Mucociliary function, ciliary ultrastructure, andciliary orientation in Young's syndrome

Robbert de Iongh, Alvin Ing, Jonathan Rutland

AbstractBackground Mucociliary clearance isimpaired in patients with Young's syn-

drome (obstructive azoospermia withrecurrent sinobronchial disease), cysticfibrosis, and primary ciliary dyskinesia.No defect of cilia or mucus has beendetected in Young's syndrome.Methods Ciliary function and ultra-structure, including ciliary orientation,were studied quantitatively in 20 patientswith Young's syndrome and 20 normalsubjects to determine the incidences ofciliary defects. Nasal ciliated epitheliumwas obtained from each subject and usedfor measurement of ciliary beatfrequency and ultrastructural analyses.Ciliary orientation was determined bymeasuring ciliary deviation in electronmicrographs; ciliary deviation is a

measure of the relative orientation ofcilia in relation to each other in whichhigh values indicate ciliary disorienta-tion.Results Ciliary beat frequency and theincidence of microtubular defects andnumbers of dynein arms did not differbetween patients with Young's syndromeand control subjects. In patients withYoung's syndrome basal ciliary deviation(16-00) was similar to that in controlsubjects (14-1°), but at the ciliary tipciliary deviation (21.90) was greater thanin healthy subjects (14.50).Conclusion The relative disorientationof the distal ciliary axoneme in patientswith Young's syndrome compared withnormal subjects may be due to a struc-tural defect but is more likely to be aconsequence ofabnormal mucus.

Respiratory Unit,Concord Hospital,Concord, New SouthWales 2139, AustraliaR de IonghA IngJ RutlandAddress for correspondence:Mr de IonghAccepted 22 November 1991

The association between male sterility andrecurrent respiratory tract infection is commonto cystic fibrosis,' primary ciliary dyskinesia,2and Young's syndrome.3 Young described 52men with obstructive azoospermia, of whomover half "had had a defect in their lungs,"including bronchitis from childhood and bron-chiectasis.' No underlying cause has beenestablished for the respiratory disease in suchpatients or the association with obstructiveazoospermia.

Mucociliary clearance is an importantdefence mechanism in the respiratory tract andalteration of either of its two main components(ciliary activity or mucus) may result inrecurrent respiratory tract disease. In cystic

Thorax 1992;47:184-187

fibrosis abnormalmucus secretion is believed tocause impaired mucociliary clearance andhence recurrent respiratory tract infection.'Azoospermia in male patients with cysticfibrosis is due to atresia or malformation of thevasa and epididymis.4 In primary ciliary dys-kinesia impaired mucociliary clearance hasbeen shown to be due to structural defects ofthe ciliary axoneme.59 Similar structuraldefects of the sperm tail axoneme in thesepatients result in immotile or poorly motilesperm. In patients with Young's syndromenasal and tracheobronchial mucociliary clear-ance is impaired or absent.' 101 No underlyingmechanism has been identified to explain eitherthe respiratory problems or the obstructiveazoospermia. Most studies of Young's syn-drome report that cilia and sperm have normalaxonemal ultrastructure, "'14 althoughmicrotubular abnormalities, 5 16 compoundcilia,'6 and reduced numbers of inner dyneinarms'5 have been reported in individualpatients. Abnormal ciliary orientation has beendescribed in two patients but not quantified.'2We report a quantitative ultrastructural

analysis of nasal ciliated epithelium in 20patients with Young's syndrome comparedwith a group of20 healthy subjects matched forage and smoking habits. In particular we

examined the orientation of cilia with a recentlydeveloped computer aided technique.'7

