Vitamin D deficiency is largely spoken of as being associated with several different diseases. Most of the associations are inferred by the levels of 25(OH)D in the plasma, which has no direct correlation to the activity of its active metabolites – i.e. 1,25(OH)D – since it follows the patterns of a steroid hormone, with negative and positive feedbacks and other molecular interactions, instead of behaving as a vitamin. Pelvic organ prolapse has also been linked to lower serum levels of D vitamin, although the exact mechanism for this increase in risk is not totally understood. One of the potential mechanisms for this increased risk is the influence vitamin D has on the architecture of the extracellular matrix, main component of the structures of the pelvic support apparatus and responsible for its mechanical properties. The aim of this study was to examine the arrangement of collagen fibers, the most prevalent fibrous protein present in the extracellular matrix at the endopelvic fascia, to capture any different morphological pattern between subjects with a normal diet and those under a vitamin D supplementation.

This aim was investigated in female healthy adult Wistar rats (8 weeks old, approximately 200-220g). The treatment group received cholecalciferol diluted in arachid oil by gavage (in the dose 37.5mcg/Kg/day, equivalent to 1,500Ui/Kg/day), and the control group received only the oil, for 18 consecutive days. Twenty-four hours after the last gavage procedure, euthanasia was performed and a surgical procedure was undertaken extracting, in bloc, the lower third of the uterus with the uterosacral ligament, the upper half of the vagina with the connective tissue surrounding and the bladder with proximal urethra. Samples were immersed in Tissue-Tek and cut in 50 micrometer thick slices and placed on the sheets for microscopic evaluation with SHG (second harmonic generation) technique, which requires no tissue preparation. The samples on the sheet contained the cervix, the upper vagina, the base of the bladder and the uterosacral ligament, confirming the representative region with the tissue of interest, the endopelvic fascia (Fig. 1) . For the purpose of interpretation, the rats were also separated into groups according to the estrous phase: high estrogenic influence (proestrus and estrus) and low estrogenic influence (metaestrus and diestrus) identified by histology under conventional light microscopy with H&E (hematoxylin and eosin). Tissues were analyzed using TCS SP8 CARS Confocal Microscope (Leica) and F and Epi-SHG were used to detect SHG from collagen fibers. Images are represented as maximum intensity projections, corresponding to the Z-series of confocal stacks and were collected by tile scan and processed with Leica LAS AF software and manipulated with ImageJ software.

We found that collagen fibers have a consistently undulated pattern and more random disposition in the treated group whereas it assumes a more taut and oriented appearance in the control group when under high estrogenic influence according to the estrous cycle. The collagen quantification showed statistical significance with a higher ammount of the substance in the treated group compared to the control group also under a high estrogenic environment (Fig. 2).

These findings suggest that there is a difference in the morphological presentation of the extracellular matrix of subjects under high dose intake of vitamin D compared to subjects under normal intake of the vitamin, pointing to a probable change in the mechanical properties of the tissue and that there might be an interaction of the metabolic pathways of the two steroid hormones cholecalciferol and estrogen at least regarding the amount of collagen deposited in the tissue.

Oral high dose intake of vitamin D changes the framework and composition of the extracellular matrix at the level of the endopelvic fascia, which can impact the properties and resilience of the tissue. Our novel findings provide new exciting insights into the mechanical influence of the vitamin D in the pelvic tissues. This may explain the higher prevalence of pelvic floor disfunctions if similar results are shown in human, given that the structure of the tissue dictates its function. In summary, new research are needed in the biomechanical field to approach a clinical significance but we have shown a tangible relation between vitamin D exposure and collagen fiber architecture.

Moalli PA, Shand SH, Zyczynski HM, Gordy SC, Meyn LA. Remodeling of vaginal connective tissue in patients with prolapse. Obstet Gynecol. 2005;106(5 Pt 1):953-63.
Parker-Autry CY, Markland AD, Ballard AC, Downs-Gunn D, Richter HE. Vitamin D status in women with pelvic floor disorder symptoms. Int Urogynecol J. 2012;23(12):1699-705.
Mostaco-Guidolin L, Rosin NL, Hackett TL. Imaging Collagen in Scar Tissue: Developments in Second Harmonic Generation Microscopy for Biomedical Applications. Int J Mol Sci. 2017;18(8).