Archives of Dermatological Research (2018) 310:77–83

https://doi.org/10.1007/s00403-017-1798-0

ORIGINAL PAPER

5α-Reductase isozymes and aromatase mRNA levels in plucked hair

from young women with female pattern hair loss
P. Sánchez1 · C. Serrano‑Falcón2 · J. M. Torres1 · S. Serrano3 · E. Ortega1

Received: 21 March 2017 / Revised: 20 November 2017 / Accepted: 21 November 2017 / Published online: 28 November 2017
© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract
Female pattern hair loss (FPHL) is an important hair disorder, especially when young women are affected. However, phar-
macological treatments are not successful in all women. Androgens, especially dihydrotestosterone (DHT), may play a role
in FPHL, but many women with this disorder have normal serum androgen levels. It therefore appears that hair follicle levels
of DHT depend on in situ testosterone (T) metabolism. Because T can be converted to DHT or estradiol (E2) by 5α-reductase
(5α-R) and aromatase, respectively, these enzymes would determine DHT and E2 concentrations and their ratio. We propose
and apply a low-invasive, sensitive and precise method for the absolute quantification of mRNA levels of aromatase and
5α-R isozymes (type 1, type 2 and type 3) in plucked hair from young women with FPHL. Normoandrogenic women with
FPHL and controls were studied. Plucked hair samples were obtained by trichogram from vertex scalp and mRNA levels
quantified by real-time RT-PCR. We revealed for the first time the presence of 5α-R3 mRNA in human hair. Interestingly,
one, two, or even three 5α-R isozymes were increased in some women with FPHL but not in others, which may explain the
lack of response to 5α-R inhibitors in some FPHL cases. Aromatase mRNA levels were significantly lower in women with
FPHL than in controls. It may therefore produce a reduction in oestrogen levels and an increase in the androgen/oestrogen
ratio in hair. The proposed low-invasive technique offers a molecular aetiologic diagnosis of FPHL for the selection of more
appropriate pharmacological treatments with early predicted effectiveness.

Keywords Female pattern hair loss · Aromatase · 5α-R isozymes · mRNA levels · Trichogram

Introduction anxiety and depression, especially when young women are

affected [49].
Female pattern hair loss (FPHL) is one of the most impor- Although the precise aetiopathogenesis of FPHL remains
tant hair disorders, and it may have significant psychoso- unknown, most hypotheses point to hormone pathways, in
cial effects, impairing quality of life and increasing rates of particular androgens [48]. Therefore, biochemical tests are
usually performed to identify a hyperandrogenic state as the
cause of FPHL, but normal androgen levels are found in sera
from most patients with this condition [32]. Androgen levels
P. Sánchez and C. Serrano-Falcón contributed equally to this of hair follicles may rely more on in situ metabolism than on
work.
circulating androgens, given that they are equipped with the
* S. Serrano necessary enzyme machinery for this purpose [5]. For this
salvio@ugr.es reason, some researchers have focused on quantifying andro-
* E. Ortega gen concentrations [20] and localizing androgen-metabo-
esortega@ugr.es lizing enzymes [3, 8, 42] in the hair follicle, although they
used invasive methods for the sampling (e.g. scalp biopsy).
1
Department of Biochemistry and Molecular Biology, Dihydrotestosterone (DHT), the most potent androgen,
Faculty of Medicine, University of Granada, Avda. de la
Investigación, 18016 Granada, Spain regulates specific genes responsible for the gradual miniatur-
2 ization of genetically programmed hair follicles [9] and for
Hospital of Guadix, Granada, Spain
proteins involved in shortening the hair growth cycle [22].
3
Department of Dermatology, Faculty of Medicine, University DHT increases the production of cytokines, which induce
of Granada, Avda. de la Investigación, 18016 Granada, Spain

