Biological sex differences play an important role in the development and progression of disease but also in the response to drugs and treatment. Sex differences have been studied at length in diseases such as cardiovascular disease, musculoskeletal disease and neuronal disease [1]. The role of sex in lung diseases, and in particular, in interstitial lung diseases (ILD), is an expanding research area. Although, the complete mechanisms behind these sex differences are unknown, differences between men and women are known to be influenced by variances in male and female biology, epigenetic differences and sex hormones. Understanding these differences are important considerations for patients, pulmonary researchers and clinicians.

Lung development and maturation differ based on biological sex and the influence of sex hormones. Female foetal lungs mature faster than their male counterparts, evidenced by earlier production of surfactant [2]. Male neonates are therefore more susceptible to the development of respiratory distress syndrome (RDS) as it is largely due to surfactant deficiency [3]. The female lung is generally smaller than the male lung at birth with physical differences persisting through to adulthood. Dysanaptic or disproportionate growth of the lung parenchyma and the airways during maturation is observed in both sexes, however, faster large airway versus parenchyma growth is observed in young girls with the opposite effect present in young boys [4]. However, in general, adult males possess larger conducting airways regardless of lung or body size [5].

ILDs are a group of heterogeneous progressive pulmonary disorders that are characterised by tissue remodelling and/or fibrotic scarring of the lung parenchyma. ILD patients often experience lung function decline with advancing symptoms, poor response to treatment and reduced quality of life. ILDs can occur due to genetic, physical or environmental factors, but some are idiopathic. Many are associated with various systemic diseases, and are classified according to specific clinical, radiological and histopathological features. The most common form of ILD is IPF but there are a large number of ILDs that are associated with CTDs.

Pulmonary fibrosis is a dysregulated wound healing process in which injured epithelium is not adequately repaired. It is characterised by fibroblast accumulation and the excessive deposition of extracellular matrix (ECM), resulting in the destruction and remodelling of the normal lung architecture, leading to the loss of lung function and finally death. Although the aetiology of pulmonary fibrosis is still poorly understood, numerous risk factors and predisposing factors have been proposed. Pulmonary fibrosis is generally accepted to be triggered by repeated subclinical injury to endothelial and alveolar epithelial cells, caused by an interaction between genetic predisposition, aging and environmental agents such as cigarette smoking (CS), gastroesophageal reflux (GER), viruses and exposures to metal, wood and silica dusts, reviewed extensively in ^[6][7][8][6,7,8].

Endothelial injury results in the activation of platelets, coagulation pathways, fibrin-rich clot formation, vasculogenesis and angiogenesis. Alveolar epithelial cell injury and apoptosis progressively disrupt the lung matrix, interfering with the integrity of the basement membrane, resulting in the activation and accumulation of myofibroblasts. Chemokine gradients recruit inflammatory cells, resulting in the infiltration of neutrophils, eosinophils, lymphocytes, and macrophages. The inflammatory cells then secrete chemokines, cytokines and growth factors, such as IL-4, IL-13, and TGF-β. These cytokines display significant pro-fibrotic activity, and act by recruiting, activating and proliferating fibroblasts, macrophages and myofibroblasts, collagen synthesis/deposition. In addition to fibroblasts, fibrocytes, a group of circulating cells can also differentiate into myofibroblasts. These myofibroblasts are resistant to apoptotic signals and in combination with the impaired capacity for alveolar epithelial type 2 cells (AEC2) to renew, lead to excess ECM deposition and aberrant alveolar epithelium regeneration and re-epithelialization, preventing injury resolution, resulting in progressive pulmonary fibrosis (Reviewed in ^[6][8][9][6,8,9]).

In addition to these mechanisms, others such as reactivation of embryologic pathways, oxidative stress, endoplasmic reticulum stress and the unfolded protein response as well as sex hormones have been proposed ^[10][11][12][10,11,12] in pulmonary fibrosis pathogenesis.

Mortality rates for IPF are high and increasing according to data from both the United States (US) National Vital Statistics System and the Office of National Statistics in the United Kingdom (UK). Age-adjusted mortality rates for IPF have increased nearly 10% from 18.81/100,000 in 2000 to 20.66 in 2017 in the US and 1.66/100,000 in 1979 to 8.29 in 2016 in the UK with mortality rates higher in men and with increasing age in both cohorts ^[13][14][13,14].

Han et al. 2008 made some important observations namely that males with IPF have a more rapid deterioration of exertional desaturation over time than females; that survival is worse in males compared to females; and that females have a better survival rate after additional adjustment for relative change in exertional desaturation and percentage forced vital capacity (FVC) on spirometry [15]. These findings were in contrast to a lesser-powered retrospective analysis of IPF patients, where sex did not appear to be a significant predictor of adjusted survival [16].

