Reduced artery diameters in Klinefelter syndrome

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  • ORIGINAL ARTICLE

    Reduced artery diameters in Klinefelter syndromeC. Foresta,* N. Caretta,* P. Palego,* A. Ferlin,* D. Zuccarello,* A. Lenzi and R. Selice*

    *Department of Molecular Medicine, Section of Clinical Pathology and Center for Human Reproduction Pathology, University of Padova, Padova,

    Italy, and Department of Experimental Medicine, Section of Medical Pathophysiology and Endocrinology, Sapienza University of Rome, Rome,

    Italy

    Introduction

    Klinefelter syndrome (KS) is the most common sex chro-

    mosomal disorder, with a prevalence of 1:600 and it is a

    frequent form of male hypogonadism and infertility

    (Lanfranco et al., 2004; Bojesen et al., 2006). The KS is

    characterized by the presence of one or more extra

    X-chromosomes; about 80% of cases have 47, XXY karyo-

    type, the remaining 20% have higher-grade chromosome

    aneuploidies (48, XXXY, 48, XXYY, 49, XXXXY), mosa-

    icisms (46, XY 47, XXY) or structurally abnormal Xchromosomes (Lanfranco et al., 2004). The chromosome

    aberration arises by maternal or paternal non-disjunction

    during meiotic division in germ-cell development, or

    rarely in early embryonic mitotic cell divisions (Lanfranco

    et al., 2004).

    The clinical features of KS commonly include hypergo-

    nadotropic hypogonadism, gynecomastia, small testes and

    azoospermia (Klinefelter et al., 1942), but Klinefelter syn-

    drome may be associated also with an increased risk of

    systemic diseases including malignancies, osteoporosis

    (Kubler et al., 1992), venous thromboembolism (Camp-

    bell & Price, 1981), diabetes and cardiovascular diseases

    (CVDs) (Swerdlow et al., 2005; Bojesen & Gravholt,

    2011). Various epidemiological studies in relatively large

    cohorts of patients with KS described the increased mor-

    bidity and mortality in KS. Focusing on CVDs, Bojesen

    et al. in 2004 found that KS was associated with a signifi-

    cant increase in mortality risk because of various causes,

    including circulatory diseases (Hazard ratio: 1.41). Succes-

    sively, Swerdlow et al., 2005found in KS a significantly

    raised mortality for diseases of the circulatory system, in

    Keywords:

    artery diameter, brachial FMD, Klinefelter

    syndrome, reproductive hormones

    Correspondence:

    Prof. Carlo Foresta, University of Padova,

    Department of Histology, Microbiology and

    Medical Biotechnologies, Section of Clinical

    Pathology and Center for Human

    Reproduction Pathology, Via Gabelli 63,

    35121 Padova, Italy.

    E-mail: carlo.foresta@unipd.it

    Received 22 December 2011; revised 8

    February 2012; accepted 21 February 2012

    doi:10.1111/j.1365-2605.2012.01269.x

    Summary

    Various epidemiological studies in relatively large cohorts of patients with

    Klinefelter syndrome (KS) described the increased morbidity and mortality in

    these subjects. Our aim was to study the structure and function of arteries in

    different districts to investigate in these subjects possible alterations. A total of

    92 patients having non-mosaic KS, diagnosed in Centre for Human Reproduc-

    tion Pathology at the University of Padova, and 50 age-matched healthy male

    controls were studied. Klinefelter syndrome subjects and controls evaluation

    included complete medical history, physical examination, measurement of con-

    centrations of the reproductive hormones, lipidic and glycidic metabolism, AR

    function and sensitivity, ultrasound examinations (diameters, carotid intima-

    media thickness and brachial flow-mediated dilation) of brachial, common

    carotid and common femoral artery and abdominal aorta. Klinefelter syndrome

    patients showed significantly reduced artery diameters in all districts evaluated.

