Molecular and Cellular Endocrinology 424 (2016) 42e49

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Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Review

Potential role of estrogen in maintaining the imbalanced sympathetic

and sensory innervation in endometriosis
Yanchun Liang, Shuzhong Yao*
Department of Obstetrics and Gynecology, First Afliated Hospital of Sun Yat-sen University, Guangzhou, China

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 9 May 2015
Received in revised form
2 November 2015
Accepted 13 January 2016
Available online 15 January 2016

Endometriosis, one of the most common benign gynecological diseases, affects millions of women of
childbearing age. Endometriosis-associated pain is a major cause of disability and compromised quality
of life in women. Neuropathic mechanisms are believed to play an important role. An imbalanced
sympathetic and sensory innervation (reduced sympathetic innervation, with unchanged or increased
sensory innervation in endometriotic lesions) has been demonstrated in endometriosis in recent studies.
And it is believed to contribute to the pathogenesis of endometriosis-associated pain. It is primarily
considered to be a natural adaptive program to endometriosis-associated inammation. However, it is
important to further clarify whether other potential modulating factors are involved in this dysregulation. It is generally accepted that endometriosis is an estrogen dependent disease. Higher estrogen
biosynthesis and lower estrogen inactivation in endometriosis can lead to an excess of local estrogen in
endometriotic lesions. In addition to its proliferative and anti-inammatory actions, local estrogen in
endometriosis also exerts potential neuromodulatory effects on the innervation in endometriosis. The
aim of this review is to highlight the role of estrogen in mediating this imbalanced sympathetic and
sensory innervation in endometriosis, through direct and indirect mechanisms on sympathetic and
sensory nerves. Theoretical elaboration of the underlying mechanisms provides new insights in supporting the therapeutic role of estrogen in endometriosis-associated pain.
2016 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:
Estrogen
Sympathetic innervation
Sensory innervation
Endometriosis
Pathophysiology

1. Introduction
Endometriosis is a chronic, benign, multifactorial gynecological
disease affecting millions of women of childbearing age. It is estimated that endometriosis is present in 6e10% of reproductive age
women (Khan et al., 2013) and in up to 20e40% of infertile women.
Among women with pelvic pain, the prevalence goes up to 50%
(Giudice, 2010). The various locations of endometriotic lesions
make the patient suffer from diffuse, dull, but poorly localized
feelings of pain, which are a major cause of disability and
compromised quality of life in women and teenage girls (D'Hooghe
and Hummelshoj, 2006).
Growing evidences tend to support the view that abnormal
innervation of endometriosis plays a crucial role in the generation
of pain (Mechsner et al., 2009; Anaf et al., 2000). There is a recently

* Corresponding author. Department of Obstetrics and Gynecology, First Afliated Hospital, Sun Yat-sen University, No. 58, The 2nd Zhongshan Road, Yuexiu
District, Guangzhou, Guangdong Province 510080, China.
E-mail address: yszlfy@163.com (S. Yao).

reported phenomenon in endometriosis that draws investigators'

interest. Arnold et al. demonstrated an imbalance between sympathetic and sensory nerve bers in peritoneal endometriosis
(Arnold et al., 2012, 2013). The density of substance P positively
stained sensory nerve bers was signicantly lower in the unaffected peritoneum compared to the endometriotic lesion, while the
density of tyrosine hydroxylase (TH) positively stained sympathetic
nerve bers was signicantly higher in the unaffected peritoneum
in comparison to sympathetic nerve ber density (NFD) in the
lesion (Fig. 1). This imbalanced sympathetic and sensory innervation is consistent with the partial results demonstrated by Ferrero
et al., in 2010 (Ferrero et al., 2010). Their research team found out
that sympathetic nerve bers were signicantly lower in the
mucosal and muscular layers near the endometriotic lesions
compared with the area far from the lesions, while sensory NFD
was not altered in the area near the endometriotic lesions. But this
phenomenon has not been investigated in ovarian endometriosis
and deep inltrating endometriosis (DIE). This imbalanced innervation is believed to contribute to the pathogenesis of peripheral
neuropathic pain in endometriosis. Therefore, it is important to

http://dx.doi.org/10.1016/j.mce.2016.01.012
0303-7207/ 2016 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).

Y. Liang, S. Yao / Molecular and Cellular Endocrinology 424 (2016) 42e49

43

DNA binding, or by recruiting steroid receptor coactivator 2 (SRC-2)

(Cvoro et al., 2008).