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the hair to enter telogen phase and the dermal papilla to
become senescent [18, 50]. DHT may also interfere with
the follicular cycle by interacting with the Wnt signalling
pathway [38].
DHT is synthesized from testosterone (T) by the enzyme
steroid 5α-reductase (5α-R). There are two well-character-
ized 5α-R isozymes in the hair follicle, type 1 (SRD5A1)
and type 2 (SRD5A2) [42]. The importance of 5α-R2 in
androgenetic alopecia is demonstrated by the absence of
this condition in males with congenital 5α-R2 deficiency
[17]; however, little is known about the role of 5α-R1 in hair
growth because a human 5α-R1 genetic deficiency syndrome
has not been identified [24]. The more recently discovered
5α-R type 3 isozyme is expressed throughout the dermis
and epidermis [2], but to our best knowledge studies on this
isozyme in the hair follicle has not been carried out.
For many decades, androgens have dominated endo-
crine research into hair growth control. However, the role
of androgens in FPHL remains controversial. Around
22–34.8% of women with polycystic ovary syndrome pre-
sent FPHL [33, 37]; however, FPHL can also occur in com-
plete androgen insensitivity syndrome [6], pointing to other
pathogenic factors besides an androgenetic aetiology [26].
Oestrogens may also play a role in scalp hair growth,
[30]. Thus, aromatase, the key enzyme required to convert
androgens to oestrogens, has been localized in scalp hair
follicles [41].
Pharmacological treatments against FPHL include the
administration of 5α-R inhibitors, but their effectiveness
remains unclear [13]. Furthermore, the therapeutic response
varies among patients, probably due to the different aetiolo-
gies of the disease. Hence, an adequate approach to FPHL
requires an understanding of androgen and oestrogen metab-
olism in the hair follicle and of the involvement of the key
enzymes 5α-R and aromatase, which may be responsible for
its androgen/oestrogen ratio.
The objective of this study was to propose and apply a
low-invasive, sensitive and precise method for the abso-
lute quantification of mRNA levels of aromatase and 5α-R
isozymes (type 1, type 2 and type 3) in plucked hair from
young women with FPHL.
Materials and methods
Subjects and sampling
This study included 10 women (age range 20–30 years)
diagnosed with grade II alopecia according to the Ludwig
classification, considered as FPHL, presenting more than
20% of miniaturized hairs, and 7 healthy women of similar
age with no hair loss or thinning, who served as controls.
All participants had serum testosterone, SHBG, 17-OH
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Archives of Dermatological Research (2018) 310:77–83
progesterone, FSH, LH, prolactin, androstenedione, DHEA-
S, and estradiol within the normal range, and none showed
signs of clinical virilization or had undergone antiandrogenic
or contraceptive hormonal therapy in the previous 6 months.
All individuals signed their written consent before inclusion
in the study.
In all participants, human hair samples were obtained by
trichogram from the vertex area of the scalp, plucking 30–40
hairs with tightly closing epilation forceps. To avoid differ-
ences in tractional (epilation) force between the inner and
outer area of the sample, all samples were taken in identical
conditions by the same dermatologist following Serrano-Fal-
con et al. [44]. The epilated hair roots were then placed on
a glass slide and covered with coverslip, using light micros-
copy to examine hair root types. The anagen/telogen ratio
upon root examination was about 80/20. Only hair roots in
anagen phase were selected for study. Examination of the
proximal end of the hair shaft helps to determine whether
a hair is in anagen, telogen, or catagen phase. Anagen hair
shafts are longer, have a uniform diameter, a rectangular
shape, and a slight distal angle. Anagen hair bulbs are seen
as darkly pigmented triangular or delta-shaped bulbs with
an angle to the hair shaft and there is presence of inner root
sheath. The sample included hair longer than 2 cm, with
some miniaturized hairs but no fuzz. The 10 anagen hairs
showing the most intact sheaths were selected for this study.
Samples were immediately transferred to individual
Eppendorf tubes prefilled with 0.5 mL RNAlater (Ambion)
to avoid RNA degradation and were stored at room tempera-
ture for 24 h followed by storage at − 80 °C.
RNA isolation
Around 10 hair roots in anagen phase per participant were
homogenized in phenol–guanidine isothiocyanate (Trizol
Invitrogen). Total RNA was extracted with chloroform
and precipitated with isopropanol by 12,000g centrifuga-
tion at 4 °C. The RNA pellet was washed twice with 75%
ethanol and resuspended in diethylpyrocarbonate-treated
water. RNA samples were then treated with Turbo DNase
(Ambion) to remove contamination with genomic DNA.
RNA yield was determined spectrophotometrically by A260/
A280 ratio using a NanoDrop ND-1000 spectrophotometer
(ThermoFisher). Isolated total RNA integrity was electro-
phoretically verified by ethidium bromide staining.
Reverse transcription and quantitative real‑time
PCR
First-strand cDNA was synthesized from 1 µg of total RNA
following Castro et al. [4]. Absolute quantification of mRNA
of 5α-R1, 5α-R2, 5α-R3 and aromatase in the plucked hairs
was performed by real-time RT-PCR using the Techne
Archives of Dermatological Research (2018) 310:77–83 79

