Significance Inguinal hernia is one of the most common disorders that affect elderly men. A major pathology underlying inguinal hernia is the fibrosis and other degenerative changes that affect the lower abdominal muscle strength adjacent to the inguinal canal. Here we describe a critical role of estrogen and its nuclear receptor that enhance fibroblast proliferation and muscle atrophy, leading to inguinal hernia. Further research may reveal a potential role of estrogen ablation to prevent muscle fibrosis or hernia in a subset of elderly men.

Abstract Inguinal hernia develops primarily in elderly men, and more than one in four men will undergo inguinal hernia repair during their lifetime. However, the underlying mechanisms behind hernia formation remain unknown. It is known that testosterone and estradiol can regulate skeletal muscle mass. We herein demonstrate that the conversion of testosterone to estradiol by the aromatase enzyme in lower abdominal muscle (LAM) tissue causes intense fibrosis, leading to muscle atrophy and inguinal hernia; an aromatase inhibitor entirely prevents this phenotype. LAM tissue is uniquely sensitive to estradiol because it expresses very high levels of estrogen receptor-α. Estradiol acts via estrogen receptor-α in LAM fibroblasts to activate pathways for proliferation and fibrosis that replaces atrophied myocytes, resulting in hernia formation. This is accompanied by decreased serum testosterone and decreased expression of the androgen receptor target genes in LAM tissue. These findings provide a mechanism for LAM tissue fibrosis and atrophy and suggest potential roles of future nonsurgical and preventive approaches in a subset of elderly men with a predisposition for hernia development.

Inguinal hernia is a common malady in elderly men, and hernia repair is the most commonly performed general surgical procedure in the United States. Although its pathogenesis is poorly understood, the lifetime risk of inguinal hernia is 27% in men and 3% in women (1). Approximately 800,000 inguinal hernia repairs are performed every year (1, 2), and annual health care costs directly attributable to inguinal hernia exceed $2.5 billion in the United States (3). Surgery is the only treatment option for an inguinal hernia. Unfortunately, complications, such as long-term postoperative pain, nerve injury, wound infection, and recurrence continue to challenge surgeons and patients (4⇓⇓–7). There are no actively utilized animal models for studying inguinal hernia or sex steroid-related muscle fibrosis and atrophy; furthermore, currently there are no experimental or approved medical options for the prevention of inguinal hernias in subsets of elderly men.

Inguinal hernias are termed “indirect” if the bowel is herniated via a defective inguinal ring or “direct” if the bowel protrudes through another weakened portion of lower abdominal muscle (LAM) wall (3, 5). The inguinal canal in male mice, which connects the abdominal cavity and the scrotum, is structurally similar to that in men and particularly vulnerable to indirect herniation of bowel (8). Therefore, experimental scrotal hernias in mice have similarities to indirect inguinal hernia in humans, which comprise two-thirds of all inguinal hernias in men (5). Previous studies in mice suggest that the development of scrotal hernias may be associated with abnormalities in the abdominal muscles, particularly those in the inguinal region (9, 10). In men, histological studies have identified myocyte (myofiber) atrophy, fibrosis, and fatty degeneration in the internal inguinal ring area from indirect hernia patients and in the abdominal wall surrounding a direct hernia border (11⇓–13). LAM tissue is composed of layers of oblique and transverse skeletal muscle made of myocytes. Stromal tissue, a mixture of well-organized fibroblasts and extracellular matrix (ECM), surrounds a single myocyte, fascicles (groups of myocytes), or the entire muscle tissue (groups of fascicles), and eventually becomes the deep fascia. An inguinal hernia occurs if both the muscle and adjacent fascia are weakened and can no longer support bowel in the abdomen.

Age is a significant risk factor for inguinal hernia formation in men with a striking increase in incidence after the age of 55 y. By the time they reach 75 y of age, nearly 50% of men will develop inguinal hernia (14, 15). Indirect inguinal hernia, which comprises 70% of all hernias, peaks between the ages of 70 and 79 y in men (16). It is a disease of primarily elderly men. The increased risk of inguinal hernia in elderly men may be related to age-linked skeletal muscle atrophy associated with fibrosis in the inguinal area. However, the mechanisms mediating skeletal muscle atrophy and fibrosis that increases the risk of hernia formation are not well understood.

