Abstract
Introduction
Early-onset hypogonadism may increase the risk of early vascular ageing (EVA).
Methods
Boys with conditions linked to intrauterine hypogonadism – Klinefelter syndrome (KS) and hypospadias – were recruited alongside healthy controls. Assessments included clinic blood pressure (BP), flow-mediated dilatation, carotid intima-media thickness (CIMT), and pulse wave velocity. Fasting blood tests measured testosterone, anti-Müllerian hormone, luteinising hormone, follicle-stimulating hormone, triglycerides, glucose, and cholesterol. Oxidative stress and epigenetic markers were evaluated using total antioxidant capacity (TAOC), peroxide, oxidative stress index (OSI), 8-hydroxydeoxyguanosine (8-OHdG), thiobarbituric acid reactive substances (TBARS), glutathione, alpha klotho protein, histone H3 modification, telomerase, and DNA methyltransferase (DNMT) activity.
Results
Eleven boys per group were studied (median age: 14.5 years). Systolic BP was higher in KS and hypospadias compared with controls. CIMT and pulse pressure were elevated in hypospadias versus controls. Mean arterial pressure was increased in KS. Oxidative stress markers (TBARS, TAOC, peroxide, and OSI) were higher in hypospadias. 8-OHdG was elevated in hypospadias versus controls but lower than in KS. DNMT activity was increased in both KS and hypospadias. No differences were observed in alpha klotho, histone H3 modification, or telomerase.
Conclusion
Adolescents with early-onset hypogonadism show evidence of EVA, with associated oxidative stress and epigenetic alterations warranting further investigation.
Keywords: Hypertension, Hypospadias, Klinefelter, Blood pressure
Plain Language Summary
Some boys are born with conditions that affect how their bodies develop male hormones before birth, such as Klinefelter syndrome (KS) or hypospadias. These conditions may increase the risk of “early vascular ageing,” where blood vessels become less healthy sooner than expected. This study explored whether boys with these conditions show early signs of vascular changes during adolescence. We compared boys with KS and hypospadias to healthy boys of a similar age. We measured blood pressure and examined blood vessel health using ultrasound and other noninvasive techniques. We also analysed blood and urine samples to look at hormone levels, markers of oxidative stress (damage caused by harmful molecules), and indicators of epigenetic changes (biological processes that affect how genes work). We found that boys with KS and hypospadias had higher blood pressure than healthy boys. Those with hypospadias also showed thicker artery walls, which can be an early sign of cardiovascular risk. Markers of oxidative stress were higher in boys with hypospadias, suggesting increased cellular damage. Both KS and hypospadias groups showed higher activity of DNA methyltransferase, an enzyme linked to epigenetic changes. Overall, these findings suggest that boys with early-onset hypogonadism may already show signs of blood vessel ageing in adolescence. Oxidative stress and changes in gene regulation may help explain why this happens. Larger studies are needed to confirm this.
Introduction
Early vascular ageing (EVA) refers to premature changes in blood vessel structure and function typically seen in older individuals [1]. Children with EVA are at high risk of cardiovascular (CV) morbidity and mortality in adulthood [2]. Vascular function is regulated by various mechanisms, including sex steroid control [3]. Increased EVA and vascular dysfunction can be mediated by a number of different factors, including increased reactive oxygen species (ROS) generation and oxidative stress or inflammation.
“Androgens” are vasoactive, with many effects on the vasculature, including influencing oxidative stress and ROS production [4]. While androgen deficiency in adult men is known to increase CV risk [5], the vascular phenotype of boys with congenital conditions linked to early-onset hypogonadism remains unclear.
Hypospadias is a congenital anomaly that is common, occurring in around 1 in 300 live male births, secondary to early-onset hypogonadism and a lack of androgen exposure in utero during a critical period of fetal programming called the Masculinisation Programming Window [6]. During this period between approximately weeks 6 and 20 in utero, boys would be expected to have a surge of testosterone, leading to typical internal and external genital development [6]. This is supported by the association of hypospadias with genetic and biochemical defects of androgen synthesis or action. Adults with a history of hypospadias have higher CV morbidity [7, 8], and adolescents may also exhibit subclinical EVA and increased oxidative stress [7]. Other conditions, such as Klinefelter syndrome (KS), affecting 1:400 live male births, can also lead to early-onset hypogonadism and increased CVD risk [9]. It is likely that the altered androgen exposure occurs at different timepoints in boys with hypospadias and those with KS. It is not clear, therefore, what effect the timing of this reduced androgen exposure has on vascular development and function.
