A C TA Obstetricia et Gynecologica
AOGS M A I N R E SE A RC H A R TI C LE
Use of hormonal therapy is associated with reduced nerve fiber density in deep infiltrating, rectovaginal endometriosis SATU TARJANNE1, CECILIA H.M. NG2, FRANK MANCONI2, JOHANNA AROLA3, MAARIT MENTULA1, BHARVI MANECK2, IAN S. FRASER2 & OSKARI HEIKINHEIMO1 1
Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, Department of Obstetrics, Gynecology and Neonatology, Queen Elizabeth II Research Institute for Mothers and Infants, University of Sydney, Sydney, New South Wales, Australia, and 3Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, Helsinki, Finland
2
Key words Deep infiltrating endometriosis, pain, nerve fibers Correspondence Oskari Heikinheimo, Department of Obstetrics and Gynecology, Helsinki University Hospital/ €opisto Hospital, P.O. Box 610, 00029K€atilo HUS, Helsinki, Finland. E-mail: [email protected] Conflict of interest ISF has received lecture fees, expenses and research support, and undertaken consultancies, for Bayer and Merck. Similarly, OH has undertaken consultancies and given lectures for fee for these companies and ST has given lectures for fee for Bayer. The other authors have no conflict of interest in relation to this work. Please cite this article as: Tarjanne S, Ng CHM, Manconi F, Arola J, Mentula M, Maneck B, et al. Use of hormonal therapy is associated with reduced nerve fiber density in deep infiltrating, rectovaginal endometriosis. Acta Obstet Gynecol Scand 2015; 94: 693–700. Received: 17 September 2014 Accepted: 5 April 2015 DOI: 10.1111/aogs.12652
Abstract Objective. To study the density of nerve fibers in cases of deep infiltrating endometriosis (DIE) of the rectovaginal septum in relation to various clinical factors. Design. A research laboratory-based study. Setting. A tertiary center together with a research laboratory. Methods. Archived DIE tissue samples from 45 women operated upon for rectovaginal septum DIE were re-examined histologically, and by immunohistochemistry. Main outcome measures. The effect of progestogens or combined oral contraceptives on the density of nerve fibers, and the expression of nerve growth factor (NGF) and its high-affinity receptor (tyrosine kinase receptor A, Trk-A). Results. The use of hormonal therapy was associated with reduced densities of sympathetic, parasympathetic and sensory nerve fibers in DIE lesions. Density of total nerve fibers (with pan-neuronal marker PGP9.5) was significantly lower (p < 0.05) in lesions collected from hormone-treated women (8.6/mm², 4.2–20.8/mm²; median density, from 25th to 75th quartiles) compared with that in lesions from untreated women (24.9/mm², 11.2–34.9/ mm²). DIE lesions stained strongly for NGF and its receptor Trk-A. Expression of NGF, but not of Trk-A, was significantly reduced during use of hormonal therapy. Conclusions. Use of hormonal therapy was associated with significantly reduced nerve fiber density in DIE lesions. This may be an important mechanism of action of hormonal therapy for controlling DIE pain symptoms. The expression of estrogen-regulated NGF and its receptor was only partially suppressed during the use of hormonal therapy, suggesting that local estrogen action is often maintained during conventional hormonal therapy in cases of DIE. COC, combined oral contraceptives; DIE, deep infiltrating endometriosis; NGF, nerve growth factor; NPY, neuropeptide-Y; PGP9.5, protein gene product 9.5; rAFS, revised American Fertility Society; RVE, rectovaginal endometriosis; SP, substance P; Trk-A, tyrosine kinase receptor A; VIP, vasoactive intestinal peptide.
Abbreviations:
Key Message Introduction Endometriosis is a benign estrogen-dependent disease occurring in 6–10% of women of reproductive age (1,2). It is frequently associated with severe pelvic pain symptoms such as dysmenorrhea and dyspareunia. The most
Progestogen and oral contraceptive therapies were associated with a greatly reduced density of small nerve fibers present in invasive endometriotic lesions of the rectovaginal septum. This may be an important mechanism of action of hormonal therapies in controlling pain symptoms in endometriosis.
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severe pain-generating form of endometriosis is deep infiltrating endometriosis (DIE), which is commonly located in utero-sacral ligaments, the cul-de-sac or rectovaginal septum (3). In the case of rectovaginal endometriosis (RVE), endometriotic lesions can penetrate bowel wall causing debilitating bowel symptoms, such as pain during defecation. The pathophysiology of endometriosis-associated pain remains unclear. It has been related to recurrent cyclic micro-bleeding in invasive endometriotic implants (3). In addition, inflammation is thought to be a major cause of pain in endometriosis as several inflammatory mediators are known to be secreted by the endometriotic glands and stroma, and by local immune cells (2,4). In recent years, new aspects have been discovered regarding endometriosis-associated pain as several researchers have identified nerve fibers in endometriotic lesions (5–8). In 2006 Tokushige et al. (5) demonstrated the presence of multiple, small unmyelinated nerve fibers of different types in peritoneal endometriotic lesions. Density of nerve fibers in these lesions was much greater than that in peritoneum from women without endometriosis. Nerve fiber density was even greater in deep-invasive lesions involving bowel (9). The clinical relevance of finding nerve fibers in endometriotic lesions is still somewhat controversial. However, in RVE lesions from women with severe pain, perineural and intraneural invasion by endometriotic lesions has been demonstrated (10), suggesting an association between presence of nerve fibers and pain. According to a recent study by Maharajaa et al. there is a significant positive correlation between nerve fiber density and perimenstrual pain score (11). It seems that inflammation associated with endometriosis induces the release of neurotrophic factors, which subsequently lead to the generation of nerve fibers and pain (11). The use of progestogens and combined oral contraceptives (COCs) has been associated with greatly reduced density of nerve fibers, and the complete disappearance of sensory nerve fibers, in eutopic endometrium in women with endometriosis (12). In addition, it has been demonstrated that hormonal therapies reduce the density of small nerve fibers in lesions of peritoneal endometriosis (13), indicating that this mechanism of action may play an important role in reducing pain symptoms among the users of hormonal therapies. In this study, we investigated the presence of different types of nerve fibers in specimens obtained from women operated upon because of RVE. The density of these nerve fibers and the intensity of the main neurotrophin [nerve growth factor (NGF)] and its receptor [tyrosine kinase receptor A (Trk-A)] were studied in relation to several clinical factors, including specifically the use of
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hormonal therapies and need for bowel resection, parameters which have not been studied previously.
Material and methods This study was approved by the Ethics Committee of Hospital District of Helsinki and Uusimaa (6.4.2004 Dnro 125/E9/06), and all women gave their informed consent for participation. Between January 2002 and May 2004 a total of 128 women underwent surgery for symptomatic RVE at the Department of Obstetrics and Gynecology, Helsinki University Central Hospital. The women were identified from the hospital database and their preoperative and perioperative characteristics, as well as the surgical technique, have been described elsewhere (14). To study the long-term outcome of surgical treatment and biological behavior of the disease, 116 of the 128 operated women were offered a new gynecological followup visit at our clinic. Two women were excluded because of severe health problems; 10 had moved to other cities. Altogether, 60 of the 116 women consented to the study (15). At the time of the clinical re-evaluation, written consent was obtained from all of these women to reexamine the RVE tissues prepared at the time of the original operation to investigate the presence of small nerve fibers in these specimens. The RVE specimens collected from a total of 45 women were analyzed. Two of the original 60 specimens could not be located and in 13 cases, tissue quality was compromised to such an extent that the nerve fiber count could not be reliably performed.
