Microneedling with glutathione versus microneedling alone in treatment of facial melasma: Split‐face comparative study

Melasma is a distressing disfiguring acquired pigmentary disorder especially affecting females and it is of high tendency of recurrence. Up till now, treatment of melasma is a challenging problem.

Patients and Methods: In this study, 29 adult females with epidermal type of melasma (confirmed with Wood's light examination) were enrolled.The affected area was subjected to microneedling using dermapen followed by application of glutathione solution on the right side only.This session was performed every 2 weeks for 3 months (six sessions for every patient).The response to therapy was measured using modified melasma area and severity index (m MASI) that were calculated on each side of the face (Hemi-m MASI) before treatment sessions.
Results: There was statistically significant reduction in the mean of Hemi-m MASI score over the sessions on both sides of the face but the right side (microneedling with glutathione) showed more reduction and earlier response to therapy than the left side (microneedling alone).On the left side, mean of Hemi-m MASI score before and after sessions was (4.06 ± 1.91, 2.31 ± 1.450) and on the right side, it was (4.21 ± 2.08, 1.96 ± 1.30), respectively and this was statistically significant.Percentage of improvement on the left side was 46.92 ± 16.30 (%) while on the right side was 55.17 ± 15.50 (%) and this was statistically significant.

Conclusions:
Microneedling is an effective promising tool in treatment of melasma and its combination with glutathione as a whitening agent, increases and accelerates its efficacy.So, combined therapy is more preferred than monotherapy in treatment of facial melasma.

K E Y W O R D S
glutathione, melasma, microneedling

| INTRODUC TI ON
People with darker skin color are more susceptible to melasma, a common pigmentary disorder.Depending on the ethnicity, the prevalence ranges between 8.8% and 40%. 1 Melasma mostly affects females throughout the reproductive cycle.It is more common in Asians and those of Hispanic descent, as well as in patients with Fitzpatrick skin Type III-V. 2 There are several pathogenic mechanisms for melasma including increased activity of melanocytes, melanin and melanosomes deposition in epidermis and dermis, solar elastosis, increased mast cells, basement membrane disruption, and increased vascularization. 3Melasma classified clinically into; Centrofacial, malar, and mandibular types and also, can be classified by wood's lamp into epidermal, dermal, and mixed forms. 4 treatment of melasma, there are already a variety of topical and systemic drugs, chemical peels, lasers, and light-based therapies that have all been attempted in various studies with diverse results. 5croneedling is a minimally invasive technique that was reported to improve melasma.Possible mechanisms include stimulating fibroblasts proliferation, enhancing collagen formation, and increasing delivery of topically applied drugs. 6Glutathione is an anti-oxidant tri-peptide, which is normally present in our bodies and has been reported as an effective treatment modality for melasma.It reduces tyrosinase activity, shifts melanogenesis from eumelanin to phaeomelanin synthesis, and acts as scavenger of UV radiation-induced reactive oxygen species. 7Few number of studies evaluated the effectiveness of each microneedling and glutathione separately in treatment of melasma.So, In this study, we aimed to evaluate the efficacy of both microneedling with glutathione versus microneedling alone to treat melasma.

| Study Design
This clinical trial was carried out at the departments of Dermatology, Venereology, and Andrology, Sohag University Hospital, Egypt.This study was conducted between June 2020 and September 2021.

The study was approved by the Institutional Ethics and Research
Committee of the Faculty of Medicine, Sohag University, Sohag, Egypt.After being informed of the study's methods, anticipated results and potential side effects, the patients who agreed to participate in the study signed a consent form.

| Patients
Females in the reproductive age (18 years old and above) presented with bilateral symmetrical epidermal melasma confirmed by wood's light were included in this study.Exclusion criteria were female with age under 18 years old, the dermal and mixed types of melasma, those taking oral or topical depigmenting medications within the previous 3 months, pregnant or nursing women, those using oral contraceptives, those with a history of hypertrophic scars or keloids, those with a history of recurrent herpes infection or current cutaneous infection, blood disorders, chronic liver disease, and those taking systemic chemotherapy, anticoagulant therapy or antiplatelet agents.A complete medical history including each patient's (age, sex, occupation, marital status, place of residence), family history, past medical history, dermatological examination (including the Fitzpatric skin phototype and the clinical pattern of melasma), and Wood's light examination to determine type of melasma were all conducted.

| Procedure
The patient face was disinfected using alcohol then application of topical anesthesia was done for 30-45 min (Pridocaine cream, contains Lidocaine 2.5% and Prilocaine 2.5%, Global Napi Pharmaceuticals, Egypt).All the affected area was subjected to microneedling then application of glutathione on the right side only.
This session was performed every 2 weeks for 3 months(six sessions for every patient).Microneedling was done using dermapen (My M pen) with 36 needles tip and needle length was adjusted according to the treated site (0.5-2 mm).It was done on the affected area until appearance of erythema or pinpoint bleeding followed by application of glutathione solution on the right side only.Glutathione will be used in solution form with concentration 2400 mg (Global cosmetics Pharma).

