Rectal prolapse (RP), the full thickness protrusion of the rectum through the anus, observed in both men and women, more prevalent among women in their 70s and often occurs with pelvic organ prolapse (POP). RP can also lead to fecal incontinence, compromising quality of life. Observational studies suggest an association between connective tissue disorders and RP, but direct analysis of connective tissue is underdeveloped. Prior studies implicate elastin homeostasis disruption in the development of RP but the studies were limited because i) the analysis was done on prolapsed skin rather than rectal tissue and ii) the percentage of elastic fibers was quantified but not morphology of elastic fibers. Understanding the morphology of elastic fibers in RP is necessary to assess the biomechanical aspects of the prolapsed rectum as well as to tailor patient specific treatments. 
Lysyl oxidase like 1 (LOXL1) is an elastin cross-linking enzyme important for elastin fiber repair after injury. Female LOXL1 knockout (KO) mice predictably develop POP and RP 1-4 months after delivery 1. The aim of this study was, for the first time, to investigate the effects of LOXL1 KO on RP prevalence and elastin morphometry in female multiparous mice. We hypothesized that the morphological parameters of elastic fibers are altered with LOXL1 KO, age and presence of RP or POP in female LOXL1 KO mice (Table 1).

LOXL1 KO female mice were backcrossed with male wild type (WT) mice (B6129SF1/J). The heterozygote female and male offspring were bred, resulting in WT, heterozygous, and KO mice. Genotype identification was done by PCR. Breeding pairs of genetically matched brother/sister WT and KO animals were established and allowed to breed ad libitum. The resultant animals were used in this study. To generate the multiparous females needed for this study, breeding pairs of genetically matched animals were established at 8–9 weeks old.
The experiment was divided into two parts:
Experiment 1. Prevalence and incidence of RP. The breeding colony of WT and LOXL1 KO mice was monitored weekly for RP stage according to a published mouse RP staging system (0, none; 1, RP present but mild; 2, RP present and severe including eversion of the anus and distal rectum)2. Mice with scores of 1 or 2 for 2 weeks in a row were considered to have RP. POP was also recorded weekly on a published scale of 0-4 where 0 is no prolapse and 4 is complete eversion of the vagina (2). Mice with scores of 2-4 for 2 weeks in a row were considered to have POP. Prolapse scoring was done every week until they were approximately 72 weeks old while breeding continued at will. Mice were euthanized at either 16 or 72 weeks old and the rectum and anus was harvested for histological assessment in Experiment 2. Fisher’s exact test for proportional differences was used to investigate differences in prevalence and incidence between LOXL1 KO and WT mice, and between female LOXL1 KO mice that had delivered or were nulliparous significant at P < 0.05.
Experiment 2. Elastin morphometry in RP.  Rectal tissues were harvested from female WT and LOXL1 KO mice at 16 and 72 weeks old as above. There were 6 animals in each sub-group listed in Table 1. Tissues were paraffin embedded, sectioned at 5 µm, and stained with a modified Hart stain (1 volume of Wiegert’s iron resorcin fuschin and 9 volumes of 1% hydrochloric acid in 70% ethanol) which stains elastic fibers in purple 3. The stained sections were scanned using a Leica whole slide scanner and morphometric analysis was performed on each section using Image Pro® software. Morphometric analysis was done according to a published method 3. In brief, the images were spatially calibrated and region of interest (ROI) was defined as the entire section. Pixels corresponding to elastin based on color of stain were selected and selection of elastin by color range and density was extended throughout the ROI. The selected elastin pixels were defined as objects and the following morphometric parameters were computed: angle, area, percent area, maximum diameter, minimum diameter, perimeter length and tortuosity.
A random effects mixed models was used to determine mouse group means and assess inter-group differences. Comparisons were made for differences in average values for area, aspect and perimeter length, totals for percent area, and measurement of variability for angle and minimum and maximum diameter (SAS v. 9.4). Custom contrasts were built for specific means comparisons between animal groups to test specific hypotheses (Table 1). The Bonferroni correction was applied to all multiple comparisons significant at P < 0.05.

Experiment 1.  RP prevalence in female LOXL1 KO mice was significantly increased vs female WT mice (p<0.0001). Prevalence of RP in nulliparous LOXL1 KO was significantly higher than in nulliparous WT mice (p=0.004). RP neither significantly preceded nor followed VP in female LOXL1 KO mice, although female mice that delivered pups developed both RP and VP earlier than nulliparous mice. No difference was seen in the incidence of RP between KO nulliparous female and WT female mice whereas the incidence of RP in 10-20 weeks old mice was significantly higher in female KO delivered mice than in WT female mice (p = 0.0003). Similarly, incidence of RP in KO female mice was significantly higher vs WT female mice of the same age group (p = 0.0015). 

Experiment 2. Elastic fibers are visibly disrupted with RP and LOXL1 KO (Figure 1). No significant difference was seen in the mean area, perimeter length and variability of maximum diameter and angle of elastin objects between the groups. Mean aspect ratio of elastin objects was significantly decreased in KO F72 VP RP vs all females. Total percent area of elastin was significantly increased in all KO female mice vs all WT female mice. Variability in minimum diameter was significantly higher in KO F72 VP RP vs KO F72 NVP NRP and WT female mice. Similarly, variability and average of tortuosity was significantly higher in all KO female vs all WT females, KO F72 VP RP vs KO F72 NVP NRP and KO F72 VP RP vs all females.

Increased prevalence of RP with LOXL1 deficiency even in nulliparous mice and increased incidence of RP in LOXL1 KO delivered mice indicates that impaired elastin fiber crosslinking and repair after injury contributes to RP. Smaller aspect ratio of elastin objects in KO F72 VP RP vs all other female mice indicates the presence of shorter fragmented elastic fibers. This is corroborated by total percent area, which was significantly increased in KO female mice vs WT female mice, suggesting presence of more elastin objects per ROI analyzed. Similarly, significantly higher variability of object minimum diameter with RP also suggests chronic elastolytic activity in the RP group leading to fiber formation.

RP in women could be a long-term consequence of chronic elastic fiber breakdown, reduced ability to repair these elastic fibers, and maintain healthy elastin homeostasis after delivery of children likely compounded by aging and other conditions that negatively impact elastin homeostasis like smoking, diabetes, and obesity. Future translational studies will seek to perform elastin morphometry in samples of women with RP. Determining the contribution of aberrant elastin homeostasis to RP could lead to development of novel therapies and personalized medicine approaches.

Lee UJ, Gustilo-Ashby AM, Daneshgari F, Kuang M, Vurbic D, Dan LL, Flask CA, Li T, Damaser MS. Lower urogenital tract anatomical and functional phenotype in lysyl oxidase like-1 knockout mice resembles female pelvic floor dysfunction in humans. Am J Physiol - Ren Physiol. 2008;295:545–555.
Rahn DD, Acevedo JF, Roshanravan S, Keller PW, Davis EC, Marmorstein LY, Word RA. Failure of pelvic organ support in mice deficient in fibulin-3. Am J Pathol. 2009;174:206–215.
Dahal S, Kuang M, Rietsch A, Butler RS, Ramamurthi A, Damaser MS. Quantitative Morphometry of Elastic Fibers in Pelvic Organ Prolapse. Ann Biomed Eng. 2021;