The effect of IGF-1 on the closure process of processus vaginalis

Abstract

Background

The processus vaginalis is a peritoneal sac that descends with the testes during the fetal period and typically undergoes obliteration in early life. However, failure of obliteration is associated with pathologies such as inguinal hernia, hydrocele, and cysts of the canal of Nuck, as well as cryptorchidism and epididymal anomalies. The Insulin-like Growth Factor (IGF) system is a signaling pathway that plays a critical role in biological processes such as cell growth, proliferation, and differentiation. This study investigates the effect of IGF-1 on the obliteration process of the processus vaginalis.

Methods

This retrospective study includes 218 cases recorded in the pathology archives between January 1, 2022, and June 30, 2024, comprising cases of inguinal hernia and hydrocele. The inguinal hernia cases include both pediatric and adult age groups, while the hydrocele cases pertain to the pediatric age group. New paraffin blocks were prepared from archived specimens using the manual microarray method, and the samples were stained with IGF-1 antibody. Evaluations were conducted semi-quantitatively under a light microscope. Data analysis was performed using the Pearson chi-square test, Mann-Whitney U test, and Friedman test.

Results

No significant differences were observed between the inguinal hernia and hydrocele groups in terms of epithelial, stromal, and muscle staining levels (P = 0.958, P = 0.669, and P = 0.529, respectively). In the hernia group, a significant difference was identified between epithelial and muscle staining levels, with muscle staining being significantly higher (P = 0.016). No significant differences were noted among epithelial, stromal, and muscle staining levels in the hydrocele group (P = 0.199).

Conclusion

Our study demonstrates that IGF-1 expression is similar in hernia and hydrocele cases, showing positive expression in vascular and muscle tissues but negative expression in epithelial cells. IGF-1 expression was higher in the pediatric group, suggesting that IGF-1 may be associated with closure defects.

Background

The processus vaginalis represents a blunt protrusion emerging from the abdominal wall during fetal development, which generally undergoes obliteration in early childhood. Open processus vaginalis is closely associated with congenital inguinal hernia and hydrocele. Despite being open in children with undescended testes, clinically significant inguinal hernia remains uncommon, manifesting in approximately 10–15% of such cases. Functioning as a peritoneal sac, the processus vaginalis descends alongside testicles during embryonic development [1]. Numerous conditions, such as acquired undescended testes, congenital indirect inguinal hernias, canal of Nuck cysts, communicating hydroceles are linked to the persistence of the processus vaginalis [2]. Furthermore, its persistence plays at least a partial role in various epididymal anomalies such as cryptorchidism and retractile testes [3].

The Insulin-like Growth Factor (IGF) system is a highly conserved signaling pathway that influences endocrine, paracrine, and autocrine mechanisms in most cell types. The IGF system is composed of two ligands, insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2); two receptors, the IGF-1 receptor (IGF1R) and IGF-2 receptor (IGF2R); and six IGF-binding proteins (IGFBP1-6). IGFs regulate various biological processes, metabolism, including cell growth, differentiation and proliferation. IGF-1 is an important component of the insulin signaling pathway. It is a key growth factor associated with several biological properties such as survival, differentiation, cell proliferation, and maturation. IGF-1 acts as an amplifier for the hormonal action of gonadotropins. It is important for cell proliferation as well as the development of embryos and is involved in the binding of IGF2 to IGF1R. Phosphorylation of the PI3K/Akt signaling pathway also occurs here. These factors function by reducing oxidative stress and increasing mitochondrial activity and membrane potential [4, 5].

Many studies have questioned what genetic, hormonal and developmental factors may be involved in PV closure defects [6]. Smooth muscle cells that fail to undergo apoptosis are blamed for this failure to obliterate, and the derepensation and loss of innervation of smooth muscle cells are blamed [7–9]. When the literature is reviewed, it is noteworthy that the relationship with IGF-1 has not been investigated. If a relationship between IGF-1 and closure defects can be established, it may become possible to eliminate the need for surgical interventions associated with conditions such as inguinal hernia and hydrocele. In this study, the effect of IGF-1 on this closure process was examined.

Methods

The study was retrospective and included cases of hydrocele and inguinal hernia recorded in the pathology laboratory of a tertiary hospital between January 1, 2022 and June 30, 2024. This study was assessed and approved by the Non-Interventional Ethics Committee of University on 18/12/2024 with protocol no: GOKAEK 2024/23/2.

Age and gender characteristics of the cases included in the study were recorded. Cases for which report information and paraffin blocks could not be obtained from the pathology laboratory archive were excluded from the study.

New paraffin blocks were prepared from archived specimens using the manual microarray method, and the samples were stained with IGF-1 antibody on a closed Leica Bond system. Different areas will be investigated in each cross-section in the assessment. Evaluation was performed using a semi-quantitative method under a light microscope. For immunohistochemical analysis, sections were examined separately for antibodies. negative IGF-1 immunostaining were defined as sparse brown-yellow particles visible in the tissue on immunohistochemical screening. To assess the severity of the immunohistochemical reaction of cells with markers, scoring ranging from (0) to (3) was used. For this purpose (0) = negative, (1) = focal mild staining, (2) = diffuse mild staining, (3) = diffuse severe staining. For evaluation, 10 different areas were examined in each section under 40X objective magnification (scores: 0, 1, 2, 3; Figs. 1, 2 and 3).

