Key words
nerve fibers - neurofilament - pelvic pain - protein gene product 9.5 - uterine leiomyoma
Schlüsselwörter
Nervenfasern - Neurofilament - Beckenschmerzen - Protein-Genprodukt 9.5 - Leiomyom
des Uterus
Introduction
Uterine leiomyomas are benign monoclonal tumors that originate from the smooth muscle
of the uterus. Although 70% of uterine leiomyomas are asymptomatic, they can cause
dysmenorrhea, menorrhagia, infertility, abortion, pelvic pressure and pain. Symptoms
depend on the location and size of the uterine leiomyomas, although not in all cases
[1].
Pelvic pain is a relatively common and important clinical symptom. Clinical studies
have reported pelvic pain complaints in about 34% of patients with uterine leiomyoma
[2]. The most commonly used scale for evaluating subjective pain is the “visual analog
scale (VAS)”. Patients rate the pain by giving it a score between 0 and 10 (i.e.,
from none to severe) [3]. We used this scale in the present study.
Endometriosis and uterine leiomyomas are common causes of pelvic pain. Increased nerve
fibers in the endometrium have been detected in a majority of patients with endometriosis
in some studies [4], [5]. Because adenomyosis and uterine leiomyomas are believed to be controlled by the
same steroid hormones as endometriosis, it was suggested that increased nerve fibers
may be involved in the mechanism of pain generation in women with uterine leiomyomas.
So far, only two studies have investigated the density of nerve fibers in the endometrium
and myometrium of patients with endometriosis, adenomyosis and uterine leiomyomas
with pain [6], [7]. These studies retrospectively evaluated nerve fibers in the endometrium and myometrium,
but not in leiomyoma tissue, and found increased nerve fibers in patients reporting
pain.
Protein Gene Product 9.5 (PGP 9.5) is a specific marker for both myelinated and unmyelinated
nerve fibers and neurofilament (NF) is a specific marker for unmyelinated nerve fibers
[8]. We used these dyes as biological neuronal markers to detect nerve fibers in tissue,
as was also done in the majority of studies in the literature.
A better understanding of the physiopathology of pain and its relationship to nerve
fibers in uterine leiomyomas could contribute to better therapeutic management. For
this reason, we aimed to prospectively compare the presence and density of nerve fibers
in patients with or without pain.
Methods
Participants and study design
Premenopausal women between the ages of 40 – 50 undergoing hysterectomy for uterine
leiomyoma were recruited prospectively into the study. The indications for hysterectomy
included enlarged uterus, chronic pelvic pain, pelvic pressure symptoms, intractable
menorrhagia or dysmenorrhea. The exclusion criteria were psychiatric or neurological
disorders, disease that can alter the perception of pain (diabetes mellitus, etc.),
hormone treatment for at least 6 months prior to surgery, and the presence of other
gynecological diseases (adnexal masses, endometrioma, endometriosis, pelvic congestion
syndrome, etc.). The study was approved by the Dr. Lutfi Kirdar Kartal Training and
Research Hospital Ethical Committee (approval number: 89513307/1009/351). Oral and
written informed consent was obtained from all patients scheduled for hysterectomy
because of uterine leiomyoma who agreed to participate in the study.
The sample size was calculated on the basis of a previous study conducted by Zhang
et al., which had reported a 69% difference in nerve fiber density between patients
with and patients without pain [6]. Based on their findings, it was estimated that 12 patients would have to be included
into each group to achieve a 69% difference between two groups with 0.05 alpha error
and 95% power.
The visual analog scale (VAS) was used to measure pain levels before surgery. The
VAS scale was administered to all patients by the same physician. Forty-eight patients
were prospectively included in the study; 25 patients with leiomyoma without pelvic
pain (VAS < 5) were assigned into Group 1, 23 patients with leiomyoma and pelvic pain
(VAS ≥ 5) were assigned into Group 2.
Collection of tissue samples
The tissue samples were obtained from the endometrium, myometrium and uterine leiomyoma.
The number and density of myelinated and unmyelinated nerve fibers were investigated
using Protein Gene Product 9.5 (PGP 9.5) and neurofilament (NF) staining ([Figs. 1] and [2]).
Fig. 1 Nerve fibers in fibroid tissue (× 200, PGP 9.5). Tissue samples were obtained from
uterine leiomyoma. The density and number of nerve fibers were investigated with Protein
Gene Product 9.5 (PGP 9.5) staining.
Fig. 2 Nerve fibers in fibroid tissue (× 200, NF). Tissue samples were obtained from uterine
leiomyoma. The density and number of nerve fibers were investigated with neurofilament
(NF) staining.
