Hypothesis / aims of study
Small animal models have been developed to study aging and ovarian failure to gain an understanding of how muscle composition, structure and function is altered in menopause. This systematic review aimed to determine 1) how aging and ovarian failure have been modeled in small animals to examine changes in the levator ani muscles (LAMs) and the external anal sphincter (EAS) and 2) how aging and ovarian failure in small animals impact the composition, structure and function of the LAMs and EAS.
Study design, materials and methods
A systematic review was conducted following Cochrane methods and the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guideline and was registered apriori with PROSPERO (ID: CRD42022350979). A search strategy was developed in consultation with a local research librarian. The search was conducted using four databases (i.e., MEDLINE, Embase, Web of Science, and Cochrane Central Register of Controlled Trials) from inception to August 31st, 2023.
Articles published in peer reviewed journals with full text available in English or French were considered eligible if they included female animals from the orders Rodentia (e.g., mice, rats, hamsters) or Lagomorpha (e.g., rabbits, hares) who were exposed to natural aging (minimum of 16 months for rodents and 24 months for lagomorphs) and/or ovarian failure through surgical (e.g., ovariectomy (OVX)) or chemical (e.g., 4-vinylcyclohexane treatment) methods. Studies were required to compare the exposed groups to younger and/or reproductively intact animals. Exclusion criteria included single-cohort study designs and the use of genetic modification (e.g., genetic knockouts). Acceptable outcome measures included any that assessed the composition, structure and/or function of the LAMs, the EAS, Articles that included outcomes associated with the urethral sphincters were also retained but are beyond the scope of the current review.
Two reviewers independently reviewed included studies and assessed their quality (via SYRCLE’s risk of bias (ROB) tool for animal studies [1]) and extracted data. Disagreements and inconsistencies were resolved by a third reviewer when required. Data were primarily summarized narratively, with heterogeneity discussed but not assessed statistically. A meta-analysis was not conducted due to the small number of included studies and their heterogeneity (design, sample, outcome measures).
Results
A total of 5661 unique abstracts underwent screening, out of which 221 proceeded to full text review. Among these, 12 articles were deemed suitable, describing outcome measures pertaining to the LAM and/or EAS (Figure 1). Eleven articles focused on the LAMs, the majority assessed the composition of the pubococcygeus portion, with two studies including the iliococcygeus portion and one study not specifying which portion of the LAMs were dissected. Three articles focused on the EAS.
In terms of models, most of the included articles used only OVX (n = 10), with one study focusing solely on aging and another using both OVX and aging. Rats (n =7) were the most common species followed by rabbits (n = 4) and mice (n = 1). Animal ages for OVX studies ranged from 6 weeks to 18 months. Aging studies had young animals at 2 or 3 months and older animals at 18 or 24 months. Only one OVX study used parous animals.
Table 1 provides a detailed list of the outcome measures used in the included studies as well as the reported effects of aging and/or OVX. In the LAMs, OVX appears to promote local estrogen production, estrogen receptor expression, and oxidative stress while impairing cell cycle arrest. Glucose uptake and glycogen storage are not affected by OVX. Additionally, OVX was associated with more isomyosin I protein content, larger myofiber cross-sectional area (pubococcygeus only, no difference with iliococcygeus), and an increase in the number of myonuclei, with no changes in gross-muscle morphology. Functional differences observed in the LAMs after OVX included a reduction in contractile tension under electrical stimulation and shifts in electromyographic power and frequency during urodynamic testing. The impact of aging on the LAMs was less clear than OVX, as only isomyosin I and the cyclin-dependent kinase inhibitor, p27kip1, were measured, with greater levels of both protein content and mRNA respectively observed in older animals compared to young.
OVX and aging were also associated with greater levels of isomyosin I and p27kip1 in the EAS, and aging was associated with an increase in myofiber cross-sectional area (CSA) and muscle CSA. No studies assessed EAS function.
The ROB was largely unclear due to a lack of descriptive information provided in articles regarding the study protocols. Attrition was the most frequently reported bias, with over half of articles acknowledging animal death or exclusion from the analyses.
Interpretation of results
The findings primarily highlight the impact of OVX on the composition, structure and function of the LAMs. Overall, study findings align with existing evidence and theories addressing the role of estrogens in other skeletal muscles. Estrogens impact oxidative stress and cellular proliferation, reflected by changes in malondialdehyde and p27kip1, a cyclin-dependent kinase inhibitor. The hypoestrogenic state triggered by OVX appears to lead to increases in local estrogen production (e.g., aromatase) and signaling sensitivity (e.g., upregulation of receptors), possibly as a compensation. Individual articles discussed these changes as being similar to observations in human studies on pelvic floor disorders.
Two studies that examined OVX in rats found it led to greater myofiber CSA in the pubococcygeus. Myofiber hypertrophy with OVX has been previously reported and attributed to an increased influence of testosterone in the absence of estrogens. However, this effect is not consistent across muscles or species. One study found no concurrent difference in the iliococcygeus between OVX and sham animal myofiber characteristics [2]. Further, two studies that examined the pubococcygeus in rabbits found no difference in myofiber CSA. The discrepancy between findings in rats and rabbits may be the result of underlying differences in response to OVX or heterogeneity between protocols.
The increase in isomyosin I observed in the LAMs in response to OVX is surprising. Aging is associated with an increase in the proportion of type I fibers in skeletal muscle due to preferential degeneration of type II fibers. However, the increase in isomyosin I in both the LAMs and EAS is counterintuitive, as reduction in estrogens should lower the preference for type I fibers. Indeed, increases in type I fiber content with OVX have been reported in other rat muscles, but there is a lack of consensus regarding the underlying mechanism for this phenomenon in rats.
Only one study examined the effects of aging and OVX together, finding that OVX in older animals led to more significant changes in both the LAMs and EAS [3] than OVX in younger animals. Importantly, none of the included studies evaluated the combined impact of age, parity and hormone status on LAM or EAS composition, structure and/or function. Future studies seeking to understand the impact of menopause on the pelvic floor muscles, particularly with the intent of assessing interventions for menopause-associated pelvic floor disorders, will benefit from detailed characterization of such combined effects.