Androgenetic alopecia (AGA), a nonscarring progressive miniaturization of the hair follicle, has a characteristic distribution pattern in genetically predisposed men and women 1,2. In women, it typically presents as a diffuse reduction in hair density over the frontal and central areas of the scalp, but the parietal and occipital regions may also be involved 3. Occasionally, it occurs in a male pattern distribution in women 4. AGA is essentially a cosmetic disorder; however, it affects the patient psychologically 5.
Approximately 10% of premenopausal women show evidence of AGA. The incidence increases with age and almost 50–75% of women 65 years or older suffer from this condition 6.
The Ludwig scale 7 divides the severity of hair density reduction over the crown into three grades (grades I, II and III). Another grading system, a five-point visual analogue scale, the Sinclair scale 8, assesses the degree of hair loss using the midline parting, which is a simplification of the widely accepted Savin and Kalamazoo 9 density scale that classifies female pattern hair loss into eight stages of increasing crown balding, in addition to a special subcategory to detect frontal anterior recession.
AGA by name describes the two dominant causal factors, genetic susceptibility and androgens 10, where, genetic factors modify the magnitude of the hair follicle response to circulating androgens 11.
Human hair possess a unique endocrine paradox; androgens through the conversion into dihydrotestosterone (DHT) cause hair follicle enlargement in certain areas while simultaneously leading to miniaturization of scalp hair follicles and balding, that is, androgens have different effects on human hair follicles depending on the body site 12.
In 2001, Hoffmann and colleagues reported that dehydroepiandrosterone sulphate (DHEA-S) might act as an important endocrine factor in the development of AGA 13.
The androgen receptor (AR) gene is located on the X-chromosome, which is passed on from the mother to the male child 14. Polymorphism of this receptor gene was first identified in association with AGA. Identification of new susceptibility genes on chromosomes 3q26 and 20p11 indicates that non-androgen-dependent pathways are also involved 15. Genetic variability in the AR gene is the cardinal prerequisite for the development of early-onset AGA 16,17.
Concurrently, AR protein expression appears to be markedly increased within the balding vertex, compared with occipital hair follicles 18. The number of these receptors in frontal hair follicles is higher than that in the occipital area 19, and is lower by 40% in women than in men 20.
The association of AGA with underlying clinical and metabolic abnormalities is common. Although several studies have addressed this association with individual components of the metabolic syndrome (MS) (abdominal obesity, dyslipidaemia, hypertension and hyperglycaemia), there are little data on the association with the MS as a whole 21.
Hyperaldosteronism could be an explanation for the prevalence of hypertension among patients with AGA, where specific mineralocorticoid receptor antagonists are of interest in the treatment 22. Therefore, blood pressure screening and assessment of serum aldosterone levels would be valuable in the early detection of hypertension in patients complaining of AGA.
Despite the recent research efforts that have aimed at elucidating the mechanisms behind hair loss in AGA, thorough understanding has not yet been achieved. The aim of the study was to estimate the serum levels of aldosterone, free testosterone and DHEA-S and to detect aldosterone and ARs in scalp biopsy specimens in female patients with AGA and healthy controls in an attempt to clarify their role in the pathogenesis of AGA.
Patients and methods
This case control study included 20 female patients with AGA and 20 age-matched healthy female controls. Both were recruited from the Dermatology Outpatient Clinic, Kasr – Alainy Hospital and National Research Centre, from February 2010 to July 2011. The ethics committee of the National Research Centre approved the study and informed consent was obtained from all patients and controls.
Only middle-aged women in the child-bearing period (20–45 years) presenting with female pattern AGA were included in the study, whereas pregnant or lactating women, or those complaining of any diseases (ovarian, adrenal, dermatological or systemic) or patients ever receiving any hormonal medications were excluded.
All patients were diagnosed clinically and subjected to assessment of complete personal and family history and history of present illness (duration, previous treatment) and age of menarche or history of any menstrual irregularities to exclude cases with abnormal results.
Evaluation of the degree of AGA was carried out using the Ludwig scale 7.
Venous blood samples were collected from patients and controls after 10–12 h of fasting left to clot and then centrifuged at 4000 rpm for 15 min to separate the serum. The serum was stored at −20°C for subsequent estimation of aldosterone, free testosterone and DHEA-S.
- Estimation of the serum level of aldosterone was carried out according to study conducted by Cartledge and Lawson 23 using the ELISA kit, which was purchased from Immunospec Corporation (Los Angeles, California, USA).
- Estimation of the serum level of free testosterone was carried out according to the study conducted by Wheeler 24 using the ELISA method; the kit was purchased from Labor Diagnostic Nord Gmbh & Co. KG (Johannesburg, Nasrec, South Africa).
- Solid-phase enzyme immunoassay for the quantitative determination of DHEA-S in human serum was carried out according to the study conducted by Dequet and Wallace 25. The kit was purchased from Inter Medical (Villaricca, Napoli, Italy).