| Post-procedure care
The patients requested that the following should be avoided: washing the face for 24 h following the procedure and being in the sun for at least 7 days.When going outside during the day, all patients were advised to use sunscreen with an SPF of at least 50 and to refrain from using any additional melasma treatments while undergoing our therapy.Patients were warned that they should anticipate some erythema, swelling, and bruising at the treatment locations that could remain for 2-3 days.

| Evaluation of the efficacy of treatment
Evaluation of the efficacy of treatment was done using Hemi-m MASI which calculated on each side of the face at the baseline and then before each session (every 2 weeks) for six sessions (total 3 months) and 3 months after stoppage of treatment.The m MASI calculations are based on assessment of two factors: darkness(D) and area of involvement (A) that divide the face into four areas forehead, right malar, left malar, and chin. 8In this study, the severity of melasma on each side of the face (right and left) was evaluated According to the calculated percent, the response to treatment was graded based on the judgment of two expert dermatologists as follows, no response (no improvement), mild response (<25% improvement), moderate response (25%-49% improvement), good response (50%-74% improvement), and very good response (>75% improvement). 9Patient satisfaction was assessed using the short assessment of patient satisfaction (SAPS) which is a short, reliable, and valid seven item scale that can be used to assess patient satisfaction with their treatment. 10Score of SAPS can be interpreted as follows: 0-10 = Very dissatisfied, 11-18 = Dissatisfied, 19-26 = Satisfied, and 27-28 = Very satisfied. 10Patients were asked to report any adverse effects such as intolerable pain, marked erythema, burning, swelling, itching, pigmentation change (hyperpigmentation and hypopigmentation), purpura, textural alteration to detect any side effect from the procedure applied.On the left side (microneedling alone) there was statistically significant reduction in the mean of Hemi-m MASI score after the third session and continued significant reduction over sessions till the last session.While, on the right side (microneedling with glutathione) there was statistically significant decrease in Hemi-mMASI score which began earlier than the left side (immediately after the first session) and significant reduction continued over the sessions.This was demonstrated in Table 3, Figure 1.

| Hemi modified MASI score changes before and after sessions
On the left side, mean of Hemi-m MASI score before and after sessions was (4.06 ± 1.91, 2.31 ± 1.450) and on the right side, it was (4.21 ± 2.08, 1.96 ± 1.30), respectively and this was statistically significant.Table 4.

| Percentage of clinical improvement of left and right sides of the face of the studied group
The percentage of improvement on the left side was 46.92 ± 16.30 (%) while on the right side was 55.17 ± 15.50 (%) and this was statistically significant.This was shown in Table 5.

| Response to treatment in left and right sides of the face of studied group.
On the left side response to treatment was moderate to good response while on the right side it was mainly good response (68.97%).
This was shown in

| Patient satisfaction according to short assessment of patient satisfaction.
A total of 18 out of 29 patients with melasma (62.07%) were satisfied.This was shown in Table 7.

| Side effects
No serious side effects reported, only slight erythema, edema, and dryness which subsided spontaneously within 2-3 days after the procedure.

| Recurrence
Patients attended our clinic for follow-up (3 months after the last session).Recurrence was recorded in 14 (48.28%) of these 29 patients (recurrence was the same on both treated sides).However, these patients did not follow instructions about protection themselves from sun exposure and use of appropriate sunscreen.