Sex steroid hormones change as men age. Age-related changes in serum estradiol (E 2 ) levels in men are conflicting, as some studies have reported increases but others have noted unchanged or even decreased E 2 levels with advancing age (17⇓⇓⇓⇓⇓⇓–24). Conversion of circulating testosterone (T) to E 2 via aromatase expression in bulky tissues—namely, skeletal muscle and adipose tissue—produces the majority of estrogen in men (25, 26). Human muscle and adipose tissue aromatase expression or activity has been found to increase with advancing age, which coincides with the incidence of inguinal hernia (25, 27⇓–29). In the 1930s, two separate laboratories reported that ∼40% of male mice that received postnatal estrogen injections or ovarian grafts developed scrotal hernias (8, 30). Estrogen injections initiated as early as postnatal day 28 or as late as 30 wk of age led to the development of scrotal hernia within a few weeks (8). These data were suggestive of a direct link between local tissue estrogen and acquired inguinal hernias and do not support the possible contribution of a congenital defect. The underlying cellular and molecular mechanisms, however, remain unknown. Conversely, serum T levels decrease by 20% by age 50 and by 50% by age 80 y in association with decreased skeletal muscle mass in men (31⇓–33). A higher ratio of E 2 to T was observed in elderly men compared with younger men (31). It remains unclear whether the age-related shift in the estrogen to T ratio in the LAM tissue causes muscle fibrosis and atrophy and predisposes a subset of older men to develop inguinal hernia.

Aromatase is the only enzyme that catalyzes the conversion of T to E 2 . The tissue distribution patterns of aromatase expression in humans and mice are markedly distinct. In male mice, aromatase is expressed only in the testes, gonadal fat, and brain via 3 promoters, whereas humans use 10 distinct promoters to express aromatase in many peripheral tissues, including skeletal muscle. We recently generated mice expressing aromatase from the human promoter (Aromhum) to mimic human physiology with respect to aromatase expression and estrogen production (34). Aromhum mice physiologically express the human aromatase gene in many peripheral tissues, including skeletal muscle (25, 35, 36). Intriguingly, male Aromhum mice exhibit increased estrogen levels in peripheral LAM tissues, low serum T levels, and scrotal hernia formation, mimicking what has been observed in a subset of older men. Thus, we used this mouse model to test the hypothesis that alterations in E 2 , T, and their nuclear receptors, estrogen receptor-α (ERα) and androgen receptor (AR), in LAM tissues lead to fibrosis, skeletal muscle atrophy, and the development of scrotal hernias. We also investigated some of the underlying cellular and molecular mechanisms behind hernia formation.

