Mycopathologia (2019) 184:505–515

https://doi.org/10.1007/s11046-019-00345-8 (0123456789().,-volV)
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ORIGINAL ARTICLE

Investigation on Microecology of Hair Root Fungi

in Androgenetic Alopecia Patients
Jinghong Huang . Yuping Ran . Sushmita Pradhan . Wei Yan .
Yaling Dai

Received: 28 March 2019 / Accepted: 30 May 2019 / Published online: 26 June 2019
Ó Springer Nature B.V. 2019

Abstract method was used to detect the fungal microecology of

Background This study focused on the differences in hairy roots at different sites. Moreover, the compar-
hairy root fungal microecology between androgenetic ison of fungal loads of Malassezia in different group
alopecia patients and healthy individuals. and scalp area were tested by PCR.
Methods Light microscopy was used to observe the Results The fungi in the hair root observed by optical
morphology of hairy roots. Morphological observa- microscopy are mainly Malassezia yeast. The pos-
tions were also performed in the positive specimens itive rate of Malassezia in the hair loss group (60%)
using scanning electron microscopy and transmission was higher than that in the control group (40%). The
electron microscopy. The high-throughput sequencing detection efficiency of Malassezia examined by
scanning electron microscopy was higher than that
by light microscopy. Results acquired from high-
Handling Editor: Stephane Ranque. throughput molecular sequencing of fungi suggested
that Ascomycota was the dominant species, whereas
J. Huang  Y. Ran (&)  S. Pradhan  W. Yan in the occipital hair roots of the control group
Department of Dermatovenereology, West China Basidiomycota was the dominant species in the hair
Hospital, Sichuan University, No. 37, Guo Xue Xiang,
Chengdu 610041, Sichuan Province, China loss group. Malassezia followed by Trichosporon
e-mail: ranyuping@vip.sina.com were the most abundant fungal genera. The changes
J. Huang in abundance at the top and occipital region of the
e-mail: sensen.006@163.com control group were more significant than those of the
S. Pradhan genus Fusarium, followed by Epicoccum and
e-mail: 1197597473@qq.com Malassezia. The load of Malassezia located on
W. Yan calvaria in the alopecia group was significantly
e-mail: yanweihappyhappy@163.com higher than that in the control group. In the alopecia
group, the load of Malassezia on the scalp was
J. Huang
higher than that on the occipital region. The load of
Department of Dermatovenereology, Medical Center of
Dujiang yan, Dujiangyan 611830, Sichuan Province, Malassezia globosa and Malassezia restricta in the
China hair loss group was higher on calvaria and occipital
areas.
Y. Dai
Conclusion Malassezia had a positive correlation
Laboratory Medicine, West China Hospital, Sichuan
University, Chengdu 610041, Sichuan Province, China with the incidence of androgenic alopecia.
e-mail: 839894198@qq.com