Immunohistochemistry Following the primary surgery, tissue specimens were routinely fixed in 10% neutral buffered formalin, then Table 1. List of antibodies and optimal dilutions determined for immunohistochemical staining.
Antibodya PGP9.5 NPY VIP SP NGF Trk-A
Antigen target retrieval pH 9 pH 6 No target retrieval pH 9 pH 9 pH 9
Dilution of primary antibody
Incubation time
Chromagen
1:1400 1:8000 1:5000
30 min 16 h 30 min
Fast Red Fast Red Fast Red
1:8000 1:500 1:500
16 h 30 min 17 h
Fast Red Fast Red Fast Red
a All antibodies were supplied by Dako, Glostrup, Denmark. PGP9.5, protein gene product 9.5; NPY, neuropeptide Y; VIP, vasoactive intestinal peptide; SP, substance P; NGF, nerve growth factor; Trk-A, tyrosine kinase receptor A.
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processed and embedded in paraffin according to a standard protocol. For the purposes of this study, tissue slides were re-evaluated by one pathologist (JA) and representative areas were chosen to illustrate different aspects of the micro-anatomy of the DIE lesions in relation to the bowel. Serial sections were cut at 4-lm and routinely stained with hematoxylin & eosin. Deparaffinized slides
(a)
for immunohistochemical staining underwent antigen retrieval for 20 min at 95–99°C in a preheated water bath. Dual Endogenous Enzyme Block (Dako, Glostrup, Denmark) was applied for 10 min followed by incubation with the primary antibodies (protein gene product 9.5 (PGP9.5), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), substance P (SP), NGF and Trk-A) for 30 min.
(b)
(e)
(d)
(c)
(f)
(g)
Figure 1. Panel of markers staining for nerve fibers in women with rectovaginal endometriosis. Protein gene product 9.5 (PGP9.5) staining in (a) women using hormone therapy and (b) women not using hormone therapy, shown at 100 9 magnification. Representative staining in women not using hormonal therapy for (c) sympathetic nerve fibers with neuropeptide Y (NPY), (d) parasympathetic nerve fibers with vasoactive intestinal peptide (VIP), and (e) sensory nerve fibers with substance P (SP), (f) the main neurotrophin with nerve growth factor (NGF) and (g) its high-affinity receptor tyrosine kinase receptor A (Trk-A); shown at 200 9 magnification using Fast Red chromagen.
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Optimal dilutions and incubation times are outlined in Table 1. REALTM Detection System – Alkaline Phosphatase/Red Rabbit/Mouse (Dako, Glostrup, Denmark) was applied with the contents of link, biotinylated secondary antibodies and streptavidin alkaline phosphatase, and incubated for 15 min, respectively. This was followed with Liquid Fast Red Substrate Chromagen System (Dako) for 10 min (in 2 9 5-min steps) producing a bright red end product at the site of the target antigen. The Dako Autostainer Plus Model S3400 (Dako) was used to perform all immunostaining. Following the immunostaining, slides were counterstained using Mayer’s hematoxylin solution, dried, and cover-slipped using Ultramount (Dako). The images were captured using an Olympus BX51 microscope (Olympus, Tokyo, Japan) and digital camera DP70 using IMAGE PRO DISCOVERY software (MediaCybernetics, Rockville, MD, USA). Once the ‘image’ features of the lesion were acquired, the lesion area contained within the image was analyzed (glandular epithelium, endometriotic stroma and a small amount of surrounding fibrotic interstitium, probably equivalent to the a-smooth muscle actin area previously described in peritoneal lesions) (16). Presence of sensory, parasympathetic and sympathetic nerve fibers was confirmed with positive staining with specific neuronal markers. An assessment of nerve fiber density and immunhistochemical staining was carried out manually twice by two independent observers (each
blinded to the other) without any knowledge of the clinical parameters. The concordance rate was >95% between observers. For each woman, the average nerve fiber count over five lesion areas was calculated (per mm2). To do this, nerve fiber counts for each lesion area were tabulated and the mean count for each specimen was calculated. The immunohistochemical staining for NGF and Trk-A was assessed in a semiquantitative manner using a fivestep protocol: 0, no staining; 1, minimal staining; 2, mild staining; 3, moderate staining and 4, intense immunostaining. Examples of immunohistochemical staining of PGP9.5, NPY, NGF and Trk-A are illustrated in Figure 1. Examples of all the markers using identical techniques in our laboratories are illustrated in previous publications (9,13,17). Special care was taken with the counting of nerve fibers in those parts of bowel (rectum or sigmoid colon), which were clearly affected by endometriosis. The complexity of the normal enteric nervous system meant that it was difficult to distinguish “endometriotic” nerve fibers from those of the normal enteric nervous system.
