Fig. 1: Schematic overview of the study design and neovaginal epithelium development.

a Multi-site longitudinal sampling in 39 MRKH syndrome patients subjected to laparoscopic peritoneal vaginoplasty. Samples from the vagina (if not otherwise indicated, MRKH patient’s vagina refers to the pre-surgery dimple and the post-surgery neovagina collectively for simplicity), stool, and tongue coating were collected 0–2 days before the surgery with no prior perturbation, and 14 days, 3 months, 6–12 months, and 2–4 years after the surgery, with time points denoted as PRE, P14D, P90D, P6/12 M, and P2/4Y, respectively. Peritoneal fluid (PF), abdominal skin samples, and control saline were collected during the laparoscopic Davydov procedure. Normal vaginal microbiota data of 472 healthy adult women were included as references for the neovaginal microbiota. b Colposcopic images of the pre-surgery dimple, neovaginal apex (P14D, P90D, P6/12 M, and P2/4Y) and healthy cervix. c Iodine staining of the pre-surgery dimple, neovaginal apex (P90D, P6/12 M, and P2/4Y) and healthy cervix. Cells of vaginal origin contain glycogen, which is stained brown or dark brown by iodine. Biopsies taken from the vaginas of three MRKH women (dimple, the upper third neovagina at P90D, P6/12 M, and P2/4Y), and the normal vagina of three control women (the upper third). After formalin fixation, paraffin embedding and sectioning (section thickness: ~3 µm), the upper, middle and lower sections of each biopsy were subjected to Hematoxylin and eosin (HE) staining (d) and PAS staining (e), and similar results were obtained. HE-stained vaginal biopsies (d) showing fibrous stroma lined with a stratified squamous epithelium in all samples. PAS-stained vaginal biopsies (e) showing glycogen-accumulated in a stratified squamous epithelium in all samples.