Vaginal birth increases the risk for developing pelvic organ prolapse (POP) among parous women and is the single most important modifiable risk factor for this condition (1).  The first vaginal birth has been shown to pose the greatest risk for POP.  This hidden epidemic indicates the urgent need to prioritize research focused on understanding mechanisms involved in pelvic floor injury and repair and to develop prevention strategies. Although we are now capable of identifying the presence and location of injury, factors involved in the repair of birth-related injury that will impact healing and eventual development of a pelvic floor disorder remain unknown.  

Pro-inflammatory cytokines such as interleukin 6 (IL-6) are known to be induced in response to skeletal muscle injury and are essential for modulating inflammation and healing following injury (2).  Altered expression of specific inflammatory cytokines could lead to altered inflammation and poor healing of pelvic floor muscle injury following vaginal delivery and to later development of pelvic floor dysfunction. The current lack of research using a human model to determine the specific markers to evaluate when investigating pelvic floor injury indicates the need for the development of an animal model.  The present study aimed to develop a mouse model to investigate the expression of inflammatory markers in mice with levator ani (LA) injury similar to that seen in vaginal delivery, and what role the pleiotropic cytokine IL-6 plays in this process.

IL-6 knockout (KO) and C57 wild type mice were used to develop a model for simulating birth injury to the LA. To simulate injury, a Word catheter was inserted into the rectum of the study mice and inflated with 0.3ml saline and affixed to a 100-gram weight hung to gravity. Injured LA muscles from these mice were harvested and evaluated histologically for injury and inflammatory response relative to mouse strain.  Expression of IL-1β, TNFα, IL-10, CCL2, CCL3, CCL4, CXCL1, CXCL2, and CXCL10 protein and mRNA was measured by multiplex protein assay and RT-PCR respectively.  

Non-parametric Mann-Whitney U exact tests were used to determine differences in cytokine expression.  The dependent variable in the model was the cytokine expression and the independent variable was mouse strain. An a priori significance level was set at an alpha 0.05 and cytokine expression between strains considered statistically significantly different when the calculated p-value was less than 0.05.

IL-6 KO mice had a greater inflammatory response following LA injury seen with histologic estimation of the number of inflammatory cells on post-injury day 5 than C57 mice (p=0.0351).  Additionally, the IL-6 KO strain displayed increased protein expression of IL-1β, CCL3, and CCL4 compared to C57 mice five days post-injury (p=0.0106, p=0.0182, and p=0.0364, respectively).

This study is the first attempt to characterize which, if any, inflammatory mediators may be important in the immune response to pelvic floor muscle injury (specifically, the levator ani muscle).  Using our model, we successfully demonstrated that, in a murine model simulating injury to the LA muscle that would be expected with childbirth, mice deficient IL-6 respond differently as compared to wild-type mice.  Specifically, the concentration of IL-1β, CCL3, and CCL4 in injured muscle tissue was significantly greater in IL-6 deficient mice than in the C57 strain as late as post-injury day 5 (Fig 1).  This would indicate a possible prolonged pro-inflammatory response in the IL-6 KO mice.  Furthermore, although not statistically significant, the IL-6 KO mice had a lower concentration of only one protein: CCL2.  This protein is known to be pro-healing (3), and thus its lower concentration raises the question of delayed anti-inflammatory/pro-healing actions in these mice (Graph 1).

IL-6 KO mice have decreased healing and increased inflammatory cell infiltration and pro-inflammatory cytokine expression following simulated birth injury to the LA muscle compared to C57 wild-type mice.

Patel DA, Xu X, Thomason AD, Ransom SB, Ivy JS, DeLancey JOCINAJOGM, et al. Childbirth and pelvic floor dysfunction: an epidemiologic approach to the assessment of prevention opportunities at delivery. American journal of obstetrics and gynecology.
Philippou A, Bogdanis G, Maridaki M, Halapas A, Sourla A, Koutsilieris M. Systemic cytokine response following exercise-induced muscle damage in humans. Clinical chemistry and laboratory medicine : CCLM / FESCC. 2009;47(6):777-82.
Warren GL, Hulderman T, Mishra D, Gao X, Millecchia L, O'Farrell L, et al. Chemokine receptor CCR2 involvement in skeletal muscle regeneration. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2005;19(3):413-5.