Until now, the etiology of keratoconus has been poorly understood.1 There seems to be a complex interaction between genetic, biomechanical, biochemical, and environmental factors in keratoconus,1 but their relative contributions are currently unknown. It has been shown that collagenase enzymes such as matrix metalloproteinases (MMPs) might be involved in the pathogenesis of keratoconus. These MMPs are upregulated in association with the dysregulation of the cortisol rhythm.2
Measurements of acute cortisol levels from blood, saliva, or urine samples can be highly volatile because they exhibit substantial ultradian and circadian rhythmicity and are influenced by numerous situational factors. In addition, they are less useful for deriving reliable information about long-term cortisol output patterns. This makes the assessment of stable long-term systemic cortisol exposure using these methods difficult.3 Hair analysis has been used for a long time to monitor exposure to certain compounds (eg, drug monitoring).4 Head hair grows at an average rate of 1 cm/month, and assessments of drugs present in hair samples can reflect changes over time.5 Because cortisol also accumulates in growing hair, hair cortisol concentrations should reflect integrated cortisol secretion over a period of months.6
In this study, we analyzed hair cortisol concentration values in patients with progressive keratoconus to evaluate the possible influence of cortisol on the progression of this disease.
Patients and Methods
In this prospective observational study, we measured the hair cortisol concentrations of patients with stable and progressive keratoconus and of healthy controls. Eighty-six participants were examined between May 2015 and November 2016 at the Department of Ophthalmology of the University Hospital Carl Gustav Carus, TU Dresden. The study protocol was approved by the Medical Ethics Committee of the TU Dresden. Written informed consent was obtained from each participant before examination.
Study participants were recruited from our ectatic disease clinic, and three study groups were formed. The progressive keratoconus group (n = 32) included patients who revealed significant changes in corneal topographic parameters as observed via a corneal topographic analysis using a Pentacam device (Oculus Optikgeräte, Wetzlar, Germany) and thus were indicated for corneal cross-linking. The significant progression of keratoconus was based on an increase in maximum keratometry value at the apex of the cornea of 1.00 diopter (D) in 1 year and/or a mean decrease in corneal thickness of 5% or greater based on corneal thickness measurements within the previous 12 months.7 The stable keratoconus group (n = 32) consisted of patients with keratoconus that was stable in regard to the above-mentioned criteria for progression. Additionally, a group of healthy controls (n = 22) was formed to obtain reference values for normal hair cortisol values for use in comparing hair cortisol levels among the three groups. In this group, the presence of subclinical ectatic corneal disease was excluded based on inconspicuous clinical examinations and tomographic and topographic values.
The inclusion criteria were a diagnosis of keratoconus in the progressive and stable keratoconus groups and, for all groups, a hair length of at least 3 cm at the back of the head. The diagnosis of keratoconus was confirmed based on a Pentacam and Ocular Response Analyzer (Reichert Technologies, Inc., Buffalo, NY) analysis; unclear cases were not enrolled in this study.
For the progressive keratoconus group, only patients who revealed significant changes in the corneal topographic values mentioned above and who were indicated for corneal cross-linking were enrolled. For inclusion in the stable keratoconus group, patients must have had stable corneal thickness values for the previous 12 months. The exclusion criterion for both keratoconus groups was previous cross-linking in both eyes. Cross-linking in one eye was permitted in either group.
The exclusion criteria for all groups were presence of other corneal pathologies, a history of ocular surgery, uveitis, glaucoma, connective tissue disease, rheumatological disorders, diabetes mellitus, psychiatric diseases, a history of medications that might affect hormone levels (eg, systemic steroids), and pregnancy. In all groups, only participants 18 years and older were enrolled.