3. Potential role of estrogen in maintaining imbalanced

sympathetic and sensory innervation in endometriosis
In addition to its proliferative and anti-inammatory actions,
are there any other pathophysiologic effects of estrogen on endometriosis? Here, the potential neuromodulatory effects of estrogen
on the nervous system in endometriosis will be presented. Moreover, as far as we know, this is the rst review that reveals the
potential role of estrogen in maintaining imbalanced sympathetic
and sensory innervation in endometriosis (Fig. 2).

Fig. 1. Imbalanced sympathetic innervation in peritoneal endometriosis. Sympathetic

nerve ber density is signicantly higher in the para-endometriotic area in comparison
to that of the endometriotic lesion.

investigate the mechanism of this imbalanced innervation. Previous studies supposed that the imbalance between sympathetic and
sensory nerve bers in endometriosis was a natural adaptive program to endometriosis-associated inammation (Arnold et al.,
2012, 2013). However, further research is needed to help us have
a better understanding of this imbalance.
Endometriosis is widely accepted to be an estrogen (E)-dependent disease. The dysregulation of the enzymes required to synthesize estrogen leads to the hyperestrogenism environment of
endometriosis. More importantly, estrogen has been demonstrated
to exert neuromodulatory effects through multiple processes
(Wharton et al., 2012). The aim of this article is to highlight the
potential role of estrogen in maintaining the imbalanced sympathetic and sensory innervation in endometriosis in detail, providing
a new pathophysiologic vision of estrogen in endometriosis.

3.1. Potential neuromodulatory effects of estrogen in endometriosis

Combined oral contraceptives (OCCs) are composed of estrogen
(estradiol) and progestogen (progestin). It was reported that cyclic
or continuous OCCs effectively reduced the baseline pain score for
dysmenorrhea (Harada et al., 2008). Other reports showed that
postoperative OCCs could also reduce the frequency and severity of
recurrent dysmenorrhea compared with surgery alone (Seracchioli
et al., 2009, 2010a, 2010b). Moreover, several studies showed that
hormonal treatment signicantly decreased the nerve ber density
in peritoneal endometriotic lesions, in comparison with lesions
from women who did not receive hormone treatment (Tokushige
et al., 2009). As a consequence, estrogen is believed to be
involved in the modulation of endometriosis-associated pain as
well as aberrant innervation in endometriosis.

2. The role of estrogen and its receptors in endometriosis

A review performed by Huhtinen et al. summarizing the results
of a series of studies indicates that all the enzymes required to
synthesize estrogen are expressed in endometriotic lesions (Rizner,
2009; Attar and Bulun, 2006). The aberrant expression of these
estrogen-metabolizing enzymes leads to higher E2 biosynthesis
and lower E2 inactivation, and to an excess of local E2, which results in further proliferation of ectopic endometrium (Giudice and
Kao, 2004). Increased production of local E2 induces prostaglandin E2 (PGE2) formation and then stimulates cyclooxygenase
type 2 (COX-2), which in turn stimulates the expression of aromatase, establishing a positive feedback cycle to enhance E2 production (Rizner, 2009).
There are two distinct isoforms of estrogen receptors (ERs): ERa
and ERb, the actions of which are regulated by ligand availability
and by the presence of various coregulators (Musgrove and
Sutherland, 2009). Estrogen stimulates the growth of endometrial
cells mainly through the activation of ERa (Shang, 2006; Vivacqua
et al., 2006). But many studies have shown that both ERs
contribute to stimulation of the cell cycle and promotion of endometriotic stromal cells proliferation (Harris et al., 2005;
Trukhacheva et al., 2009). Estrogens also exert anti-inammatory
functions by repressing several inammatory genes including IL8, IL-6, TNF-a. ERa and ERb, mainly ERa, mediate the antiinammatory action of estrogens by inhibiting NF-kB activity and

Fig. 2. Estrogen modulates the imbalanced sympathetic and sensory innervation in

multifactorial mechanisms. High level of estrogen (top) acts directly on both sympathetic and sensory nerves in endometriotic lesion, partly leading to reduced sympathetic innervation and increased/unchanged sensory innervation. Estrogen also exerts
up-regulation on specic molecules (such as Sema 3F, NGF, BDNF, Ntm, etc.) and induces degeneration of sympathetic nerves with preserved or increased sensory nerves.
Moreover, SMCs, acting as a functional target, also respond to estrogen leading to a
restricting environment for sympathetic nerve growth, but an permissive or a stabilizing environment for sensory nerve growth. In para-endometriotic area, opposite
effects are induced by low level of estrogen (bottom). NGF: nerve growth factor; BDNF:
brain derived neurotrophic factor; VEGF: vascular epithelial growth factor; Sema 3F:
Semaphorin 3F; Ntm: neurotrimin; ANG II: Angiotensin II; SMCs: smooth muscle cells.

