic sperm injection (ICSI) (Brugo et al., 1997Go; Chemes et al., 1998Go). We report a successful pregnancy achieved by in-vitro fertilization (IVF) using spermatozoa from a patient with Kartagener's syndrome.


    Case report
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
The patient was a 29 year old man who had a history of chronic sinusitis and respiratory disease, previously investigated by respiratory physicians. The following features were found on investigation: situs invertus with marginal dilation of the bronchial tree on computerized axial tomography scanning, normal ciliary function (normal beat frequency and pattern of beating), as were sweat electrolytes. He was negative for the nine most common cystic fibrosis mutations. Transmission electron microscopy of nasal and bronchial cilia showed absence of dynein arms on the peripheral microtubule doublets. Thus, the diagnosis of Kartagener's syndrome was made.

He and his partner presented with a 5 year history of primary infertility to the Infertility Clinic, Royal of Infirmary Edinburgh. His spouse was shown to have hyperprolactinaemia due to a micro-adenoma of the pituitary gland. Following treatment with bromocriptine she had ovulatory levels of progesterone on day 21 of her menstrual cycle. Otherwise she had an uneventful history. Her investigations, including pelvic ultrasound, diagnostic laparoscopy and hydrotubation, were normal. His history provided no further evidence of medical conditions which might affect fertility. On examination he had normal secondary sex characteristics and both testicular volumes were normal. He was noted to have a large right-sided varicocele, which was treated by percutaneous trans-catheter embolization. His serum prolactin, follicle-stimulating hormone (FSH) and testosterone were all within normal limits.

Semen analysis
Viscosity, liquefaction and pH were normal. Semen samples were assessed for ejaculate volume, morphology, concentration and percentage motile spermatozoa using the protocols described by the World Health Organization (WHO, 1992). The results of eight semen analyses assessed over a 4 year period were normal ejaculate volume and sperm concentration but mean total motility was only 25% and mean progressive motility was only 2.5% (Table IGo). Semen was negative on bacteriological culture and immunological screening.


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Table I. Results of eight semen analyses
 
Automated semen analysis
Detailed motility studies were performed using a Hamilton Thorn Motility Analyser (HTMA-IVOS version 10®; Hamilton Thorn Research Inc., Beverly, MA, USA) with an operating frequency of 50 Hz. The HTMA-IVOS settings employed during analysis were minimal contrast 10, minimum size 5, low and high head size gates 0.18 and 2.18 respectively, non-motile head size 5, non-motile head intensity 80, magnification factor 2.53, minimum number of data points 13 and frames captured 20. Of the 1713 spermatozoa analysed only 14% were motile. Of the remaining motile spermatozoa all had a slow straight trajectory, with reduced track speed, lateral head deviation and beat frequency. No spermatozoa showed hyperactivation as previously defined (Burkman, 1984Go; Table IIGo).


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Table II. Results of computer-assisted semen analyses
 
Transmission electron microscopy
Spermatozoa were fixed in 3% gluteraldehyde in 0.1 mol/l sodium cacodylate/HCl buffer pH 7.2–7.4. Secondary fixation was performed using 1% osmium tetroxide, following which spermatozoa were dehydrated in graded alcohol and embedded in Emix resin. Ultra-thin sections (90 nm) were cut, stained with uranyl acetate and lead citrate and examined using a Jeol 100CX II® Jeol UK Ltd., Herts, UK transmission electron microscope.

On ultrastructural examination sperm heads had intact nuclei and acrosomal regions. However, all flagella examined were grossly abnormal with axonemal disruption surrounded by a thick uncondensed fibrous sheath. Figures 1 and 2GoGo are transverse sections of sperm flagella. In Figure 1Go, microtubular pairs can be identified with one set translocated outside the fibrous sheath. Outer dynein arms are mostly absent, though the number of inner dynein arms is reduced. In Figure 2Go there is complete disorganisation of the microtubules with no recognizable pattern. Transmission electron microscopy was repeated on two separate occasions and on both occasions all spermatozoa analysed had defects in the dynein arms.



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Figure 1. Transverse section of sperm tail from the reported case with Kartagener's syndrome. Most outer dynein arms are absent, there are reduced inner dynein arms and one set of microtubules is translocated outside the fibrous sheath.

 


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Figure 2. Transverse section of sperm tail from the reported case with Kartagener's syndrome. There is a thickened irregular fibrous sheath and complete disorganization of the microtubules, with no dynein arms.