Statistical analysis The data were analyzed using IBM SPSS 19.0 Statistics (Armonk, NY, USA). Differences in continuous variables for skewed data were analyzed by means of Mann–Whitney
Table 2. Nerve fiber densities in different clinical situations. Clinical feature
n
PGP9.5
NPY
VIP
SP
NGF
TRKA
Age ≤ 32 years Age > 32 years p BMI ≤ 25 kg/m2 BMI > 25 kg/m2 p Nulliparous Parous p HT+ HT p Bowel symptoms no Bowel symptoms yes p Bowel resection No resection p rAFS st 2 rAFS st 3 or 4 p
26 19
15.1 (4.1–33.3) 13.0 (8.0–27.0) 0.79 11.0 (4.6–27.0) 20.7 (16.6–33.4) 0.20 11.1 (4.7–25.5) 28.2 (14.5–46.3) 0.06 8.6 (4.2–20.8) 24.9 (11.2–34.9) 0.01 15.6 (5.1–27.2) 10.2 (2.3–27.4) 0.45 19.4 (5.0–31.5) 12.0 (5.5–26.0) 0.73 19.8 (8.2–30.2) 10.5 (4.2–23.1) 0.18
4.1 (2.4–13.8) 5.8 (3.2–10.2) 0.41 5.4 (2.8–10.8) 7.1 (3.6–15.0) 0.64 5.0 (2.7–10.7) 6.4 (4.6–24.6) 0.13 4.0 (2.6–6.8) 9.8 (3.0–18.6) 0.02 5.6 (2.8–11.0) 4.0 (2.2–14.2) 0.97 5.8 (2.4–13.8) 5.0 (3.0–9.4) 0.76 6.8 (3.2–17.0) 3.7 (2.6–9.3) 0.10
3.1 (1.0–8.2) 4.8 (3.6–9.0) 0.18 3.8 (1.6–8.3) 7.9 (1.3–11.9) 0.57 3.8 (1.6–8.2) 9.6 (5.7–15.3) 0.03 3.0 (1.0–7.0) 8.1 (3.6–9.7) 0.02 4.1 (1.7–8.9) 3.0 (0.6–7.5) 0.27 3.6 (1.2–9.1) 4.5 (2.4–8.2) 0.56 4.8 (1.8–8.6) 3.5 (1.6–8.5) 0.70
1.9 (0.4–5.3) 3.3 (1.2–5.4) 0.15 2.6 (1.2–5.0) 2.1 (1.0–7.3) 0.86 2.0 (1.1–4.4) 7.0 (2.9–10.5) 0.03 1.2 (0.8–3.3) 4.9 (2.2–7.0) 0.00 2.5 (1.2–5.3) 1.2 (0.5–4.9) 0.33 2.0 (0.9–6.7) 2.7 (1.2–4.4) 0.77 3.0 (1.2–4.3) 2.1 (1.0–6.4) 0.73
2 (1.0–3.0) 3 (2.0–3.0) 0.30 2 (1.0–3.0) 3 (3.0–3.0) 0.09 2 (1.3–3.0) 3 (2.5–4.0) 0.11 2 (1.0–3.0) 3 (2.0–4.0) 0.01 3 (2.0–3.0) 2 (1.5–3.0) 0.51 2 (1.0–3.0) 3 (2.0–4.0) 0.14 3 (2.0–3.0) 2.5 (1.0–4.0) 0.86
2 (1.0–3.3) 2 (2.0–3.0) 0.37 2 (1.0–3.0) 2 (1.0–4.0) 0.99 2 (1.0–3.0) 2 (0.5–3.0) 0.35 2 (1.0–3.0) 2 (1.0–4.0) 0.98 2 (1.0–3.0) 2 (1.5–3.5) 0.84 2 (1.0–3.0) 2.5 (1.0–4.0) 0.38 2 (1.0–3.0) 2.5 (1.0–3.3) 0.41
39 6 40 5 27 18 40 5 21 24 19 26
Values are presented as medians/mm² (from 25th to 75th quartile). Expression of NGF and Trk-A is shown using a scale from 1 to 4. PGP9.5, protein gene product 9.5; NPY, neuropeptide Y; VIP, vasoactive intestinal peptide; SP, substance P; NGF, nerve growth factor; Trk-A, tyrosine kinase receptor A; BMI, body mass index; HT, hormone therapy; rAFS st, revised American Fertility Society stage.
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U test. The data are presented as medians (25th to 75th quartile range). Statistical significance was defined as p < 0.05.
Results The median (range) age of the women at the time of the original surgery was 32 (23–42) years. All women were symptomatic with severe pain. Altogether, 27 women received hormonal treatment at the time of the operation. Two of them were using progestogens alone [one orally and another with the levonorgestrel-releasing intrauterine system (LNG-IUS)] and 25 used COCs with progestogen dominance. Altogether 18 women were not using any hormonal treatment. Five women were parous, two (2/27, i.e. 7%) in the group using hormonal therapy, and three (3/18; 17%) in the group not using hormonal therapy. Bowel resection was performed at the time of the original surgery for 21 women (21/45, 47%). Nineteen (42%) women were classified according to revised American Fertility Society (rAFS) scoring (18) to have rAFS class II endometriosis, while 11 (24%) women had rAFS III endometriosis, and 15 (33%) had rAFS class IV endometriosis. 40
Nerve fibers/mm2
35
p = 0.005
30 25
p = 0.018
20 15
p = 0.019
p = 0.001
10 5 0
HT– HT+
HT– HT+
HT– HT+
HT– HT+
PGP9.5
NPY
VIP
SP p = ns
p = 0.01
4 3 2 1 0
HT+
HT–
NGF
HT+
HT–
Trk-A
Figure 2. Nerve fiber densities (top panel) and immunohistochemical staining for nerve growth factor (NGF) and tyrosine kinase receptor A (Trk-A) (lower panel) in women not using hormone therapy (HT–) (n = 18) and using hormone therapy (HT+) (n = 27). Values are presented as medians (and 25th to 75th quartiles). PGP9.5, protein gene product 9.5; NPY, neuropeptide Y; VIP, vasoactive intestinal peptide; SP, substance P.
Nerve fiber densities and intensity of NGF and Trk-A expression in different clinical situations are all summarized in Table 2 and representative immunohistochemical staining is shown in Figure 1. These nerve fiber densities were all assessed in the stromal and surrounding lesionspecific regions of deep endometriotic lesions. The median density (from 25th to 75th quartile range) of nerve fibers stained for PGP9.5 was significantly lower (p = 0.01) in stroma from lesions collected from hormone-treated women (8.6/mm², 4.2–20.8 mm²) than that in stroma from untreated women (24.9/mm², 11.2– 34.9 mm²) (Table 2). Similarly, serial sections of RVE stained for NPY, VIP and SP showed that the density of all three sympathetic, parasympathetic and sensory nerve fibers was significantly lower (p values from 0.01 to 0.02) in women treated with hormonal therapy compared with those untreated (Figure 2, Table 2). In addition, NGF and its high-affinity receptor (Trk-A) were strongly expressed in RVE lesions (Figure 2, Table 2). The expression of NGF persisted, but was significantly reduced (2 compared with 3; p < 0.01) during the use of hormone therapy. In contrast, the expression of Trk-A was not decreased during the use of hormone therapy (Figure 2, Table 2). Significantly greater density of parasympathetic (median 9.6/mm2, compared with 3.8/mm2, p = 0.03) and sensory (7.0/mm2, compared with 2.0/mm2, p = 0.03) fibers (but not sympathetic fibers) was identified in endometriotic stroma among parous women compared with those not having a history of giving birth (Table 2). The nerve fiber densities did not differ significantly among women with rAFS II classification compared with those with rAFS III or IV. Similarly, there was no significant difference in the density of nerve fibers, or in the intensity of expression of NGF and Trk-A, between women with more serious invasive disease requiring bowel resection (median 19.4/mm2) and those who had not required bowel resection (12.0/mm2; p = 0.73; Table 2). Examination of these tissues was focused on areas of lesions primarily affecting the submucosal region of bowel, so that more realistic comparisons of different nerve fiber densities could be made between hormonally treated and non-treated women. This takes into account the conventional pattern of the enteric nerve plexus. As controls (n = 10), nerve fiber densities (using PGP9.5) were assessed in the submucosal plexus region of normal rectum [3.2 0.9/mm2 (mean SD), range: 2.4–4.2 mm2].