Histopathological examination
Formalin-fixed paraffin-embedded tissue samples were incubated for 30 minutes with
rabbit polyclonal Anti-PGP 9.5 (CELL MARQUE, Code CMC 31811021, 1/200 dilution, USA)
or Anti-neurofilament (2F11) mouse monoclonal antibody (CELL MARQUE, Code CMC 30221021,
1/200 dilution, USA). Post-primary antibody, polymer antibody and diaminobenzidine
(DAB) were added, respectively. Paraffin sections were stained using Mayerʼs hematoxylin.
All samples were evaluated by the same pathologist using an Olympus BX53 microscope.
The diagnosis of uterine leiomyoma was confirmed in all patients by histopathologic
examination.
Statistical analysis
Data were analyzed using SPSS version 22.0. Mean standard deviation, median (lowest
and highest), frequency and ratio values were used for descriptive statistics. Histogram,
normality plots and Shapiro-Wilk normality test were used to analyze data distribution.
Mann-Whitney U test was used to analyze quantitative data. χ2 test or Fisherʼs exact test was used to analyze qualitative data. Cohenʼs kappa coefficient
was used to measure agreement between the two staining techniques. Statistical significance
was established at p < 0.05.
Results
Demographic data
There were no statistically significant differences between the two groups with regard
to age, number of pregnancies, and size and location of uterine leiomyomas. There
was a statistically significant difference between groups with regard to VAS pain
scores and duration of pain (p < 0.05) ([Table 1]).
Table 1 Patient demographics.
|
Group 1 (n = 25)
|
Group 2 (n = 23)
|
p
|
|
Age (years)
|
47 ± 2.86
|
46.21 ± 3.27
|
0.06
|
|
Gravida
|
3 (1 – 11)
|
3 (1 – 7)
|
0.63
|
|
Size of uterine fibroid (cm)
|
7.68 ± 4.02
|
8.91 ± 4.99
|
0.53
|
|
Duration of pain (months)
|
2 (0 – 24)
|
12 (2 – 72)
|
< 0.005
|
|
VAS (visual analog scale) pain scores
|
2 (0 – 4)
|
7 (6 – 8)
|
< 0.005
|
Endometrial samples
None of the endometrial samples from either of the groups stained with PGP 9.5 or
NF. The distance of nerve fibers to the endometrial lining was similar in both groups
(p > 0.05).
Myometrial samples
The percentage of nerve fibers stained with PGP 9.5 in myometrial tissue was 84% (n = 21)
in Group 1 and 70% (n = 16) in Group 2 (p = 0.23). The percentage of nerve fibers
stained with NF in myometrial tissue was 88% (n = 22) in Group 1 and 74% (n = 17)
in Group 2 (p = 0.28). The density of nerve fibers stained with PGP 9.5 in myometrial
tissue was 0.1 ± 0.22 per mm2 in Group 1 and 0.1 ± 0.21 per mm2 in Group 2 (p = 0.39). The density of nerve fibers stained with NF in myometrial
tissue was 0.06 ± 0.09 per mm2 in Group 1 and 0.07 ± 0.12 per mm2 in Group 2 (p = 0.83). There was no statistically significant difference between
the two groups ([Table 2]).
Table 2 Comparison of presence and density of nerve fibers between groups.
|
PGP 9.5
|
p
|
NF
|
p
|
|
Group 1 (n = 25)
|
Group 2 (n = 23)
|
|
Group 1 (n = 25)
|
Group 2 (n = 23)
|
|
|
PGP 9.5 = protein gene product; NF = Neurofilament
|
|
Presence of nerve fibers in endometrium
|
–
|
–
|
|
–
|
–
|
|
|
Presence of nerve fibers in myometrial tissue (%)
|
84
|
70
|
0.23
|
88
|
74
|
0.28
|
|
Density of nerve fibers in myometrial tissue (per mm2)
|
0.1 ± 0.22
|
0.1 ± 0.21
|
0.39
|
0.06 ± 0.09
|
0.07 ± 0.12
|
0.83
|
|
Density of nerve fibers in fibroid tissue (per mm2)
|
0.001 ± 0.006
|
0.001 ± 0.003
|
0.29
|
0.004 ± 0.013
|
0.002 ± 0.007
|
0.65
|
|
Percentage of nerve fibers in uterine fibroid tissue (%)
|
4 (n = 1)
|
13 (n = 3)
|
> 0.05
|
12 (n = 3)
|
9 (n = 2)
|
> 0.05
|
Leiomyoma tissue samples
The percentage of nerve fiber tissue stained with PGP 9.5 in uterine leiomyoma tissues
was 4% (n = 1) in Group 1 and 13% (n = 3) in Group 2 (p > 0.05). The percentage of
nerve fiber tissue stained with NF in uterine leiomyoma tissues was 12% (n = 3) in
Group 1 and 9% (n = 2) in Group 2 (p > 0.05). The density of nerve fibers stained
with PGP 9.5 in uterine leiomyoma tissues was 0.001 ± 0.06 per mm2 in Group 1 and 0.001 ± 0.003 per mm2 in Group 2 (p = 0.29). The density of nerve fibers stained with NF in uterine leiomyoma
tissues was 0.004 ± 0.013 per mm2 in Group 1 and 0.002 ± 0.007 per mm2 in Group 2 (p = 0.65). There was no statistically significant difference between
the two groups ([Table 2]).