IPF is among many fibrotic disorders in which researchers have found effects of sexual dimorphism in terms of prevalence and progression. Women are less likely to develop IPF even in the setting of genetic disease with the same mutations ^[17][55]. They also have a better survival rate when they do develop the disease ^[17][55]. Similar findings have also been found in other fibrotic associated disorders such as kidney and liver disease. Men with chronic kidney disease have been shown to progress more rapidly than women to end-stage kidney failure requiring dialysis ^[18][56] and end-stage liver disease or cirrhosis is more common in men than women ^[19][57].

The understanding of the molecular basis for the male predominance of IPF is currently lacking. However, it is thought that in contrast to the adverse effects of estrogens in chronic inflammatory lung diseases such as asthma and cystic fibrosis, estrogens may be protective against airway fibrosis ^[20][58], whilst androgens are detrimental ^[21][59]. Curiously, results from experimental fibrosis studies in vivo are species dependent. For example, male mice develop more severe bleomycin-induced pulmonary fibrosis than age-matched females ^[22][60]. In contrast, bleomycin instillation to rats is more severe for females versus males, alleviated by ovariectomy, and exacerbated by estradiol replacement ^[23][61].

A change in the ratio of estrogen receptor alpha (ERα):ERβ can alter cell function. Interestingly, Elliot et al., 2019 found that ERα expression was upregulated in male IPF lung tissue and fibroblasts at both the mRNA and protein levels, that IPF fibroblasts responded better to estrogen in comparison with controls, and that blocking ER lessened this effect ^[24][62]. They also showed in a mouse model of bleomycin-induced fibrosis that pharmacologic inhibition and mimicry of ERα and ERβ, respectively attenuate fibrosis. However, mice with a mutation in the AF2 estrogen ligand-binding domain of ERα still developed bleomycin-induced fibrosis, indicating that other ligands could be responsible for activation of ERα to mediate fibrosis.

In addition to the effects of sex hormones and their receptors, the loss of the transcriptional repressor ELK1 has been shown to enhance α5β6 integrin (ITGB6) gene expression and fibrosis; this integrin is dramatically increased in IPF. Interestingly, ELK1 is an X chromosome gene and therefore this may potentially contribute to the sex imbalance in IPF ^[25][63].

5. Gender Differences in Clinical Research

It is clear that at a biochemical and cell level, sex hormones and gender differences play a role in disease development. However, there are under recognised gender biases to consider that can affect diagnosis, research and treatment, and therefore affect clinical outcomes. Much of the analysis of the IPF epidemiological data in the literature are retrospective and therefore susceptible to bias including referral bias. While it is recognised that women tend to seek healthcare earlier than men, in general as a group they are less likely to be referred to sub-specialty services ^[26][127]. In addition, even when referred to a respiratory service there is evidence for bias when using sex to make the diagnosis of an ILD. Assayag et al. conducted their own analysis of data from a large online evaluation of ILD clinical cases by an international group of respiratory physicians. In the original study, physicians were asked to give up to five diagnoses along with diagnostic likelihood in order to measure their level of confidence in their ILD diagnosis ^[27][128]. Results showed that there was a bias when sex was used to diagnose ILD, which could result in men being over diagnosed and women being underdiagnosed with IPF. Therefore, the exact prevalence of IPF in men and women may be difficult to estimate and subject to multiple sources of bias. Documents, meta-analysis, reviews, articles not published in English, trial protocols, research on the same trial participants in separate publications and missing gender data were excluded. This revealed how the male: female ratio of trial participants compared in each respiratory condition of interest. CTD has an estimated gender bias ratio of 7:1 to 10:1 female: male ^[28][65], but for participants in clinical trials with a diagnosis of CTD associated ILD the male: female ratio was much higher. Across 19 trials that reported gender demographic data, there were a total of 1982 female participants and 697 male participants enrolled, resulting in a ratio of 2.84:1 female: male. Similarly, IPF is estimated to have a gender bias ratio of 1:1.45, female: male ^[29][17], but across 134 clinical trials in the past ten years the female: male ratio of participants was 1:2.82. These findings may be confounded by pathophysiological factors (where not all those with CTD develop ILD); factors in the trial design, referral or recruitment bias; and behavioural differences of those who are willing to participate in trials (volunteer bias). Moreover, the epidemiological prevalence may be erroneous due to similar biases. Further to this, many trials failed to report demographics on gender, despite evidence of gender playing a significant role in disease development.