| DISCUSS ION
Melasma is a common pigmentary condition.Patients who have melasma experience severe emotional and psychosocial discomfort.
There are numerous modalities to treat melasma.This current study aimed to evaluate the efficacy of microneedling with glutathione versus microneedling alone in treatment of melasma.In this splitface study, it was demonstrated that there was statistically significant reduction in the mean Hemi-m MASI score over the sessions on both sides of the face but the right side (microneedling with glutathione) showed more reduction and earlier response to therapy (appeared after first session immediately) than the left side (microneedling alone) which showed statistically significant decrease in the mean Hemi-m MASI score after the third session.The percentage of clinical improvement of melasma was more on the right side of the face than the left one.
In the present study, combined therapy of both microneedling and glutathione was used on the right side and this resulted in more significant reduction of the mean Hemi-m MASI score and more percent of improvement than the left one (microneedling alone).
In agreement with our results,  which was applied to the left half of the face and they found that both mixures had significant effectiveness in reduction of modified MASI score in each side but significantly more reduction of m MASI score with mixure A (containing glutathione) than mixure B (without glutathione). 11Also, Puri (2020) who examined the efficacy of mesotherapy using glutathione and vitamin C for the treatment of melasma in 40 patients showed reduction in MASI score. 12The role of glutathione as a skin-bleaching molecule is carried out through the inhibition of the enzyme tyrosinase during the melanogenesis process, either by directly binding to the copper-containing active site in the enzyme or by indirectly eliminating peroxides and other free radicals. 13Limited number of studies on the use of glutathione as a lightening agent are available, and there is a high need for more studies to evaluate the effectiveness of glutathione in skin hyperpigmentation disorders.
Our results are in agreement with Bosamiya and Jain (2021)   who conducted A prospective study on 40 patients with melasma, 20 patients in each group; one group were treated with combined microneedling and depigmenting cream (0.05% tretinoin + 2% hydroquinone + 0.01% floucinolone acetonide) while the other group treated with depigmenting cream alone and the outcome was evaluated using the m MASI score which showed Significant reduction in the combined treatment. 14 earlier split-face study using 20 patients with melasma found that the side treated with combined treatment (skin needling plus depigmenting serum) displayed a statistically significant reduction in MASI score and luminosity index (L) levels compared to the side treated with depigmenting serum alone. 15Microneedling allows better delivery of the therapeutic agents to the skin and so, better effect will be obtained.It results in a deeper and more even placement of the medication to the epidermis and dermis.Topicals that were applied along with microneedling showed a considerable improvement in MASI scores. 16 this study, microneedling alone is used on the left side of the face of patients with melasma and led to reduction of the mean Hemi-mMASI score to lesser and more delayed response than the right side (microneedling with glutathione).Our findings are in line with a previous study that was done on 17 female melasma patients.
Each patient received split-face treatment for facial melasma over the course of five treatments, separated by two-week intervals.
There was a significant difference in mMASI score between baseline and after sessions in the right side, where each patient received microneedling (dermapen) than the left side received a placebo. 17 using the Hemi-m MASI score because to the split-face structure of our study.This score is calculated by rating the darkness and area of involvement of three locations on each half of the face.After being entered into a calculation, these numbers produce the final Hemi-mMASI score for both half of the face.Equation: chin (0.05 D.A.) + malar (0.30 D.A.) + forehead (0.15 D.A.) In order to compare the mean percentage change, the mean value of the data collected from each half of the face was determined.When the mean Hemi-m MASI score at each side of the face decreased, improvement was deemed to have occurred.Digital photograghs were taken from both sides of the face using smart phone in the first baseline visit and in follow up visits.
Data was analyzed using STATA version 14.2 (Stata Statistical Software: Release 14.2 College Station, TX: Stata Corp LP.).Quantitative data was represented as mean, standard deviation, median, and range.Data was analyzed using student t-test to compare means of two groups and ANOVA for comparison of the means of three groups or more.When the data was not normally distributed Kruskal-Wallis test for comparison of three or more groups and Mann-Whitney test was used to compare two groups.Qualitative data was presented as number and percentage and compared using Chi square test.Spearman correlation analysis was used.Graphs were produced by using Excel or STATA program.p Value was considered significant if it was less than 0.05.

3. 1 |
Hemi modified MASI score changes of left and right sides of the face of treated patients over sessions.

TA B L E 3 F I G U R E 1
Hemi modified MASI score changes of left and right sides of the face of treated patients over sessions Hemi modified MASI score changes of left and right sides of the face of treated patients over sessions.

TA B L E 5 TA B L E 6
Percent of clinical improvement between left and right sides of the face of the studied group.Response to treatment in left and right sides of the face of studied group combination therapy for the treatment of melasma that included tranexamic acid (TA), vitamin C, with or without glutathione and conducted a split-face randomized clinical experiment on 30 patients with melasma received mixure A (TA 4 mg/ mL, vitamin C 3%, and glutathione 2%), which was applied to the right half of the face, and mixure B (TA 4 mg/mL, vitamin C 3%), Also,In a pilot split face study of 20 patients received microneedling on one side and mesoneedling on another side of their face.Treatment was repeated on a monthly basis for 4 months.Both treatments were effective in treating melasma, without producing any notable side-effects or complications.18Our outcomes are consistent F I G U R E 2 (A,B) Right side before and after microneedling with glutathione.(C,D) Left side before and after microneedling alone, respectively.with those reported by Iriarte et al. (2017) who claimed that the improved transdermal medication absorption shown with microneedling has produced superior results than skin lightening treatments alone in the treatment of melasma. 19Previous study conducted on 22 patients with intractable melasma were previously treated with microneedling alone, without the use of any active medicine, found that all participants showed a lightening of their skin pigmentation and concluded that microneedling alone is a promising modality for treatment of recalcitrant melasma. 20Microneedling encourages fibroblast growth and upper dermal collagenesis.It can also repair F I G U R E 3 (A,B) Right side before and after microneedling with glutathione.(C,D) Left side before and after microneedling alone, respectively.F I G U R E 4 (A,B) Right side before and after microneedling with glutathione.(C,D) Left side before and after microneedling alone, respectively.
This split-face study included 29 female patients with bilateral symmetrical epidermal type of melasma with mean age of 38.21 ± 6.36.Sociodemographic criteria and clinical features of melasma of the studied group were demonstrated in Tables1, 2, respectively.Sociodemographic criteria of studied population.Clinical features of melasma of the studied group.

modified MASI score Baseline Last session Difference between baseline and last session p Value* Mean ± SD median (range) Mean ± SD median (range) Mean ± SD median (range)
Hemi modified MASI score before and after sessions.
Iraji et al (2019)who assessed the TA B L E 4 *Mann-Whitney test was used p value < 0.05 was significant.