|Year : 2021 | Volume
| Issue : 2 | Page : 75-81
Diagnosis of endometriosis by detection of nerve fibers using protein gene product 9.5 immunohistochemistry
Shafqat Bano1, Dilleswari Pradhan2, Pradeep Kumar Behera2, Asaranti Kar2, Akruti Mishra2, Tushar Kar3
1 Department of Pathology, Chacha Nehru Bal Chikitsalaya, New Delhi, India
2 Department of Pathology, S.C.B. Medical College, Cuttack, Odisha, India
3 Department of Obstetrics and Gynecology, S.C.B. Medical College, Cuttack, Odisha, India
|Date of Submission||14-Apr-2021|
|Date of Decision||13-Aug-2021|
|Date of Acceptance||16-Aug-2021|
|Date of Web Publication||30-Dec-2021|
Bajrakabati Canal Road, Cuttack - 753 012, Odisha
Source of Support: None, Conflict of Interest: None
Introduction: Endometriosis is a benign, chronic, multifactorial, debilitating gynecological disease defined by the presence of ectopic foci of endometrial glands and stroma. Diagnostic laparoscopy and histopathological study of hysterectomy specimens are the definitive tool for diagnosis which carries associated risks. The objective of this study is to analyze the presence of protein gene product 9.5 (PGP9.5) antibody-positive nerve fibers by immunohistochemistry (IHC) in endometrial biopsy samples and endometriotic lesions in suspicious cases of endometriosis.
Materials and Methods: This was a case–control study carried out in a tertiary care center. It comprised of cases of endometriosis interna (adenomyosis), endometriosis externa (endometriosis), and age- and parity-matched women without endometriosis taken as control. Clinically/radiologically diagnosed cases of endometriosis and those undergoing laparoscopy for infertility with confirmation after histopathology were included in the study. Endometrial biopsy samples were subjected to IHC using PGP9.5 antibody and examined for The presence of nerve fibers. Statistical analysis was done using SPSS software, and analysis of nerve fibers stained by PGP9.5 was done by the Kruskal–Wallis test.
Results: A total of 60 cases are included in this study broadly divided into three groups (30 women with adenomyosis, 10 with endometriosis externa, and 20 women without endometriosis). Majority (26/30 = 65%) belonged to the third to fourth decade of life with pain being the most specific symptom. Small nerve fibers were noted in endometrial foci of the endometriosis cases but not in women without endometriosis (P = 0.001).
Conclusion: The presence of nerve fibers can be taken as a novel marker of clinical endometriosis, and thus, it could replace hysterectomy as a definitive modality for diagnosing endometriosis.
Keywords: Adenomyosis, endometriosis, histopathological study, immunohistochemistry, nerve fibers, protein gene product 9.5 antibody
|How to cite this article:|
Bano S, Pradhan D, Behera PK, Kar A, Mishra A, Kar T. Diagnosis of endometriosis by detection of nerve fibers using protein gene product 9.5 immunohistochemistry. J Curr Res Sci Med 2021;7:75-81
|How to cite this URL:|
Bano S, Pradhan D, Behera PK, Kar A, Mishra A, Kar T. Diagnosis of endometriosis by detection of nerve fibers using protein gene product 9.5 immunohistochemistry. J Curr Res Sci Med [serial online] 2021 [cited 2022 May 24];7:75-81. Available from: https://www.jcrsmed.org/text.asp?2021/7/2/75/334451
| Introduction|| |
Endometriosis is an enigmatic gynecological disease defined by the presence of ectopic foci of endometrial glands and stroma. It is of two types - Endometriosis interna (adenomyosis) and externa (endometriosis). Adenomyosis is the presence of endometrial glands and stroma within the myometrium while endometriosis externa is defined by the presence of ectopic foci of endometrial glands and stroma outside the uterine cavity. Falcone and Lebovic showed that the exact prevalence of endometriosis in the general population is not clear, the prevalence in women of reproductive age is estimated to range between 10% and 15%. Being a complex disease, its origin remains unclear. It creates many health and social problems that warrant intensive scientific trials to manage this disorder. At present, endometriosis is considered a