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Keywords Androgenetic alopecia  Fungal
microecology  Malassezia  High-throughput
sequencing
Introduction
Androgenetic alopecia (AGA) remains one of the most
common hair loss diseases, which is characterized by a
progressive decrease in hair density, becoming thin-
ner, sparse, and with lighter color hair. It has been
divided into male pattern alopecia and female pattern
alopecia based on gender difference [1]. The occur-
rence of AGA has been considered to have a great
negative impact on the patient’s psychology and the
quality of life [2, 3]. Therefore, research related to
AGA has attracted much attention. In past studies,
AGA was considered to be a polygenic hereditary
disease caused by interactions between multiple genes
and environmental factors [4, 5]. It was confirmed that
the two major genetic risk loci are the X chromosome
AR/EDA2R locus and the PAX1/FOXA2 locus resid-
ing on chromosome 20 [6–8]. In addition, androgen
receptor regulatory genes modulated the sensitivity of
the androgen receptor [9]. Recently, researches have
proposed that the micro-inflammatory reaction of hair
follicles in the pathology of AGA patients with scalp
was distinctive from the typical inflammation and
destruction process of scar alopecia, and it was
involved in the development of AGA and its hair
follicle inflammation rendered the slower process,
which was hidden and difficult to detect. This may be a
chemical stress response [10] caused by UV, cosmet-
ics, cosmetic agents, contaminants, and photochemi-
cal damage. More severely, the process of AGA
folliculitis may be related to the local inflammatory
response [11] to the microbial toxins associated with
Propionibacterium, Staphylococcus, Malassezia, or
Demodex.
Malassezia spp. is a resident flora on the skin of
humans and animals and a kind of lipophilic yeast. At
present, 11 out of the above 17 species of Malassezia
have been demonstrated to be involved in the human
skin microecology [12]. Since the growth of Malasse-
zia is dependent on lipids (except for M. pachyder-
matis), they are mainly distributed in areas rich in
sebum, such as the scalp, face, and chest and back,
which account for about 50–80% of the total number
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Mycopathologia (2019) 184:505–515
of colonized fungi in healthy people. But the turbu-
lence of the percentage of microorganisms will
contribute to AGA. As early as 1998, Piérard et al.
[11] found that use of 2% ketoconazole shampoo in
androgenetic alopecia increased the hair’s density,
diameter, and proportion of hair follicles where the
effect was almost equivalent to the treatment by
topical 2% minoxidil solution. The mechanistic study
indicated that ketoconazole has anti-infective effects
against the microflora on the scalp, especially
Malassezia, which played therapeutic roles in andro-
gen alopecia. In recent years, studies have shown that
micro-inflammatory reactions brought about in the
hair follicles of androgenetic alopecia patients, which
may be related to hair loss [11, 12]. This hair follicle
microinflammation differs from the process of inflam-
mation and destruction in typical scar alopecia. It
progresses slowly, concealed, and is unnoticeable.
Propionibacterium, Staphylococcus, Malassezia, or
Demodex are related to microorganisms. Organism
toxins may be involved in inflammatory responses.
The influence of microbiota on androgenetic alopecia
has received abundant attention [12, 13]. Keisha et al.
[14] collected 14 samples from ten healthy adults and
sequenced DNA of the fungus samples. The study
found abundant Malassezia in the head and trunk and
further confirmed that elucidating of fungal and
bacterial ecosystems on the human body will con-
tribute to a more comprehensive understanding of skin
diseases.
The present study focused on differences in the
hairy root fungal microecology between androgenetic
alopecia patients and healthy people. Through the
high-throughput sequencing method technique, we
obtained more precise data about hairy root fungal
microecology and have further understood the patho-
genic mechanism of AGA.
Materials and Methods
Hair Loss Group Inclusion Criteria
1. According to ‘‘Chinese Clinical Dermatology’’
[1], the clinical manifestations of androgenetic
alopecia can be described as male appearance of
the forehead on both sides with less, thin, sparse
hair gradually extended toward the top of the head,
Mycopathologia (2019) 184:505–515 507