Relevant sociodemographic variables (sex, age, body mass index [BMI], and hormonal contraception) and ocular and medical histories were recorded. In addition, self-reports of participants' individual stress levels within the previous 3 months were obtained using a 10-point visual analogue scale (1 = “no stress,” 10 = “maximum stress”). The Trier Inventory for the Assessment of Chronic Stress screening scale was employed as a self-reported measure of perceived stress within the previous 3 months.8
Hair strands from all participants were analyzed. Based on an average hair growth rate of 1 cm/month, the hair cortisol concentration in a hair segment 3 cm in length can be considered to represent the cortisol profile during the previous 3 months. We investigated hair strands with a diameter of approximately 3 mm, which were taken near the scalp and from a posterior vertex location. Cortisol concentrations were determined from the 3-cm hair segment most proximal to the scalp. Cortisol levels were determined at the Institute of Biopsychology, TU Dresden using a commercially available immunoassay kit with chemiluminescence detection (CLIA; IBL International GmbH, Hamburg, Germany). Detailed methods of the hair cortisol analysis have been described elsewhere.9
Because the hormone values were usually not normally distributed, the cortisol values obtained underwent a logarithmic transformation.
The comparison of the three groups was performed using an analysis of covariance (ANCOVA) that considered several factors (sex, age, BMI, and hormonal contraception) as covariates, followed by Sidak's post hoc tests. The adjusted mean values were back-transformed, and 95% confidence intervals (CIs) were determined.
The statistical analysis was performed using SPSS software (version 21; IBM Corporation, Armonk, NY) and a P value of less than .05 was considered statistically significant.
The progressive and stable keratoconus groups included 32 eyes each, and the healthy control group included 22 eyes. Patients with keratoconus were 27.3 ± 10.0 years old, and the participants in the healthy group were 31.5 ± 9.6 years old (P > .05).
In patients with progressive keratoconus, hair cortisol concentrations were higher than those in patients with stable keratoconus (mean value: 29.11 [95% CI: 22.13 to 8.28] vs 15.88 [95% CI: 12.25 to 20.65] pg/mg, P = .007). In healthy controls, hair cortisol concentrations were significantly lower than those in the progressive keratoconus group (mean value: 29.11 [95% CI: 22.13 to 38.28) vs 16.98 [95% CI: 12.30 to 23.44) pg/mg, P = .049). However, there was no difference between the healthy controls and patients with stable keratoconus (mean value: 16.98 [95% CI: 12.30 to 23.44) vs 15.88 [95% CI: 12.25 to 20.65) pg/mg, P = .991) (Figure 1).
Absolute values of hair cortisol in the two groups: patients with progressive keratoconus had higher levels than the stable keratoconus group and healthy controls.
The ANCOVA found no influence of age, sex, BMI, or hormonal contraception.
Normal hair cortisol concentrations range from 5 to 25 pg/mg,10 so a threshold of 25 pg/mg was assumed. Values above this threshold were more frequently found in the progressive keratoconus group than in the stable keratoconus group (50% vs 25%, P = .039), whereas there was no difference between the stable keratoconus group and healthy controls (25% vs 23%, P > .05) (Figure 2).
Distribution of hair cortisol values between the three groups with a cut-off of 25 pg/mg. Elevated levels were more common in patients with progressive keratoconus than in the stable keratoconus group and healthy controls.
Regarding sociodemographic parameters, BMI values were significantly higher for patients with progressive keratoconus than for those with stable keratoconus and for healthy controls (27.56 ± 7.4 vs 24.75 ± 4.8 vs 22.38 ± 2.5 kg/m2, P = .007), and there was no difference between patients with stable keratoconus and healthy controls (P > .05).
No differences were found in age, sex, hormonal contraception, or subjective stress levels measured via the Trier Inventory for the Assessment of Chronic Stress among the three groups. There was also no correlation between Trier Inventory for the Assessment of Chronic Stress scores and hair cortisol values (P = .535, Table 1).