probable precancerous lesion. In addition, its behavior and association with certain ovarian cancers are categorized as endometriosis-associated ovarian cancers. It is commonly associated with a wide range of symptoms such as chronic pelvic pain and abdominal pain, chronic dysmenorrhea, back pain, dyspareunia, and infertility. However, the relationship between different pains and endometriosis is not well understood, and there is a poor correlation between the severity of pain symptoms and anatomical staging of the disease. The definitive diagnostic tool incorporates a major surgical procedure that is done under general anesthesia and is associated with risks of anesthesia, injury to vessels, morbidity, significant delay in treatment with a heavy financial and mental burden to patients and their relatives. There is no simple, less invasive, and less expensive way to diagnose endometriosis, and a number of studies are being done to identify biomarkers of this disease. Aberrant neuronal growth within the uterus has been identified as a factor that may contribute to abnormal fertility and uterine dysfunction. Recently, analysis of neural innervation in endometrial biopsy samples as a minimally invasive diagnostic tool for endometriosis and adenomyosis has been proposed by some researchers. Moreover, it is shown that endometriotic lesions are innervated by protein gene product 9.5 (PGP9.5) positive nerve fibers. PGP9.5 is a very specific pan-neuronal marker for myelinated and unmyelinated nerve fibers.
The objective of this study was to analyze the presence of nerve fibers by immunohistochemistry (IHC) using PGP9.5 antibody in endometrial biopsy and endometriotic foci samples in diagnosed cases to assess the efficacy of nerve fiber detection in diagnosing endometriosis.
| Materials and Methods|| |
This is an observational case–control study; done over a period of 2 years in the Departments of Pathology and Obstetrics and Gynecology. Women of the reproductive age group, i.e., between 18 and 45 years were included who were undergoing laparoscopy for pelvic pain or infertility and those who were not receiving any hormonal treatment 3 months prior to radiological investigation. The major surgical procedure such as hysterectomy or excision of endometriotic foci was conducted in these patients for confirmation of the diagnosis of adenomyosis/endometriosis. Exclusion criteria for the study included pregnant women, women having malignancy, and unwillingness on the part of participants. Age and parity matched women were taken as control. Ethical approval was obtained from the Institutional Ethical Committee of the Medical College.
After obtaining written informed consent, the clinical details, investigation findings, and imaging data were collected. The demographic details included age, parity, basal metabolic index, clinical details such as chief complaints (dysmenorrhea/dyspareunia/infertility), obstetric history (uneventful/eventful), medical history (diabetes mellitus/hypertension/thyroid disorders), and history of any addiction, ultrasonography (USG) was carried out and findings were recorded [Figure 1]a and [Figure 2]a. Endometrial biopsies were obtained by endometrial pipelle after taking the aseptic precautions. The hysterectomy specimens and tissue from endometriotic foci were collected. All samples were fixed in 10% neutral buffered formalin for 18–24 h, processed, and embedded in paraffin wax according to a standard protocol. In the case of endometriosis externa like ovarian or scar endometriosis, the most representative part of tissue was grossed [Figure 2]b and processed histotechnically [Figure 2]c. Those cases which showed evidence of endometriosis were included in the study group, and standard hematoxylene and eosin (H and E) staining followed by IHC using PGP9.5 antibody was employed. IHC staining was done using polyclonal rabbit antibodies. H and E stained sections of endometrium were diagnosed as proliferative [Figure 1]b, secretory, nonsecretory (if it is neither proliferative nor secretory and does not have definitive findings of either), and bleeding phase endometrium.