with the frontal part of the hair retrusion; the of healthy and adolescents with androgenetic alopecia,
forehead becomes high, forming a ‘‘high amount,’’ and on hair roots in different parts of healthy scalp.
frontal hairline with M-shaped, starting to fall off The subjects were divided into four groups: alopecia
from the top of the head. With progressive develop- areata group, alopecia occipital group, control top
ment, the frontal area and the top hair loss area would group, and control occipital group. Each group had ten
integrate with each other. The hair loss area consisted specimens. High-throughput sequencing was per-
of smooth skin with no atrophy, and visible fine formed on the four groups of samples for sequencing
armpit hair. It may be accompanied by increased analysis.
sebum secretion with no other symptoms. According
to the Hamilton classification, male patients with Subject Recruitment
alopecia grades III–IV were recruited. Females
demonstrated the phenotype that the hair on the top The tests were conducted on the patients and healthy
of the head gradually became less, sparse and volunteers, and they were informed about the test.
generally did not involve the forehead. According
to the Ludwig classification, female patients with Sample Collection
grade II hair loss were selected. Perms and hair dyes
were not used 2 months prior the treatment. Anti-
1. Subjects were prohibited from shampooing for
hair loss shampoo was also not used. Oral or topical
48 h prior testing.
antifungal preparations were not given within
2. Gentle pull of selected hair, and remove the top
1 month prior the treatment. No scalp-related dis-
and occipital loose hair.
eases such as scalp folliculitis, head lice, and alopecia
3. With the help of sterile eye occlusion clip hair
areata were detected in the experimental subjects.
distal and a ruler, the hair from hair follicle
The control group included the following criteria: measuring 1.5 cm long was selected. It was stored
(1) the age and sex of healthy volunteers were in a sterile EP tube, and it was preserved at
basically matched with those who participated in the - 20 °C for further examination.
hair loss group; (2) perms and hair dyes were not used
2 months prior the treatment. Anti-hair loss shampoo
DNA Extraction and PCR Amplification
was also not used; (3) oral or topical antifungal
preparations were not given within 1 month prior the
Microbial DNA was extracted from hair follicle
treatment; (4) no scalp-related diseases such as scalp
samples using the E.Z.N.A.Ò soil DNA Kit (Omega
folliculitis, head lice, and alopecia areata were
Bio-tek, Norcross, GA, USA) according to the man-
observed in the individuals.
ufacturer’s protocol. The final DNA concentration and
purity were determined by NanoDrop 2000 UV–Vis
Observation of Fungi
spectrophotometer (Thermo Scientific, Wilmington,
USA), and DNA quality was checked by 1% agarose
The morphological observation of fungi was per-
gel electrophoresis. The ITS region of the fungal
formed by using hair roots samples. Electron micro-
rRNA gene was amplified with primers ITS1F (50 -
scope (Hitachi, Japan) observations were divided into
CTTGGTCATTTAGAGGAAGTAA-30 ) and 2043R
two groups: alopecia group and control group. Each
(50 -GCTGCGTTCTTCATCGATGC-30 ) by thermo-
group had ten specimens. Three cases of Malassezia
cycler PCR system (GeneAmp 9700, ABI, USA).
positive specimens were selected for further observa-
The PCRs were conducted using the following
tion by using scanning electron microscopy and
program: 3 min of denaturation at 95 °C, 27 cycles
transmission electron microscopy.
of 30 s at 95 °C, 30 s for annealing at 55 °C, 45 s for
elongation at 72 °C, and a final extension at 72 °C for
Sequencing Analysis for Fungi
10 min. PCRs were performed in triplicate 20 lL
mixture containing 4 lL of 5 9 FastPfu Buffer, 2 lL
The diversity analysis of fungal microbiota (FM) was
of 2.5 mM dNTPs, 0.8 lL of each primer (5 lM),
performed on hair roots in different parts of the scalp
0.4 lL of FastPfu Polymerase, and 10 ng of template

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DNA. The resulted PCR products were run on and

extracted from a 2% agarose gel and further purified
using the AxyPrep DNA Gel Extraction Kit (Axygen
Biosciences, Union City, CA, USA) and quantified
using QuantiFluorTM-ST (Promega, USA) according
to the manufacturer’s protocol.

Illumina MiSeq Sequencing

Purified amplicons were pooled in equimolar and

paired-end sequenced (2 9 300) on an Illumina
MiSeq platform (Illumina, San Diego, USA) accord-
ing to the standard protocol provided by Majorbio Bio-
Pharm Technology Co. Ltd. (Shanghai, China).

Statistical Analysis

Malassezia positive rate of hair loss group and normal

group was compared using Chi-squared tests.