    On the contrary no statistically significant difference was found in cIMT and

    brachial FMD values between KS patients and controls. Furthermore, we found

    no statistically significant correlation of artery diameters with reproductive hor-

    mones, metabolic parameters, anthropometric measures and weighted CAG

    repeats. To our knowledge, this is the first study finding a reduced artery diam-

    eter in several districts in KS patients compared with that of normal male

    subjects and overlapping to that of female subjects. We have not an explana-

    tion for this phenomenon, even if a possible involvement of genes controlling

    the development of vascular system might be hypothesized, and further

    research is required to verify this hypothesis.

    international journal of andrology ISSN 0105-6263

    2012 The Authors International Journal of Andrology, 16International Journal of Andrology 2012 European Academy of Andrology 1

  • particular for pulmonary embolism [Standardized Mortal-

    ity Ratio (SMR): 5.7; 95% CI 2.511.3], cerebrovascular

    disease (SMR: 2.2; 95% CI 1.63.0), peripheral vascular

    disease (SMR: 7.9; 95% CI 2.917.2), subarachnoid haem-

    orrhage (SMR: 3.1; 95% CI 1.26.8).

    In recent years, a large body of data focused on the

    possible role of diverse ultrasound measurements to

    detect early signs of endothelial dysfunction, which in

    turn has been considered as the primum movens with

    respect to the onset and development of CVDs.

    Several studies reported the predictive role of carotid

    intima-media thickness (cIMT) in cardiovascular (CV)

    risk stratification and future CV events (Chambless et al.,

    1997; Lorenz et al., 2007) but such relationship is weak

    and adds little to CVD prediction by traditional risk fac-

    tors (Simon et al., 2010) and remains a topic of debate,

    especially in young subjects.

    Also flow-mediated dilation (FMD) has been consid-

    ered as a predictor of CVD and other CV events, but its

    value for risk stratification of CV events is still debatable

    and further studies are needed to resolve such controversy

    (Peters et al., 2011).

    Recently, few reports documented the correlation

    between brachial artery diameter (BAD) and CV risk fac-

    tors and or CV events in the general population (Yeboahet al., 2007; Holewijn et al., 2009) and has been suggested

    a more promising tool in CV risk prediction (Kullo et al.,

    2007; Holewijn et al., 2009). On the basis of these recent

    findings, considering that subjects affected by KS have an

    increased risk of morbidity and mortality for CVD, we

    studied the structure and function of arteries (diameter,

    IMT and FMD) in different districts to investigate possible

    alterations in these subjects and to correlate these mea-

    sures with testosterone levels and function of the andro-

    gen receptor (AR) CAG length and inactivation status.

    Material and methods

    Patients and clinical analysis

    We studied, in a prospective study, a total of 92 patients

    having non-mosaic KS (mean age 31.5 8.7 years; range

    1552 years), diagnosed in Centre for Human Reproduc-

    tion Pathology at the University of Padova and 50 age-

    matched healthy male controls (mean age 30.8 8.2 years;

    range 1949 years) from January 2010 to July 2011.

    Patients had been referred to our centre for fertility prob-

    lems, testicular hypotrophy, or referred to us for clinical

    evaluation by other specialists. Healthy controls were

    recruited through andrologic primary prevention check up

    for fertility, premature ejaculation, erectile dysfunction

    and lower urinary tract symptoms. The study was

    approved by the Hospital Ethics Committee and each par-

    ticipant gave his written informed consent.

    All subjects (patients and controls) underwent periph-

    eral karyotype analysis, evaluating al least 50 peripheral

    blood lymphocyte metaphases. Each men had never

    received testosterone substitution at the time of evalua-

    tion.

    Subjects evaluation included complete medical history

    (pubertal history, lifestyle, physical activity, smoking, alco-

    hol misuse), physical examination [weight, height, body

    mass index (BMI), waist circumference, arm span, body

    surface area calculated by the DuBois-DuBois formula;

    0.007184 height (cm)0.725 weight (kg)0.425, blood pres-sure] measurement of concentrations of the reproductive

    hormones (LH, FSH, total testosterone, estradiol), lipidic

    and glycidic metabolism (total cholesterol, HDL, triglyce-

    rides, fasting glucose). Patients with more than one super-

    numerary X chromosome, mosaicisms, or with any

    endocrine dysfunction different from hypogonadism and

    subjects assuming any drug were excluded from the study.

    Blood pressure was measured in fasting conditions,

    between 08.00 and 10.00 h, avoiding cigarette smoking

    for a minimum of 12 h. Patients and controls reported a

    regular lifestyle.