|Figure 1: (a) Ultrasonography of abdomen and pelvis showing bulky uterus, (b) photomicrograph showing endometrial glands and stroma (H and E, ×100) (c) PGP9.5 positive nerve fibers in the endometrium (PGP9.5, ×100) (d) PGP9.5 positive nerve fibers in the endometrium (PGP9.5, ×400). PGP9.5: Protein gene product 9.5|
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|Figure 2: (a) Ultrasonography of the pelvis showing ovarian cyst, (b) gross photograph of ovarian cyst with brownish-black deposits, (c) photomicrograph showing ovarian stroma and hemosiderin-laden macrophages (H and E, ×100), (d) photomicrograph showing ovarian stroma with positively stained nerve fibers (PGP9.5, ×400, ×100) (d) indicated by yellow arrows|
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Calculation of nerve fiber density
The method of microvascular density quantification described by Weidner N (1995) was followed to count the number of nerve fibers identified by PGP9.5 staining in endometriotic lesions. After immunostaining, the entire section was scanned at low power (×100) (Mic; Olympus, India) to identify the hot spots, which represent the areas of highest innervation [Figure 1]c. Then, under high power (×400) individual nerve fibers were counted to obtain a nerve count in a given defined area [Figure 1]d and [Figure 2]d. An average number of nerve fibers per hotspot was obtained by dividing the total number of nerve fibers by the total number of hot spots (each hot spot with a square of 1 mm × 1 mm). In each specimen from all endometriotic lesion samples, the results are denoted as the mean number of nerve fibers per mm (standard derivation [SD]). The average nerve count in five hotspots was calculated because there were no significant differences in the total number of hotspots between study groups, and most slides did not show more nerve fiber hot spots.
Results obtained were analyzed using statistical methods (SPSS software (IBM, Chicago, USA)). Kruskal–Wallis test was applied for the analysis of nerve fibers stained by IHC with PGP9.5 antibody, because their distribution was not normal.
| Results|| |
This study comprised of 60 consecutive cases including 30 cases of adenomyosis, 10 cases of endometriosis (externa) and 20 cases were of women without endometriosis taken as control. Among 10 cases of endometriosis, 8 (80%) were of ovarian endometriosis and 2 (20%) were of scar endometriosis at the incision site of cesarean section. The groups were distributed as follows:
Group A = endometriosis interna/adenomyosis (30 cases), Group B = endometriosis externa/endometriosis (10 cases: Ovarian-80%, skin-20%), Group C = control.
In the present study, most of the patients with adenomyosis (13 cases = 43%) were in between 27–35 years, followed by 37% of patients in 36–45 years [Table 1]. Endometriosis was seen most commonly in the 27–35 years of age group (5 cases = 50%). The least number of cases were seen between 18 and 26 years of age group. The majority of the patients belonged to the lower middle class of socioeconomic status (23 cases = 38.3%) which is followed by patients belonging to the upper-middle-class (22 cases = 36.7%). Parity distribution among endometriosis externa patients revealed a maximum number of cases (43 cases), i.e., 66.4% cases to be multiparous. Of them, the equal incidence was seen in para 2 and above [Table 1]. The analysis of medical history (diabetes mellitus/hypertension/thyroid disorder) in the above study showed that most of the patients had no such medical history (53%), followed by diabetes mellitus in 25% of cases [Table 2]. The incidence of eventful obstetric history (tubectomy/cesarean section/endometrial curettage) was maximum observed in 16 cases (=53.3%) of adenomyosis. Endometriosis cases showed an equal number of patients with eventful and uneventful obstetric history (5 cases each = 50%). Imaging studies showed, in the group with adenomyosis, a bulky uterus was the most common USG finding [Figure 1]a of the abdomen and pelvis (13 cases = 43.3%) followed by inhomogeneous myometrium (9 cases = 30%). [Table 3] shows Kruskal-Wallis test for nerve fiber density in differed groups. The observed values were 7.58 & 8.56 in endometriosis interna/endometriosis and endometriosis externa/endometriosis respectively which is much higher from control group (10.68). The difference was found statistically significant having p value 0.001.