Results

Alteration in Morphology of Hair Follicle Roots

Between Androgenetic Alopecia and Healthy
Individuals
In order to understand the morphological changes of
hair follicle roots between androgenetic alopecia and
healthy individuals, they were observed by using
optical microscopy, transmission electron micro-
scopy, and scanning electron microscopy, respec-
tively. In detail, ten specimens from each subject’s
hair follicle specimens were randomly selected and
were then placed on glass slides. The specimens were
stained with methylene blue and covered with cover-
slips and observed with an optical microscope. It
indicated that the fungi observed in the root of the hair
follicle by an optical microscope are mainly yeast cells
of Malassezia spp. (Fig. 1).
If the number of Malassezia roots in the hair follicle
reached 5/high magnification and three consecutive
fields of view are regarded as positive for Malassezia,
in the specimens that are positive for Malassezia, a
large number of spherical hair roots were observed
with an optical microscope. An oval shaped Malasse-
zia spores or buds were observed. The positive rate in
the alopecia group (60%) was significantly higher than
that in the control group (40%) (P \ 0.01, N = 20)
Fig. 1 Observation of scalp condition from healthy individuals
and alopecia patients. a The patient male, 39 years old, had hair
loss for 6 years and showed a delayed extension of the hairline.
b Healthy volunteers male, 32 years old, with partial oily hair.
c The patient female, 33 years old, had hair loss for 4 years.
d Healthy volunteer female, 28 years old, healthy hair
(Table 1). The specimens confirmed to be positive
under the optical microscopy were sent to the scanning
electron microscope and transmission electron micro-
scope for further observations. Scanning electron
microscopy revealed that more Malassezia yeast cells
were detected in the hair roots of AGA patients
(Fig. 2). Transmission electron microscopy observa-
tion proved that the yeast cells of Malassezia were
embedded in the superficial hair root tissue of AGA
patients (Fig. 3).
The Differences in Fungal Phylum Between
Control Group and Hair Loss Group
The results of high-throughput sequencing suggested
that hairy root fungi of the TCG (the top of the control
group) in the control group were mainly Ascomycota

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Table 1 Microscopic observation of fungal microscopic examination of hair roots

Number Control group Age Microscopic Hair loss Age Microscopic examination
examination results group results

1 Male 32 ? Male 39 ?
2 Male 45 - Male 54 ?
3 Male 38 - Male 44 ?
4 Female 28 - Female 33 -
5 Male 40 - Male 30 --
6 Female 35 ? Female 26 ?
7 Male 52 ? Male 40 ?
8 Female 35 - Female 37 -
9 Female 46 ? Female 48 ?
10 Male 56 - Male 54 -
Positive % 40% 60%*
*P \ 0.01

Fig. 2 SEM observation of hair root in AGA patients. There are more Malassezia yeast cells (characteristic collar-like
SEM 9 1000 (a), 9 2000 (b), 9 5000 (c), 9 10,000 structures at the bud) observed at the hair root in AGA patients
(d) 9 4000 (e) male patients, 39 years old, hair loss for 6 years.

(red mark, 73.16%) and Basidiomycota (green mark, mainly composed of Ascomycota (red mark, 63.78%)
24.94%), and Zygomycota (blue marker, 0.49%); other and Basidiomycota (green mark, 34.37%), and Zy-
categories accounted for 1.41%. In the control group, gomycota (blue logo, 0.62%); other categories
the occipital area of the control group (OCG) was accounted for 1.23%. The hair roots of the scalp