Biomarkers of oxidative stress are strongly associated with cardiovascular diseases (CVDs) and EVA [10]. On a cellular level, our group has previously identified increased levels of ROS in vascular smooth muscle cells obtained from boys with hypospadias compared to those from control boys undergoing religious circumcision. Incubation with the ROS scavenger, N-acetylcysteine, improved the altered vascular reactivity (increased U46619-mediated contractility and reduced acetylcholine and sodium nitroprusside-mediated vasodilatation) seen in resistance arteries from boys with hypospadias, suggesting that this is ROS-mediated [7]. In addition, assessment of biological ageing via the measurement of telomerase activity and other epigenetic changes in children at risk of EVA may also provide information about the mechanisms resulting in vascular changes in these populations. As such, this study aimed to assess whether noninvasive vascular phenotyping and markers of oxidative stress or epigenetic change differ among healthy boys, those with hypospadias, and those with KS.
Methods
Patient Selection
Boys with hypospadias or KS aged 5.0–18.9 years were invited to participate from the endocrine and paediatric surgery clinics at the Royal Hospital for Children, Glasgow, between 2020 and 2023. In addition, healthy volunteers of similar ages to the patients already recruited (healthy children of colleagues) were also invited. Exclusion criteria included any severe concurrent medical condition or medications that may alter CV dynamics. Tanner staging was performed for pubertal status, and an external masculinisation score [11] at birth was calculated for all. To assess physical activity and fitness levels, boys completed the Physical Activity Questionnaire for Adolescents (PAQA, for those aged 12 years or above) or the corresponding Physical Activity Questionnaire for Children (PAQC, for those aged 11.9 years or below) with the assistance of the researcher [12].
Blood Collection and Analysis
Fasting samples were collected for serum testosterone (T), anti-Müllerian hormone, luteinising hormone, follicle-stimulating hormone, triglycerides, glucose, and total cholesterol as described elsewhere [7].
Vascular Phenotyping
Blood pressure. Blood pressure (BP) was recorded using a Welch Allyn DS55 (Welch Allyn, UK) sphygmomanometer. It was taken in the right arm with the boy seated, and the arm supported on a table. The BP cuff varied according to the size of the boy, with the width of the cuff covering at least 75% of the upper arm from the acromion to the olecranon. Prior to BP measurement, the boys were asked to sit quietly for a minimum of 5 min and a mean of 3 BP readings was taken, from which BP standard deviation scores (SDSs) were calculated according to gender, height, and age [13].
Carotid intima-media thickness. Carotid intima-media thickness (CIMT) measurements were obtained with the Acuson Sequoia C512 (Siemens, Germany) ultrasound machine, using the 9.0-MHz transducer probe. The participant was asked to lie flat with his head turned to 90°. Ultrasound gel was applied, and a longitudinal view of the common carotid artery was taken, 1–2 cm from the carotid bulb. The mean of ten consecutive CIMT measurements was obtained, and the procedure was repeated on the contralateral side, then a mean value for both sides was calculated and converted to SDSs for age and sex [14].
Flow-mediated dilatation. Flow-mediated dilatation (FMD) was measured whilst the patient was fasting using the UNEX EF (UNEX Corporation, Japan) machine. This is a semi-automated device that uses B-Mode ultrasound to simultaneously capture longitudinal and cross-sectional views of the brachial artery. Participants were asked to lie supine with their right arm outstretched on the machine apparatus for 5 min in a quiet, temperature-controlled room for 5 min. During this time, an occlusion cuff was placed on the right forearm, and two ECG cuffs were attached to each wrist. Thereafter, a brachial cuff was placed on the left arm to obtain a resting BP. This was used to determine how much occlusion was required for analysis, with the occlusion cuff being inflated 50 mm Hg above systolic blood pressure (SBP). The ultrasound probe was placed approximately 5 cm proximal to the elbow and secured using a probe holder once an adequate image of the brachial artery was obtained. The UNEX EF software calculated the brachial artery’s resting diameter from the intima-intima via a tracking gate. The occlusion cuff was then inflated for 5 min. During this time, the participant was reminded not to move their head or body, despite any sensations of paraesthesia. Afterwards, the cuff was deflated, and the brachial artery was tracked by the software for 2 min. The diameter of the brachial artery per beat was synchronised with the R waves measured by the ECG cuffs. Automated outputs for rest diameter, maximum diameter, and % FMD were recorded.