    The AR function and sensitivity was studied through

    the evaluation of the CAG repeat length and inactivating

    status. The AR gene is located on the X chromosome and

    therefore in KS is present in double copy. The inactiva-

    tion rate of the two X chromosomes, and the effective

    CAG repeat value in heterozygous KS men was calculated

    as an X-weighted biallelic mean. This analysis was based

    on Methylation-Specific PCR (MS-PCR) at the human

    AR locus, with primers spanning the (CAG)n polymor-

    phism region, as previously described (Kubota et al.,

    1999).

    Hormone assays

    Blood was collected in the fasting state between 08.00 and

    10.00 h. Serum FSH, LH, total testosterone (T) and estra-

    diol (E) were evaluated by commercial electrochemilumi-

    nescence immunoassay methods (Elecsys 2010; Roche

    Diagnostics, Mannheim, Germany). For all parameters the

    intra- and interassay coefficient of variation were

  • multifrequency linear probe. Intra-observer variability was

    estimated to be less than 10%. Images were recorded and

    measured in telediastole.

    The inner to inner wall diameter (first echoic line in

    the lumen, respectively, of near and far wall) of the bra-

    chial artery was measured 4 cm above the antecubital

    region using a dedicated semiautomatic software available

    in the ultrasound machine. The arithmetic mean of six

    consecutive measurements was considered for statistical

    analysis.

    The common carotid and common femoral artery were

    measured with the same semiautomatic software and with

    same procedure, both in the trait between 2 and 4 cm

    before the bifurcation.

    Abdominal aorta was measured using a high resolution

    multifrequency convex probe 36 MHz: the mean of

    three inner to inner wall measurements in the longitudi-

    nal plan, from the infrarenal site to the aorto-iliac bifur-

    cation, was considered for statistical analysis.

    The IMT was measured in longitudinal scan, bilaterally,

    in the distal tract, at 30 mm before the bifurcation of

    common carotid artery, choosing the most linear tract of

    arterial wall as by operators judgement. For each side

    three semiautomatic measurements of the far wall IMT,

    defined as the distance from the first echoic line between

    lumen and wall vessel to the medio-adventitial margin,

    were performed and the mean value of these measure-

    ments was considered for statistical analysis.

    Vascular endothelial function was evaluated with bra-

    chial artery FMD (FMD) assessed in the morning, after an

    overnight fasting. Longitudinal B-mode ultrasound images

    of the brachial artery, 46 cm proximal to the antecubital

    crease were taken. Images were obtained after 10 min of

    supine relaxation during reactive hyperaemia, which was

    induced by inflation for 5 min to suprasystolic pressure

    (E20 mmHg) of an occlusion cuff placed around the fore-

    arm. Increased blood flow and shear stress during hypera-

    emia leads to NO-dependent FMD of the brachial artery.

    End-diastolic images were stored and arterial diameters

    were measured as the distance between the arterial wall

    intimamedia interfaces. The FMD was defined as the

    maximum per cent change in arterial diameter from 10 to

    120 s postdeflation of the occlusion cuff.

    Statistical analysis

    Differences between KS patients and controls were evalu-

    ated using unpaired two-sided Students t-test. The same

    test was used to evaluate differences between KS group

    with hypogonadism and KS group with normal testoster-

    one plasma levels. Relationships between continuous vari-

    ables were assessed using non-parametric Spearmans qcorrelation test.

    The significance level was set to p < 0.05. Variables are

    given as mean SD.

    Results

    Table 1 reports that KS patients had, as expected, signifi-

    cantly higher weight, waist circumference, BMI, body sur-

    face and gonadotropins with respect to controls and

    significantly reduced levels of total testosterone.

    Age, height, arm span, estradiol, weighted CAG repeat,

    blood pressure, history of smoking and metabolic param-

    eters (total and HDL cholesterol, triglycerides, glycaemia)

    did not show statistically significant differences between

    patients and controls.

    Table 2 reports brachial, common carotid, common

    femoral artery and abdominal aorta diameters in Klinefel-

    ter patients and in controls. KS patients showed signifi-

    cantly reduced artery diameters in all districts evaluated.

    On the contrary no statistically significant difference was

    found in cIMT and brachial FMD values between KS

    patients and controls.

    We then investigated in Klinefelter patients possible

    associations between artery diameters, hormonal, meta-

    bolic parameters and anthropometric measures. We found

    Table 1 Clinical, metabolic, hormonal parameters and anthropomet-

    ric measures in 92 Klinefelter patients compared with 50 controls

    Variable

    KS

    (n = 92)

    Controls

    (n = 50) p

    Age (years) 31.5 8.7 30.8 8.2 n.s.