[Table 4] shows mean nerve fiber density in this study to be 7.58 and 8.56 in adenomyosis and endometriosis, respectively which was much higher than the control group (0.3) and it was statistically significant having a P = 0.001. [Table 4]A shows a mean density of nerve fibers in Group A 19.93 which was close to the mean density of nerve fibers in Group B (22.20). The P = 0.612 which was statistically insignificant. [Table 4] also shows the mean density of nerve fibers in Group A 35.38 which was much higher than the mean density of nerve fibers in Group C (10.68). The P = 0.001 which was statistically significant. The mean density of nerve fibers in Group B was 25.50 and was much higher than the mean density of nerve fibers in Group C (10.50) with a statistically significant P = 0.001.
|Table 4: Mann–Whitney U-test comparing mean density of nerve fibers among different study groups|
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This study showed a linear relationship between age and the mean density of nerve fibers as the mean density of nerve fibers in Group A is 5.7 and 6.65 in the age group 18–26 and 27–35 years, respectively. In Group B, the mean density of nerve fibers is 9.08 in the 18–26 years of age group, while it was 9.20 in the 27–35 years of age group, respectively. However, the study did not depict any relationship between mean nerve fibers and body mass index (BMI). In Group A, mean nerve fibers were 6.01, 9.17, and 7.80, and in Group B, it was 5.5, 9.24, and 10.03 in underweight, normal, and overweight cases. Relating mean nerve fibers with parity, in Group A, it was found to be 6.58, 6.28, and 8.88, and in Group B, it was 8.33, 8.53, and 8.75 in nulliparous, primipara, and multipara females, respectively.
| Discussion|| |
Endometriosis is a common gynecologic disorder in reproductive-age women. It is a relatively common and potentially debilitating condition and a significant public health issue requiring increased surveillance, clinical awareness, and management. Prevalence is difficult to determine, firstly because of variable clinical presentations, and second because the only reliable diagnostic test is laparoscopic visualization of endometriotic deposits followed by histologic confirmation. However, in a study among 13,508 Australian women followed for 20 years, one in nine women had clinically confirmed or suspected endometriosis by the age of 44 years, with most diagnosed during their early thirties. Hence, efforts to expand knowledge on the etiology of the disease and optimal methods for disease diagnosis and management are crucial to women's health.
Although the detailed molecular mechanism of etiology remains unclear, recent studies have revealed both genetic and epigenetic backgrounds of the development of endometriosis. In clinical practice, endometriosis has been recognized as a precursor lesion of several types of malignancies and endometriosis-associated carcinoma. An imbalance between reactive oxygen species and local antioxidants has been reported to contribute to the development of endometriosis-associated carcinoma as well as the pathophysiology of this disease.
The diagnosis of endometriosis remains challenging over the years. At present, there are no simple noninvasive diagnostic tests despite an extensive search for new laboratory tests and advances in imaging technologies. The standard procedure for the diagnosis of endometriosis is imaging/laparoscopic surgery combined with a thoughtful interpretation of histological examination of excised lesions, which is an invasive surgical procedure associated with complications. Furthermore, laparoscopy has some limitations in terms of false-negative findings, such as mistaking lesions for corpus luteum cyst or missing a peritoneal or deep lesion in difficult locations, and thus, the sensitivity is not always reliable. It is possible that some cases where laparoscopy does not reveal endometriosis may be found to have endometriosis by a method of nerve fiber detection. Few spots of adhesion in deep areas may not be convincing for endometriosis in laparoscopy. Moreover, laparoscopy and hysterectomy are surgical procedures that can be associated with risks with delay in treatment. Studies have indicated that small demyelinated nerve fibers in the endometrium are found in higher density in comparison with women without endometriosis.