Pulse wave velocity. Carotid femoral pulse wave velocity (PWV) was measured using the SphygmoCor XCEL (Atcor Medical, Australia). A femoral cuff was placed around the right thigh of the participant, who was asked to lie supine. The right carotid artery was then palpated, and the distances between the location of the strongest pulse point and the jugular notch and between the jugular notch and the top of the femoral cuff were measured and inputted into the SphygmoCor XCEL software. The carotid pulse was then acquired using applanation tonometry, and the femoral pulse was measured by assessing the volumetric displacement of the cuff [15] and the carotid-femoral PWV reported by the SphygmoCor XCEL software. PWV SDSs were calculated according to age and height using standard reference data [16]. Where there was a discrepancy between the SDS for age or height, the highest centile was used.
Pulse wave analysis. Pulse wave analysis was undertaken using the SphygmoCor XCEL (Atcor Medical, Australia) device. A brachial cuff, sized as per manufacturer’s instructions, was applied as the participant was lying supine. The SphygmoCor XCEL software then generated a brachial pressure waveform, whilst measuring brachial SBP and diastolic blood pressure (DBP), and this was transformed into central aortic waveforms automatically.
Measurements were converted to SDSs as appropriate for birthweight, body mass index, BP, and CIMT. SDSs are reported as per standard guidance in lieu of absolute values in most cases due to differences in reference values according to age, height, and sex.
Questionnaires. To assess physical fitness and health-related quality of life (HRQoL), which may affect vascular status, all participants were asked to complete the KIDSREEN 52 questionnaire for self-perceived quality of life[17], and the PAQA was used to assess physical activity [18].
Markers of Oxidative Stress/Epigenetic Change
Urine samples were collected and stored at −80°C prior to batch analysis. ELISAs were conducted using commercial kits according to manufacturer’s instructions: 8-hydroxydeoxyguanosine (8-OHdG) (Thermofisher, UK; catalogue no.: EEL004), thiobarbituric acid reactive substances (Abbexa, UK; catalogue no.: abx097981), total antioxidant capacity (TAOC) (Abbexa, UK; catalogue no.: abx295022), glutathione (Sigma Aldrich, UK; catalogue no.: MAK517), total peroxide (Thermofisher, UK; catalogue no. 23280), oxidative stress index (total peroxide/TAOC), alpha klotho protein (Abbexa, UK; catalogue no.: abx257320), histone H3 modification (Abcam, UK; catalogue no.: ab233495), telomerase (Abcam, UK; catalogue no.: ab285288); and DNA methyltransferase (DNMT) activity (Thermofisher, UK; catalogue no.: ab113467). All ELISAs were analysed using the SPARKControl plate reader (serial number: 2204002323).
Ethics Approval
The study was approved by the West of Scotland Research Ethics Committee 5 (REC reference: 17/WS/0063) and by the West of Scotland Research and Development Office (study reference GN17CA060P). This study was performed in accordance with the Declaration of Helsinki. All parents, guardians or next of kin provided written informed consent for the minors to participate in this study.
Statistical Analysis
GraphPad Prism 8.0 (GraphPad Software Inc, USA) was used for all analyses. Normality of data was tested using the Shapiro-Wilks normality test. Continuous variables were analysed using Brown-Forsythe and Welch ANOVA. Multiple comparisons were undertaken using Holm-Sidak’s multiple comparisons tests. A p value of <0.05 was considered statistically significant.
