BACKGROUND: Several disorders might require vaginal reconstruction, such as congenital abnormalities, injury, or cancer. Reconstructive techniques for which non-vaginal tissue is used can be associated with complications. We assessed the use of engineered vaginal organs in four patients with vaginal aplasia caused by Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS). METHODS: We invited to participate four consecutive patients who presented over a 3-year period with congenital vaginal aplasia due to MRKHS. Patients were aged 13-18 years. We obtained a vulvar biopsy of autologous tissue from every patient. We cultured, expanded, and seeded epithelial and muscle cells onto biodegradable scaffolds. The organs were constructed and allowed to mature in an incubator in a facility approved for human-tissue manufacturing. We used a perineal approach to surgically implant these organs. We recorded history, physical examination, vaginoscopy, serial tissue biopsies, MRIs, and self-administered Female Sexual Function Index questionnaire results for all patients, with a follow-up of up to 8 years. FINDINGS: We noted no long-term postoperative surgical complications. Yearly serial biopsies showed a tri-layered structure, consisting of an epithelial cell-lined lumen surrounded by matrix and muscle, with expected components of vaginal tissue present. Immunohistochemical analysis confirmed the presence of phenotypically normal smooth muscle and epithelia. The MRIs, which showed the extent of the vaginal aplasia before surgery, showed the engineered organs and the absence of abnormalities after surgery, which was confirmed with yearly vaginoscopy. A validated self-administered Female Sexual Function Index questionnaire showed variables in the normal range in all areas tested, such as desire, arousal, lubrication, orgasm, satisfaction, and painless intercourse. INTERPRETATION: Vaginal organs, engineered from the patient's own cells and implanted, showed normal structural and functional variables with a follow-up of up to 8 years. These technologies could be useful in patients requiring vaginal reconstruction. FUNDING: Wake Forest University and Hospital Infantil de México Federico Gómez.