Discussion Aromhum mice represent a unique and pathologically relevant experimental model to study the relationship between aromatization of androgen to estrogen, the downstream estrogenic and androgenic effects in muscle tissue, and LAM fibrosis and atrophy, leading to hernia development. Human aromatase expression driven by its alternatively used cognate promoters (I.4 and to a lesser extent PII) in Aromhum mice resembled the human patterns of age-related increase in aromatase expression and estrogen formation in peripheral tissues, including LAM tissue, and an accompanying decrease in circulating T levels (25). Our data strongly indicate that locally produced E 2 acting on highly estrogen-sensitive and ERα-rich LAM fibroblasts led to stromal fibrosis, myocyte atrophy, and eventually inguinal hernia formation. The role of E 2 in this phenomenon is clear because the inhibition of E 2 production by an aromatase inhibitor prevented hernia formation. The possibly contributing roles of decreased T levels that are observed in Aromhum mice and restored to normal with an aromatase inhibitor, however, are less clear. Decreased T levels are possibly due to increased brain E 2 levels via the brain expression of human aromatase, leading to decreased gonadotropin and then testicular T secretion. As the incidence of inguinal hernia and peripheral aromatase expression increase with age in men, our findings have a particular clinical significance not only for understanding the mechanisms underlying maintenance of skeletal muscle mass in various body sites, but also for assessing hernia risk and developing strategies for hernia prevention in a subset of elderly men (27⇓–29). Previous studies showed that postnatal systemic administration of exogenous estrogen or ubiquitous overexpression of a full-length aromatase cDNA with strikingly high circulating estrogen levels led to the formation of scrotal hernias in mice; in utero estrogen exposure was not necessary for this phenotype (9, 54). Our humanized aromatase mouse model is unique in that the estrogenic effect on LAM tissue and hernia formation are primarily mediated via local estrogen production by aromatase activity from the human CYP19A1 (aromatase) gene expressed in the skeletal muscle tissue, with normal circulating E 2 levels. This model of muscle atrophy and hernia development is therefore more physiologically relevant to these common pathologies observed in a subset of elderly men. Estrogen exerts its physiological functions by binding to its receptors ERα, ERβ, and GPR30 (39⇓–41). Studies of ER knockout mice show that ERα is primarily involved in the classic actions of estrogens (i.e., sexual differentiation, fertility, uterine function, and lactation) (39, 40). ERβ has been shown to play biological roles in the central nervous system, the immune system, the ovary, and the prostate (55, 56). GPR30, on the other hand, has been linked to certain physiological and pathological effects regulated by estrogen on the central nervous, immune, renal, reproductive, and cardiovascular systems (41, 57⇓–59). Because ERβ and Gpr30 mRNA levels in LAM or UAM tissues are either barely detectable or extremely lower than ERα in our hands, the estrogenic effects on LAM fibrosis and hernia are most likely mediated primarily by ERα signaling. Indeed, we found that ERα mRNA and protein were predominantly present in the prominent perimuscular stromal fibroblast compartment of LAM tissue in both WT and Aromhum mice. Additionally, ERα levels in LAM tissue fibroblasts were higher than those in UAM or QM tissues. Moreover, ICI 182780 (which opposes E 2 action via degradation of the ER), MPP (which is an ERα-selective E 2 antagonist), or ERα knockdown diminished estrogenic gene expression in LAM fibroblasts. Thus, we conclude that locally produced estrogen in Aromhum LAM tissue is mediated via fibroblastic ERα, giving rise to increased stromal cell proliferation, fibrosis, muscular atrophy, and hernia development. This explains, at least in part, why atrophy and fibrosis develop in LAM tissue of Aromhum mice, but not in other muscle groups (i.e., UAM and QM). Gene-expression profiling allowed us to identify a number of molecular pathways and target genes activated early by E 2 /ERα in LAM tissues before the hernias