 
In-vitro fertilization
Ovarian stimulation was carried out using gonadotrophin-releasing hormone (Buserelin®; Hoeschst, Brussels, Belgium) 600 mµg/day in divided doses in association with human menopausal gonadotrophin (Metrodin®; 75 IU/day FSH, Serono Laboratories, Herts, UK) 2 ampoules (150 IU/day) daily for 15 days. Human chorionic gonadotrophin (HCG, Profasi®; Serono) 10 000 IU was given when nine follicles over 16 mm were identified on ultrasound scan. Aspiration was performed 36 h after HCG administration and one oocyte was obtained. The majority of cumulus mass was removed using a sterile needle.

Sperm preparation was carried out using 50–100% discontinuous Percoll gradient (1 ml/1 ml, centrifuged for 17 min at 500 g) followed by two washes (5 min at 230 g). A swim-up from the final pellet was then performed using Earle's medium (Sigma, Bornem, Belgium) supplemented with 0.5% human serum albumin (Irvine Scientific, Zellick, Belgium) in an atmosphere of 5% CO2 for 50 min. After preparation 1.6x106 spermatozoa were present in 0.3 ml culture medium. A total of 88% were motile and 56% had abnormal morphology. No hyperactivated spermatozoa were seen. Spermatozoa were diluted so that ~ 100x103 in 1 ml Ham's F10 medium were added to the oocyte and incubated for 22 h. At this stage two pronuclei were present.

The embryo was transferred without difficulty 49 h following oocyte recovery. Luteal support was carried out with progesterone suppositories (Cyclogest®; Hoechst, Brussels, Belgium) 400 mg for 10 days. Blood was taken for HCG on 14 days post-oocyte recovery and was 147 IU. A pelvic scan was performed 5 weeks following embryo transfer and a single intra-uterine pregnancy was identified. A male infant was delivered at a gestation of 40 weeks weighing 3.9 kg. On examination of the infant there was no evidence of situs invertus and he has remained well to date and is currently over 18 months old.


    Discussion
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
In order to achieve fertilization, spermatozoa must undergo an organized series of events. These include capacitation, the demonstration of hyperactivated motility, acrosome reaction, penetration of the zona pellucida and fusion with the vitelline membrane of the oocyte (Yanagimachi, 1981Go). Sperm motility, in particular hyperactivation, is thought to aid sperm migration through the epithelial folds by preventing entrapment and increasing the probability of cumulus contact (Suarez et al., 1987; Katz et al., 1989Go). Hyperactivated motility is important in promoting penetration of the zona pellucida (Katz et al., 1989Go), though this has not been shown necessary for sperm–oocyte fusion (Fleming and Yangimachi, 1982).

Evidence to date suggests that spermatozoa from men with ICS have a normal ability to capacitate and acrosome react in vitro and can fuse with the vitelline membrane if micromanipulated to lie adjacent to the oocyte (Aitken et al., 1983Go). In view of the poor sperm motility and in particular the absence of hyperactivation, spermatozoa from men with ICS are unlikely to achieve fertilization in vivo.

The impaired sperm motility in ICS is due to axonemal disruption. The sperm tail has dynein arms, which link microtubular doublets in the axonemal complex (Eliasson et al., 1977Go). These dynein arms are the site of ATPase activity and mediate the sliding between doublet microtubules, essential for normal ciliary motion (Afzelius, 1986Go). When dynein arms are lacking, a variety of ineffective ciliary motion patterns can occur, resulting in poor or no sperm motility. Several other ultrastructural defects have been noted in this condition, including lack of radial spokes (Sturges et al., 1979Go), absent central pair of microtubules (Bacetti et al., 1979Go), transposition of microtubules (Sturges et al., 1980Go) and dysplasia of the fibrous sheath (Chemes et al., 1987Go). In this reported case many of these abnormalities could be identified, varying from minor defects with transposition of one doublet of microtubules outside the fibrous sheath and lack of dynein arms to severe defect with no recognizable pattern of microtubules.

Assessment of sperm physiology from men with ICS is necessary in order to devise a rational approach to treatment of this disorder. ICSI would appear to be an appropriate treatment as it avoids the need for spermatozoa to attach and penetrate the zona pellucida. It has been shown that sperm motility does not affect success rates with ICSI (Mansour et al., 1995Go). Pregnancies have been reported following ICSI in men with totally immotile spermatozoa (Terriou et al., 1993Go; Nijs et al., 1996Go) and there have been reported pregnancies resulting from ICSI using immature sperm precursor cells (Tesarik et al., 1995Go; Barak et al., 1998Go). In men with ICS, pregnancy has been reported following subzonal insemination (Wolf et al., 1993Go; Nijs et al., 1996Go) and there have been live births following ICSI (Brugo et al., 1997Go). In a recent publication (Chemes et al., 1998Go), a mosaic variant of ICS with dysplasia of the fibrous sheath was treated with IVF and ICSI. There were no pregnancies with IVF, however there was successful fertilization following ICSI in six patients with two resultant pregnancies. However, both subzonal insemination and ICSI require expertise not available in all units and there are concerns over costs and outcomes.