Discussion In the present study we have demonstrated a rich innervation of RVE lesions by a mixture of sensory (PGP9.5
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and SP), parasympathetic (VIP) and sympathetic (NPY) nerve fibers, and have defined this with more detail and greater precision than shown previously (9). The use of hormonal therapy (progestogens or COCs) was associated with significantly reduced density of all nerve fiber types in DIE of the rectovaginal septum. These findings may give new insights into understanding the pathophysiology of endometriosis-associated pain. Our findings have demonstrated that use of COCs and progestogens is associated with over 65% reduction of nerve fibers in RVE specimens, a suggestion previously noted in superficial peritoneal endometriosis (13). Even though the effects of COCs or progestogens on endometriosis symptoms were not systematically evaluated in the present study, the markedly decreased number of nerve fibers in hormonal therapy users suggests that this might also be one of the important therapeutic components in women suffering from DIE. Reduction in NGF expression suggests that suppression of sensory neural sensitivity may also be important. Hormonal treatments, including COCs and progestogens, have been widely used in the symptomatic treatment of endometriosis (19), in the prevention of recurrences (20,21), and have also been reported to be efficacious in cases of DIE (22–25). Our findings further encourage the use of progestogens or COCs as first-line therapy for pain control among women with DIE. However, the key mechanisms of action of hormonal therapies in alleviation of the endometriosis symptoms are unclear. Suppression of ovarian follicular development and estrogen secretion, reduction of uterine bleeding and direct effects on ectopic endometriosis lesions are possible contributors to pain alleviation among users of hormonal therapies. Furthermore, reduced numbers of sensory and other nerve fibers may play a role in accomplishing good pain control in this group of women. The main neurotrophin, NGF, supports the growth and survival of neurons and plays an important role in sensitization of nociceptors, in pain transmission and hyperalgesia (26). NGF and its specific high-affinity receptor Trk-A were strongly expressed in RVE lesions. NGF is thought to induce the in-growth of nerve fibers and branching of neurites into endometriotic tissue, so potentially contributing to pain generation in women with endometriosis (27). The increased expression of Trk-A may further contribute to the sensitization of nerve endings and triggering of pain signals (10). NGF continued to be expressed, but at a greatly reduced level, in women on hormonal therapy, whereas there was no difference in the level of expression of Trk-A in women currently using or not using hormone treatment. Partial suppression of NGF may be one of the mechanisms resulting in decreased nerve fiber density. Interestingly, estrogen is
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known to upregulate NGF expression and the finding of continued NGF expression in spite of hormone-use may indicate that some local estrogen action was maintained during hormonal therapy in these women; the capacity for autonomous estrogen synthesis within endometriotic lesions has been considered a fundamental feature in the pathogenesis of endometriosis (28). The women in this study continued to have pain sufficient to merit major surgery. Therefore, hormonal therapy may not have been working effectively in this group of women. In addition, it may be quite illogical to use estrogen-containing hormonal therapies, such as COCs, to treat an estrogen-sensitive disease. This should encourage the planning of effective randomized trials of progestogens and their delivery systems, and perhaps other agents, over longer periods of time than previously reported. Significantly greater densities of parasympathetic (VIP) and sensory (SP) nerve fibers were observed in specimens obtained from parous women compared to nulliparous women. It has been suggested that endometriosis may be a result of damage to uterine innervation during childbirth (denervation–reinnervation theory) (29). It has also been postulated that in nulliparous women the same damage, with subsequent re-innervation, could possibly result after sustained voluntary efforts to achieve defecation during constipation. As there were only five parous women in our study, the significance of this finding is difficult to assess. Severity of disease as assessed by higher rAFS scores, or the need for bowel resection, did not correlate directly with alterations in nerve fiber density, including density of sensory nerve fibers. We hypothesized that women with deep bowel involvement, requiring partial bowel resection, would have developed some degree of interaction between the high density of nerve fibers in the endometriosis lesions and the intrinsic enteric nerve plexus already present in the large bowel. We believed that this linkage might have led to substantially increased tenderness and local pain. However, the difference was not significant regarding densities of total nerve fibers or sensory nerve fibers alone between women with RVE who needed bowel resection and women with RVE who did not have bowel involvement and did not require bowel resection. This finding is in accordance with one previous report, which did not find a correlation between nerve fiber density and DIE severity (10). There are certain well-defined strengths in our study: the major strength being that the tissue samples collected from women with RVE occurred during surgery in one institution, and with consistent detailed clinical, surgical and pathological records. All women were confirmed to suffer from severe, symptomatic RVE. This is also the first
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comprehensive Finnish DIE population database and the first to study nerve fiber densities in RVE and their possible roles in pain generation in RVE. It is not known whether there are cultural or ethnic differences in the micro-architecture of such nerve fibers in these deep lesions (17). Our study also has certain limitations mainly being partly retrospective. We were, for example, unable to reliably assess the duration of hormonal therapy. Also, we did not have healthy contemporaneous Finnish control women (with completely normal bowel anatomy and function). Archived healthy bowel specimens from women with no pathology have been very difficult to locate. However, we have reported brief data for healthy sigmoid colon and rectum as controls in a previous study (9). These are in line with rectal submucosal counts taken from current endometriosis specimens at a distance of at least 4 cm from the lesion. The tissue specimens were analyzed for the present study on average 4 years after the original operation so possibly compromising, in some cases, the tissue expression of antigens. Furthermore, neuronal markers used in immunostaining are not fully specific for only one type of nerve fiber, hence creating minor uncertainties in interpreting the exact nerve fiber type. Some weak crossreaction may also sometimes occur with other non-neural cell types, such as neuroendocrine epithelial cells (30). Nerve fibers were only counted if their histological appearance was consistent with nerve structure. The complex disruptions of the enteric nervous system in cases of deep invasive endometriosis involving bowel are the subject of ongoing detailed study. In conclusion, RVE lesions are extensively innervated by sensory, sympathetic and parasympathetic nerve fibers. This may be a basic requirement for generation of pain among these women. The use of hormonal therapies was associated with significantly reduced nerve fiber density in RVE lesions, so potentially contributing towards better symptom control among many users of COCs or progestogens. More studies on the effects of different hormonal therapies (especially progestogens alone), dosages and delivery routes on symptom control and disease progress are warranted.
Acknowledgments The authors wish to thank Dr Alison Hey-Cunningham for critical advice on analysis and interpretation and Dr Georgina M. Luscombe for statistical analysis and advice.
