Abstract: 【Abstract】 Objective To observe the in vivo microscopic histological changes during the treatment of melasma using reflectance confocal microscopy （RCM） combined with optical coherence tomography （OCT）, and to explore its application value in evaluating therapeutic efficacy. Methods Clinical data were collected from 30 melasma patients treated at the Department of Dermatology, Wuhan Hospital of Traditional Chinese and Western Medicine, Tongji Medical College, Huazhong University of Science and Technology, from January to October 2023. Retrospective analyses were performed on clinical photographs taken before treatment and at weeks 4, 8, 12, and 16 after the start of treatment, and the efficacy was evaluated by using the melasma area and severity index （MASI） scores. Meanwhile, changes in RCM imaging indicators, such as pigment distribution patterns, melanocyte morphology and spatial distribution patterns, and inflammatory infiltration in the superficial dermis in melasma lesions, were retrospectively analyzed. For clear elucidation of concepts and subsequent quantitative analysis, characteristic microscopic histological changes in melasma lesions before treatment were classified into several patterns based on RCM findings and previous literature reports. Additionally, optical coherence signals collected by OCT were used to asses characteristic vessel parameters in the lesions before and after treatment. Finally, correlations between various microscopic histological changes and clinical efficacy were analyzed using Spearman's correlation analysis. Results With the increase in treatment duration, the proportions of pigment distribution patterns Ⅱ and Ⅲ observed by RCM gradually decreased, while the proportion of the nearly normal pattern Ⅰ markedly increased; the proportions of the 4 heterogeneous morphological patterns of melanocytes all decreased, while the pattern Ⅱ melanocytes were still focally distributed in the lesions at the last time of observation; meanwhile, the proportions of melanocyte spatial distribution patterns Ⅱ and Ⅲ decreased, which gradually restored to the normal pattern Ⅰ; the infiltration of melanophages and inflammatory cells in the superficial dermis diminished, and the proportion of the pattern Ⅲ inflammatory infiltration markedly decreased at week 4, but it lasted for a long time. The decrease rate of MASI scores was significantly correlated with the decrease rate of the proportions of pigment distribution patterns Ⅱ and Ⅲ （r = 0.82, 0.97, respectively, both P < 0.05）, melanocyte morphological patterns Ⅰ, Ⅲ, and Ⅳ （r = 0.84, 0.83, 0.83, respectively, all P < 0.05）, and inflammatory infiltration patterns Ⅰ and Ⅲ （r = 0.85, 0.81, respectively, both P < 0.05）, as well as the increase rate of the proportion of melanocyte spatial distribution pattern Ⅰ （r = 0.89, P < 0.05）, indicating that these indicators can be sensitive evaluation indicators for clinical efficacy. Moreover, OCT revealed a decrease in the average diameter and density of vessels in the lesions after treatment, and the decrease rates were correlated with the decrease rate of MASI scores （r = 0.76, 0.78, respectively, both P < 0.05）; there was no significant difference between the pre- and post-treatment average depth of vascular plexuses. Conclusion During the treatment of melasma, RCM combined with OCT could serve as a non-invasive approach with objective and robust evidence for evaluating the therapeutic efficacy.

Key words: Chloasma, Microscopy, confocal, Optical coherence tomography, Histology, Therapeutic effect evaluation