The processus vaginalis is a diverticulum of the peritoneum, inside the gubernaculum. The gubernacular mesenchyme gives rise to smooth muscles, which aids in the descent of the testis into scrotum. The processus vaginalis obliterates after the descent of the testis and the obliterated processus vaginalis is devoid of smooth muscle cells. Inguinal hernia and hydrocele in children are closely associated with the presence of the smooth muscle bundles in processus vaginalis. The patent processus vaginalis in hydrocele seems to allow only the passage of fluid in contrast to inguinal hernia. The patent processus vaginalis in undescended testis usually is not associated with inguinal hernia or hydrocele. This suggests that the patent processus vaginalis differs in these three entities. Therefore, we evaluated the processus vaginalis sacs in these three pathologies using histopathology, immunohistochemistry and electron microscopy to understand the phenotypical differences in these conditions.

In our study, smooth muscles were present in 76% of inguinal hernia and 86% of hydrocele specimens, but only 20% of specimens of UDT had the presence of smooth muscles and this difference was statistically significant. Moreover, the smooth muscle cells were organised as multiple bundles in inguinal hernia and hydrocele specimens compared to UDT specimens, where the smooth muscles were sparse and scattered, if present. Our study partially concurs with the studies by Tanyel, et al. wherein the authors reported smooth muscle was present in inguinal hernia sacs but only sparse smooth muscles were seen in hydrocele and UDT sacs [6, 9]. Whereas, a study by Piçarro, et al. observed smooth muscles in 68% of the sacs in UDT cases [10]. In all these studies, the smooth muscle cells were present in fewer patients of UDT as compared to inguinal hernia. The apparent differences in the presence of smooth muscles might be due to the age of the patients enrolled in the study. The mean (SD) of the patients enrolled in our study were 45 (± 37.7), 45.3 (± 29.8) and 44.1 (± 29.2) in inguinal hernia, hydrocele and UDT, respectively. The mean ages of the patients in the study by Tanyel, et al. [9] and Piçarro, et al. [10] were both higher. This could suggest that although processus vaginalis persists in these three conditions, the processus undergoes changes as the child grows. The smooth muscle content increases when there is failure of obliteration of processus vaginalis.

Immunohistochemical staining was done to evaluate the smooth muscle phenotype in the processus vaginalis specimens. Our study showed positive staining for desmin and SMA in 68%, 86% and 20% of the processus vaginalis sac of inguinal hernia, hydrocele and UDT, respectively and this difference was statistically significant. There was no vimentin-positive smooth muscle in any specimen. This contrasts with the report by Mouravas, et al., wherein the authors reported that vimentin was expressed by all sacs of hydrocele and half of the inguinal hernia sacs [11]. Another study by the same centre reported that vimentin was expressed in more than 90% of the UDT specimens [12]. Since vimentin is not expressed by mature smooth muscle cells, therefore the smooth muscles in processus vaginalis identified in our study were of mature phenotype. In our study, although the quantity of smooth muscle cells decreased in sacs of UDT, there was no change in the smooth muscle phenotype. Therefore, the processus vaginalis in UDT specimens are deficient in smooth muscles. Since smooth muscles in the processus vaginalis contribute to the descent of the testis, therefore the deficient smooth muscle in these specimens might contribute to the disease pathology. As the obliteration of processus vaginalis starts after the descent of the testis, the de-differentiation and apoptosis of smooth muscle cells have not started in these sacs, as evident by the mature phenotypic staining.

Myofibroblasts are intermediary cells in the process of dedifferentiation of smooth muscle cells to fibroblasts. The presence of the myofibroblasts were noted in 95%, 67% and 15% of the processus vaginalis specimens of inguinal hernia, hydrocele specimens and UDT. The presence of myofibroblasts in the majority of the specimens of hernia and hydrocele supports the theory of de-differentiation of smooth muscle cells to fibroblasts. This concurs with the results by Tanyel et al., wherein myofibroblasts were observed in all sacs of the inguinal hernia [13]. However, there is no previous literature available regarding the presence of myofibroblasts in specimens of hydrocele and UDT. The scarce presence of myofibroblasts in UDT specimens suggests that de-differentiation has not yet started in the sacs of UDT, thereby indicating the processus vaginalis undergoes de-differentiation once the descent of testis is completed. The presence of myofibroblasts on electron microscopy significantly correlated with the presence of smooth muscle in light microscopy and the expression of desmin and smooth muscle actin on immunohistochemistry in our study.

