Changes in advanced glycation content, structural and mechanical properties of vaginal tissue during pregnancy and in prolapse

Introduction: Pelvic organ prolapse is present in up to 50% of women. It is commoner in older women, often above 50 years of age and associated with hormonal e.g. oestrogen decline, and stiffer vaginal tissue. Pregnancy, on the contrary is a physiologic condition with higher oestrogen level and exhibits reversible structural and mechanical changes in pelvic tissues. Advanced glycation products, the ageing markers, stiffen connective tissues. There has been no previous systematic study on the relationship and action mechanisms of oestrogen, glycation level and mechanical property of vaginal tissues. Aim: The aim of this project was to study the ultrastructural and mechanical properties of vaginal tissues, and understand the relationship and possible mechanisms of accumulation of glycation (pentosidine), vaginal wall mechanics and oestrogen receptor (ER- expression in the vaginal tissues in both pregnancy and prolapse. It was hypothesised that prolapse is a disease of accelerated ageing and that mechanical and ultrastructural changes in prolapsed tissues and oestrogen decline are related to the elevation of glycation content in the tissues. Methods: Following ethical approval, vaginal tissues from 49 women with prolapse and 16 controls were obtained and proformas containing information on known and suspected prolapse associations were completed for each participant. Female Sprague Dawley rats‘ vaginal tissues were used for the pregnancy study. Nano-scale, micro-scale and tissue level mechanical characterizations of the tissues were performed using ball indentation technique, scanning electron microscopy, peakforce nanomechanical property mapping atomic force microscopy, and optical coherence elastography, which was applied for the first time to the study of vaginal tissues. The glycation contents of vaginal tissues in pregnancy and prolapse were quantified by high performance liquid chromatography and values obtained were analysed in comparison to medical comorbidities. Tissues were qualified by histological and immunological staining for structure (haematoxylin &eosin, trichrome and picrosirus red stainings), glyoxalase I, ER- elastin and neural stain. A sulphated glycosaminoglycan (sGAG)-collagen model was used to study the role of sGAG in collagen fibrillogenesis. Results: Rat vaginal tissues in pregnancy contained significantly lower amounts of pentosidine, higher oestrogen receptor- and glyoxalase I (antioxidant enzyme) expression with larger creep, lower elastic modulus, larger fibril diameter and higher sGAG content than their non-pregnant counterparts. Observed morphological changes of the collagen fibrils in pregnancy were attributed to sGAG, which was noted to influence collagen fibril aggregation and bundling. Skin pentosidine content was reflective of vaginal tissue pentosidine in the same subjects. Pentosidine was significantly higher in prolapsed tissues and increased with age; with more age-dependent increase observed in the prolapse population and also significantly different between the 6th and 7th decades. Glyoxalase I and ER- were poorly expressed in the prolapsed tissues in comparison to controls. Prolapsed tissues had notably disorganized ultrastructure and higher collagen fibril modulus. At all levels of tissue organization, prolapsed tissues were stiffer than controls, with increased stiffening at the more superficial layers of the tissue. Hypertension and smoking were associated with higher glycation and prolapse. In both pregnancy and prolapse, higher expression of glyoxalase I and ER- were associated with lower glycation content of the vaginal tissues and lower modulus. These observations have led to the suggestions that oestrogen plays an important role in increase or reduction of glycation through an oestrogen-gluthathione-glyoxalase (antioxdant) pathway, which directly affects vaginal tissues‘ mechanics. Future implications: These findings have implications to the current understanding of how prolapse may occur and can inspire future translational research on improved treatment of women with prolapse. Oestrogen may significantly influence the temporary and permanent mechanics of pelvic tissues such as the vaginal wall through its modulation of glycation accumulation within the tissues. Oestrogen thus shows promise of a potential future medical treatment for early stages of prolapse. The knowledge of new prolapse comorbidities can aid the early detection and possible prevention of prolapse through a high index of suspicion

Advanced glycation products' levels and mechanical properties of vaginal tissue in pregnancy

Objectives Non-enzymatic glycation is closely associated with altered mechanical properties of connective tissue. Pregnancy, marked with high levels of female hormones, confers unique alteration to the mechanical properties of pelvic connective tissues in order to meet their physiological demands. However, there are few studies on glycation content and its influence on the mechanical properties of pelvic connective tissues during pregnancy. We hypothesise that the glycation content in pelvic tissues will change with a corresponding alteration in their mechanical properties, and that these changes are influenced by hormone levels. This study aims to investigate the correlation of vaginal tissue glycation content and mechanical property changes during pregnancy in association with the expression of a key pregnancy hormone (oestrogen) receptor, and an antioxidant enzyme, glyoxalase I. Study design A rat vaginal tissue model (tissues from non-pregnant and E15-E18 (last trimester) pregnant rats) was used in this study. Mechanical characteristics of vaginal tissues were analysed by a ball-indentation technique while modulus and morphology of the collagen fibrils within the tissues were measured with atomic force microscopy. A glycation marker, pentosidine, was quantified by a high performance liquid chromatography. The expression of oestrogen receptor and glyoxalase I in the tissue was qualified by immunochemical staining. The glycosaminoglycan (GAG) concentration difference in the tissues were quantified by a biochemical assay. Results Pregnant rat vaginal tissue was characterised by significantly lower amounts of pentosidine, higher oestrogen receptor and glyoxalase I expression with larger creep, lower elastic modulus, larger fibril diameter and higher GAG content than their non-pregnant counterpart. There was a negative correlation between pentosidine and vaginal tissue creep. Conclusion There was a reduction in vaginal tissue pentosidine in pregnancy with an associated increase in oestrogen receptor and glyoxalase I immunoexpression. Reduced glycation was associated with increased creeping of vaginal tissue. Oestrogen may therefore play a role in the increase of the vaginal wall’s capacity to stretch through glyoxalase I up-regulation and subsequent glycation reduction. The new insight of the correlation of women’s oestrogen level, glycation reaction and pelvic tissue mechanical property from this study may enhance our understanding of some pelvic organ diseases

Staphylococcus aureus α-toxin impairs early neutrophil localization via electrogenic disruption of store-operated calcium entry

The pore-forming S. aureus α-toxin (Hla) contributes to virulence and disease pathogenesis. While high concentrations of toxin induce cell death, neutrophils exhibit relative resistance to lysis, suggesting that the action of Hla may not be solely conferred by lytic susceptibility. Using intravital microscopy, we observed that Hla disrupts neutrophil localization and clustering early in infection. Hla forms a narrow, ion-selective pore, suggesting that Hla may dysregulate calcium or other ions to impair neutrophil function. We found that sub-lytic Hla did not permit calcium influx but caused rapid membrane depolarization. Depolarization decreases the electrogenic driving force for calcium, and concordantly, Hla suppressed calcium signaling in vitro and in vivo and calcium-dependent leukotriene B4 (LTB4) production, a key mediator of neutrophil clustering. Thus, Hla disrupts the early patterning of the neutrophil response to infection, in part through direct impairment of neutrophil calcium signaling. This early mis-localization of neutrophils may contribute to establishment of infection