It is proved that pelvic pain is associated with an increase in the density of nerve fibers directly in the area of endometrial tissue damage which contributes to its generation. A number of biologically active triggers have been distinguished, which probably cause the intensification of local growth of nerve fibers and also directly or indirectly contribute to an increase in spontaneous contractility of smooth myocytes of the uterus. Among the secreted neuronal markers, oxytocin, which is a key factor regulating uterine contractility, must be identified. This is evidenced by an increase in oxytocin receptor expression in myometrium by 2.5 times (92.2% ±2.1% vs. 36.3% ±2.4%, P < 0.05) and in adenomyosis foci – by 2.09 times (73.7% ±1.8% vs. 35.2% ±1.4%, P < 0.05). The myometrial innervation of patients with pelvic pain is characterized by a significantly higher expression of nerve growth factor (NGF) – 2.4 times (57.2 ± 2.3 vs. 23.1 ± 1.5; P < 0.05); compared to the painless form of adenomyosis which increases the area of the local innervation field and triggers the pain syndrome. Locally increased estrogen levels and inflammation cause increased NGF production in the uterus of patients with adenomyosis. Endometriosis creates an inflammatory environment, and research shows the role of inflammation in stimulating peripheral nerve sensitization. Hence, an interaction between lesions and nerve fibers, mediated by inflammation, may be important in endometriosis-associated pain. Yan et al. pointed out that endometriotic lesions are wounds undergoing repeated tissue injury and repair, the relationship between endometriotic lesions and nerve fibers are not simply unidirectional, i.e., lesions promote hyperinnovation. Rather, it is bidirectional, i.e., endometriotic lesions and nerve fibers engage active cross-talks, resulting in the development of endometriosis and pain. That is, nerve fibers and endometriotic lesions are actually partners in crime in inflicting pains in women with endometriosis, aided possibly by other culprits, some yet to be identified. Hence, compiling these facts, it can be suggested that local tissue damage with repair, inflammation, biological triggers such as oxytocin and estrogen contribute to increased nerve fiber density in adenomyosis and endometriosis.
Nerve fibers can be detected in many gynecological disorders associated with pain like endometritis, endometrial hyperplasia, endometrial carcinoma, polyps, leiomyomas along with endometriosis. However the fibers are consistently present in the superficial layer of the endometrium compared to nonendometriotic lesions. The endometrium appears to be the only mucosal tissue in the body that is not normally innervated. However, researchers have demonstrated small C nerve fibers in both layers of the endometrium and in increased density in myometrium in virtually all women with endometriosis. Tokushige et al. reported that in women with laparoscopy-confirmed endometriosis, small nerve fibers were stained positive for a PGP9.5 in the functional layer of the eutopic endometrium, but PGP9.5 was not detected in the endometrium of women without endometriosis. Evidence suggests that pelvic innervation is altered in endometriosis-affected women, and nerve fibers are demonstrated in eutopic endometrium (of women with endometriosis) and in endometriotic lesions play roles in the generation of chronic pelvic pain.
A study done by Elbohoty et al. shows nerve fibers in the functional layer of endometrium were detected in 13.7% of nonendometriotic gynecological pathologies in contrast to 100% of endometriotic patients (P < 0.001). Studies have confirmed the presence of nerve fibers by IHC with 98% sensitivity and 83% specificity. Others have demonstrated sensitivity and specificity of 92% and 80%, respectively. Researchers believe that the presence of nerve fibers in the functional layer of endometrium contributes to the generation of pain. In a study of peritoneal endometriotic lesions, a positive correlation was obtained between the intensity of pain and the density of nerve fibers. Nerve fibers were more in deep endometriotic lesions compared to normal peritoneum.
This work was designed to study the role of pan-neuronal marker PGP9.5 as an immunomarker which can help in the definitive diagnosis of endometriosis and to correlate their expression with various clinicopathological parameters.