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Fig. 3 SEM observation of hair root in AGA patients.
TEM 9 0.5 K (a), 9 1.5 K (b), 9 4.0 K (c), 9 6.0 K (d),
male patient, 28 years old, 5-year hair loss. The superficial
alopecia area (TBG, the top of bald group) of the hair
loss group were mainly Ascomycota (green marking,
35.58%) and Basidiomycota (red marking, 61.03%)
and Zygomycota (blue logo, 0.40%), and other cate-
gories accounted for 2.99%. The hairy root fungus of
the OBG (the occipital of bald group) was dominated
by Ascomycota (green marker, 41.18%) and Basid-
iomycota (red marker, 54.21%), and phyla (Zygomy-
cota (blue logo, 1.87%); other categories accounted
for 2.74% (Fig. 4).
High-Throughput Sequencing Indicated Changes
of Fungal Genera in Hairy Root Between Hair Loss
Group and the Control Group
In order to understand the changes of fungal genera in
hair root at different parts of the scalp in the hair loss
group and the control group, we used high-throughput
sequencing methods to detect the fungal genera. Some
of the fungal species that varied by more than 1% were
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Mycopathologia (2019) 184:505–515
tissue (red arrow) has Malassezia yeast cells embedded in hair
roots of AGA patients (white arrows, with a distinctive zigzag
structure on the inside of the cell wall)
Fusarium: 13.49% at the top of the control group and
3.20% at the occipital region; 2.3% at the top of the
alopecia group and 4.11% at the occipital region.
Malassezia: 8.64% at the top of the control group and
11.64% at the occipital area; 31.44% at the top of the
alopecia group and 20.57% at the occipital region.
Epiconc (Epicoccum): 8.69% at the top of the control
group and 11.30% at the occipital area; 0.31% at the
top of the alopecia group and 0.42% at the occipital
region. Trichosporon: 5.81% at the top of the control
group and 8.53% at the occipital region; 13.45% at the
top of the hair loss group and 15.22% at the occipital
region. Alternaria: 4.48% at the top of the control
group and 5.99% at the occipital region; 1.14% at the
top of the alopecia group and 1.01% at the occipital
region. Cladosporium: 3.55% at the top of the control
group and 5.21% at the occipital region; 2.26% at the
top of the alopecia group and 1.99% at the occipital
region. Cryptococcus: 2.78% at the top of the control
group and 4.11% at the occipital region; 3.78% at the
Mycopathologia (2019) 184:505–515
Fig. 4 Distribution column of hairy root fungi in different parts
of the scalp of the control group and the hair loss group. The top
of control group (TCG) and the occipital of control group (OCG)
top of the hair loss group and 6.91% at the occipital
region. Candida: 2.21% at the top of the control group
and 4.53% at the occipital region; 5.32% at the top of
the alopecia group and 5.87% at the occipital region.
Aspergillus: 1.91% at the top of the control group and
3.68% at the occipital region; 5.56% at the top of the
alopecia group and 5.03% at the occipital region.
Exophiala: 2.15% at the top of the control group and
1.75% at the occipital region; 1.92% at the top of the
hair loss group and 1.83% at the occipital region
(Fig. 5).
The Comparison of Fungal Loads of Malassezia
in Different Groups and Scalp Areas
The load of Malassezia in different parts of the scalp is
shown in Tables 2, 3, and 4. The load of Malassezia
located on calvaria in the alopecia group
(6.7E ? 06(6.8E ? 05, 9.0E ? 06)) was significantly
higher than that in the control group (2.6E ?
05(1.2E ? 05, 1.2E ? 06)) (P \ 0.05) (Table 2).
No significant difference was detected in the load of
Malassezia located on occipital between the hair loss
group (1.1E ? 06(5.7E ? 05, 2.6E ? 06)) and con-
trol group (5.3E ? 05(9.0E ? 04, 7.5E ? 05))
(P [ 0.05) (Table 2). In the alopecia group, the load
511
are mainly Ascomycota (red mark) and the top of bald group
(TBG). The occipital of bald group (OBG) is dominated by the
Basidiomycota (green marker). (Color figure online)
of Malassezia on the scalp (6.7E ? 06(6.8E ? 05,
9.0E ? 06)) was higher than that on the occipital
region (1.1E ? 06(5.7E ? 05, 2.6E ? 06)) (P \ 0.05).
In the control group, no significant difference was
observed in the amount of Malassezia between
calvaria area group (2.6E ? 05(1.2E ? 05, 1.2E ?
06)) and the occipital area group (5.3E ?
05(9.0E ? 04, 7.5E ? 05)) (P [ 0.05) (Table 2).
The load of Malassezia globosa in the hair loss
group on calvaria area (5.2E ? 05(2.0E ? 05,
2.2E ? 06)) was higher than that in control group
(6.7E ? 04(2.2E ? 04, 2.4E ? 05)), and occipital
area (3.4E ? 05(1.1E ? 05, 6.7E ? 05)) of hair loss
group was also higher than that of control group
(8.1E ? 04(3.0E ? 04, 2.6E ? 05)) (P \ 0.05). The
load of Malassezia restricta in the hair loss group on
calvaria area (5.2E ? 05(2.2E ? 05, 3.1E ? 06))
was higher than that in control group (1.1E ?
05(1.0E ? 04, 2.1E ? 05)), and the load of Malasse-
zia restricta in the hair loss group on occipital area
(4.4E ? 05(2.0E ? 05, 8.3E ? 05)) was also higher
than that in control group (1.4E ? 05(3.1E ? 04,
2.0E ? 05)) (P \ 0.05). And no significant difference
was detected in the amount of Malassezia globosa or
Malassezia restricta between calvaria area and occip-
ital area (P [ 0.05) (Tables 3, 4).
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Fig. 5 Horizontal distribution bar graph of hairy root fungal occipital of bald group (OBG) compared to the control group,
genus of hair loss group and control group. Compared with the followed by Trichosporon (green flag) increase, and Fusarium
control group, the alopecia group had a marked increase in (Fusarium, yellow flag) and Epicoccum (Epicoccum, blue flag)
Malassezia (red mark) from the top of bald group (TBG) and the showed a decreasing trend. (Color figure online)