IVF has been previously tried as a treatment for ICS with no success (Chemes, 1993). Our case is the first documented successful IVF treatment of ICS. Unfortunately, only one oocyte was obtained and therefore we are unable to comment on the fertilization rate using this patient's spermatozoa.

It is difficult to understand how spermatozoa with such severe axonemal disruption and impaired motility can penetrate the zona pellucida. Paternity was not proven directly in this case, however routine IVF safety procedures were performed by the embryologist to ensure correct paternity. It could be argued that not all spermatozoa were affected by axonemal disruption in this case. However, in all spermatozoa assessed at repeated electron microscopy there were axonemal defects. When assessed manually and with computer-assisted motility analysis, there were greatly reduced mean motility parameters (total motility, progressive motility, mean path velocity, tract speed, lateral amplitude and beat frequency). There was overlap between the sample population and the normal range, and it is therefore possible that a few spermatozoa had motility parameters within the normal range. However, no spermatozoa displayed hyperactivation. It is possible that despite no hyperactivated motility spermatozoa can penetrate the zona pellucida. Lastly in view of the heterogeneous nature of Kartagener's syndrome, it is difficult to be certain that this case truly has Kartagener's syndrome rather than severe asthenozoospermia with other abnormalities being coincidental. However, this seems unlikely as all the clinical features and most of the laboratory features of Kartagener's syndrome are present in this patient.

There are many incongruities concerning male infertility in ICS. Spontaneous pregnancies have been reported in 12 men affected by Kartagener's syndrome, though paternity was not proven (Rott et al., 1979). Kartagener's syndrome has been diagnosed in men with normal ciliary and flagellar structure (Greenstone et al., 1985Go; Escudier et al., 1987Go), in cilia without dynein arms and normal flagella (Jonsson et al., 1982Go) and in flagella without dynein arms and normal cilia (Wilton et al., 1986Go). A variant of ICS has been identified (Chemes et al., 1998Go), in which there is dysplasia of the fibrous sheath associated with lack of dynein arms in respiratory and sperm flagella. Two subgroups were recognized: a complete form in which all spermatozoa were affected and an incomplete form with alterations in 70–80% of spermatozoa. It is clear that ICS is a heterogeneous group of disorders with similar clinical presentations but varied structural defects. This may be due to ciliary and flagellar dynein–microtubular binding factors being under separate genetic control or perhaps due to chimeras with mutations occurring during embryogenesis with only certain cells being affected. Lastly, it is possible that in certain cases situs invertus has occurred as an isolated defect and the infertility and chronic respiratory symptoms developed coincidentally due to an unrelated cause.

The heterogeneous nature of ICS makes the planning of treatment difficult. It has been shown that there is a correlation between structural and functional ciliary abnormalities in sperm samples from men with ICS (Afzelius, 1986Go). Until more is known with regard to the genetic control of ICS, we would suggest that treatment should be individualized depending on sperm motility. In cases where there is no sperm motility, ICSI may be the most appropriate treatment. However, if sperm motility is present a trial of IVF should be considered.

One concern regarding the fertility treatment of men with ICS is the possibility that the resultant child has the risk of being affected by the same condition. Although the exact genetic control of ICS is not understood, it is thought to be an autosomal recessive condition (Afzelius, 1986Go). Until the genetic basis for this condition is known, it is necessary to counsel couples regarding the possibility of genetic risks and to follow-up children fathered by men affected by ICS.

This case report demonstrates that spermatozoa from a man with ICS can penetrate the zona pellucida and fertilize oocytes in vitro despite impaired motility due to severe axonemal defects. IVF should be considered as a treatment option for infertile men with ICS when sperm motility is present. This case has important implications both for the treatment of infertile men with ICS and to further our understanding of the role of sperm motility in the fertilization process.


    Acknowledgments
 
The writers are grateful to Dr MacIntyre (Consultant Pathologist at the Western General Hospital, Edinburgh) for the excellent electron microscopy.


    Notes
 
3 To whom correspondence should be addressed Back


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 Abstract
 Introduction
 Case report
 Discussion
 References
 
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Submitted on December 19, 1998; accepted on October 5, 1999.