The majority of cases belonged to 27–35 years of age group-18/40 cases (46.5%) with the mean age being 31.73 ± 0.2 [Table 1]. This was in concordance with the previous studies which showed that endometriosis is almost always detected in women of reproductive age; the mean age at diagnosis ranges from 25 to 29 years. In a study done by Yu et al. over a period from 2006 to 2015, the highest incidence was observed among women aged 41–45 years and was higher for black (highest 44.6/10,000 woman-years in 2011) versus white women (highest 27.9/10,000 woman-years in 2010). Both the incidence of adenomyosis and endometriosis externa was observed more in multiparous patients than nulliparous patients. It was in concordance with the study done by Parazzini et al. who also reported that the frequency of adenomyosis was higher in parous women in comparison with nullipara. The odds ratio for the disease was 1.8 and 3.1, respectively in women reporting one and two or more births (P < 0.01). However, according to a review by Desai et al. there is a decreased likelihood of endometriosis in women who have been pregnant which may be due to a protective effect of pregnancy. They reported that the risk of endometriosis externa is inversely related to the number of term pregnancies. This significant difference among different researchers needs further analysis and more studies.
In the present study, the majority of patients were underweight (26 cases = 43%) closely followed by those of normal BMI [Table 1]. Regarding body habitus, weak inverse associations with weight and BMI have been found and an increased risk with taller height has been reported for endometriosis as taller women may have higher follicular-phase estradiol levels. Overweight women have been suggested to be at lower risk of endometriosis. Women with an increased body mass index have more irregular menstrual cycles and increased rates of anovulatory infertility.
This might explain the association with endometriosis. The bulky uterus was the predominant (38.3%) USG finding in endometriosis and adenomyosis and 43.3% of adenomyosis only. This was similar to but slightly less than data obtained by Bromley et al., who showed a majority of the patients with adenomyosis had bulky globular uterus (90%). 30% of cases showed ovarian cyst and 30% of cases showed normal uterus on USG of endometriosis cases. Bulky uterus and inhomogenous myometrium were present in 20% cases each.
KruskalWallis test [Table 3] was applied for the analysis of nerve fibers stained by immunomarker PGP9.5. The comparison of the mean density of nerve fibers between endometriosis patients and control shows higher density in cases of endometriosis which was statistically significant. The mean density of nerve fibers was 2.2 SD 4.7 in the group with endometriosis (P < 0.001). Aghaey Meibody et al. observed that nerve fibers were detected by IHC using PGP9.5 in all endometrial biopsies from all women with endometriosis but detected only in few cases without endometriosis.
There was a linear relationship between age and the mean density of nerve fibers, but no relation could be seen between the BMI, parity, and the mean nerve fibers. Elbohoty et al. in a cross-sectional study on women with different gynecologic pathology found that patients with PGP9.5 positive endometrial nerve fibers were significantly older (P = 0.003), but with comparable parity (P = 0.742) and BMI (P = 0.449) with those without endometrial nerve fibers.
| Conclusion|| |
Endometriosis is a common chronic gynecologic disease considered as a huge burden on women psychologically, financially, and physically. Due to the difference in clinical presentation, it can be difficult to diagnose and treat. In the present study, we have detected small nerve fibers in endometrium and endometriotic lesions using immunomarker PGP9.5. The difference in nerve fibers between women with and without endometriosis was found statistically (P < 0.05). Therefore, we suggest that the noninvasive technique like endometrial biopsy followed by PGP9.5 IHC should be employed to definitely diagnose endometriosis instead of doing major surgery like hysterectomy.
MicroRNAs have emerged more recently, but their utility to detect endometriosis remains uncertain. The search for a biomarker or a set of biomarkers is still open and may benefit from novel molecular biology and bioinformatics approaches to disease diagnosis and uncover molecular signatures specifically associated with the disease.
Small number of cases is a limitation in this study.
We sincerely acknowledge the technical help and support from the staff of the Department of Pathology, S. C. B. Medical College, Cuttack.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]