    Height (cm) 181.5 7.5 179.9 6.1 n.s.

    Weight (kg) 84.3 16.8 77.3 12.0

  • no statistically significant correlation between artery

    diameters (brachial, common carotid, common femoral

    and abdominal aorta) and reproductive hormones (LH,

    FSH, total testosterone, estradiol, LH total T, T E),anthropometric measures (height, weight, BMI, body sur-

    face, waist, arm span) and metabolic parameters. Further-

    more, we found no statistically significant correlation

    between artery diameters and weighted CAG repeats (data

    not shown).

    We then subgrouped KS patients in subjects with

    reduced total testosterone plasma levels (

  • 24) and the common carotid artery diameter between 5.4

    and 5.7 mm (Jensen-Urstad et al., 1999; Ruan et al.,

    2009; Dengel et al., 2011), Gonzales et al. report in young

    women a diameter for femoral artery of 7.1 mm (Gonz-

    ales et al., 2010). Our results showed that KS patients

    have a diameter of brachial, common carotid and com-

    mon femoral artery overlapping to women despite the

    difference in height. Whether this is an effect of the extra

    X-chromosome remains unknown, although intriguing. In

    our study the height, as the other anthropometric mea-

    sures, did not differ between KS patients and controls,

    therefore the finding of smaller artery diameters is not

    influenced by them.

    Recently, few reports documented the correlation of

    brachial artery diameter (BAD) with CV risk factors

    and or CV events in the general population (Yeboahet al., 2007; Holewijn et al., 2009). Holewijn et al., 2009

    showed a negative correlation between BAD and CVD in

    337 subjects aged 5070 years enrolled from general pop-

    ulation, whereas Yeboah et al., 2007 found that the event-

    free survival rate for CV events for subjects with BAD

    below or equal to the median was higher than for those

    with BAD above the median. Furthermore, Kullo et al.,

    2007 studied asymptomatic subjects drawn from the com-

    munity (mean age 61 years) finding that greater BAD was

    associated with the presence and quantity of coronary

    artery calcium, independent from coronary heart disease

    risk factors, although this association was weak.

    To our knowledge, this is the first study finding in KS

    patients a reduced artery diameter in several districts

    (brachial, common carotid, common femoral and

    abdominal aorta) compared with that of normal male

    subjects and overlapping to that of female subjects.

    Furthermore, an involvement of hormone imbalance,

    anthropometric measures, metabolic parameters, andro-

    gen receptor sensitivity as causative for this difference has

    been excluded.

    We do not have an explanation for this phenomenon,

    even if a possible involvement of genes controlling the

    development of vascular system might be hypothesized.

    Considering that in the opposite condition of KS, Turner

    syndrome (45, X) there is a general dilatation of the same

    arterial beds (Mortensen et al., 2011), we could hypothe-

    size that a gene or genes on the X chromosome and a

    possible paralogue on the Y needs to be present in two

    copies to control normal growth of arteries, whereas hav-

    ing only one gene leads to dilatation, while three copies

    leads to insufficient growth. This of course especially

    implicates pseudoautosomal genes like for instance the

    SHOX gene, which has not so far been implicated in the

    growth of arteries, but has BNP as one of its transcrip-

    tional targets. Further research is required to verify this

    hypothesis.

    A limitation of our study is the lacking of echocardiog-

    raphyc data to correlate our findings with cardiac func-

    tion parameters. Recently Andersen et al. (Andersen et al.,

    2008) found a higher left ventricular mass index in KS

    subjects with respect to controls. This observation sug-

    gests a possible increase of peripherical resistance related

    to a reduced artery diameters. However, our patients

    showed systolic and diastolic blood pressure levels not

    statistically significant different with respect to controls,

    probably as a consequence of a reduced resistance at

    small arterial and arteriolar level.

    In conclusion, our study provides new insight into rela-

    tions between Klinefelter syndrome and CVD. For the

    first time in KS patients we observed a reduced artery

    diameter in several districts that should be implicated in

    increased CV morbidity and mortality. Further studies are

    necessary to clarify the pathogenesis and clinical implica-

    tions of our findings.

    Conflict of interest

    The authors have nothing to disclose.

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    International Journal of Andrology, 16 2012 The Authors6 International Journal of Andrology 2012 European Academy of Andrology