Results
Characteristics of the Study Population
Eleven boys were recruited per group with an overall median age of 14.5 years (6.2, 16.9). There were no differences between the groups in terms of birthweight SDS, gestation at birth, presence of co-existing conditions, or Tanner stage (Table 1). None had received testosterone therapy. Boys with hypospadias had lower external masculinisation scores compared to controls (median 7 vs. 12, p = 0.001) or boys with KS (median 7 vs. 12, p = 0.009). There were no differences in serum biochemistry or physical activity levels between the groups (Table 1).
Table 1.
Clinical characteristics of cohorts
| | Controls, n = 11 | KS, n = 11 | Hypospadias, n = 11 |
|---|---|---|---|
| Clinical characteristics – median (minimum, maximum) | |||
| Age, years | 15.5 (6.8, 16.5) | 14 (6.2, 15.4) | 14.5 (7, 16.9) |
| Tanner stage | 3 (1, 5) | 2 (1, 3) | 2 (1, 5) |
| EMS at birth | 12 (12, 12) | 12 (11, 12) | 7 (3, 10) |
| Birthweight SDS | −0.9 (−1.3, 0.5) | −0.4 (−0.03, 1) | −1.6 (−2.8, −0.8) |
| Gestation at birth, weeks | 40 (38, 40) | 39 (38, 40) | 40 (33, 41) |
| Co-existing conditions | 0 (0) | 1 (1) | 1 (1) |
| Body mass index SDS | −0.4 (−3.2, 2.1) | 0.9 (−1.2, 1.8) | −0.3 (−3.1, 1.5) |
| PAQ score | 39 (20, 83) | 34 (22, 64) | 43 (20, 58) |
| Biochemistry – (minimum, maximum) | |||
| Testosterone, nmol/L | 5.3 (0.5, 10.1) | 6.3 (0.9, 9.4) | 4.5 (0.5, 11.6) |
| AMH, pmol/L | 160 (42, 1,060) | 76 (25, 135) | 60 (11, 1,080) |
| FSH, U/L | 0.9 (0.6, 1.3) | 4.1 (1.3, 11.2) | 16.9 (1.1, 46.1) |
| LH, U/L | 0.7 (0.2, 1.1) | 3.2 (0.1, 4.6) | 2.9 (0.1, 10.6) |
| Glucose, mmol/L | 4.5 (4.3, 4.7) | 4.9 (4.0, 7.7) | 4.9 (4.2, 5.1) |
| Triglycerides, mmol/L | 0.5 (0.4, 0.5) | 0.7 (0.4, 1.3) | 0.7 (0.5, 1.7) |
| Cholesterol, mmol/L | 4.1 (3.5, 4.6) | 3.8 (3.1, 4.9) | 4.0 (3.8, 5.3) |
AMH, anti-Müllerian hormone; EMS, external masculinisation score; FSH, follicle-stimulating hormone; LH, luteinising hormone; PAQ, Physical Activity Questionnaire; SDS, standard deviation score.
Vascular Phenotyping
In boys with hypospadias and KS, median (range) SBP SDS was 1.2 (−0.5, 3.0) and 1.5 (−0.5, 2.6), respectively, compared to 0.5 (−1.0, 2.0) in controls (p = 0.04 and 0.03) (Fig. 1). There were no differences in DBP SDS. CIMT SDS and pulse pressure were also raised in boys with hypospadias (median 2.0 [0.0, 2.5] and 34 mm Hg [28, 52], respectively) compared to controls (0.0 [−1.0, 2.0]; p = 0.01 and 30 mm Hg [19, 20]; p = 0.01, respectively) but with no differences identified between boys with KS and controls. Median mean arterial pressure was increased in boys with KS with a median of 93 mm Hg (72, 100) compared to 80 mm Hg in controls (57, 95) (p = 0.04), but with no difference compared to boys with hypospadias (88 mm Hg [65, 97]; p = 0.1). FMD, heart rate, and central pressure were similar between the 3 groups.
Fig. 1.
Noninvasive vascular phenotyping of healthy control boys, boys with KS, and boys with hypospadias. N = 11 per group. Analyses via ANOVA. a Age (years). b Systolic blood pressure (SBP). c Systolic blood pressure standard deviation score (SBP SDS). d Diastolic blood pressure (DBP). e Diastolic blood pressure standard deviation score (DBP SDS). f Carotid intima media thickness standard deviation score (CIMT SDS). g Flow-mediated dilatation (FMD). g Pulse pressure. i Mean arterial pressure. j Heart rate. k Central pressure.