Patients and methodsTwenty patients with azoospermia (mean age44 (SD 12) and a history of recurrent lowerrespiratory tract infection or sinusitis, or both,were studied. All patients had a clinicalinterview, sinus and chest radiography, andphysical examination. Primary ciliary dys-kinesia was excluded by measurement of ciliarybeat frequency and ciliary ultrastructure. Noneof the patients had a family history of cysticfibrosis. These patients did not have sweat tests,and those in whom the possibility of cysticfibrosis was considered were excluded from thisstudy. Apart from one patient whose brotheralso had features ofYoung's syndrome, none ofthe patients had a family history of respiratorytract infection. None of the patients had anygastrointestinal or pancreatic manifestations ofcystic fibrosis. Obstructive azoospermia was

documented in all patients, and eight patientshad testicular biopsies showing normal spermmaturation. Two patients had bilateral micro-surgical epididymovasostomies, which were

successful in one. At the time of investigationnone of the patients had current or recent

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Mucociliary function, ciliary ultrastructure, and ciliary orientation in Young's syndrome

Table 1 Respiratory function in patients with Young's syndrome (n= 14). All valuesare percentages ofpredicted values in an Australian population

FEV, FVC FRC RV TLC TLCO

Mean 804 91 6 83-9 105 93-6 85-3SD 15-4 152 15-7 29 12-6 13-6Range 54-108 77-126 48-106 66-186 76-118 64-105

FEV, = forced expiratory volume in one second; FVC = forced vital capacity; FRC =

functional residual capacity; RV = residual volume; TLC = total lung capacity; TLCO =carbon monoxide transfer factor.

respiratory tract infections (either bacterial orviral).

Chest radiographs in 16 patients showedabnormalities in 13, including cystic changes,bronchiectasis, calcified lymph nodes andpleural shadows. Radiographs of the paranasalsinuses were abnormal in 11 of the 16 patientsexamined. Changes included mucosal thicken-ing, fluid levels, and polyps. Results of res-piratory function tests are listed in table 1.Twenty volunteers (mean age 42 (12)) with

no history of recurrent respiratory tract diseasewere also studied. They were matched with thepatients for age and smoking habits (table 2).The study was approved by the ethics commit-tee at Concord Hospital, and all subjects gaveinformed consent.

INVESTIGATIONSNasal mucociliary clearance time was measuredby the saccharin test.'8 When no clearance wasdemonstrated after one hour a value of 60minutes was assigned for calculation of thegroup mean clearance time. Nasal mucociliaryclearance time was not measured in controlsubjects, as a previous study established thatnasal mucociliary clearance time by this tech-nique is less than 30 minutes in normalsubjects.'8 Respiratory function tests were per-formed in only 14 patients.

Samples of nasal ciliated epithelium wereobtained by nasal mucosal brushingl920 from allsubjects for photometric determination'9 ofciliary beat frequencies and for electronmicroscopy. 1720 The following features ofciliary ultrastructure were analysed quanti-tatively from electron micrographs of trans-versely sectioned cilia: the incidence ofcompound cilia, the incidence of central andperipheral microtubule defects, the numbers ofinner and outer dynein arms per cilium, andciliary orientation. Peripheral tubule defectswere defined as missing or displaced peripheral

Table 2 Smoking history and mucociliary function in patients with Young's syndrome

Age (years) Pack years NMCC (min) CBF (Hz)

Normal subjects (n = 20)Mean 44 10 7 10* 116SD 12 16-8 2 10Range 28-68 0-50 <30 10-137

Patients with Young's syndrome (n = 20)Mean 42 11-7 33t 11 9SD 12 179 21 1-3Range 29-71 0-54 9-60+ 10-2-15-1

NMCC = nasal mucociliary clearance time; CBF = ciliary beat frequency.*Data from Rutland and Cole.'8tUnderestimation of true mean-an arbitrary value of 60 min was assigned for NMCC> 60 min.

