Cell growth and development
Based on the high degree of staining for BDNF in both glands and stroma of PE and DIE, this neurotrophin appears to play a role in the innervation of endometriosis. TrkB, the main receptor of BDNF is expressed more highly in glands than in stroma, and highest in the stroma of DIE, but with high variability. The other receptor for BDNF, p75, showed similar expression, namely high expression in glands of PE and DIE, moderate expression in stroma of DIE and relatively low expression in stroma of PE. BDNF mRNA expression has previously been shown in eutopic endometrium of women with endometriosis [
20] and also in normal human and mammalian uterus as well as endometrium (glandular epithelium and stroma) [
21]. We confirm, for the first time in histological sections, that BDNF is present in endometriosis lesions, both DIE and in peritoneal lesions.
NGF and BDNF are both important for axonal growth of sensory neurons, but each induces growth of a different type of sensory neuron. In vitro studies in chick dorsal root ganglion showed that neuronal growth cones turned and migrated under NGF-coated beads through the expression of TrkA receptors [
22]. Another study showed that visceral afferent neurons in the nodose/petrosal sensory ganglion complex innervated vascular afferents that express high levels of BDNF in the development of arterial baroreceptors. The survival of these neurons was reduced by TrkB-Fc blocking [
23]. Analogous mechanisms may be responsible for inducing sensory neuron growth in endometriosis lesions via both TrkA and TrkB.
BDNF and NGF play different roles in sensory nerve development, with BDNF influencing axonal branching and the growth of lathellipodia and NGF influencing axonal elongation of sensory neurons from the dorsal root ganglion [
24]. Furthermore, endometriosis is an estrogen-dependent disease in which lesions stimulate their own growth by producing estrogen via aromatase activity [
25] and also via the mechanism of tissue injury and repair [
26]. Wessels et al. showed that estrogen exposure may activate BDNF-TrkB pathways in a mouse model, exerting wide ranging effects such as neural development, cell differentiation, growth and maintenance, angiogenesis, proliferation, and resistance to apoptosis [
17]. In clinical studies in humans, Wessels et al. reported that that plasma BDNF concentrations were significantly higher in women with endometriosis than in controls, whereas other neurotrophins, NGF and NT4/5, were not different [
18].
It is known that estrogen may have local proliferative actions as well as neuromodulatory effects on the innervation of endometeriosis [
19]. In this retrospective study, we were not able to fully account for the menstrual cycle phase at which the histological sample was obtained. However, in a recent report, there was no effect of menstrual cycle phase on circulating BDNF levels in women with endometriosis [
18]. In mice, estrogen exposure after ovariectomy significantly increases uterine BDNF, but the hormonal fluctuations of the murine estrous cycle do not [
17]. Importantly, none of the women in this study used hormonal medication for at least 3 months prior to sample collection, as hormonal treatment has been shown to decrease the nerve fiber density in peritoneal endometriosis lesions [
27].
We found that NGF was higher in PE than in DIE, in both glands and stroma. Previous studies looked only at endometrioma, adenomyosis, peritoneal lesions [
13,
28,
29] and eutopic endometrium from endometriosis patients [
20]. The main receptor of NGF, TrkA was not different between PE and DIE in both locations. These results differ from those of Anaf et al. who found that deep adenomyotic lesions had higher expression of NGF immunohistochemically than peritoneal endometriosis [
13] and imply that DIE and adenomyosis are not comparable entities when it comes to neurotrophin expression.
We found a low NGF expression as well as low nerve fiber density in DIE, especially in non-bowel DIE. Perhaps, the loss of NGF-TrkA signaling transduction causes failure of NGF-dependent neuron to survive [
30]. Furthermore, in sensory developmental studies, NGF is important for nociceptor development and BDNF is important for mechanoreceptor development [
31]. Our results are consistent with those of Arellano et al. who implicated the peritoneum as an important location for pathogenesis and pain generation in endometriosis [
32]. Thus, our results support the concept that NGF is involved in neuronal development and likely pain generation from peritoneum in endometriosis [
12,
13,
32].
Many studies have evaluated the role of Trk receptors in the invasiveness or degree of progressiveness in cancer. TrkA receptors appear to promote the growth and metastasis in breast cancer [
33,
34], while TrkB receptors promote invasion in choriocarcinoma cells [
35]. We found that TrkB expression was high in DIE, especially in stromal cells. This pattern is similar to that seen for p75 expression, but not for TrkA expression. This finding could be secondary to an effect of estrogen [
17]. Therefore, we speculate that p75 receptor is involved in survival rather than apoptosis in endometriosis.
Bowel innervation
On first analysis, it appears that there is no difference in mean nerve density between PE and DIE. However, critical differences appear when DIE from bowel and non-bowel locations was evaluated separately, as also shown by Wang [
8]. We found that the sub-group of non-bowel DIE endometriosis actually had a lower density of innervation than the PE.
The DIE from bowel showed markedly higher innervation, both total nerve density as well as the density of sympathetic nerves stained with TH. The innervation of bowel is highly complex, stemming from both intrinsic (from the enteric nervous system) as well as extrinsic nerves (from autonomic nervous system—parasympathetic and sympathetic nervous system). In addition, the intestine has sensory afferents originating from the vagus nerve (nodose ganglion) [
36,
37] and sensory afferents originating from dorsal root ganglion [
38]. A previous study likewise demonstrated that normal bowel has a rich innervation base on nerve fiber density area stained with PGP9.5 and other markers [
39,
40]. The plexus of Auerbach and Meissner (submucosal plexus) likewise express PGP9.5, and may be damaged by the invasion of an endometriosis lesion, as we also showed [
41‐
43]. Thus, it is very difficult and likely error-prone to compare the endometriosis-influenced innervation of PE to bowel endometriosis because the intrinsic and extrinsic innervations of bowel cannot be differentiated with immunohistochemistry. In future studies, investigators should focus on DIE from non-bowel sources when making comparisons to PE.
Table
3 summarizes the relative abundance of NGF, BDNF, TrkA, TrkB, p75, and nerve fibers densities for PE, non-bowel and bowel DIE.
Table 3
Relative abundance of the neurotophins NGF and BDNF, their receptors and nerve fibers in PE and DIE from non-bowel sources
Correlation between cell expressing neurotrophins and the nerve fibers density
BDNF showed a negative correlation to total nerve fibers stained with PGP9.5 in glands of DIE non-bowel only, but not in other sites and not in the stroma. These results may reflect the phenomenon of axonal pruning, through which BDNF may induce pruning in neurons and thus lead to a lower nerve fiber density in the tissue. Our results are consistent with those of Singh et al. who showed that BDNF via p75 receptor influenced the development of sympathetic axon pruning, despite the simultaneous presence of NGF at the same site [
44].
We found no correlation between NGF and nerve fiber density, as also reported by others [
45,
46]. NGF competes for the same axons as BDNF to determine the fate of nerve fibers/neurons. In in vitro studies, the pruning/apoptosis effects of BDNF override the growth effects of NGF. Induction of pruning is stronger than the induction of growth [
44]. This would explain why NGF-positive immunostaining did not correlate to a higher density of nerve fibers but instead there was a negative correlation between BDNF and nerve fiber density seen in DIE.
We did find a negative correlation between BDNF-positive staining and the density of nerve fibers stained with TH in the gland of non-bowel DIE. These results are consistent with the study by Krizsan-Agbas et al. which showed in a rat model that BDNF suppresses sympathetic neurite growth and that this effect is mediated by estrogen [
45].