TICS Scores and Hair Cortisol Concentrations
One of the first theories proposed to explain the pathogenesis of keratoconus was the release of proteolytic enzymes that degrade stromal collagen and weaken the cornea. Generally, the level of antioxidants is reduced in keratoconus, resulting in more oxidative stress and oxidative damage, which in turn induces a cascade of elevated enzyme activity, leading to alterations in proteins and a further worsening of keratoconus. Cortisol is able to upregulate enzyme levels. An increase in endogenous cortisol levels is caused by the activation of the hypothalamic–pituitary–adrenal (HPA) axis (eg, via acute and chronic stress). Increased cortisol levels could lead to increases in the production and activation of MMPs and collagenolytic enzymes11 and to accelerated matrix degeneration,12 potentially leading to the development of keratoconus. Brown et al.13 showed significant increases in the lytic action of collagenases in rabbit corneas caused by cortisol. Nemet et al.14 found a strong association between keratoconus and certain immune disorders. This is explained through potential inflammatory mechanisms leading to corneal thinning mediated via increased proteinase activity15 of MMPs within corneas of patients with keratoconus.
There is evidence in the literature that conditions such as those experienced during Ramadan fasting, which may alter a variety of physiological parameters, may also affect the ocular system.16 An increase in cortisol levels has been well-documented during fasting.16 In addition, several studies report elevated stress hormone levels in association with sleep apnea,17 which is more prevalent in patients with keratoconus.18
Monk et al.19 measured elevated cortisol concentrations in equine tears after stimulation with adrenocorticotropic hormone. The effect of cortisol on ocular pathologies needs to be investigated.19 In the current study, we hypothesized that elevated cortisol levels are an important risk factor for the progression of keratoconus, and we tested this hypothesis in the context of the possible pathogenic mechanisms illustrated below (Figure 3).
Hypothetical scheme for the possible influence of increased cortisol levels on keratoconus.
We found a significantly higher HCC in patients with progressive keratoconus than in those with stable keratoconus and healthy controls. This suggests that cortisol could play an important role in the pathogenesis of keratoconus. Until now, there has been no published in vivo evidence suggesting that the progression of keratoconus could be influenced by elevated cortisol levels. Spoerl et al.20 observed that high cortisol levels altered corneal biomechanics in their study using porcine eyes. In addition, Yu et al.21 demonstrated a reduced corneal stiffness in rabbit eyes that had been treated in advance with cortisol eye drops. Additional associations between cortisol and biomechanical changes have also been reported in other biological tissues, such as blood vessels, skin, lungs, tendons,22 and cartilage.
Priyadarsini et al.23 showed an association between elevated prolactin levels and keratoconus. Estrogens are known to regulate the levels of certain MMPs24 and to stimulate gene expression, inducing changes in corneal biomechanical properties25 and leading to the progression of keratoconus.26 There are estrogen receptors on the keratocytes of the cornea, and high estrogen levels during pregnancy can impair corneal biomechanics.25,27,28
Elevated cortisol levels could also lead to the progression of keratoconus due to alterations of the thickness and biomechanics of the cornea. In this study, there was no difference in cortisol levels between healthy controls and patients with stable keratoconus; therefore, not every patient with keratoconus seems to develop elevated cortisol levels. However, although measurements of hair cortisol levels have been suggested as a new biomarker for chronic stress,3 we did not observe any influence of subjective stress levels measured via the Trier Inventory for the Assessment of Chronic Stress between the three groups. It has been shown in multiple studies that there is no consistent correlation between hair cortisol concentrations and self-reports of perceived stress. This is another example of a “lack of psychoendocrine covariance,” or at least of a high degree of inconsistency in self-reported measures of stress, which has also been reported for other measures in association with cortisol levels, such as stress reactivity29 or the cortisol awakening response. One explanation could be that stress-related questionnaire data can be negatively affected by the variability in individuals' “awareness of their affective states” and by social desirability and retrospection bias.30 This supports the fact that an objective method of measuring chronic stress, such as using hair cortisol concentrations, is needed because subjective methods are not sufficient for detecting individual stress levels.
Chronic stress alone does not appear to significantly influence cortisol levels in all people equally, which might be due to different personality types and, hence, different responses to chronic stress. A further explanation for this hypothesis has been suggested by Trachtman,31 who described specific connections between the hypothalamus and the visual system, the so-called retinohypothalamic tracts, suggesting a further influence of the hypothalamus on the eye. This is also consistent with the findings of the current study showing that elevated cortisol concentrations seem to be associated with the progression of keratoconus, although the exact pathomechanism underlying this association needs to be further investigated.