Funding statement This study was supported by the Helsinki University Central Hospital research funds and by the research funds of
Nerve fibers in rectovaginal endometriosis
the Department of Obstetrics and Gynaecology, and Neonatology of the University of Sydney. References 1. Lebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril. 2001;75:1–10. 2. Giudice LC, Kao LC. Endometriosis Lancet. 2004;364:1789–99. 3. Fauconnier A, Chapron C. Endometriosis and pelvic pain: epidemiological evidence of the relationship and implications. Hum Reprod Update. 2005;11:595–606. 4. Anaf V, Chapron C, El Nakadi I, De Moor V, Simonart T, Noel JC. Pain, mast cells, and nerves in peritoneal, ovarian, and deep infiltrating endometriosis. Fertil Steril. 2006;86:1336–43. 5. Tokushige N, Markham R, Russell P, Fraser IS. Nerve fibers in peritoneal endometriosis. Hum Reprod. 2006;21:3001–7. 6. Tamburro S, Canis M, Albuisson E, Dechelotte P, Darcha C, Mage G. Expression of transforming growth factor b1 in nerve fibers is related to dysmenorrhea and laparoscopic appearance of endometriotic implants. Fertil Steril. 2003;80:1131–6. 7. Tokushige N, Markham R, Russell P, Fraser IS. High density of small nerve fibers in the functional layer of the endometrium in women with endometriosis. Hum Reprod. 2006;21:782–7. 8. Tokushige N, Markham R, Russell P, Fraser IS. Different types of small nerve fibers in eutopic endometrium and myometrium in women with endometriosis. Fertil Steril. 2007;88:795–803. 9. Wang G, Tokushige N, Russell P, Dubinovsky S, Markham R, Fraser IS. Hyperinnervation in intestinal deep infiltrating endometriosis. J Minim Invasive Gynecol. 2009;16:713–9. 10. Anaf V, Simon P, El Nakadi I, Fayt I, Buxant F, Simonart T, et al. Relationship between endometriotic foci and nerves in rectovaginal endometriotic nodules. Hum Reprod. 2000;15:1744–50. 11. Maharajaa SPK, Black KI, Manconi F, Ng CHM, HeyCunningham A, Fraser IS, et al. An investigation of the relationship between pelvic pain and density of nerve fibers in peritoneal lesions of endometriosis. J Endometr. 2014;6:84–91. 12. Tokushige N, Markham R, Russell P, Fraser IS. Effects of hormonal treatment on nerve fibers in endometrium and myometrium in women with endometriosis. Fertil Steril. 2008;90:1589–98. 13. Tokushige N, Markham R, Russell P, Fraser IS. Effect of progestogens and combined oral contraceptives on nerve fibers in peritoneal endometriosis. Fertil Steril. 2009;92:1234–9. 14. Tarjanne S, Sj€ oberg J, Heikinheimo O. Rectovaginal endometriosis – characteristics of operative treatment and
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AOGS M A I N R E SE A RC H A R TI C LE
Use of hormonal therapy is associated with reduced nerve fiber density in deep infiltrating, rectovaginal endometriosis SATU TARJANNE1, CECILIA H.M. NG2, FRANK MANCONI2, JOHANNA AROLA3, MAARIT MENTULA1, BHARVI MANECK2, IAN S. FRASER2 & OSKARI HEIKINHEIMO1 1
Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, Department of Obstetrics, Gynecology and Neonatology, Queen Elizabeth II Research Institute for Mothers and Infants, University of Sydney, Sydney, New South Wales, Australia, and 3Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, Helsinki, Finland
2
Key words Deep infiltrating endometriosis, pain, nerve fibers Correspondence Oskari Heikinheimo, Department of Obstetrics and Gynecology, Helsinki University Hospital/ €opisto Hospital, P.O. Box 610, 00029K€atilo HUS, Helsinki, Finland. E-mail: [email protected] Conflict of interest ISF has received lecture fees, expenses and research support, and undertaken consultancies, for Bayer and Merck. Similarly, OH has undertaken consultancies and given lectures for fee for these companies and ST has given lectures for fee for Bayer. The other authors have no conflict of interest in relation to this work. Please cite this article as: Tarjanne S, Ng CHM, Manconi F, Arola J, Mentula M, Maneck B, et al. Use of hormonal therapy is associated with reduced nerve fiber density in deep infiltrating, rectovaginal endometriosis. Acta Obstet Gynecol Scand 2015; 94: 693–700. Received: 17 September 2014 Accepted: 5 April 2015 DOI: 10.1111/aogs.12652
Abstract Objective. To study the density of nerve fibers in cases of deep infiltrating endometriosis (DIE) of the rectovaginal septum in relation to various clinical factors. Design. A research laboratory-based study. Setting. A tertiary center together with a research laboratory. Methods. Archived DIE tissue samples from 45 women operated upon for rectovaginal septum DIE were re-examined histologically, and by immunohistochemistry. Main outcome measures. The effect of progestogens or combined oral contraceptives on the density of nerve fibers, and the expression of nerve growth factor (NGF) and its high-affinity receptor (tyrosine kinase receptor A, Trk-A). Results. The use of hormonal therapy was associated with reduced densities of sympathetic, parasympathetic and sensory nerve fibers in DIE lesions. Density of total nerve fibers (with pan-neuronal marker PGP9.5) was significantly lower (p < 0.05) in lesions collected from hormone-treated women (8.6/mm², 4.2–20.8/mm²; median density, from 25th to 75th quartiles) compared with that in lesions from untreated women (24.9/mm², 11.2–34.9/ mm²). DIE lesions stained strongly for NGF and its receptor Trk-A. Expression of NGF, but not of Trk-A, was significantly reduced during use of hormonal therapy. Conclusions. Use of hormonal therapy was associated with significantly reduced nerve fiber density in DIE lesions. This may be an important mechanism of action of hormonal therapy for controlling DIE pain symptoms. The expression of estrogen-regulated NGF and its receptor was only partially suppressed during the use of hormonal therapy, suggesting that local estrogen action is often maintained during conventional hormonal therapy in cases of DIE. COC, combined oral contraceptives; DIE, deep infiltrating endometriosis; NGF, nerve growth factor; NPY, neuropeptide-Y; PGP9.5, protein gene product 9.5; rAFS, revised American Fertility Society; RVE, rectovaginal endometriosis; SP, substance P; Trk-A, tyrosine kinase receptor A; VIP, vasoactive intestinal peptide.
Abbreviations:
Key Message Introduction Endometriosis is a benign estrogen-dependent disease occurring in 6–10% of women of reproductive age (1,2). It is frequently associated with severe pelvic pain symptoms such as dysmenorrhea and dyspareunia. The most
Progestogen and oral contraceptive therapies were associated with a greatly reduced density of small nerve fibers present in invasive endometriotic lesions of the rectovaginal septum. This may be an important mechanism of action of hormonal therapies in controlling pain symptoms in endometriosis.
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severe pain-generating form of endometriosis is deep infiltrating endometriosis (DIE), which is commonly located in utero-sacral ligaments, the cul-de-sac or rectovaginal septum (3). In the case of rectovaginal endometriosis (RVE), endometriotic lesions can penetrate bowel wall causing debilitating bowel symptoms, such as pain during defecation. The pathophysiology of endometriosis-associated pain remains unclear. It has been related to recurrent cyclic micro-bleeding in invasive endometriotic implants (3). In addition, inflammation is thought to be a major cause of pain in endometriosis as several inflammatory mediators are known to be secreted by the endometriotic glands and stroma, and by local immune cells (2,4). In recent years, new aspects have been discovered regarding endometriosis-associated pain as several researchers have identified nerve fibers in endometriotic lesions (5–8). In 2006 Tokushige et al. (5) demonstrated the presence of multiple, small unmyelinated nerve fibers of different types in peritoneal endometriotic lesions. Density of nerve fibers in these lesions was much greater than that in peritoneum from women without endometriosis. Nerve fiber density was even greater in deep-invasive lesions involving bowel (9). The clinical relevance of finding nerve fibers in endometriotic lesions is still somewhat controversial. However, in RVE lesions from women with severe pain, perineural and intraneural invasion by endometriotic lesions has been demonstrated (10), suggesting an association between presence of nerve fibers and pain. According to a recent study by Maharajaa et al. there is a significant positive correlation between nerve fiber density and perimenstrual pain score (11). It seems that inflammation associated with endometriosis induces the release of neurotrophic factors, which subsequently lead to the generation of nerve fibers and pain (11). The use of progestogens and combined oral contraceptives (COCs) has been associated with greatly reduced density of nerve fibers, and the complete disappearance of sensory nerve fibers, in eutopic endometrium in women with endometriosis (12). In addition, it has been demonstrated that hormonal therapies reduce the density of small nerve fibers in lesions of peritoneal endometriosis (13), indicating that this mechanism of action may play an important role in reducing pain symptoms among the users of hormonal therapies. In this study, we investigated the presence of different types of nerve fibers in specimens obtained from women operated upon because of RVE. The density of these nerve fibers and the intensity of the main neurotrophin [nerve growth factor (NGF)] and its receptor [tyrosine kinase receptor A (Trk-A)] were studied in relation to several clinical factors, including specifically the use of
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hormonal therapies and need for bowel resection, parameters which have not been studied previously.