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Y. Liang, S. Yao / Molecular and Cellular Endocrinology 424 (2016) 42e49

3.2. Effects of estrogen on sympathetic nerves

3.2.1. Direct neuromodulatory effects of estrogen on sympathetic
nerves
Sympathetic nerves within female reproductive system are
affected by changes in the internal endocrine environment. For
example, the density of myometrial sympathetic nerve bers is
greatly decreased during pregnancy and axonal regeneration is
obtained soon after parturition (Haase et al., 1997). In adult female
rodents, widespread degeneration of sympathetic nerves in the
myometrium is observed when the levels of estrogen peak during
proestrus and estrus. However, during the metestrus and diestrus
phases of the cycle, the myometrium obtained axonal regeneration
with a decreasing level of estrogen (Zoubina and Smith, 2000).
Furthermore, these uctuations of level of estrogen and consequential imbalance of sympathetic and sensory innervation in
endometriotic lesions contribute to changes in endometriosisinduced vaginal hyperalgesic severity of the rats (Zhang et al.,
2008). Therefore, there is strong evidence supporting the modulatory effects of estrogen on sympathetic nerve bers and
endometriosis-associated pain.
In vivo injection of estrogen in ovariectomized (OVX) donor rats
showed diminished neurite outgrowth. And ganglia obtained from
the rats were responsive to estrogen added to the culture medium;
while those obtained from estrogen untreated OVX rats were unresponsive. These ndings indicate that some factors present in
non-OVX rats with low E2 levels act on the ganglion to prevent
estrogen from inhibiting neurite outgrowth. Neuritogenesis was
normal in culture of ganglia from hypophysectomized OVX donors,
but neurite outgrowth was attenuated once estrogen was added to
the culture medium (Krizsan-Agbas and Smith, 2002). It is
considered that high level of estrogen inhibits the release of prolactin from the pituitary gland (Ronchetti et al., 2013), thus
enhancing the responsiveness of sympathetic nerve bers to estrogen, and, eventually promoting sympathetic neurite outgrowth.
In addition, a slight repellent effect by low concentrations of 17bestradiol was also observed in cultured sympathetic nerve bers
(Klatt et al., 2012).
In mice with low circulating estrogen, or ERa knockout mice
with high estrogen, the uterus contains abundant sympathetic
nerves (Zoubina and Smith, 2001). High circulating estrogen reduces the numbers of intact sympathetic nerve bers in wild type
mice, whereas single injection of estradiol is completely ineffective
in altering nerve density in ERa knockout mice (Zoubina et al.,
2001). Studies have shown that both ERa and ERb are expressed
in sympathetic neurons (Zoubina and Smith, 2002, 2003). But estrogen acting through ERa determines the sympathetic nerve terminal branches within uterine smooth muscle target. Taken
together, estrogen regulates the degeneration of sympathetic
nerves partly through acting directly on sympathetic nerve bers
by combining with ERa when the myometrium is involved
(Zoubina and Smith, 2001).
3.2.2. Indirect neuromodulatory effects of estrogen on sympathetic
nerve
(1) Modulation through actions on target matrix
Sympathetic neurites were observed to grow closely associated
to myometrial smooth muscle cells. The preferential association
indicated that there existed interactions between growing neurites
and specic molecules present in the cell membranes and/or surrounding the extracellular matrix (ECM) and basal lamina (Brauer
et al., 1995, 1999, 2000).
Except for the endometrial like cells, endometriosis-associated