doublets or extra microtubules (single or dou-ble) outside the peripheral ring of doublets.Central tubule defects included missing centralmicrotubules or extra single microtubuleswithin the peripheral ring of doublets. Profilesof cilia that were clearly sectioned near the tipor base of the cilium were not included in thecounting procedures as the normal terminationof ciliary microtubules at these levels may leadto an overestimation of abnormal cilia. Therelative orientation of cilia was also measured.'7The ciliary beat axis is perpendicular to a linedrawn through the centres of the two centralmicrotubules (figure 1). The angle subtendedby this line to an arbitrary reference line wasmeasured from electron micrographs with agraphics tablet interfaced to a computer.'7 Themean ciliary angle and the standard deviation(ciliary deviation) from this mean angle weredetermined. The ciliary deviation provides ameasure of the variation in the ciliary beat axis.Ciliary deviation was measured at two levels:near the tips and near the bases of the cilia. Intransverse sections of cilia the presence ofmicrovilli indicates a plane of section near thebases of the cilia and the absence of microvilli

q.w, *

Figure I Variation in ciliary orientation along theciliary shaft in a patient with Young's syndrome. Theorientation of cilia at the ciliary tips (B) was moredisorganised (ciliary deviation 31 1') than at the ciliarybases (A) (ciliary deviation 12 5').

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de Iongh, Ing, Rutland

Table 3 Ciliary ultrastructure in patients with Young's syndrome

Ciliary deviation (degrees)

CC (%) CT (%) PT (%) DAilcilium DAo/cilium Base Tip Total

Normal subjects (n = 20)Mean 03 1'0 18 56 84 141 145 148SD 05 18 13 1 1 04 42 35 41Range 0-16 0-78 0-50 37-80 76-89 74-267 93-239 97-24

Patients with Young's syndrome (n = 20)Mean 1 4 1-7 3-3 5-7 8-5 16 6 21 9* 20 4*SD 20 18 48 1.1 05 75 87 70Range 0-75 0-66 0-215 37-76 70-90 95-214 104-401 117-334

*p < 001 (t test).CC = compound cilia; CT = central tubule defects; PT = peripheral tubule defects; DAi = inner dynein arm numbers;DAo = outer dynein arm numbers.

indicates a plane of section nearer the tips.After separate analyses of ciliary deviation atthese two levels data were pooled to provide atotal measure of ciliary deviation for eachpatient.

ANALYSISWhen data were distributed normally (nasalmucociliary clearance time, ciliary beatfrequency, ciliary deviation, and numbers ofdynein arms per cilium) analysis of varianceand t tests were used. The incidence of com-pound cilia and of central and peripheralmicrotubule defects and the numbers of innerand outer dynein arms per cilium were com-pared by the Mann-Whitney U test.

ResultsMean nasal mucociliary clearance time inYoung's syndrome patients was significantlyprolonged (p < 0001) compared with normalvalues. Mean ciliary beat frequency was notsignificantly different (p > 0-05) from normalsubjects. Ciliary beat frequency in all patientswith Young's syndrome was within normallimits. Ten patients had nasal mucociliaryclearance times greater than 60 minutes(table 2).

Cilia from patients with Young's syndromehad normal ciliary ultrastructure with the nor-mal complement of microtubules (9 + 2) anddynein arms (table 3). Although not quantified,radial spokes were also visible and seemed to bepresent in normal numbers. Some patients hadhigher numbers of compound cilia and peri-pheral microtubule defects than had normalsubjects, but as a group there was no significantdifference. Mean ciliary deviation for patientswith Young's syndrome was within normallimits,'7 but there was a significant differencebetween normal subjects and patients withYoung's syndrome (p < -0 1). Seven patientshad mean ciliary deviation (25.50, 26.6°, 29.30,33.6°, 33.70, 36.70, 39.60) outside the range fornormal subjects. Random ciliary orientation,often seen in patients with primary ciliarydyskinesia,"7 was not found in any patient withYoung's syndrome. Comparison of ciliarydeviation at two levels along the length of theaxoneme showed that at the ciliary base ciliarydeviation was not significantly different from

that in normal subjects, although at the tips ofthe cilia ciliary deviation was significantlygreater (p < 0(01) than that in normal subjects.The different orientation of cilia at these twolevels is shown in figure 1. The distribution ofciliary angles in patients with Young's syn-drome (figure 2) showed a small increase in thenumbers of cilia with large angles of deviationfrom the mean angle compared with normalsubjects.