become manifest. These pathways include ERα-driven fibroblast activation and fibrosis pathways (44⇓–46, 60, 61). Consistent with these findings, E 2 is found to induce fibrosis in pathologic tissues, including gynecomastia, uterine fibroid tumors, and the skin of patients with systemic sclerosis (62⇓–64). Several known E 2 /ERα target genes were highly expressed in LAM tissue of Aromhum mice. Moreover, some of these genes, (e.g., Greb1 and Pgr) were selectively induced in primary fibroblasts of LAM tissue, suggesting that these effects took place primarily in the fibroblast compartment of LAM tissue. Greb1 is a chromatin-bound ER coactivator and is essential for ER-mediated transcription (61). Greb1 is also one of the most highly estrogen-inducible genes and correlates well with changes in ER activity following breast cancer treatment (43, 65). Thus, Greb1 may also contribute to fibroblast proliferation in LAM. Moreover, the estrogen-responsive fibrotic genes, including Kiss1, Krt8, Krt7, Spon2, Krt18, Tnc, Plod2, and Eln, were also increased in LAM of Aromhum mice (Table 1), as has been reported for several other tissues (66⇓⇓⇓⇓⇓–72). On the other hand, several other well-known estrogen response genes (e.g., Adora1, Tff1, and Susd3) were not induced by aromatase expression in LAM tissue (44, 45, 73), suggesting epigenetic differences between tissues or cell types may account for E 2 /ERα induction of a select group of genes. T is the major substrate of the aromatase enzyme. T is not only converted by aromatase to E 2 in target tissues for estrogenic action, but is also converted by 5α-reductatase-1 or -2 into a potent and nonaromatizable androgen, DHT (74). mRNA levels of 5α-reductase-1 or -2 were undetectable or barely detectable in LAM tissue. Thus, in LAM, androgen action must be provided primarily via an interaction of T with AR. In fact, in double 5α-reductase knockout mice (for both types 1 and 2), T was shown to exert androgenic action via AR (74). T and AR exert anabolic effects on skeletal muscle, resulting in muscle protein synthesis and increased muscle mass (75, 76). Overall, AR-FL seems to be the predominant AR type in both LAM and UAM tissue, suggesting a more important role of AR-FL in both WT and Aromhum LAM tissues. The precise role of AR45 in abdominal muscle tissue, however, needs further investigation. We also found that serum T was significantly lower in Aromhum mice, which may contribute to decreased androgen action. These results indicate that low circulating T levels, together with local estrogen excess, shift the steroid balance from androgen to estrogen in Aromhum mice or in a subset of elderly men, leading to muscle atrophy and hernia, possibly via decreased muscle mass. In summary, the lower portion of the LAM tissue adjacent to the scrotal opening is particularly enriched with ERα-expressing fibroblasts and thus sensitive to E 2 . Expression of aromatase in mice, which directly converts circulating T to E 2 locally in the muscle or in the brain, leads to stromal proliferation, muscle atrophy, and hernia development in LAM tissue via up-regulation of estrogenic action and down-regulation of androgenic action (Fig. 8). All Aromhum mice uniformly developed fibrosis, myocyte atrophy, and hernia, which was entirely blocked and prevented by an aromatase inhibitor. There are currently no experimental or approved medical options for the prevention of inguinal hernias associated with skeletal muscle fibrosis and atrophy in a subset of men. In particular, the surgical repair of a recurrent hernia is quite problematic and carries a high risk of treatment failure or recurrence. Intriguingly, our findings uncover a previously unrecognized mechanism for LAM fibrosis and atrophy and inguinal hernia formation and open new horizons for drug development to prevent hernia, especially recurrent hernia after surgery in vulnerable populations, such as elderly men. Currently available aromatase inhibitors or analogs of androgen may provide alternative or complementary management modalities in addition to surgical repair. Fig. 8. Schematic demonstrating the effect of a shift from androgen to estrogen action induced by human aromatase gene expression in LAM tissue on fibrosis, myocyte atrophy, and hernia formation in mice.