smooth muscle cells (SMCs) are found to be present in peritoneal

endometriosis (Anaf et al., 2000; Mechsner et al., 2005), ovarian
endometrioma (Mechsner et al., 2005; Fukunaga, 2000), and deep
inltrating endoemtriosis (van Kaam et al., 2008). These cells
constitute a considerable portion (up to 80%) of the endometriotic
lesions (Mechsner et al., 2005; Barcena et al., 2011a). It is demonstrated that estrogen receptors are expressed on these cells,
providing the possibility that estrogen acts on SMCs through a
ligandereceptor interaction pathway (Barcena et al., 2011a).
Previous studies have shown that sympathetic ganglion explant
outgrowth is enhanced in the presence of different types of target
tissues, including heart, blood vessels, intestine and vas deferens
(Rawdon and Dockray, 1983; Southwell et al., 1985; Rawdon, 1991).
The presence of myometrial target from OVX donor rats in cultured
sympathetic ganglion markedly increased the number of neurites
compared to sympathetic ganglion cultured alone. But the neuritogenesis inducing effect exerted by myometrium is markedly
inhibited by in vivo treatment of E2, which is consistent with the
in vivo observations that E2 prevented normal developmental increases in sympathetic innervation in developing rats (Brauer et al.,
1999) and attenuated collateral sprouting of iris sympathetic
nerves into myometrial explants in oculus (Brauer et al., 2000).
Myometrial substrate-bound signals play a crucial role during
development and continue to function during maturity to regulate
neuronal plasticity and regeneration (Hou et al., 2008). Further
study demonstrated that alterations of myometrial substratebound signals induced by estrogen contribute to diminished myometrial supportive ability to sympathetic nerve growth (Richeri
et al., 2010). In addition, ERa is expressed in myometrial smooth
muscle cells (Hiroi et al., 1999; Wang et al., 1999) and is required for
E2 to elicit modulation of uterine sympathetic remodeling (Zoubina
and Smith, 2001). SMCs contained in endometriotic lesions are also
similar to the myometrial cells in the uterus. Similar to that exerted
by myometrium, interactions between these SMCs and sympathetic
nerves may lead to a sympathetic neuritogenesis facilitating effect,
thus regulating the abnormal innervation of sympathetic nerve in
endometriosis under abnormal levels of estrogen.
A possible mechanism may be involved in the induction of
pathological outgrowth of sympathetic nerve bers and subsequent hyper-innervation by SMCs. Mechanical stretch of SMCs is
produced by the chaotic/irregular contractions mediated by
oxytocin receptor, leading to pathophysiological stimulus within
the lesions. More importantly, mechanical stretch is able to induce
the growth of sympathetic neurocytes by enhancing the expression
of nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF)
in an autocrine/paracrine manner (Rana et al., 2010). It is believed
that SMCs are more likely to originate from smooth muscle metaplasia (SMM), out of stromal cells or epithelialemesenchymal
transition (EMT) (Hayashida et al., 2011) of the endometriotic cells,
other than from basal stem cells or reactivated coelomic epithelial
cells. As a result, SMCs close to the endometriotic lesion (intrastromal SMCs/surrounding SMCs) seem to be immature, whereby
the peripheral SMCs are fully differentiated (Barcena et al., 2011a).
The contractility and subsequent mechanical stretch induced by
well differentiated peripheral SMCs is much stronger than that
induced by immature SMCs because mature SMCs possess a welldeveloped system of contractile myolaments (Mouravas et al.,
2010). Therefore, sympathetic neurite outgrowth is increased to a
greater degree in the peripheral area of the endometriotic lesion
than that in the endometriotic lesion itself. Most interestingly, Rak
Hachani et al. demonstrated that sympathectomy of hypercholesterolemic rats not only increased sensory innervation, but also
induced vascular SMC dedifferentiation with higher expression of
vimentin and lower expression of h-caldesmon. This phenomenon
is extremely similar to the decreased sympathetic and increased

Y. Liang, S. Yao / Molecular and Cellular Endocrinology 424 (2016) 42e49

sensory innervation in peritoneal endometriotic lesion (Hachani

et al., 2010). And immature SMCs resulting from inadequate sympathetic innervation exert decreased mechanical stretch within the
lesion. In contrast, it is believed that sympathetic innervation
promotes SMCs differentiation in a positive feedback in paraendometriotic areas (Damon, 2005) (Fig. 3). In a word, SMCs also
act as a target of estrogen in endometriosis, leading to the loss of
sympathetic innervation within the endometriotic lesions and an
increased sympathetic nerve density peripheral to the lesions.
Taken together, these ndings support the notion that estrogen
acts directly on estrogen receptors expressed on the target endometriotic tissue, regulating sympathetic neuritogenesis in a
cellecell interaction manner.
(2) Modulation through acting on specic molecules