Quantica™ Real-time PCR system with SYBR Green PCR Table 1  Primers for PCR amplification of each gene studied and sizes
Master Mix (Promega). The final optimized concentra- of amplified fragments
tion used in the reactions was 0.4 µM for both the forward Gene Sequence (5′–3′) Size frag-
and reverse 5α-R1, 5α-R2, 5α-R3 and aromatase primers. ment (refer-
Twenty-two microliters of the prepared PCR master mix ence)
was added to each well of the M ­ icroAmp® Optical 96-well SRD5A1
reaction plate (Thermo Scientific). Three microliters of each Sense AGC​CAT​TGT​GCA​GTG​TAT​GC 163
reverse-transcribed RNA sample was added to the appropri- Antisense AGC​CTC​CCC​TTG​GTA​TTT​TG
ate well to make a final reaction volume of 25 µl. SRD5A2
Absolute quantification of mRNA is achieved by extrap- Sense TGA​ATA​CCC​TGA​TGG​GTG​G 154
olation of fluorescence signals from test samples against Antisense CAA​GCC​ACC​TTG​TGG​AAT​C
standard curves representing the initial copy numbers for SRD5A3
a defined fluorescence signal; an external in vitro synthesis Sense TCC​TTC​TTT​GCC​CAA​ACA​TC 212
cRNA standard was used for their construction. Synthesis Antisense CTG​ATG​CTC​TCC​CTT​TAC​GC
of cRNAs was performed by in vitro transcription with T7 CYP19A1
RNA polymerase followed by reverse transcription and Sense TAT​TAG​GGC​CCT​GTG​TCT​GC 193
real-time PCR, using the method described by Fronhoffs Antisense TGG​GTT​GGG​ACT​TTT​CCT​CC
et al. [10]. The standard curve was generated by performing
five independent serial dilutions of the cRNA standard and
assaying each dilution in duplicate. To maximize the accu-
racy, dilutions were made over the range of copy numbers
that included the amount of target mRNA expected in the
experimental RNA samples. Samples and cRNA standards
were reverse-transcribed and amplified in a parallel proce-
dure using identical primer pairs and hybridization probes.
This process allows the parallel amplification of cRNA
standards with a sequence identical to the target gene itself.
In comparison to relative quantification, this method offers
the advantage of giving an absolute copy number for a spe-
cific target. The amount of mRNA was expressed as number
of mRNA copies per micrograms of total RNA.
The PCR profile was as follows: denaturation at 94 °C
for 30 s; annealing at 55 °C for the SRD5A1 gene, 54 °C
for the SRD5A2 gene, 60 °C for the SRD5A3 gene, 60 °C
for the CYP19A1 gene for 30 s; and extension at 72 °C for Fig. 1  mRNA levels of 5α-reductase type 1 (5α-R1) in anagen hairs
30 s. The number of cycles was 40 in all cases. At the end plucked from young women with female pattern hair loss (FPHL)
(n = 10) and controls (n = 7). Data are expressed as means ± SD
of the amplification phase, a melting curve analysis was car-
ried out on the products formed in order to confirm that a
single PCR product was detected by the SYBR Green dye. non-normal by the Kolmogorov–Smirnov test. GraphPad
All reactions were run in triplicate and no cDNA was added Prism 5.0 software (San Diego, CA) was used for the statis-
to the negative reactions. tical analysis. P < 0.05 was considered significant.
Primers for 5α-R1 (SRD5A1 mRNA, Genbank accession
no. NM_001047.3), 5α-R2 (SRD5A2 mRNA, GenBank
accession no. NM_000348.3), 5α-R3 (SRD5A3 mRNA Results
GenBank accession no. NM_024592.4) and aromatase
(CYP19A1 mRNA GenBank accession no. NM_000103.3) In plucked hair samples from the global series (young
were designed using Primer 3 software. The primer women with FPHL and controls), the highest mRNA levels
sequences (5′–3′) are given in Table 1. were for 5α-R1 followed by 5α-R3 and then by 5α-R2.
Mean mRNA levels of 5α-R1 (Fig. 1), 5α-R2 (Fig. 2)
Statistical analysis and 5α-R3 (Fig. 3) isozymes were higher in the women with
FPHL than in the controls, but statistical significance was
The non-parametric Mann–Whitney U test was used for not reached. There was a wide variability of results for these
comparisons because the data distribution was found to be isozymes among the women with FPHL, as can be observed