Material and methods This study was approved by the Ethics Committee of Hospital District of Helsinki and Uusimaa (6.4.2004 Dnro 125/E9/06), and all women gave their informed consent for participation. Between January 2002 and May 2004 a total of 128 women underwent surgery for symptomatic RVE at the Department of Obstetrics and Gynecology, Helsinki University Central Hospital. The women were identified from the hospital database and their preoperative and perioperative characteristics, as well as the surgical technique, have been described elsewhere (14). To study the long-term outcome of surgical treatment and biological behavior of the disease, 116 of the 128 operated women were offered a new gynecological followup visit at our clinic. Two women were excluded because of severe health problems; 10 had moved to other cities. Altogether, 60 of the 116 women consented to the study (15). At the time of the clinical re-evaluation, written consent was obtained from all of these women to reexamine the RVE tissues prepared at the time of the original operation to investigate the presence of small nerve fibers in these specimens. The RVE specimens collected from a total of 45 women were analyzed. Two of the original 60 specimens could not be located and in 13 cases, tissue quality was compromised to such an extent that the nerve fiber count could not be reliably performed.
Immunohistochemistry Following the primary surgery, tissue specimens were routinely fixed in 10% neutral buffered formalin, then Table 1. List of antibodies and optimal dilutions determined for immunohistochemical staining.
Antibodya PGP9.5 NPY VIP SP NGF Trk-A
Antigen target retrieval pH 9 pH 6 No target retrieval pH 9 pH 9 pH 9
Dilution of primary antibody
Incubation time
Chromagen
1:1400 1:8000 1:5000
30 min 16 h 30 min
Fast Red Fast Red Fast Red
1:8000 1:500 1:500
16 h 30 min 17 h
Fast Red Fast Red Fast Red
a All antibodies were supplied by Dako, Glostrup, Denmark. PGP9.5, protein gene product 9.5; NPY, neuropeptide Y; VIP, vasoactive intestinal peptide; SP, substance P; NGF, nerve growth factor; Trk-A, tyrosine kinase receptor A.
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processed and embedded in paraffin according to a standard protocol. For the purposes of this study, tissue slides were re-evaluated by one pathologist (JA) and representative areas were chosen to illustrate different aspects of the micro-anatomy of the DIE lesions in relation to the bowel. Serial sections were cut at 4-lm and routinely stained with hematoxylin & eosin. Deparaffinized slides
(a)
for immunohistochemical staining underwent antigen retrieval for 20 min at 95–99°C in a preheated water bath. Dual Endogenous Enzyme Block (Dako, Glostrup, Denmark) was applied for 10 min followed by incubation with the primary antibodies (protein gene product 9.5 (PGP9.5), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), substance P (SP), NGF and Trk-A) for 30 min.
(b)
(e)
(d)
(c)
(f)
(g)
Figure 1. Panel of markers staining for nerve fibers in women with rectovaginal endometriosis. Protein gene product 9.5 (PGP9.5) staining in (a) women using hormone therapy and (b) women not using hormone therapy, shown at 100 9 magnification. Representative staining in women not using hormonal therapy for (c) sympathetic nerve fibers with neuropeptide Y (NPY), (d) parasympathetic nerve fibers with vasoactive intestinal peptide (VIP), and (e) sensory nerve fibers with substance P (SP), (f) the main neurotrophin with nerve growth factor (NGF) and (g) its high-affinity receptor tyrosine kinase receptor A (Trk-A); shown at 200 9 magnification using Fast Red chromagen.
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Optimal dilutions and incubation times are outlined in Table 1. REALTM Detection System – Alkaline Phosphatase/Red Rabbit/Mouse (Dako, Glostrup, Denmark) was applied with the contents of link, biotinylated secondary antibodies and streptavidin alkaline phosphatase, and incubated for 15 min, respectively. This was followed with Liquid Fast Red Substrate Chromagen System (Dako) for 10 min (in 2 9 5-min steps) producing a bright red end product at the site of the target antigen. The Dako Autostainer Plus Model S3400 (Dako) was used to perform all immunostaining. Following the immunostaining, slides were counterstained using Mayer’s hematoxylin solution, dried, and cover-slipped using Ultramount (Dako). The images were captured using an Olympus BX51 microscope (Olympus, Tokyo, Japan) and digital camera DP70 using IMAGE PRO DISCOVERY software (MediaCybernetics, Rockville, MD, USA). Once the ‘image’ features of the lesion were acquired, the lesion area contained within the image was analyzed (glandular epithelium, endometriotic stroma and a small amount of surrounding fibrotic interstitium, probably equivalent to the a-smooth muscle actin area previously described in peritoneal lesions) (16). Presence of sensory, parasympathetic and sympathetic nerve fibers was confirmed with positive staining with specific neuronal markers. An assessment of nerve fiber density and immunhistochemical staining was carried out manually twice by two independent observers (each
blinded to the other) without any knowledge of the clinical parameters. The concordance rate was >95% between observers. For each woman, the average nerve fiber count over five lesion areas was calculated (per mm2). To do this, nerve fiber counts for each lesion area were tabulated and the mean count for each specimen was calculated. The immunohistochemical staining for NGF and Trk-A was assessed in a semiquantitative manner using a fivestep protocol: 0, no staining; 1, minimal staining; 2, mild staining; 3, moderate staining and 4, intense immunostaining. Examples of immunohistochemical staining of PGP9.5, NPY, NGF and Trk-A are illustrated in Figure 1. Examples of all the markers using identical techniques in our laboratories are illustrated in previous publications (9,13,17). Special care was taken with the counting of nerve fibers in those parts of bowel (rectum or sigmoid colon), which were clearly affected by endometriosis. The complexity of the normal enteric nervous system meant that it was difficult to distinguish “endometriotic” nerve fibers from those of the normal enteric nervous system.