Striated muscles were observed in a few specimens of processus vaginalis in all three pathologies and this was statistically significant. The striated muscle believed to develop within the gubernaculum is the cremaster muscle. The striated muscle may have also developed through trans differentiation of the smooth muscle. Innervation appears to play a role in trans differentiation.

The processus vaginalis sacs from four female patients of inguinal hernia did not differ statistically from the processus vaginalis sacs from thirty-three male patients with inguinal hernia with respect to the presence of smooth muscles, distribution of smooth muscle bundles, expression of desmin, smooth muscle actin, presence of striated muscles and presence of myofibroblasts. This contrasts with the results of Tanyel, et al., wherein the authors reported that striated muscles were observed in sacs from girls and not the sacs from boys with inguinal hernia [9]. However, we observed striated muscles in sacs from hydrocele and UDT as well. The study by Tanyel, et al. [9] had not evaluated the other characteristics of the sacs, unlike our study. In both the studies the sample size of girls with inguinal hernia was small. Therefore, larger studies on this aspect may yield a conclusive answer regarding sexual dimorphism of the processus vaginalis sacs.

Processus vaginalis is a special structure with smooth muscle content. If the smooth muscle does not disintegrate, it may hinder the obliteration and give rise to hydrocele and inguinal hernia. The amount of persisting smooth muscle determines the clinical outcome as hernia, hydrocele or UDT. The genitofemoral nerve has been proposed to respond to androgens by release of calcitonin gene-related peptide (CGRP) from its sensory nerve endings to control growth of the gubernaculum to the scrotum. Smooth muscles develop as a result of this signal to help in the descent of the testis. The processus vaginalis obliterates after the descent of testis. The exact mechanism of obliteration of the processus vaginalis is unknown. Failure of the obliteration of the processus vaginalis results in an inguinal hernia, hydrocele or UDT.

Our study highlights the differences in tissue composition in the processus vaginalis sacs in inguinal hernia, hydrocele and UDT, thereby resulting in the varied clinical presentation. Inguinal hernia and hydrocele sacs contain more smooth muscle cells and myofibroblasts and express desmin and SMA in greater magnitude than the sacs from UDT. Newer studies have shown the role of cytokines such as hepatocyte growth factor (HGF) and transforming growth factor beta 1 (TGF-b1) in the closure of processus vaginalis. The presence of HGF receptors in the processus vaginalis may indicate the role of HGF in triggering epithelial-mesenchymal transformation during inguinal hernia closure [14]. TGF-b1 is a potent fibrogenic agent and stimulates fibrosis of processus vaginalis. Mosavi, et al. have observed that the amount of TGF-b1 was higher in communicating hydrocele fluid than in non-communicating hydrocele [15]. Further studies on the role of cytokines may provide insight into the exact mechanism of obliteration of processus vaginalis.

Ours is the first study to analyze the processus vaginalis sacs of inguinal hernia, hydrocele and UDT using histology, immunohistochemistry and electron microscopy simultaneously. We evaluated the presence and maturity of smooth muscle cells using histology and immunohistochemistry and confirmed the presence of myofibroblasts using electron microscopy in all the specimens. We also analyzed the processus vaginalis sacs from female hernia patients; albeit in a smaller number. Due to the implementation of uniform protocol and accurate tests employed to evaluate the stated objectives, our study carried out in a larger sample size adds significantly to the available literature on the characteristics of the processus vaginalis in inguinal hernia, hydrocele and UDT. However, there are a few limitations of our study. The sample size of female inguinal hernia patients was small. Therefore, the validity of our results need to be studied in a larger sample before generalizing the results in this subset. We conducted a qualitative and semi-quantitative analysis on the smooth muscle presence in the processus vaginalis specimens due to technical limitations. In spite of these limitations, our study highlights the differences in the characteristics of the smooth muscle cells in inguinal hernia, hydrocele and UDT which lead to the variation in presentation in these conditions.