When we look at the consistency between the two dyeing methods, nerve fibers in uterine
leiomyoma tissues showed fair agreement (p < 0.05/κ = 0.388) and nerve fibers in myometrial
tissues showed substantial agreement (p < 0.05/κ = 0.622). The distribution of nerve
fibers in myometrial and leiomyoma tissue was similar in both groups with both dyes.
Discussion
Physiologically, uterine innervation is regulated by estrogen and progesterone through
neurotrophins (nerve growth factor, prostaglandins, etc.) [9], [10]. In estrogen-dependent diseases such as endometriosis, adenomyosis and uterine leiomyoma,
estrogen may play a role in the increase of nerve fibers in the endometrium and myometrium
by regulating neurotrophins. It is known that there are no nerve fibers in the functional
layer of the endometrium. In recent years, however, many publications have identified
nerve fibers in endometrial specimens of endometriosis patients who present with pain
complaints [5], [6], [11], [12], [13]. Based on these findings, we thought that investigating the presence of nerve fibers
in uterine leiomyoma patients could shed light on the etiopathogenesis of leiomyoma-associated
pain.
There is only one study on the innervation of endometrial and myometrial tissue in
patients with painful uterine leiomyomas [7]. In this study, the density of PGP 9.5-stained nerve fibers was significantly higher
in the functional and basal layer of the endometrium in patients with uterine leiomyoma
and pain complaint. According to the authors, nerve fibers in the functional layer
of the endometrium could play a role in the pathogenesis of pain in patients with
endometriosis, adenomyosis and uterine leiomyomas with pain complaint. In contrast
to their results, in our study nerve fibers could not be detected in endometrial tissue
of any of the patients using PGP 9.5 and NF dye. The absence of nerve fibers in the
endometrium may be due to endometrial compression atrophy associated with large uterine
leiomyomas (mean size: 8.27 ± 4.50 cm) in our study group.
In recent years, several studies have indicated that the amount of nerve fibers in
the myometrium of patients with pelvic pain is higher than in patients without pain
[14] – [16]. Lerner and his co-workers reported on the proliferation and hypertrophy of unmyelinated
nerve fibers in the myometrium of patients with chronic pelvic pain [13]. In another study, nerve fiber proliferation was detected in myometrial tissue around
the arteries and veins of patients with endometriosis-associated dysmenorrhea and
dyspareunia [15]. Sun et al. investigated increased nerve fiber density in pseudo-capsules using
staining with anti-oxytocin and PGP 9.5 and compared the result to findings for the
adjacent myometrium. However they did not compare the nerve fiber density of patients
with and patients without pain [17]. Their findings indicate that pelvic pain could be correlated with increased myometrial
nerve fibers. In our study, nerve fibers in the myometrium stained with PGP 9.5 and
NF were detected in 77 and 81% of all patients, respectively. But there was no significant
difference between patients with and patient without pain with regard to nerve fiber
density in myometrial tissue detected by staining with PGP 9.5 (p = 0.39) and NF (p = 0.83).
In the literature the only study related to innervation in leiomyomas is a retrospective
qualitative study which demonstrated that leiomyomas in multiparous women do not contain
nerves while leiomyomas in nulliparous women contain nerves [18]. We investigated the density of nerve fibers in uterine leiomyoma tissue. Nerve
fibers stained with PGP 9.5 and NF in leiomyoma tissue were detected in 8 and 10%
of patients, respectively. We found that there was no significant difference between
patients with and patients without pain in terms of nerve fiber density in uterine
leiomyoma tissue based on staining with PGP 9.5 (p = 0.29) and NF (p = 0.65).
The diagnosis of uterine leiomyoma was confirmed in all patients by histopathologic
examination. The strength of this study which investigated nerve fiber density in
uterine leiomyoma tissue, endometrial and myometrial tissue was its prospective design
and quantitative approach. To the best of our knowledge, the current study is the
first quantitative study investigating nerve fiber density in uterine leiomyoma tissue.
The relatively small sample size can be viewed as a limitation.
In conclusion, the mechanisms of dysmenorrhea and pelvic pain in patients with uterine
leiomyoma are not yet fully understood. In our study, NF and PGP 9.5 immunoreactive-staining
nerve fibers were not detected in the endometrium of any patients with uterine leiomyoma
and pain complaint. The quantity and density of nerve fibers in myometrial and leiomyoma
tissue in patients with pain was similar to that detected in patients without pain.
A population-based study is needed to investigate the mechanism of pain in uterine
leiomyomas.