3.2.2.1. Nerve growth factor. Nerve growth factor (NGF), known to

be a neuron growth-activating factor, is highly expressed in endometriotic lesions and in peritoneal uid from patients with endometriosis. Previous reports have indicated that NGF is required for
the regenerative remodeling of the uterine sympathetic innervation after pregnancy (Varol et al., 2000). More importantly, higher
NGF expression has been proven to be correlated with a higher ratio
of sensory to sympathetic nerve bers (Arnold et al., 2012). Under
optimal conditions preferring the binding of NGF and TrkA, NGF
mediates sympathetic neuronal survival and axonal growth (Kohn
et al., 1999). However, under suboptimal conditions, sympathetic
neuronal apoptosis and growth cone inhibition are promoted by
the combining of NGF and p75NTR (Kohn et al., 1999; Bamji et al.,
1998). Growing evidence indicates that estrogen is able to regulate the synthesis and release of NGF and its receptors (TrkA and
p75NTR) through interactions with estrogen response elements
that are present in promoter regions of the target gene (Zoubina
and Smith, 2001; Krizsan-Agbas et al., 2003a). And the regulation
by estrogen changes the neurotrophic environment required for
maintaining the sympathetic and sensory innervation. Studies have
demonstrated that estrogen reduces NGF protein content in

Fig. 3. Positive feedback loop between smooth muscle cells (SMCs) differentiation and
sympathetic innervation in para-endometriotic areas. Mature SMCs exerts a stronger
mechanical stretch inducing the growth of sympathetic neurocytes by enhancing the
expression of nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF); while
an increased sympathetic neuritogenesis also induces differentiation of SMCs, with
increased h-caldesmon, a-SMA and desmin expression but decreased vimentin
expression.

45

sympathetic vascular targets, which may lead to decreased sympathetic innervations to these targets (Kaur et al., 2007). Interestingly, opposite up-regulation of NGF in the uterus is observed when
long-term estrogen is applied (Krizsan-Agbas et al., 2003b; Bjorling
et al., 2002). Acute and chronic estrogen treatment also reduced
TrkA and p75NTR expression in sympathetic neurons (Hasan et al.,
2005) and in endometriotic lesions (Tokushige et al., 2008). However, there is also evidence supporting that the expression of
p75NTR in uterine-projecting sympathetic neuron is promoted by
estrogen (Richeri et al., 2005). As a result, there are many controversies in different regulatory effects of estrogen on NGF and its
receptors. Further studies are required to identify the distinct
functions of estrogen on NGF and its receptors.
NGF is synthesized as a precursor, proNGF, which undergoes
processing posttranslationally to generate mature NGF (NGF-b)
(Fahnestock et al., 2004). Studies showed that proNGF and its
proteolytically processed protein products may differentially activate pro- and antiapoptotic cellular responses through preferential
activation of TrkA or p75NTR (Lee et al., 2001). Highly accumulated
ProNGF in intermyometrial connective tissue layers at term pregnancy where sympathetic axon degeneration occurs may
contribute to the pregnancy-related uterine degeneration (Lobos
et al., 2005). ProNGF has been shown to be neurotoxic when
bound in a heterotrimer with the p75 receptor and the receptor
sortilin. In vitro experiments show that proNGF is indeed neurotoxic for NGF-responsive sympathetic neurons and that blockade of
sortilin rescues proNGF-induced cell death (Al-Shawi et al., 2007).
Therefore, increased proNGF in intermyometrial layers combined
with increased sortilin expression in projecting sympathetic neurons may contribute to neuronal atrophy and degeneration.
Since TrkA and p75NTR are required for carrying out the
modulatory effects on sympathetic neurons of NGF, downregulation of both receptors by estrogen attenuates the functions
of NGF because of the limitation of saturated ligandereceptor interactions. Markedly up-regulation of NGF may be a compensatory
response to the decreased sympathetic neurite outgrowth. Another
explanation is that the expression pattern of NGF, TrkA and p75 is
more correlated to progesterone than estrogen (Bjorling et al.,
2002; Shi et al., 2006), which indicates that estrogen and/or progesterone may act synergistically with NGF in modulating sympathetic nerve development and growth. Thirdly, up-regulated NGF
prefers to promote axonal growth of sensory nerve bers, other
than inhibits mature sympathetic ganglia outgrowth in estrogenrich conditions (Krizsan-Agbas et al., 2003a; Latini et al., 2008).
Moreover, there may be other factors that can repress NGF-TrkA
signaling in sympathetic neurons, limiting its neurite outgrowth
promoting functions (Atwal et al., 2003).
3.2.2.2. Semaphorin 3F. Semaphorin 3F (Sema 3F), a member of
class-3 secreted semaphorins that are originally identied to as
axonal guidance molecules through repulsion during the development of the nervous system (Serini et al., 2013), has been
demonstrated to be repellent for sympathetic nerves by binding to
its receptor neuropilin-2 (NRP2) and co-receptor plexin A2 (Fassold
et al., 2009). Interestingly, Sema 3F can also antagonize NGF-TrkA
signaling in sympathetic neurons (Atwal et al., 2003). In vivo and
in vitro studies have shown that NRP2 is required in mediating the
repulsive axonal responses to Sema 3F (Fassold et al., 2009; Chen
et al., 2000; Waimey et al., 2008).
Estrogen provokes a tissue-specic induction of Sema 3F, which
prefers to locate in the hypertrophic connective tissue separating
smooth muscle bundles and surrounding major blood vessels
(Richeri et al., 2011). Estrogen treatment of OVX rats decreased
uterine NRP2 mRNA levels (Northern blot analysis) approximately
40% compared with those without treatment (Pavelock et al., 2001).