Table 2 Malassezia loads in different groups and different scalp areas

Group n Calvaria Occipital Z P

Hair loss 12 6.7E ? 06(6.8E ? 05, 9.0E ? 06) 1.1E ? 06(5.7E ? 05, 2.6E ? 06) - 2.194 0.028
Control 12 2.6E ? 05(1.2E ? 05, 1.2E ? 06) 5.3E ? 05(9.0E ? 04, 7.5E ? 05) - 0.115 0.908
Z - 3.233 - 1.848
P 0.001 0.065

Table 3 Malassezia globosa loads in different groups and different scalp areas
Group n Calvaria Occipital Z P

Hair loss 12 5.2E ? 05(2.0E ? 05, 2.2E ? 06) 3.4E ? 05(1.1E ? 05, 6.7E ? 05) - 1.155 0.248
Control 12 6.7E ? 04(2.2E ? 04, 2.4E ? 05) 8.1E ? 04(3.0E ? 04, 2.6E ? 05) - 0.115 0.908
Z - 2.944 - 2.309
P 0.003 0.021

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Table 4 Malassezia restricta loads in different groups and different scalp areas 0.007
Group n Calvaria
Hair loss 12 5.2E ? 05(2.2E ? 05, 3.1E ? 06)
Control 12 1.1E ? 05(1.0E ? 04, 2.1E ? 05)
Z - 2.829
P 0.005
Discussion
The skin is the largest organ in the human body and
covers the entire body. It keeps the first barrier of the
human immune system that not only resists the
invasion of foreign pathogens, but also protects
internal tissues and organs. Price et al. [15] have
classified microbes on the surface of the skin into two
categories: resident microorganisms and transient
microorganisms. The resident microorganisms mainly
include actinomycetes, thick-walled bacteria, mem-
bers of proteobacteria, and yeasts (mostly Malasse-
zia). And the growths of these microorganisms are
affected by the stratum corneum, water content,
sebaceous gland and sweat gland secretion, which
leads to the formation of different micro-ecological
distributions on different areas of body skin [16]. The
studies conducted by Clavaud et al. [17] on scalp
microorganisms revealed that the main bacterial flora
on the scalp surface was Propionibacterium acnes and
Staphylococcus epidermidis, while the fungal
microorganisms were mainly Malassezia.
Malassezia plays significant role in the scalp
microecology. The number of Malassezia roots
reached 5/high magnification, and three fields of
view were regarded as positive for Malassezia. It was
observed that the positive rate of AGA alopecia
group (60%) was higher than that of the control group
(40%) (Table 1). We speculated that this difference
may be due to the richness of scalp lipids in AGA
patients. Because this type of scalp environment is
suitable for the growth of Malassezia with lipophilic-
ity, high-throughput molecular sequencing was
applied to detect the fungal microflora in normal
human calvaria and occipital hair roots. The pre-
dominant fungi were Malassezia, and no significant
difference was seen in the abundance of Malassezia
between the top of scalp (8.64%) and occipital
(11.64%) (Fig. 5).
513
Occipital Z P
4.4E ? 05(2.0E ? 05, 8.3E ? 05) - 0.115 0.908
1.4E ? 05(3.1E ? 04, 2.0E ? 05) - 0.404 0.686
- 2.714
0.007
The fungal microflora in the calvaria and occipital
hair roots of AGA patients was detected (Figs. 1, 2, 3)
and furthered investigated by the high-throughput
sequencing method to explore the fungal microecol-
ogy of hairy roots at different sites (Fig. 5). It revealed
that the Malassezia genus still occupied a large
proportion, but interestingly, the abundance at the
top of the calvaria (31.44%) was significantly higher
than that at the occipital region (20.57%). Then, we
speculated that it was due to the uneven distribution of
oils and fats in the scalp of AGA patients that caused
regional imbalance in growth of Malassezia spp.
Therefore, further investigations are warranted to
elucidate the regulatory mechanism.
In Malassezia, most bacteria contain invasive
enzymes, such as lipase that can break down triacyl-
glycerols into free fatty acids, causing local irrigative