In addition to the influence of chronic stress on hair cortisol concentration, there are further factors that can alter cortisol secretion, such as pregnancy,9 alcoholism,32 Cushing's syndrome, Addison's disease,33 and aerobic endurance sports.
The hormone thyroxin also plays a crucial role in the development of keratoconus. It has been demonstrated that thyroid gland dysfunction is prevalent among patients with keratoconus, where thyroxin and its receptors in keratocytes are significantly elevated, leading to alterations of collagen and cytokeratins.34 Therefore, thyroid disease seems to have a strong effect on corneal biomechanics.35
According to the results of this study, possible therapeutic options for preventing the progression of keratoconus include the suppression of the HPA axis, which can be accomplished via the application of cyclosporine A, which is known to reduce plasma cortisol concentrations. Shetty et al.36 reported that topical treatments with cyclosporine A reduce MMP and inflammatory cytokine levels, with a concomitant arrest of disease progression in patients with keratoconus. They suggest that cyclosporine might be a novel treatment option for keratoconus.36
Certain confounders influencing hair cortisol concentration have been reported, such as BMI, smoking status, and hormonal contraception.6 Dettenborn et al.37 demonstrated that oral contraceptive use does not influence hair cortisol levels. The results of their study further revealed a lack of any effect of smoking status, natural hair color, and the frequency of hair washing on hair cortisol levels. Interestingly, Spoerl et al.38 showed that cigarette smoke may lead to a cross-linking of collagen, possibly preventing the development and progression of keratoconus.
In this study, BMI was significantly higher for patients with progressive keratoconus than for those with stable keratoconus and healthy controls. Body fat is associated with the activation of the HPA axis, leading to elevated long-term cortisol secretion and, hence, to obesity and metabolic syndrome, as demonstrated in a study by Manenschijn et al.39 BMI and hair cortisol levels have previously been shown to be positively correlated with each other. Nevertheless, in a different study, no such correlation was found. This is consistent with the results of the current study because no influence of BMI on hair cortisol levels was found in an ANCOVA.
According to the results of this study, there might be an association between increased hair cortisol levels and the progression of keratoconus. MMPs, which are upregulated via the effects of cortisol and the HPA axis, seem to play a key role in this pathophysiologic progress; however, there seem to be additional factors influencing keratoconus progression, such as thyroid hormones. Therefore, considering a patient's individual endocrinological status when interpreting corneal topographic and biomechanical parameters should be an important factor in patient assessment.
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- Szymanowski A, Nijm J, Kristenson M, Jonasson L. Elevated levels of circulating matrix metalloproteinase-9 are associated with a dysregulated cortisol rhythm: a case-control study of coronary artery disease. Psychoneuroendocrinology. 2011;36:139–143. doi:10.1016/j.psyneuen.2010.06.012 [CrossRef]
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- Gaillard Y, Vayssette F, Balland A, Pépin G. Gas chromatographic-tandem mass spectrometric determination of anabolic steroids and their esters in hair: application in doping control and meat quality control. J Chromat B Biomed Sci App. 1999;735:189–205. doi:10.1016/S0378-4347(99)00416-8 [CrossRef]