Fig. 1.

Pediatric age group hernia (epithelial, muscle, and stromal staining) (IGF-1X). A.Stromal Vasculer×40 (grade1), B.Epithelial×40 (grade1), C. Muscle×40 (grade1), D. Stromal×40 (grade1)

Fig. 2.

Pediatric age group hydrocele (epithelial, muscle, and stromal staining) (IGF-1X). A Stromal×40(grade1), B Muscle×40(grade2)

Fig. 3.

Adult age group hernia (epithelial, muscle, and stromal staining) (IGF-1X), A. Epithelial×40(grade1), B. Muscle×40(grade1)

Statistical analysis

All data were recorded in a data set and analyzed using SPSS version 26 (IBM, Armonk, New York, USA). The distribution of age and sex between inguinal hernia and hydrocele cases was compared using the Pearson chi-square test. For immunohistochemical results, 1, 2, and 3 scores were all considered as positive, and chi-square test were performed to compare the rates. Additionally, the Mann-Whitney U test was used to compare epithelial, stromal, and muscle staining levels between diagnoses, while the Friedman test was employed for separate comparisons of epithelial (mesothelial), stromal (including vascular structures), and muscle staining levels within each diagnosis. P values of < 0.05 were considered as statistically significant.

Results

A total of 218 cases were included in this study. Among the pediatric inguinal hernia cases, 138 were males and 26 were females. In the pediatric hydrocele group, 36 were males and 7 were females. The pediatric group age range was 0–14 years. The adult inguinal hernia group comprised 11 cases, with 4 females and 7 males, and the age range was 43–85 years.

Distribution of demographic characteristics and staining grades by diagnosis are shown in Table 1. The distribution of sex and age groups was found to be similar in both groups (p values: 0.758 and 0.092, respectively). No significant differences were observed between the hernia and hydrocele groups in terms of epithelial, stromal, and muscle staining levels (p values: 0.958, 0.669, and 0.529, respectively). In the hernia group, a significant difference was found between epithelial and muscle staining levels, with muscle staining being significantly higher (p = 0.016). No other significant differences were identified. In the hydrocele group, no significant differences were found among epithelial, stromal, and muscle staining levels (p = 0.199). Comparison of staining grades by age group in the hernia group are shown in Table 2. In the hernia group, no significant differences in IGF-1 epithelial, IGF-1 stromal, or IGF-1 muscle staining levels were observed between younger and older age groups (p > 0.05). IGF-1 Expression Staining Grades of Study Groups are shown in Fig. 4.

Table 1.

Distribution of demographic characteristics and staining grades by diagnosis

		Diagnosis				P Value
		Hernia		Hydrocele
		n	%	n	%
Gender	Male	143	81.7	36	83.7	0.758
	Female	32	18.3	7	16.3
Age group	Young	164	93.7	43	100	0.092
	Elderly	11	6.3	0	0
IGF-1 Epithelial Scores	0	167	95.4	41	95.3
	1	8	4.6	1	2.3	0.958
	2	0	0	1	2.3
IGF-1 Stromal Scores	0	155	88.6	37	86
	1	18	10.3	6	14	0.669
	2	2	1.1	0	0
IGF-1 Muscles Scores	0	153	87.4	36	83.7
	1	12	6.9	4	9.3	0.529
	2	10	5.7	3	7

IGF-1: Insulin-like growth factor-1

Table 2.

Comparison of staining grades by age group in the hernia group

		Pediatric Age Group		Adult Age Group		Total	P Value
		n	%	n	%	n
IGF-1 Epithelial Scores	0	156	95.1	11	100	167	0.455
	1	8	4.9	0	0	8
IGF-1 Stromal Scores	0	144	87.8	11	100	155	0.220
	1	18	11	0	0	18
	2	2	1.2	0	0	2
IGF-1 Muscle Scores	0	143	87.2	10	90.9	153	0.681
	1	11	6.7	1	9.1	12
	2	10	6.1	0	0	10

IGF-1: Insulin-like growth f

Fig. 4.

IGF-1 Expression staining grades of study groups

Discussion

The processus vaginalis is a peritoneal diverticulum formed during the descent of the testicle into the scrotum following the gubernaculum, which is then obliterated and the connection between the inguinal canal and the abdomen is lost [10]. The anomalies linked to the processus vaginalis are usually related to its persistence. Different inguinal anomalies such as inguinal hernia, hydrocele, Nuck’s duct cysts occur due to non-closure of the processus vaginalis [9, 11]. The persistence of the processus vaginalis is linked to the large amounts of its abnormalities. In order to appreciate the abnormalities that occur when obliteration fails, it is essential to find out the embryological development of the processus vaginalis. Although obliteration of the processus vaginalis can occur prenatally, it remains present at birth in up to 80% of males and 60% of females. By eight weeks of age, persistent processus vaginalis is observed in 63% of males, with obliteration occurring at any time up to two years of age. Beyond two years, up to 40% of males may still have a persistent processus vaginalis, approximately half of which remain asymptomatic throughout life. These mechanisms demonstrate the possibility of cases being observed across different age groups [9, 12]. The age distribution in our study supports this conclusion. In this study, a male predominance was observed. However, the study’s limitations include the lack of data on right or left side localization. Additionally, no cases were included in the study more than once.