DiscussionThe aetiology of Young's syndrome is un-known.'0'21-24 Repeated studies have shownsweat electrolyte concentrations to be nor-mal.'416212426 We have confirmed the finding

Normal subjects(n = 4593 cilia)

50-

40'

30

20

10

cJ

CU)L.U,

0'

60 1

50 -

40 -

30 -

20 -

10-

0-

10 20 30 40 50 60 70 80 90

Young's syndrome(n = 4700 cilia)

10 20 30 40 50 60 70 80 90

Ciliary deviation (0)

Figure 2 Pooled distribution histograms of ciliary anglesof deviation in normal subjects and in patients withYoung's syndrome. The distributions were similar, but thepatients had slightly increased numbers of cilia withdeviationsfrom the mean angle in the range of 40-90' andthefrequency of deviations of less than 10° was reduced by5%.

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Mucociliary function, ciliary ultrastructure, and ciliary orientation in Young's syndrome

that mucociliary clearance may be impaired butwith normal ciliary beat frequency and internalciliary ultrastructure." There was no evidenceof reduced numbers of inner dynein arms or anincreased incidence of microtubule defects asshown in case reports.'5 16 The increasedincidence ofcompound cilia and the occurrenceof microtubule defects that we found confirm aprevious report,'6 although these defects areunlikely to be a primary defect as they occur innormal subjects and as non-specific defectsafter respiratory tract infection.27"Abnormal ciliary orientation has been des-

cribed previously in Young's syndrome but hasnot been quantified.'2 Ciliary deviation in ourgroup of patients was significantly greater thanthat in normal subjects but was not as great asthat seen in primary ciliary dyskinesia.'7 Ciliarydisorientation may be the only defect inpatients with recurrent respiratory disease."2This finding of moderate ciliary disorientationcould offer a possible explanation for thereduced mucociliary clearance found inYoung's syndrome. It is possible that thetwisting of the cilia may be a manifestation of asubtle structural microtubule defect not detec-table by electron microscopy. The inconsis-tency ofthis finding suggests, however, that thisis probably an epiphenomenon and more likelyto be a secondary effect of recurrent respiratorytract infection than a primary cause of therespiratory tract disease.Young's syndrome has been proposed as

being a forme fruste of cystic fibrosis thatresults in abnormal respiratory mucus andinspissated seminal secretions. AlthoughYoung's syndrome is probably not a variant ofcystic fibrosis, because sweat electrolyte con-centrations are normal, the pancreas is notaffected, and the clinical picture and genitaldysfunction are different, the rheologicalproperties of mucus may be abnormal inYoung's syndrome. Lopez-Vidriero et alreported the viscoelastic properties ofmucus inYoung's syndrome and considered them con-sistent with a "chronic hypersecretorydefect."" The finding of ciliary disorientationonly at the tips of cilia in our group of patientsmight be consistent with this suggestion ofabnormal mucus. Viscid secretions may causecilia to beat inconsistently or become trappedin the mucus layer, resulting in twisting of thedistal parts of the ciliary axoneme. A mucusdefect of varying severity might explain thevariable occurrence of ciliary disorientation inYoung's syndrome. A trial of "mucoregulatoryagents" in Young's syndrome showed no effecton mucociliary clearance, sperm counts, orpulmonary function.'4We have found more variable orientation of

the distal ciliary axoneme in patients withYoung's syndrome than in normal subjects.Though this might be due to a subtle structuraldefect, it could also be a consequence ofabnormal mucus.

We thank Associate Professor R Newland and Mr J R Perkins ofthe Department of Anatomical Pathology, Concord Hospital,for their help and access to the facilities of the electronmicroscopy laboratory. This work was supported by a researchgrant from the Department ofVeterans' Affairs, Commonwealthof Australia, and the Clive and Vera Ramaciotti Foundation.

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