Real-time PCR analyses for androgen and aldosterone receptor gene expression: skin biopsy specimens using a 2 mm punch were taken from involved areas (any site), noninvolved areas and controls from occipital areas and were kept in RNA lysis buffer. The method involved the following steps.
RNA isolation and reverse transcription: RNA isolation was carried out by means of the Qiagen Tissue Mini Kit (Qiagen, Valencia, California, USA). In the beginning, the skin tissue biopsy was homogenized under liquid nitrogen using a mortar and pestle, then the cells were lysed and the RNA was released by centrifugation of the cell-homogenate through a biopolymer shredder (Qiashredder; Qiagen). The quality and yield of the RNA were determined by spectrophotometry at 260 nm and the integrity was examined by agarose gel electrophoresis with ethidium-bromide staining. The quantification of the total RNA was carried out using a NanoDropND-1000 UV-Vis Spectrophotometer (NanoDropTechnologies, Wilmington, Delaware, USA).
cDNA was generated from 5 μg of total RNA extracted with 1 μl (20 pmol) antisense primer and 0.8 μl superscript AMV reverse transcriptase for 60 min at 37°C.
Polymerase chain reaction – real-time PCR: The relative abundance of mRNA species was assessed using the CYBR Green method on an ABI prism 7500 sequence detector system (Applied Biosystems, Foster City, California, USA). PCR primers were designed using Gene Runner Software (Hasting Software Inc., Hasting, New York, USA) from RNA sequences from GenBank. All primer sets had a calculated annealing temperature of 60°C. Quantitative RT-PCR was performed in duplicate in a 25 μl reaction volume consisting of 2× CYBR Green PCR Master Mix (Applied Biosystems) 900 nmol/l of each primer (Table 1) and 2–3 μl of cDNA. Amplification conditions were 2 min at 50°C, 10 min at 95°C and 40 cycles of denaturation for 15 s and annealing/extension at 60°C for 10 min. Data from real-time assays were determined using the v1·7 Sequence Detection Software (PE Biosystems, Foster City, California, USA). The relative quantification (relative expression) data were expressed in cycle threshold (Ct). The PCR data sheet included Ct values of the assessed gene (aldosterone and androgen mRNA) and the housekeeping (reference) gene, the gene that is continuously and normally expressed in the cell (β-actin). In order to measure the gene expression of a certain gene, a negative control (reference) sample should be used 26. Therefore, target gene expression was assessed and related to the reference (internal control) gene.
Relative quantification was calculated according to the following equation:
Data were statistically described in terms of range, mean±SD, median, frequencies (number of cases) and percentages when appropriate. Comparison of numerical variables between the study groups was carried out using the Mann–Whitney U-test for independent samples. Comparison between involved and noninvolved data was carried out using the Wilcoxon signed rank test for paired (matched) samples. A correlation study was carried out to establish the relationship between different variables using Spearman’s correlation coefficient. A P value less than 0.05 was considered statistically significant. All statistical calculations were carried out using computer program statistical package for the social science (SPSS Inc., Chicago, Illinois, USA) version 15 for Microsoft Windows.
The study included 20 female patients complaining of AGA and 20 healthy age-matched nonpregnant women. The age of the patients ranged from 20 to 45 years, with a mean of 32.9±6.12 years. The duration of the disease ranged from 1 to 18 years, with a mean of 5.15±4.36 years. Seven patients had a grade I Ludwig score, seven had a grade II score and six had a grade III score. Almost half of the patients had a positive family history and two patients complained of menstrual irregularities.
Serum levels of aldosterone, free testosterone and DHEA-S showed an increase in patients in comparison with controls; serum aldosterone and free testosterone showed a significant increase, P value=0.001 and 0.019, respectively, whereas serum DHEA-S showed no significance, P value=0.176 (Table 2).
Estimation of androgen and aldosterone receptors in patients (involved and noninvolved areas) and controls showed a significant increase on comparing involved areas in patients with controls P=0.001 (both) (Table 3) and noninvolved areas in patients with controls P=0.001 (both) (Table 4), and on comparing both involved and noninvolved areas in terms of AR (P=0.002) and aldosterone receptors (P=0.001) (Table 5).
According to Spearman’s correlation coefficient, no correlation was found between the duration of AGA and each of serum levels of testosterone (r=−0.18), DHEA-S (r=−0.04) and aldosterone (r=0.03), aldosterone receptors in involved areas (r=−0.09) and noninvolved areas (r=−0.17), ARs in involved areas (r=−0.06) and in noninvolved areas (r=−0.42) (P>0.05), nor between serum levels of free testosterone and AR levels in scalp biopsy specimens from involved (r=−0.124) and noninvolved areas (r=−0.001) (P>0.05).
