# Contrast-enhanced Micro-CT imaging of a foetal female pelvic floor reveals anatomical details

## TL;DR

This paper uses contrast-enhanced micro-CT imaging to reveal detailed anatomy of a fetal female pelvic floor, clarifying muscle structures and their connections.

## Contribution

The study introduces a non-destructive method for high-resolution imaging of long-fixed fetal tissue using Lugol staining and µCT.

## Key findings

- Lugol staining effectively enhanced contrast in long-fixed tissue without disadvantages compared to short-fixed tissue.
- Detailed 3D reconstructions revealed interconnections between pelvic floor muscles like the external anal sphincter and bulbospongiosus muscle.
- No skeletal muscle was found corresponding to the deep transverse perineal muscle; instead, smooth muscle fibers in connective tissue were observed.

## Abstract

The pelvic floor is a highly important structure for the stability of the pelvis, providing support for the organs that lie within it. Until today, the detailed anatomy of the female perineal centre and the exact course of surrounding muscles remain controversial. We demonstrate a method to non-destructively obtain high-resolution contrast-enhanced x-ray microtomography images from a long-fixed sample and thereby aim to contribute to the detailed anatomical knowledge about the female pelvic floor.

A human foetal pelvis of 20–21 weeks gestational age, formalin-fixed for 4 years, was immersed in Lugol solution and tomographically scanned periodically to document the staining process. The influence of the former fixation time was addressed by comparison with a short time fixed mouse pelvis. High-resolution imaging was performed using µCT, with detailed anatomical analysis supported by segmentation and 3D reconstruction.

Lugol staining of long-fixed tissue was effective and showed no disadvantages compared to short-fixed tissue. Lugol staining and high-resolution µCT images provided a nicely stained image-stack with clearly identifiable tissue types. The anatomy of the foetal pelvis and its structures were resolved in detail. Interconnections between the external anal sphincter, the bulbospongiosus muscle and the superficial transverse perineal muscle could be shown within the perineal centre. There was no evidence for a skeletal muscle that corresponded to the formerly described deep transverse perineal muscle, instead there was cloudy-looking tissue, most likely smooth muscle fibres dispersed in connective tissue in a 3-D arrangement.

X-ray microtomography of Lugol-stained tissue is an excellent method to gain anatomical details in high resolution, in a non-invasive and non-destructive way, independently of the fixation time. Using this method, the topographical relationships of the pelvic floor muscles could be illustrated, showing their linkage within the perineal centre.