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Y. Liang, S. Yao / Molecular and Cellular Endocrinology 424 (2016) 42e49

In contrast, another study suggested that estrogen induced the

expression of NRP2 (immunohistochemistry analysis) in uterine
nerves (Richeri et al., 2011). At the end of chronic estrogen treatment, no TH positively stained intrauterine perivascular nerves
were visible. And simultaneous immunostaining of NRP2 and TH
demonstrated no TH-immunoreactivity in these NRP2 positively
stained nerve bers, which suggested NRP2 might be expressed in
sympathetic nerves only after the loss expression of TH during the
estrogen-induced sympathetic degeneration (Kitawaki et al., 2002).
Therefore, up-regulation of Sema 3F and aberrant modulation of
NRP2 also contribute to estrogen inducing sympathetic nerve ber
repulsion, possibly through antagonizing potential axonal promoting factors or complicated sequential loss and gain of modulators on sympathetic nerves.
3.2.2.3. Brain derived neurotrophic factor. Brain derived neurotrophic factor (BDNF) can exert potential suppression of sympathetic neuritogenesis in myometrium, but the underlying
mechanisms are unclear (Krizsan-Agbas et al., 2003b). One possible
pathway is that BDNF activates p75NTR, leading to the production
of ceramide, which induces neuronal apoptosis (Bamji et al., 1998)
and inhibits axonal elongation (Rice et al., 1998). Estrogen injection
of OVX rats could increase the level of BDNF in myometrium and
endometrium. Further in vitro experiments showed that BDNF was
required for estrogen to suppress sympathetic neurite outgrowth
(Krizsan-Agbas et al., 2003a). However, BNDF neutralization fails to
fully reverse estrogen's effects, suggesting that additional targetderived factors also participate.
3.2.2.4. Neurotrimin. Neurotrimin (Ntm) is a neural cell adhesion
molecule that is present in the uterus. In uteri of rats at estrus,
when sympathetic innervation density is low, Ntm staining was
markedly increased in circular and longitudinal smooth muscle.
Moreover, in vivo experiments showed that myometrial Ntm mRNA
and protein were up-regulated 6 h after estrogen administration.
Estrogen also decreased neurite outgrowth in smooth
muscleeneuron cocultures, indicating an unsuitable condition for
sympathetic neurite growth induced by estrogen. Furthermore,
Ntm knockdown impaired estrogen's ability to reduce sympathetic
innervation (Krizsan-Agbas et al., 2008). Collectively, Ntm may also
potentially act as a target mediated by estrogen that exerts repellent effects on sympathetic innervation in endometriosis.
3.2.2.5. Artemin. Artemin, a member of the glial cell-derived neurotrophic factor (GDNF) (Warnecke et al., 2010), acts as a guidance
molecule for the growth of sympathetic nerve bers along blood
vessels in many tissues (Honma et al., 2002). The migration and
axonal projection of sympathetic nerve bers, other than sensory
nerve bers, are severely impacted with knockouts of artemin or its
receptor GFRa (Honma et al., 2002). The expression of artemin in
endometriosis is not reported yet, but it is demonstrated to be
increased in endometrial carcinoma (Kang et al., 2010). Recently,
the expression of artemin is shown to be positively correlated to the
ER status in human mammary carcinoma, indicating an estrogeninducible characteristic of artemin. The circumstantial evidence
suggests that estrogen might also play a role in regulating artemin
in endometriosis. Therefore, the alteration of artemin expression
results in the inhibition of sympathetic neuritogenesis in endometriotic lesions. However, this hypothesized theory needs further
verication.
3.2.2.6. Other potential specic molecules. Vascular epithelial
growth factor (VEGF)some inammatory factors such as IL-6
(Canellada et al., 2008) or TNF-a (Velasco et al., 2006) are also
regulated by estrogen, and may also play important roles in