- Wennig R. Potential problems with the interpretation of hair analysis results. Forensic Sci Int. 2000;107:5–12. doi:10.1016/S0379-0738(99)00146-2 [CrossRef]
- Stalder T, Kirschbaum C. Analysis of cortisol in hair: state of the art and future directions. Brain Behavior Immunity. 2012;26:1019–1029. doi:10.1016/j.bbi.2012.02.002 [CrossRef]
- Raiskup F, Spoerl E. Corneal crosslinking with riboflavin and ultraviolet A: Part II. Clinical indications and results. Ocul Surf. 2013;11:93–108. doi:10.1016/j.jtos.2013.01.003 [CrossRef]
- Schulz P, Schlotz W. The Trier Inventory for the Assessment of Chronic Stress (TICS): scale construction, statistical testing, and validation of the scale work overload. Diagnostica. 1999;45:8–19. doi:10.1026//0012-19220.127.116.11 [CrossRef]
- Kirschbaum C, Tietze A, Skoluda N, Dettenborn L. Hair as a retrospective calendar of cortisol production: increased cortisol incorporation into hair in the third trimester of pregnancy. Psychoneuroendocrinology. 2009;34:32–37. doi:10.1016/j.psyneuen.2008.08.024 [CrossRef]
- Albar WF, Russell EW, Koren G, Rieder MJ, van Umm SH. Human hair cortisol analysis: comparison of the internationally-reported ELISA methods. Clin Invest Med. 2013;36:e312–e316. doi:10.25011/cim.v36i6.20629 [CrossRef]
- Hillegass JM, Villano CM, Cooper KR, White LA. Glucocorticoids alter craniofacial development and increase expression and activity of matrix metalloproteinases in developing zebrafish (Danio rerio). Toxicol Sci. 2008;102:413–424. doi:10.1093/toxsci/kfn010 [CrossRef]
- Cury PR, Araujo VC, Canavez F, Furuse C, Araujo NS. Hydrocortisone affects the expression of matrix metalloproteinases (MMP-1, -2, -3, -7, and -11) and tissue inhibitor of matrix metalloproteinases (TIMP-1) in human gingival fibroblasts. Journal of Periodontology. 2007;78:1309–1315. doi:10.1902/jop.2007.060225 [CrossRef]
- Brown SI, Weller CA, Vidrich AM. Effect of corticosteroids on corneal collagenase of rabbits. Am J Ophthalmol. 1970;70:744–747. doi:10.1016/0002-9394(70)90494-0 [CrossRef]
- Nemet AY, Vinker S, Bahar I, Kaiserman I. The association of keratoconus with immune disorders. Cornea. 2010;29:1261–1264. doi:10.1097/ICO.0b013e3181cb410b [CrossRef]
- Collier SA. Is the corneal degradation in keratoconus caused by matrix-metalloproteinases?Clin Exp Ophthalmol. 2001;29:340–344. doi:10.1046/j.1442-9071.2001.d01-17.x [CrossRef]
- Javadi MA, Assadi M, Einollahi B, Rabei HM, Afarid M, Assadi M. The effects of Ramadan fasting on the health and function of the eye. J Res Med Sci. 2014;19:786–791.
- Crawford-Achour E, Saint Martin M, Roche F. Stress hormones in obstructive sleep apnea complications: the role of cortisol. Sleep Med. 2014;15:3–4. doi:10.1016/j.sleep.2013.10.004 [CrossRef]
- Gupta PK, Stinnett SS, Carlson AN. Prevalence of sleep apnea in patients with keratoconus. Cornea. 2012;31:595–599. doi:10.1097/ICO.0b013e31823f8acd [CrossRef]
- Monk CS, Hart KA, Berghaus RD, Norton NA, Moore PA, Myrna KE. Detection of endogenous cortisol in equine tears and blood at rest and after simulated stress. Vet Ophthalmol. 2014;(17 suppl 1):53–60. doi:10.1111/vop.12128 [CrossRef]
- Spoerl E, Zubaty V, Terai N, Pillunat LE, Raiskup F. Influence of high-dose cortisol on the biomechanics of incubated porcine corneal strips. J Refract Surg. 2009;25:S794–S798. doi:10.3928/1081597X-20090813-06 [CrossRef]
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TICS Scores and Hair Cortisol Concentrations
|Characteristic||Progressive Keratoconus||Stable Keratoconus||Healthy Controls||P|
|No. of patients||32||32||22||–|
|Mean age ± SD (y)||31.1 ± 9.8||32.0 ± 9.6||27.3 ± 10.0||.202|
|Mean TICS score ± SD||13.8 ± 6.4||16.5 ± 8.9||14.0 ± 7.8||.359|
|Hair cortisol (pg/mg) (95% CI)||29.11 (22.13 to 38.28)||15.88 (12.25 to 20.65)||14.0 (12.30 to 3.44)||.003|