A positive nonsignificant (P>0.05) correlation was found between the serum levels of aldosterone and aldosterone receptor levels in scalp biopsy specimens from both involved and noninvolved areas (r=0.54 and 0.72), respectively, and between grade of AGA and serum levels of aldosterone (r=0.06), free testosterone (r=0.39) and DHEA-S (r=0.57).
A negative nonsignificant (P>0.05) correlation was found between the grade of AGA and levels of aldosterone and ARs in scalp biopsy specimens from both involved (r=−0.2 and −0.03, respectively) and noninvolved areas (r=−0.17 and −0.19, respectively).
AGA is one of the most common types of hair loss in women that intensifies with age. It is the complicated and combined result of an androgen-dependent process on the basis of the genetic susceptibility of the ARs to androgens and intrafollicular conversion of weak androgens into potentially stronger ones using numerous steroid-converting enzymes 27.
The current study showed a significant increase in the serum level of aldosterone in patients in comparison with controls (P<0.001). The results were in agreement with two previous studies; one was carried out on 40 male AGA patients in whom aldosterone levels were significantly higher in patients versus controls (P<0.007) and among patients, and those with hypertension had higher aldosterone levels than normotensive patients. However, no difference was found between normotensive and hypertensive controls 22. The second study included 40 female patients with AGA in whom aldosterone levels were significantly higher in patients than controls (P<0.002), with a positive correlation between the aldosterone levels and both systolic and diastolic blood pressure 28.
The serum level of free testosterone also showed a statistically significant increase in patients compared with controls (P<0.019), whereas serum DHEA-S also showed an increase but with no statistical significance (P<0.176). These results were in agreement with previous studies 29,30 that showed that male AGA patients have high serum levels of 5α-reductase, free testosterone and total free androgens including DHT and low serum level of total testosterone. Elevated urinary DHEA and serum DHEA-S were also reported in balding young men, indicating that DHEA-S is an important endocrine factor in the development of AGA 13. A retrospective study 31 on 43 adolescent patients (12–18 years) presenting with AGA showed normal serum levels of testosterone and DHEA-S, except in one 18-year-old woman, in whom the levels were elevated. AGA is a common form of hair loss in adolescents, and can be the presenting sign of an underlying endocrine disorder. An accurate and timely diagnosis is essential for appropriate medical and psychosocial intervention when warranted 32.
Several studies 33,34 have reported that women with AGA (moderate–severe) are more likely to have elevated androgen levels and/or show an increased frequency of other features of androgen excess. However, female AGA has been reported in a patient with complete androgen insensitivity syndrome, where this was explained either by the presence of sufficient AR function to the circulating testosterone and DHT or androgens acting through non-AR mechanisms, that is, androgens aromatized to oestrogens or female AGA are not necessarily androgen dependent 35. In agreement with these assumptions, some studies 36,37 failed to find evidence of increased androgens in female AGA.
Another study 38 showed that women with AGA do not have higher levels of circulating androgens. However, they have been found to have higher levels of 5α-reductase, more ARs and lower levels of cytochrome P450 (which converts testosterone into oestrogen).
There are few reported genetic association studies for AGA in women compared with men. This might be explained by the difficult interpretation as the AR is presented on the X-chromosome that is randomly inactivated in X-chromosome inactivation 39.
ARs in this study were significantly higher in involved and noninvolved areas of patients than in controls, thus pointing to the importance of ARs in the pathogenesis of AGA. This was in agreement with previous studies, 18,20 which showed that AR protein expression appears to be markedly increased within the balding vertex, compared with occipital hair follicles.
Early-onset AGA in men may be associated with obesity, insulin resistance, hypertension, dyslipidaemia, MS and early-onset coronary heart disease. However, there are only a few studies addressing these associations in women with AGA during the child-bearing period 21.
When comparing between aldosterone receptor levels in involved areas, noninvolved areas and controls, it was found that they were significantly higher in involved and noninvolved areas of patients than in controls, thus pointing to the importance of aldosterone receptors in the pathogenesis of AGA together with increased levels of serum aldosterone in the same study. To the best of our knowledge, this is the first report on the importance of aldosterone receptors in AGA female patients.
The nonsignificant correlations of the studied hormones and receptors with each other, age and duration of AGA could be attributed to the small sample size, which is a limitation in this study.
The significant increase in the serum levels of aldosterone and free testosterone in female patients with AGA in comparison with controls, together with a significant increase in the levels of aldosterone and ARs in skin biopsy specimens taken from involved areas in comparison with noninvolved areas, both higher than their levels in the controls, indicate the suggested role of these hormones and their receptors in the pathogenesis of AGA that may lead to new lines of therapy. Therefore, it is recommended to routinely determine the aldosterone levels in AGA to identify patients prone to development of hypertension. Aldosterone antagonist therapy may have a dual beneficial effect in hypertensive patients with AGA by controlling the blood pressure and preventing the progression of alopecia. Further studies are required to verify the mechanism by which aldosterone and its receptor induce AGA.
Conflicts of interest
There are no conflicts of interest.
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