Materials and Methods Aromhum Mouse Maintenance, Hernia Assessment, and Letrozole Treatment. The Aromhum mouse (FVB/N background) was generated and genotyped in our laboratory, as previously described (34). Aromhum transgenic mice contain the complete human aromatase coding region, a >75-kb promoter region including promoters I.4, I.7, I.f, I.6, I.3 and PII, and the 3′-polyadenylation site. Mice were maintained on a 14-h light:10-h dark cycle with standard chow (7912; Harlan Teklad) and water available ad libitum. All animal experiments were approved by and conducted in accordance with guidelines established by the Institutional Animal Care and Use Committee at Northwestern University. Animals were randomly used for all experiments in a blinded manner. Hernia development in 32 Aromhum male mice was monitored by weekly visual inspection and palpation from 3 to 26 wk of age. Age-matched WT male littermates were used as controls. Hernia dimensions were measured using a digital caliper, and hernia area was calculated by the formula, area (mm2) = length (mm) × width (mm). At the designated endpoints, UAM, LAM, and QM from each individual mouse were resected, with one-half of the tissue snap-frozen in liquid nitrogen, and the other half fixed in 4% phosphate-buffered paraformaldehyde for histological and IHC analyses. All tissue and serum samples were collected from mice between 10:00 AM and 12:00 PM (noon) to avoid possible variability in daily hormone fluctuations. In some experiments, male mice were randomly treated with letrozole (10 µg/d per mouse) using 90-d continuous-release pellets (Innovative Research of America) or control pellets starting at 3 wk of age (77). Pellets were implanted subcutaneously on the lateral side of the neck between the ear and the shoulder. Human Subjects. We recruited six hernia-free men (aged 50–68 y) and six men with hernias (aged 60–77 y) from the First Affiliated Hospital of Nanjing Medical University in China. Men were excluded if they had chronic debilitating disease, had undergone chemotherapy or radiation therapy, or had received any hormonal treatments within the past 3 mo. Muscle biopsy specimens from the inguinal area were obtained and preserved in 10% formalin and embedded in paraffin. The human study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Nanjing Medical University and complied strictly with the national ethical guidelines of China. Written informed consent was obtained from all participants before inclusion in the study. All participants were identified by assigned numbers. Primary Mouse Skeletal Muscle Tissue Fibroblast Culture, Hormonal Treatments, and Small-Interfering RNA Knockdown. Isolation and culture of primary skeletal muscle tissue fibroblasts were performed based on a modified version of an established protocol for culturing human or mouse adipose fibroblasts and mouse skeletal myoblasts (78⇓–80). In brief, UAM and LAM tissues from six WT and six Aromhum mice were minced and digested with collagenase D (1.5 U/mL), dispase II (2.4 U/mL), and CaCl 2 (2.5 mM) at 37 °C for 30–60 min. Single-cell suspensions were prepared by filtration through a 75-μm sieve. Fibroblasts in cell suspensions were allowed to attach collagen-coated Petri dishes for 15 min and unattached myoblasts were removed. Primary fibroblasts were obtained by growing cells in DMEM/F-12 medium with 10% FBS. Under such conditions, there was preferential growth of fibroblastic cells which comprised 99% of the total population within 2 wk (80). Cells were grown to 80% confluence and placed in serum-free and phenol-free medium for 16 h before treatment. Primary fibroblasts were incubated in serum-free and phenol-free DMEM/F-12 in the absence or presence of physiological doses of E 2 (0.1 nM, 1 nM, and 10 nM; Sigma-Aldrich) for 24–48 h. Cells were pretreated with ICI 182780 (100 nM) or MPP (10 µM) for 2 h before the addition of E 2 . Cell extracts were prepared for real-time RT-PCR analysis or immunoblotting. To knock down endogenous ERα expression, primary fibroblasts were transfected with two separate ON-TARGETplus mouse ERα siRNAs (Dharmacon) using DharmaFECT 1 transfection reagent (Dharmacon) for 48 h. ON-TARGETplus nontargeting control siRNA (Dharmacon) was transfected as a negative control. Data Availability. The RNA microarray data that support the findings of this study have been deposited to the National Center for Biotechnology Information Gene Expression Omnibus database under the accession no. GSE92748 (https://www.ncbi.nlm.nih.gov/geo/). Statistical Analysis. Results are expressed as mean ± SEM, unless otherwise indicated. Statistically significant differences at P < 0.05 were determined using two-tailed Student’s t test, one-way ANOVA, or two-way ANOVA. All statistical tests were performed using the GraphPad Prism software. Extended method and information about RNA isolation and quantitative real-time PCR, exon-specific RT-PCR amplification, histology, Masson’s trichrome staining, IHC, scoring of immunoreactivity, protein extraction and immunoblotting, serum and tissue hormone levels, and microarrays, and data analyses are described in SI Appendix, Materials and Methods.

Acknowledgments We thank Dr. Elizabeth M. McNally, Dr. Pin Yin, Dr. Alexis R. Demonbreun, Dr. Matthew J. Schipma, and Dr. Matthew T. Dyson at Northwestern University for all their help and insight; the Ligand Assay & Analysis Core at the University of Virginia Center for Research in Reproduction for measuring serum sex steroid hormones and gonadotrophins; the Mouse Histology & Phenotyping Laboratory and the Pathology Core Facility Laboratories at Northwestern University for performing immunohistochemistry; and Dean Evans from Novartis for providing the aromatase inhibitor letrozole. This work was supported by the NIH Grant R37-HD36891 (to S.E.B.).

Footnotes Author contributions: H.Z., F.J.D., W.G.T., and S.E.B. designed research; H.Z., L.Z., L. Li, J.C., R.T.C., D.C.B., E.J., and X.X. performed research; H.Z. and L. Li contributed new reagents/analytic tools; L. Liu collected the human muscle samples; H.Z., L.Z., L. Liu, Z.D., J.J.S., and S.E.B. analyzed data; and H.Z. and S.E.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo/ (accession no. GSE92748).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1807765115/-/DCSupplemental.