Markers of Oxidative Stress
Four markers of oxidative stress were higher in boys with hypospadias compared to controls or boys with KS: thiobarbituric acid reactive substances (p = 0.03 and 0.003, respectively), TAOC (p = 0.008 and p = 0.01), peroxide (p = 0.001 and p = 0.0002), and oxidative stress index (p = 0.0006 and p = 0.0007) (Fig. 2). In addition, boys with hypospadias had increased 8-OHdG levels with a median of 0.123 ng/mL (0.121, 0.132) compared to controls (median 0.119 [0.110, 0.136]; p = 0.03) but lower levels compared to boys with KS (median 0.131 [0.130, 0.132]; p = 0.005). Boys with KS had a median TAOC of 342 (279, 367) compared to a median of 272 in controls (163, 392) (p = 0.01) and a median peroxide level of 0.11 (0.4, 0.17) compared to 0.14 (0.09, 0.19) in controls (p = 0.04).
Fig. 2.
Markers of oxidative stress in healthy controls, boys with KS and hypospadias. N = 11 per group. Analyses via ANOVA. a 8OHdG. b TBARs. c Total antioxidant capacity. d Glutathione. e Peroxide. f Oxidative stress index. g Histone H3 modification. h Alpha klotho protein. i Telomerase activity. j DNMT activity. 8OHdG: 8-oxo-7,8-deoxyguanosine; KS: Klinefelter Syndrome; TBARs: thiobarbituric acid reactive substances; DNMT, DNA methylastransferase.
Markers of Epigenetic Change
No differences were demonstrated in concentrations of alpha klotho protein, histone H3 modification, or telomerase concentration between the 3 groups of boys. In boys with hypospadias and KS, median DNMT activity was 0.9 (0.5, 1.7) and 0.8 (0.3, 1.7), respectively, compared to 0.3 (0.1, 1.5) in controls (p = 0.004 and 0.01, respectively) (Fig. 2).
Discussion
This study demonstrates that boys and adolescents with different conditions associated with early-onset male hypogonadism have subclinical evidence of EVA from a young age. This is concerning because EVA is known to be associated with increased risk of CV events in later life [21]. A longitudinal study reviewing nearly 3,000 individuals over a 30-year period from childhood to mid-life demonstrated that higher SBP or DBP in early life significantly increased the risk of CV morbidity in midlife (OR 1.67–2.75) [22]. Blood vessels in youth demonstrate a degree of plasticity, whereby the signs of EVA can be reversed with appropriate lifestyle modification and intervention [23], and as such, it is essential to identify groups at risk of EVA in childhood and adolescence.
That said, different patterns of changes in vascular function were demonstrated between boys with hypospadias and boys with KS, suggesting that while both are at increased risk of EVA, the underlying causes may vary. In boys with hypospadias, there is an inadequate rise in testosterone in utero [24], as demonstrated by a reduction in anogenital distance, a marker of prenatal androgen exposure in boys with hypospadias [6]. The masculinisation programming window coincides with the timing of development of the CV system [19], which may explain the early susceptibility to EVA, given the importance of testosterone on normal vascular function. In contrast, the timing of hypogonadism in boys with KS is variable and adult men with KS are reported as having similar anogenital distance levels to 46,XY men [17], suggesting adequate prenatal androgen exposure. As such, the development of EVA in boys with KS may be secondary to a different mechanism, for example, an effect of the additional X chromosome [18, 25].
Whilst the link between hypertension and hypospadias has previously been reported [7, 8], previous studies of boys with KS have shown no increase in BP [9], albeit with increased rates of metabolic syndrome [26, 27] and CV morbidity and mortality by adulthood [28]. CV morbidity in boys with KS has also been linked to inflammation [29, 30]. Future research should therefore focus on gathering more information on anthropometry, including waist circumference, and biomarkers of CV inflammation, such as hs-C-reactive protein, asymmetric dimethylarginine, and neutrophil-to-lymphocyte ratio.
