Bio-identical progesterone (PRG) is an exogenous female steroidal hormone which is used for treatment of polycystic ovary syndrome (PCOS). However, it suffers from poor bioavailability due to hepatic metabolism and poor solubility. The target of this work was to evaluate and statistically optimize PRG-loaded nanovesicle transethosomes (NVTEs) based in mucoadhesive gel for transvaginal delivery of PRG as potential luteal-phase support. A 2 full factorial design was used to explore the effect of phosphatidylcholine (PC), Tween 80, cetyltrimethyl ammonium bromide and ethanol concentration on particle size, entrapment efficiency (EE%), % in vitro PRG release after 24 h and transvaginal flux. PRG-loaded NVTEs were prepared by injection sonication method. The results revealed that the mean particle sizes ranged from 133.3 ± 3.42 to 349.5 ± 1.24 nm, zeta potential ranged from -23.5 ± 3.84 to +74.6 ± 4.97 mV, EE% ranged from 87.93 ± 3.58 to 97.05 ± 2.61%, % PRG release ranged from 50.9 ± 2.75 to 90.69 ± 2.07 and transvaginal flux ranged from 0.274 ± 0.03 to 0.531 ± 0.04 mg/cm/h. The optimized formulation was subjected to transmission electron microscope for morphological examination and then incorporated in the mucoadhesive vaginal gel using Carbopol 974, hydroxyl propyl methylcellulose and sodium alginate. The optimized formulation was clinically studied in anovulatory PCOS and showed a significant increase in the serum PRG, endometrial thickness, echogenicity degree and the pregnancy rate. Briefly, PRG-loaded NVTEs vaginal gel might be a promising formulation for luteal phase support and increase pregnancy rate in anovulatory PCOS.