reactions, and bacteria also contain a small amount of
proteases [18]. What is more, researchers have iden-
tified that seven strains of Malassezia standard have
proteases activity [19]. Proteases can decompose
keratin and collagen and provide nitrogen sources
for the growth and reproduction of bacteria. These
properties allow Malassezia to adhere to host cells and
colonize on the skin and in deep tissues. The
phenomenon observed by the scanning electron
microscope and the transmission electron microscope
also confirmed this finding (Figs. 1, 2, 3). Scanning
electron microscopy observed that the surface of the
hair roots of positive specimens had more yeast cells
adherence to Malassezia, and the superficial hair roots
were observed under transmission electron micro-
scope (Fig. 2). The tissue contains Malassezia yeast
cells. In addition, the previous studies found that
Malassezia would increase the levels of IL-8, IL-10,
and TGF-b1 in keratinocytes [20]. These cytokines are
critical in the morphological changes and apoptosis of
keratinocytes and have chemotactic activity on neu-
trophils and lymphocytes and the increase in invasive
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enzyme activity, and the expression of inflammatory
cytokines will cause erosive damage to hair roots.
Malassezia increases the secretion of cytokines, which
may cause micro-inflammatory reactions around the
scalp and around the hair follicles. It is significantly
involved in the process of androgenic alopecia.
Moreover, it was demonstrated that glands and
inflammatory cell infiltration around hair follicles
were caused by Malassezia by the previous research
published in British Journal of Dermatology [21].
Compared with healthy people, the abundance of
fungal microecological in the hair roots of androge-
netic alopecia patients has led to the increase in
Malassezia genus species such as Malassezia globosa
and Malassezia restricta species most significantly
seen in hair loss areas (Tables 2, 3, 4). Malassezia
played opposite role in healthy people and AGA
patients because of the different scalp microecology
environments. It maintains a harmonious and balanced
symbiotic relationship with the host in healthy people.
When AGA occurs, because of the increase in local
scalp oil content, the original microbial environment
has changed and then leads to the growth and
reproduction of Malassezia. Invasive enzymes pro-
duced by Malassezia and interleukins produced by
keratinocytes will change the abundance of Malasse-
zia species (such as the increase in Malassezia globosa
and Malassezia restricta) and break the original
micro-ecological balance. It will produce the micro-
inflammatory reaction around the hair follicle and
continues to participate in the entire process of
androgenetic alopecia.
Conclusion
The load of Malassezia was significantly increased in
the patients with androgenic alopecia, and it had a
positive correlation with the incidence rate of andro-
genic alopecia. We hypothesized that Malassezia is
involved in the hair follicles inflammation of AGA
patients and the strategy of controlling the fungal
ecology of scalp will have more vital value in the
treatment of AGA.
Acknowledgements We are grateful to all the team members
who worked hard for the experiment, and all the volunteers who
provided their hair specimens. Moreover, the measurements of
the colonies of Malassezia globosa or Malassezia restricta
123
Mycopathologia (2019) 184:505–515
between calvaria area and occipital area are accomplished by
Research Core Facility of West China Hospital.
Compliance with Ethical Standards
Conflict of interest All authors declare that they have no
conflict of interest.
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