While PWV, a marker of arterial stiffness, did not differ among groups, boys with hypospadias had higher pulse pressure and those with KS had elevated mean arterial pressure – both early signs of vascular change [31–33]. Of note, the boys also had evidence of increased CIMT and pulse pressure in the absence of an increase in measured central pressure. CIMT may be affected by local haemodynamic forces within the carotid artery, which may not be fully captured by central pressure estimates measured via the device used in this study. In addition, central pressure measurements may not rise proportionally due to differences in vascular compliance and timing of wave reflections and amplification [34]. Elevated pulse pressure, even without a rise in central pressure, can lead to increased mechanical strain on the carotid artery, promoting intima-media thickness [35]. Finally, shear stress, turbulence and regional stiffness can also alter CIMT independently of central pressure [36].
Oxidative stress has a key role in the pathogenesis of EVA. Four different markers of oxidative stress were raised in boys with hypospadias, suggesting that oxidative stress is associated with subclinical vascular dysfunction seen in our cohort. In addition, both boys with hypospadias and KS had higher levels of 8-OHdG compared to controls. 8-OHdG is an indirect measure of DNA oxidation, the main consequences of which include atherogenesis, diabetes and even carcinogenesis, and studies have reported links between oxidative stress and 8-OHdG with hypogonadism [37, 38]. Indeed, 8-OHdG has been suggested as a biomarker for hypogonadism and erectile dysfunction [39] and may therefore be a useful way to monitor vascular function in children and young people with conditions such as hypospadias and KS, although further research is required.
Another consistent finding was that both boys with hypospadias and boys with KS had altered DNMT activity. Embryological cardiac and vascular development is in part regulated by DNA methylation [40], and where this is altered, there is a known association with the development of CVD [40]. Dysregulated DNA methylation has previously been described in hypospadias and KS [41, 42]. Oxidative stress can, however, also induce DNA hypermethylation by increasing DNMT expression [20]. Therefore, further research is required to confirm whether altered DNA methylation is a cause or consequence of oxidative stress or early-onset hypogonadism.
Although this study shows interesting associations with vascular parameters and possible underlying mechanisms, limitations of the study include the small sample size, single timepoint assessment, and lack of data on testosterone treatment effects. Overall, larger longitudinal studies are required to consider the clinical significance of these findings and whether they have any potential impact on clinical care for boys and men.
In conclusion, this study demonstrates that boys with conditions associated with early-onset hypogonadism, such as those with hypospadias and KS, exhibit subclinical vascular changes from a young age. Elevated SBP, increased carotid intima-media thickness and higher pulse and mean arterial pressures suggest EVA and dysfunction in these groups. Variable patterns of alterations in the vascular phenotype and in markers of oxidative stress suggest different mechanisms for EVA in boys with hypospadias and KS, and currently it is not clear whether any changes are secondary to androgen deficiency during critical periods of development, oxidative stress, altered DNA methylation or other mechanisms. Overall, the findings suggest evidence of early CV risk in populations not routinely monitored for such issues. This merits further evaluation with larger longitudinal studies, to clarify mechanisms and guide prevention of CV disease in these at-risk groups.
Acknowledgments
We are grateful to the patients and their families who participated in the study.
Conflict of Interest Statement
The authors have no conflicts of interest to disclose.
Funding Sources
A.K.L.-H. was funded by a National Education Scotland/Chief Scientist Office Clinical Lectureship (PCL/21/05).
Author Contributions
A.K.L.-H. and S.F.A. designed the study. A.K.L.-H. recruited patients and undertook study assessments, analysis of data, and writing the draft. J.M. undertook data analysis. A.K.L.-H., S.F.A., and J.M. edited versions and approved the final draft prior to submission.
Funding Statement
A.K.L.-H. was funded by a National Education Scotland/Chief Scientist Office Clinical Lectureship (PCL/21/05).
Data Availability Statement
Some or all datasets generated during and/or analysed during the current study are not publicly available due to privacy reasons, but are available from the corresponding author on reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Some or all datasets generated during and/or analysed during the current study are not publicly available due to privacy reasons, but are available from the corresponding author on reasonable request.