A particular structure with smooth muscle composition is the processus vaginalis. It can prevent obliteration and result in inguinal hernia and hydrocele if the smooth muscle stays intact. Emerging research suggests that androgens stimulate the genitofemoral nerve to release calcitonin gene-related peptide (CGRP) from sensory nerve endings, thereby regulating gubernaculum growth into the scrotum. Consequent smooth muscle development facilitates testicle descent, with processus vaginalis typically obliterating post-descent. However, the precise obliteration mechanism remains enigmatic. Processus vaginalis obliteration failure can result from multiple factors, including smooth muscle cell dedifferentiation, impaired innervation, and compromised apoptotic processes. Krishnan et al. demonstrated significant variations in processus vaginalis sac tissue composition across different clinical presentations, revealing distinct cellular characteristics. Comparative analyses reveal that hydrocele and inguinal hernia sacs exhibit higher smooth muscle cell and myofibroblast concentrations, with elevated desmin and SMA expression compared to undescended testicle (UDT) sacs [13]. Recent investigations have illuminated the pivotal role of transforming growth factor beta 1 (TGF-b1) and cytokines like hepatocyte growth factor (HGF) in processus vaginalis closure. HGF receptor presence suggests its potential in initiate epithelial-mesenchymal transformation during inguinal hernia closure [14]. As a potent fibrogenic agent TGF-b1, induces fibrosis of the processus vaginalis. Mosavi et al. reported that the concentration of TGF-β1 was higher in the fluid of communicating hydroceles than in non-communicating hydroceles [15].

A study conducted by Yang et al., diaphragm dysfunction and skeletal muscle function in mechanically ventilated patients were examined, showing that IGF-1 preserves striated muscle function and is inversely related to apoptotic mechanisms [16]. Guan et al. found that IGF-1 signaling is involved in mitochondrial remodeling during myogenic differentiation and affects myogenic differentiation [17]. In this study, surface epithelium, muscle layers, and stromal components were evaluated, revealing increased staining in the muscle component of the hernia group. These findings were statistically significant. In the hydrocele group, no significant differences were found among epithelial, stromal, and muscle staining levels. This finding can be attributed to the physiological role of IGF-1 in smooth muscle homeostasis. IGF-1 is a potent mitogen and survival factor for smooth muscle cells, promoting protein synthesis and inhibiting apoptosis. The processus vaginalis contains a smooth muscle component within its wall, which is essential for its structural integrity prior to obliteration. The differential expression likely reflects the higher metabolic and proliferative demands of the smooth muscle layer compared to the mesothelial epithelial lining. This suggests that while IGF-1 is constitutively involved in the maintenance of the PV wall’s muscular component, its expression pattern across layers is conserved regardless of the clinical outcome (hernia vs. hydrocele) [18].

Numerous studies have investigated the effects of IGF-1 on vascular structures. Some of these studies have suggested strong relationships among FGFR1, IGF-1, IGF-1R, and WNT-1 in blood vessels, contributing to their pro-angiogenic effects on endothelial cells. In our study, IGF-1 expression in vascular structures was observed to be higher than in mesothelial cells, though this difference was not statistically significant. This partially aligns with previous studies. Kaulins et al., in their study on congenital diaphragmatic hernia, observed a relationship between IGF and angiogenic mechanisms, with minimal or no impact on surface epithelium. These findings correlate with the results of our study on hernia and hydrocele cases [19, 20].

Conclusion

IGF-1 expression was found to be similar in hernia and hydrocele cases, with negativity observed in epithelial cells and positivity observed in vascular and muscle tissues. Based on the differences in the mechanisms of formation, IGF-1 expression in older hernia cases was markedly lower than in pediatric hernia cases. Although not statistically significant, we believe there may be clinical associations between IGF-1 and PV closure defects. To strengthen this hypothesis, further studies with larger case groups and additional analyses would be beneficial.

Authors’ contributions

Conception: HE, OY, IEG, OO; Data curation: OY, IEG, DK; Fundings: OY, HE, OO; Materials: HE, IEG, DK; Design: OY, HE, KK; Validation: OY, KK, AA, IEG; Supervision: OY, IC, AA; Analysis: YK, IC; Literature Review: HE, OY, AA, IC; Writing – original draft: HE, OY, IEG; Writing – review & editing: AA, IC.

Funding

No specific grant was received from any funding agency in the public, commercial, or not-for-profit sectors.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

This study was approved by the Non-Interventional Ethics Committee of University (18/12/2024, GOKAEK 2024/23/2). As this was retrospective research, no informed consent was obtained from the participants.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.