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Fig. 2  mRNA levels of 5α-reductase type 2 (5α-R2) in anagen hairs
plucked from young women with female pattern hair loss (FPHL)
(n = 10) and controls (n = 7). Data are expressed as means ± SD
Fig. 3  mRNA levels of 5α-reductase type 3 (5α-R3) in anagen hairs
plucked from young women with female pattern hair loss (FPHL)
(n = 10) and controls (n = 7). Data are expressed as means ± SD
in the figures. Aromatase mRNA levels were significantly
lower (p < 0.05) in the women with FPHL than in the con-
trols (Fig. 4).
Discussion
This study presents a low-invasive methodology that avoids
biopsy and obtains plucked hair samples trichogram, allow-
ing the absolute quantification of mRNA levels of aromatase
and 5α-R isozymes by real-time RT-PCR.
Previous reports have postulated that the dermal papilla
(DP) cells are a main target for androgen action [7, 14].
Thus, it has been shown that 5α-R activity was concentrated
in the DP, whereas the root sheath of hair follicle expressed
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Archives of Dermatological Research (2018) 310:77–83
Fig. 4  mRNA levels of aromatase in anagen hairs plucked from
young women with female pattern hair loss (FPHL) (n = 10) and con-
trols (n = 7). Data are expressed as means ± SD. *P < 0.05 vs. Control
high levels of 17β-hydroxysteroid-dehydrogenase and only
minor 5α-R activity [7]. However, other authors have shown
androgen receptor, 5α-R1 and 5α-R2 expression in the root
sheath of hair follicle pointing that it plays an important role
in androgen regulation [42]. Besides, aromatase has also
been located in root sheath of hair follicles [41]. Plucked
anagen hairs are lacking of DP, however they are mainly
constituted by keratinocytes cells forming both outer and
inner root sheaths [31]. In view of this, plucked anagen hairs
may be an appropriate tissue to study key enzymes in andro-
gen/oestrogen metabolism in a low-invasive manner.
Here, we proposed a sensitive approach able to detect
as few as 10 initial template copies, and several intra- and
inter-assays have demonstrated its precision [10, 34, 39]. In
addition, the standard curves (external in vitro synthesized
cRNA standards) used in this study, offers some important
advantages. Thus, samples and cRNA standards with a
sequence identical to the target gene can be reverse-tran-
scribed and amplified in a parallel procedure using identical
primer pairs, and samples and standard curve show equal
amplification efficacies. This permits comparisons of data
gathered on different days or in distinct laboratories and
allows early evaluation of the effectiveness of a drug treat-
ment in a patient.
Our study revealed for the first time the presence of 5α-R3
mRNA in human plucked hair, although the possible con-
tamination cannot be completely ruled out during plucking
using this technique. 5α-R3 may contribute to DHT produc-
tion in hair follicles, as proposed in other tissues [51]. Inter-
estingly, 5α-R3 was overexpressed in some prostate cancer,
and various studies have suggested an association between
male pattern baldness and prostate cancer [53].
The high 5α-R1 mRNA levels observed in some women
may explain why endogenous 5α-R activity in hairs from
Archives of Dermatological Research (2018) 310:77–83
women with FPHL can be suppressed by a dual 5α-R1
and 5α-R2 inhibitor but not by a selective 5α-R2 inhibi-
tor [11], suggesting a role for 5α-R1 in FPHL. In line with
our results, immunohistochemical studies have described a
greater expression of 5α-R1 than of 5α-R2 in the outer root
sheath of hair follicles in both sexes [42]. Furthermore, a
study using relative quantification of mRNA levels in hair
from women found high mRNA levels of 5α-R1 but did not
detect 5α-R2 [31]. On the contrary, other authors have found
higher expression of 5α-R2 and relatively lower of 5α-R1
in DP from AGA [15, 19]. In the present study, although
5α-R2 mRNA levels were lower than those for 5α-R1, it
should be borne in mind that the affinity of 5α-R2 for T is
much higher than that of 5α-R1 [36]. 5α-R2 may therefore
play a major role in FPHL, as reported in male androgenetic
alopecia [17]. In this paper we have quantified mRNA but
unfortunately, due to the low starting material obtained in
plucking hair, enzymatic activity have not been measured,
this being one of the limitations of the study.
In the present series, mean 5α-R1, 5α-R2 and 5α-R3
mRNA levels appeared to be higher in the normoandrogenic
women with FPHL than in the controls. However, the differ-
ences did not reach statistical significance, which is likely
attributable to the wide variability in levels observed among
the women with FPHL. We highlight the very high mRNA
levels of one, two, or even three 5α-R isozymes in some
of the women with FPHL, while none of these levels were
increased in others (Table 2). Probably, FPHL is a complex
trait in which sex steroids are important in some individuals
but not in others. Therefore, the pharmacological approach
to this disorder should take account of this disparity among
the women, which would explain why treatment with 5α-R