regulating abnormal sympathetic and sensory innervation in

endometriosis. Additional investigations need to be performed to
further verify these hypotheses.
3.3. Effects of estrogen on sensory nerves
3.3.1. Direct effects of estrogen on sensory nerves in endometriosis
Nociceptive sensory nerve hyperinnervation is implicated in
some female pain syndromes, including interstitial cystitis, vulvodynia, irritable bowel syndrome and breast pain (Pang et al., 1995;
Bohm-Starke et al., 1998; Gopinath et al., 2005). ERa and ERb are
also expressed on sensory neurons, which indicates that estrogen
may also affect sensory axonal outgrowth through acting directly
on the neuron (Taleghany et al., 1999; Papka and Storey-Workley,
2002). In fact, there are only a few studies that specially investigate direct effects of estrogen on sensory nerves. Blacklock et al.
demonstrated that chronic estrogen elevation induced selective
proliferation of rat mammary gland calcitonin gene related peptide
(CGRP) positively stained sensory nerve bers (Blacklock and
Smith, 2004). But this proliferative effect was restricted to nerves
around the blood vessels, which served as nociceptors that induced
a vasodilatory function. However, the sensory of the nipple and
adjacent skin was not signicantly affected, which was consistent
with the ndings from the study of Traurig et al. (Traurig et al.,
1984). These observations suggested that sensory nerves might
respond to uctuation of estrogen levels in a target specic pattern.
Later on, Blacklock et al. found that estrogen exerted by direct effects on nociceptor neurons to promote axon outgrowth via an
NGF-independent mechanism, instead of eliciting nociceptor hyperinnervation by promoting targeted release of neurotrophic
factors (Chakrabarty et al., 2008). Accordingly, cellular and molecular mechanisms responsible for estrogen-regulating sensory
innervation are still poorly understood.
3.3.2. Indirect neuromodulatory effects of estrogen on sensory
nerves in endometriosis
3.3.2.1. Smooth muscle cells. However, regulatory effects of estrogen on target tissue or the target derived trophic factors may also
inuence sensory neuritogenesis (Bjorling et al., 2002;
Albuquerque et al., 1998). The irregular contraction of
endometriosis-associated smooth muscle cells caused by local
inammation stimulates the nociceptors (sensory nerve bers)
within the lesion, thus generating endometriosis-associated pain
(Mechsner et al., 2005; Odagiri et al., 2009). Similar to the myometrial cells in the uterus, estrogen receptors are also expressed on
these smooth muscle cells, indicating that these cells might also act
as a direct functional target of estrogen (Barcena et al., 2011a).
3.3.2.2. Nerve growth factors. The overexpression of nerve growth
factor (NGF) in peritoneal uid from women with endometriosis
also plays an important role in sensory neurite outgrowth. Treatment with NGF inhibitor (both anti-NGF and K252a) can inhibit
neuritogenesis of dorsal root ganglion (DRG) (Barcena et al., 2011b).
Another research demonstrated that transfection of rats with surgically induced endometriosis using specic b-NGF siRNA
decreased the density of CGRP and SP immunoreactive sensory
nerve bers in the implants (Chen et al., 2014) as well as density of
nerve bers stained with NGF, TrkA, and NGFRp75. In fact, estrogen
was shown to increase levels of the TrkA in sensory nerves
(Sohrabji et al., 1994; Lanlua et al., 2001). As a result, up-regulation
of NGF in endometriosis induced by estrogen can stimulate the
sensory neurite outgrowth, which may be involved in the increased
sensory innervation in peritoneal endometriotic lesions.
3.3.2.3. Semaphorins. Semaphorin 3F is known to be a repellent of