Statistical analysis The data were analyzed using IBM SPSS 19.0 Statistics (Armonk, NY, USA). Differences in continuous variables for skewed data were analyzed by means of Mann–Whitney
Table 2. Nerve fiber densities in different clinical situations. Clinical feature
n
PGP9.5
NPY
VIP
SP
NGF
TRKA
Age ≤ 32 years Age > 32 years p BMI ≤ 25 kg/m2 BMI > 25 kg/m2 p Nulliparous Parous p HT+ HT p Bowel symptoms no Bowel symptoms yes p Bowel resection No resection p rAFS st 2 rAFS st 3 or 4 p
26 19
15.1 (4.1–33.3) 13.0 (8.0–27.0) 0.79 11.0 (4.6–27.0) 20.7 (16.6–33.4) 0.20 11.1 (4.7–25.5) 28.2 (14.5–46.3) 0.06 8.6 (4.2–20.8) 24.9 (11.2–34.9) 0.01 15.6 (5.1–27.2) 10.2 (2.3–27.4) 0.45 19.4 (5.0–31.5) 12.0 (5.5–26.0) 0.73 19.8 (8.2–30.2) 10.5 (4.2–23.1) 0.18
4.1 (2.4–13.8) 5.8 (3.2–10.2) 0.41 5.4 (2.8–10.8) 7.1 (3.6–15.0) 0.64 5.0 (2.7–10.7) 6.4 (4.6–24.6) 0.13 4.0 (2.6–6.8) 9.8 (3.0–18.6) 0.02 5.6 (2.8–11.0) 4.0 (2.2–14.2) 0.97 5.8 (2.4–13.8) 5.0 (3.0–9.4) 0.76 6.8 (3.2–17.0) 3.7 (2.6–9.3) 0.10
3.1 (1.0–8.2) 4.8 (3.6–9.0) 0.18 3.8 (1.6–8.3) 7.9 (1.3–11.9) 0.57 3.8 (1.6–8.2) 9.6 (5.7–15.3) 0.03 3.0 (1.0–7.0) 8.1 (3.6–9.7) 0.02 4.1 (1.7–8.9) 3.0 (0.6–7.5) 0.27 3.6 (1.2–9.1) 4.5 (2.4–8.2) 0.56 4.8 (1.8–8.6) 3.5 (1.6–8.5) 0.70
1.9 (0.4–5.3) 3.3 (1.2–5.4) 0.15 2.6 (1.2–5.0) 2.1 (1.0–7.3) 0.86 2.0 (1.1–4.4) 7.0 (2.9–10.5) 0.03 1.2 (0.8–3.3) 4.9 (2.2–7.0) 0.00 2.5 (1.2–5.3) 1.2 (0.5–4.9) 0.33 2.0 (0.9–6.7) 2.7 (1.2–4.4) 0.77 3.0 (1.2–4.3) 2.1 (1.0–6.4) 0.73
2 (1.0–3.0) 3 (2.0–3.0) 0.30 2 (1.0–3.0) 3 (3.0–3.0) 0.09 2 (1.3–3.0) 3 (2.5–4.0) 0.11 2 (1.0–3.0) 3 (2.0–4.0) 0.01 3 (2.0–3.0) 2 (1.5–3.0) 0.51 2 (1.0–3.0) 3 (2.0–4.0) 0.14 3 (2.0–3.0) 2.5 (1.0–4.0) 0.86
2 (1.0–3.3) 2 (2.0–3.0) 0.37 2 (1.0–3.0) 2 (1.0–4.0) 0.99 2 (1.0–3.0) 2 (0.5–3.0) 0.35 2 (1.0–3.0) 2 (1.0–4.0) 0.98 2 (1.0–3.0) 2 (1.5–3.5) 0.84 2 (1.0–3.0) 2.5 (1.0–4.0) 0.38 2 (1.0–3.0) 2.5 (1.0–3.3) 0.41
39 6 40 5 27 18 40 5 21 24 19 26
Values are presented as medians/mm² (from 25th to 75th quartile). Expression of NGF and Trk-A is shown using a scale from 1 to 4. PGP9.5, protein gene product 9.5; NPY, neuropeptide Y; VIP, vasoactive intestinal peptide; SP, substance P; NGF, nerve growth factor; Trk-A, tyrosine kinase receptor A; BMI, body mass index; HT, hormone therapy; rAFS st, revised American Fertility Society stage.
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U test. The data are presented as medians (25th to 75th quartile range). Statistical significance was defined as p < 0.05.
Results The median (range) age of the women at the time of the original surgery was 32 (23–42) years. All women were symptomatic with severe pain. Altogether, 27 women received hormonal treatment at the time of the operation. Two of them were using progestogens alone [one orally and another with the levonorgestrel-releasing intrauterine system (LNG-IUS)] and 25 used COCs with progestogen dominance. Altogether 18 women were not using any hormonal treatment. Five women were parous, two (2/27, i.e. 7%) in the group using hormonal therapy, and three (3/18; 17%) in the group not using hormonal therapy. Bowel resection was performed at the time of the original surgery for 21 women (21/45, 47%). Nineteen (42%) women were classified according to revised American Fertility Society (rAFS) scoring (18) to have rAFS class II endometriosis, while 11 (24%) women had rAFS III endometriosis, and 15 (33%) had rAFS class IV endometriosis. 40
Nerve fibers/mm2
35
p = 0.005
30 25
p = 0.018
20 15
p = 0.019
p = 0.001
10 5 0
HT– HT+
HT– HT+
HT– HT+
HT– HT+
PGP9.5
NPY
VIP
SP p = ns
p = 0.01
4 3 2 1 0
HT+
HT–
NGF
HT+
HT–
Trk-A
Figure 2. Nerve fiber densities (top panel) and immunohistochemical staining for nerve growth factor (NGF) and tyrosine kinase receptor A (Trk-A) (lower panel) in women not using hormone therapy (HT–) (n = 18) and using hormone therapy (HT+) (n = 27). Values are presented as medians (and 25th to 75th quartiles). PGP9.5, protein gene product 9.5; NPY, neuropeptide Y; VIP, vasoactive intestinal peptide; SP, substance P.
Nerve fiber densities and intensity of NGF and Trk-A expression in different clinical situations are all summarized in Table 2 and representative immunohistochemical staining is shown in Figure 1. These nerve fiber densities were all assessed in the stromal and surrounding lesionspecific regions of deep endometriotic lesions. The median density (from 25th to 75th quartile range) of nerve fibers stained for PGP9.5 was significantly lower (p = 0.01) in stroma from lesions collected from hormone-treated women (8.6/mm², 4.2–20.8 mm²) than that in stroma from untreated women (24.9/mm², 11.2– 34.9 mm²) (Table 2). Similarly, serial sections of RVE stained for NPY, VIP and SP showed that the density of all three sympathetic, parasympathetic and sensory nerve fibers was significantly lower (p values from 0.01 to 0.02) in women treated with hormonal therapy compared with those untreated (Figure 2, Table 2). In addition, NGF and its high-affinity receptor (Trk-A) were strongly expressed in RVE lesions (Figure 2, Table 2). The expression of NGF persisted, but was significantly reduced (2 compared with 3; p < 0.01) during the use of hormone therapy. In contrast, the expression of Trk-A was not decreased during the use of hormone therapy (Figure 2, Table 2). Significantly greater density of parasympathetic (median 9.6/mm2, compared with 3.8/mm2, p = 0.03) and sensory (7.0/mm2, compared with 2.0/mm2, p = 0.03) fibers (but not sympathetic fibers) was identified in endometriotic stroma among parous women compared with those not having a history of giving birth (Table 2). The nerve fiber densities did not differ significantly among women with rAFS II classification compared with those with rAFS III or IV. Similarly, there was no significant difference in the density of nerve fibers, or in the intensity of expression of NGF and Trk-A, between women with more serious invasive disease requiring bowel resection (median 19.4/mm2) and those who had not required bowel resection (12.0/mm2; p = 0.73; Table 2). Examination of these tissues was focused on areas of lesions primarily affecting the submucosal region of bowel, so that more realistic comparisons of different nerve fiber densities could be made between hormonally treated and non-treated women. This takes into account the conventional pattern of the enteric nerve plexus. As controls (n = 10), nerve fiber densities (using PGP9.5) were assessed in the submucosal plexus region of normal rectum [3.2 0.9/mm2 (mean SD), range: 2.4–4.2 mm2].