inhibitors is not always successful [43]. Furthermore, these
drugs are associated with undesirable adverse effects [12],
making their inappropriate administration a highly relevant
issue.
Table 2  FPHL patients with increased (⇑) 5α-R isozymes or
decreased (⇓) aromatase
FPHL 5α-R1 5α-R2 5α-R3 Aromatase
1 ⇑ ⇑ ⇓
2 ⇑ ⇑ ⇑ ⇓
3 ⇓
4 ⇑
5 ⇑ ⇓
6 ⇑ ⇑ ⇓
7
8 ⇑ ⇑ ⇓
9
10 ⇑ ⇑ ⇑ ⇓
81
FPHL has been associated with high androgen levels
[48] and/or elevated androgen receptor sensitivity [40],
explaining the administration of androgen receptor inhibi-
tors in these cases. However, FPHL has also been observed
in women with complete androgen insensitivity syndromes
[6], suggesting that another mechanism may contribute to
its aetiopathogenesis. In this study, aromatase mRNA lev-
els were significantly lower in hair follicles from women
with FPHL than in controls. In line with our results, aro-
matase levels in women were higher at the frontal hairline
than in other areas of the scalp, in agreement with the
relative preservation of the frontal hairline in FPHL [27].
The decrease in aromatase may lead to a decrease in oes-
trogens in situ, unfortunately not measured in this study. The
role of oestrogens in FPHL is controversial. Thus, ex vivo
studies in an animal model reported that oestrogens may
inhibit hair growth [30]. However, other studies may point to
the contrary suggesting that oestrogens may prevent FPHL
by modulating cytokines and growth and transcription fac-
tors [30]. Oestradiol increases the secretion of both IGF-1
and VEGF [21, 35]. IGF1 is a potent stimulator of hair folli-
cle growth and prevents the follicle from entering a catagen-
like state [35], while VEGF promotes perifollicular vascu-
larization and regulates the hair follicle blood supply [52].
Conversely, oestrogen inhibits the release of TNF-alpha,
IL-6 and TGF-beta [25, 29], whose overexpression leads to
hair loss [45, 47]. In this sense, it has been found that preg-
nant women (period with known elevated estrogens levels)
present a higher number of hairs in anagen phase [28], while
women with lower oestrogen levels during menopause, post-
partum, treatment with aromatase inhibitor or selective oes-
trogen receptor modulators are more likely to develop FPHL
[1]. However, in spite of these observations, Kanty et al.
[23] have reported that the aromatase inhibitor tamoxifen
did not affect hair growth parameters. On the other hand,
many physiological changes during menopause pregnancy
and postpartum should be also kept in mind. Thus, it is very
difficult to strictly dissociate the effects of estrogens in the
hair growth from other factors in these situations.
Probably, the key issue for FPHL is the 5α-R/aro-
matase ratio in hair follicle. Thus, elevated 5α-R levels
and decreased aromatase levels may divert T to DHT
in situ rather oestrogen and vice versa. In this context, it
is important to consider the regulation of aromatase and
5α-R isozymes. Thus, it has been demonstrated that oes-
trogen induces aromatase activity in intact human anagen
hair follicles [16], and may also lead to a direct inhibition
of 5α-R [30]. On the other hand, DHT itself may induce
the expression of 5α-R isozymes by a feed-forward mecha-
nism [46]. The fact that both DHT and E2 may activate the
key enzymes implicated in their own biosynthesis compli-
cates even further our understanding of the mechanisms
involved.
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Conclusion
We here report for the first time, at least to our best knowl-
edge, a study showing a low-invasive methodology for the
absolute quantification of mRNA levels of aromatase and
5α-R isozymes in human plucked hair. Results of its appli-
cation in women with FPHL and controls indicate the pos-
sible implication of these enzymes in the pathogenesis of
FPHL through their regulation of the levels and balance of
androgens and oestrogens. The variability observed in the
mRNA levels 5α-R isozymes in hair follicles from women
with FPHL may explain, at least in part, reported differences
in their response to pharmacologic treatments. Therefore,
a proper aetiological diagnosis of FPHL is necessary to
improve the effectiveness of treatment and minimize adverse
effects. These findings open up a new research line that may
lead to a better understanding of the molecular mechanisms
underlying FPHL in young women as the basis for treatment
improvements. However, due to the small sample size, fur-
ther research is warranted to investigate this relevant issue
in greater depth.
Acknowledgements The authors thank R. Davies for revising the Eng-
lish text.
Compliance with ethical standards
Funding This study was supported in part by the Andalusian Regional
Government (Endocrinology and Metabolism Group).
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval All procedures involving human participants were
in accordance with the ethical standards of the national research com-
mittee and the 1964 Helsinki declaration and its later amendments or
comparable ethical standards.
Informed consent Informed consent was obtained from all individuals
who participated in the study.
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