Y. Liang, S. Yao / Molecular and Cellular Endocrinology 424 (2016) 42e49

sympathetic nerve bers (Chen et al., 2000, 1998), but there are no
studies exploring its role in regulating sensory neurite outgrowth
in endometriosis. However, expressions of other semaphorins
(such as Sema 3A, a repellent factor of both sympathetic and sensory nerve bers (Kawasaki et al., 2002; Long et al., 2009; Graf et al.,
2012)) in endometriosis may play potent roles in regulation the
abnormal sensory innervation in endometriosis. Additional studies
are needed to gure out the relationship of estrogen and these
repellent factors, and whether the aberrant expression of these
factors contributes to the imbalance of sympathetic and sensory
innervation in endometriosis.

47

important potent modulator that maintains this imbalanced sympathetic and sensory innervation in endometriosis. Estrogen regulates multiple aspects of the endometriotic microenvironment to
selectively induce the repulsion of sympathetic axon while preserving sensory innervation. However, other mechanisms underlying this complicated pathophysiological process still remain to be
further studied. It is also important to discover new neuromodulatory targets of estrogen in the future, providing more evidence elucidating this dysregulation of sympathetic and sensory
innervation in endometriosis.
Conicts of interest

3.3.2.4. Angiotensin . Angiotensin II (ANGII), the major biologically active endogenous ligand of renin-angiotensin system (RAS),
is present in the lumber dorsal root ganglion (DRG) of adult rats
(Patil et al., 2010; Smith et al., 2013; Muralidharan et al., 2014).
Locally synthesized ANGII can induce estrogen-mediated sensory
DRGs neurite outgrowth through activating ANGII type 2 receptor
(AT2R), a receptor co-localized with DRG sensory neurons and upregulated by estrogen (Chakrabarty et al., 2008). ANGII signaling
via the AT2R induces neurite outgrowth in cultured cells of
neuronal origin via a mechanism involving nerve growth factor
(NGF)-independent phosphorylation of the neurotrophic tyrosine
kinase A receptor (TrkA) (Plouffe et al., 2006). PD123,319, a selective AT2R blocker, can eliminate estrogen-mediated DRG neurons
sprouting in vitro, further supporting that AT2R plays a central role
in E2-induced axon sprouting (Chakrabarty et al., 2008). Moreover,
AT2R up-regulation by estrogen also promotes hyperinnervation in
cutaneous, glandular, and vascular targets in vivo (Blacklock and
Smith, 2004; Blacklock et al., 2004). Eutopic endometrium can
produce ANGII through local angiotensin-converting enzyme (ACE)
activity (Ahmed et al., 1995), but the role of locally synthesized
ANGII remains speculative. After ectopic transplantation, ANGIIactivating AT2R may also play an essential role in estrogenpromoting sensory hyperinnervation in endometriotic lesions.
3.3.2.5. Brain derived neurotrophic factor. BDNF inhibits the axonal
outgrowth from small dorsal root ganglion neurons in culture
(Gavazzi et al., 1999) through acting on the p75NTR expressed on
sensory nerves (Mu et al., 1993). But the sensory nerves do not
degenerate in the uterine after the level of estrogen was elevated,
which makes it uncertain how estrogen affects the sensory
innervation.
3.3.2.6. Other potential specic molecules. Ntm promotes
outgrowth of resident sensory nerves as well as stabilizes sensory
axons, thus ensuring the preservation of sensory innervation
(Krizsan-Agbas et al., 2008). Inammatory factors as well as immune factors in response to up-and-down levels of estrogen also
play an important role in regulating sensory neurite outgrowth.
Due to a large number of these factors and various complicated
functions involved in this process, we will not present and discuss
them here in further detail.
4. Conclusion
Locally higher E2 biosynthesis and lower E2 inactivation, as well
as aberrant expression of ERa and ERb in endometriosis further
support the estrogen-dependent characteristic of endometriosis.
Except for its proliferative and anti-inammatory actions, estrogen
also exerts neuromodulatory functions through direct or indirect
mechanisms on sympathetic and sensory nerves. The major
outcome is reduced sympathetic innervation, with unchanged or
increased sensory innervation in endometriotic lesions. Taken all
these evidences together, we propose that estrogen acts as an

None declare.
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