Discussion In the present study we have demonstrated a rich innervation of RVE lesions by a mixture of sensory (PGP9.5
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and SP), parasympathetic (VIP) and sympathetic (NPY) nerve fibers, and have defined this with more detail and greater precision than shown previously (9). The use of hormonal therapy (progestogens or COCs) was associated with significantly reduced density of all nerve fiber types in DIE of the rectovaginal septum. These findings may give new insights into understanding the pathophysiology of endometriosis-associated pain. Our findings have demonstrated that use of COCs and progestogens is associated with over 65% reduction of nerve fibers in RVE specimens, a suggestion previously noted in superficial peritoneal endometriosis (13). Even though the effects of COCs or progestogens on endometriosis symptoms were not systematically evaluated in the present study, the markedly decreased number of nerve fibers in hormonal therapy users suggests that this might also be one of the important therapeutic components in women suffering from DIE. Reduction in NGF expression suggests that suppression of sensory neural sensitivity may also be important. Hormonal treatments, including COCs and progestogens, have been widely used in the symptomatic treatment of endometriosis (19), in the prevention of recurrences (20,21), and have also been reported to be efficacious in cases of DIE (22–25). Our findings further encourage the use of progestogens or COCs as first-line therapy for pain control among women with DIE. However, the key mechanisms of action of hormonal therapies in alleviation of the endometriosis symptoms are unclear. Suppression of ovarian follicular development and estrogen secretion, reduction of uterine bleeding and direct effects on ectopic endometriosis lesions are possible contributors to pain alleviation among users of hormonal therapies. Furthermore, reduced numbers of sensory and other nerve fibers may play a role in accomplishing good pain control in this group of women. The main neurotrophin, NGF, supports the growth and survival of neurons and plays an important role in sensitization of nociceptors, in pain transmission and hyperalgesia (26). NGF and its specific high-affinity receptor Trk-A were strongly expressed in RVE lesions. NGF is thought to induce the in-growth of nerve fibers and branching of neurites into endometriotic tissue, so potentially contributing to pain generation in women with endometriosis (27). The increased expression of Trk-A may further contribute to the sensitization of nerve endings and triggering of pain signals (10). NGF continued to be expressed, but at a greatly reduced level, in women on hormonal therapy, whereas there was no difference in the level of expression of Trk-A in women currently using or not using hormone treatment. Partial suppression of NGF may be one of the mechanisms resulting in decreased nerve fiber density. Interestingly, estrogen is
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known to upregulate NGF expression and the finding of continued NGF expression in spite of hormone-use may indicate that some local estrogen action was maintained during hormonal therapy in these women; the capacity for autonomous estrogen synthesis within endometriotic lesions has been considered a fundamental feature in the pathogenesis of endometriosis (28). The women in this study continued to have pain sufficient to merit major surgery. Therefore, hormonal therapy may not have been working effectively in this group of women. In addition, it may be quite illogical to use estrogen-containing hormonal therapies, such as COCs, to treat an estrogen-sensitive disease. This should encourage the planning of effective randomized trials of progestogens and their delivery systems, and perhaps other agents, over longer periods of time than previously reported. Significantly greater densities of parasympathetic (VIP) and sensory (SP) nerve fibers were observed in specimens obtained from parous women compared to nulliparous women. It has been suggested that endometriosis may be a result of damage to uterine innervation during childbirth (denervation–reinnervation theory) (29). It has also been postulated that in nulliparous women the same damage, with subsequent re-innervation, could possibly result after sustained voluntary efforts to achieve defecation during constipation. As there were only five parous women in our study, the significance of this finding is difficult to assess. Severity of disease as assessed by higher rAFS scores, or the need for bowel resection, did not correlate directly with alterations in nerve fiber density, including density of sensory nerve fibers. We hypothesized that women with deep bowel involvement, requiring partial bowel resection, would have developed some degree of interaction between the high density of nerve fibers in the endometriosis lesions and the intrinsic enteric nerve plexus already present in the large bowel. We believed that this linkage might have led to substantially increased tenderness and local pain. However, the difference was not significant regarding densities of total nerve fibers or sensory nerve fibers alone between women with RVE who needed bowel resection and women with RVE who did not have bowel involvement and did not require bowel resection. This finding is in accordance with one previous report, which did not find a correlation between nerve fiber density and DIE severity (10). There are certain well-defined strengths in our study: the major strength being that the tissue samples collected from women with RVE occurred during surgery in one institution, and with consistent detailed clinical, surgical and pathological records. All women were confirmed to suffer from severe, symptomatic RVE. This is also the first
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comprehensive Finnish DIE population database and the first to study nerve fiber densities in RVE and their possible roles in pain generation in RVE. It is not known whether there are cultural or ethnic differences in the micro-architecture of such nerve fibers in these deep lesions (17). Our study also has certain limitations mainly being partly retrospective. We were, for example, unable to reliably assess the duration of hormonal therapy. Also, we did not have healthy contemporaneous Finnish control women (with completely normal bowel anatomy and function). Archived healthy bowel specimens from women with no pathology have been very difficult to locate. However, we have reported brief data for healthy sigmoid colon and rectum as controls in a previous study (9). These are in line with rectal submucosal counts taken from current endometriosis specimens at a distance of at least 4 cm from the lesion. The tissue specimens were analyzed for the present study on average 4 years after the original operation so possibly compromising, in some cases, the tissue expression of antigens. Furthermore, neuronal markers used in immunostaining are not fully specific for only one type of nerve fiber, hence creating minor uncertainties in interpreting the exact nerve fiber type. Some weak crossreaction may also sometimes occur with other non-neural cell types, such as neuroendocrine epithelial cells (30). Nerve fibers were only counted if their histological appearance was consistent with nerve structure. The complex disruptions of the enteric nervous system in cases of deep invasive endometriosis involving bowel are the subject of ongoing detailed study. In conclusion, RVE lesions are extensively innervated by sensory, sympathetic and parasympathetic nerve fibers. This may be a basic requirement for generation of pain among these women. The use of hormonal therapies was associated with significantly reduced nerve fiber density in RVE lesions, so potentially contributing towards better symptom control among many users of COCs or progestogens. More studies on the effects of different hormonal therapies (especially progestogens alone), dosages and delivery routes on symptom control and disease progress are warranted.
Acknowledgments The authors wish to thank Dr Alison Hey-Cunningham for critical advice on analysis and interpretation and Dr Georgina M. Luscombe for statistical analysis and advice.
Funding statement This study was supported by the Helsinki University Central Hospital research funds and by the research funds of
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