Introduction
The word melasma has its origin in Greek, where melas means black. It is a common acquired melanosis represented on the face by symmetric hyperpigmented patches with irregular, serrated, and geographical boundaries. Because of its cosmetic impact melasma causes emotional and psychosocial distress and adversely affects quality of life [1].
Melasma is multifactorial in nature and can be caused by ultraviolet light (UV) exposure, hormones, drugs, oral contraceptive pills and systemic disorders [2].
Melasma management is difficult since the disease is refractory and recurrent. Numerous treatment options have been tried.
Hydroquinone (HQ) inhibits the enzyme tyrosinase and prevents the conversion of dihydroxy phenylalanine (DOPA) to melanin. HQ affects the development, melanisation, degradation of melanosomes and causes necrosis of melanocytes. HQ remains the gold standard treatment for melasma and is the most commonly prescribed depigmenting agent in the world [3].
An upcoming modality of treatment in the practice of dermatology is platelet-rich plasma (PRP). PRP is an autologous human plasma preparation with an elevated platelet content that is created by centrifuging a greater volume of the patient’s own blood [4].
Platelets possess three types of granules. The alpha granules are essential as they contain various growth factors (GF). Some of the most studied GFs are platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), platelet-derived angiogenic factor (PDAF), transforming growth factor β (TGF-β) among others [5].
PRP in dermatology has been widely used for androgenetic alopecia, alopecia areata, acne scars, facial rejuvenation, wound ulcers, and striae distensae [6].
The pigment lightening effect of platelets can be attributed to a variety of factors including basement membrane repair by laminin, collagen IV and tenascin stimulation by TGF-β released upon platelets activation.
In melasma, a role of TGF-β1 has been proposed. TGF-β1 suppresses melanogenesis by inhibiting the expression of microphthalmia associated transcription factor (MITF) promoter and the paired-box homeo-c gene (PAX3). When these signal transduction pathways are inhibited, the synthesis of tyrosinase-related protein 1, 2 and tyrosinase is reduced. The epidermal growth factor (EGF) decreases prostaglandin E2 (PGE 2) expression and tyrosinase enzyme activity, hence decreases melanin production in melanocytes [7].
Objective
So far only a few studies on the efficacy of PRP in melasma have been published in the literature, but none of them could clearly identify its efficacy. This prompted us to undertake this study.
Material and methods
This was a hospital-based, prospective and comparative study, conducted on a total of 112 patients attending the outpatient department (OPD) of dermatology in our hospital. The duration of study was 2 years from Jan 2020 to Jan 2022 (table 1). The ethical clearance was obtained from the Institutional Ethical Committee. Proper written and informed consent was obtained from participating patients.
Patients with melasma not taking any treatment for past 4 weeks and aged more than 18 years, were included in the study. Patients with local skin infection, blood clotting disorders or any hemodynamic instability, patients on anticoagulants or non-steroidal anti-inflammatory drugs, patients taking topical or systemic glucocorticosteroids, or with any chronic medical illness (diabetes, chronic infection, blood dyscrasias), pregnancy and lactation, patients who refuse to consent, patients with exogenous ochronosis and those having known hypersensitivity to hydroquinone were excluded.
Patients were enquired about their age, sex, education, occupation, marital status, residence, mode of onset, progression, duration and history of chronic drug intake and details were recorded on a predesigned proforma (table 2). The site, distribution and type of melasma were documented. Dermoscopy and Wood’s lamp examination were done to confirm diagnosis, classify epidermal, dermal and mixed melasma and to differentiate melasma from common mimickers. Routine laboratory investigations were done in all patients: complete blood count, bleeding and coagulation time.
Scoring was done using modified Melasma Area and Severity Index (mMASI). The mMASI score was proposed by Pandya et al. in 2011 [8]. It was devised to remove the homogeneity component in MASI score and to decrease the inter-observer variability [9].
For calculating mMASI score, face is divided into four regions. Severity of melasma in each of the four regions is evaluated: forehead (f), right malar region (rm), left malar region (lm) and chin (c). The total score is calculated by using the formula – 0.3A(f) D(f) + 0.3A(lm) D(lm) + 0.3A(rm) D(rm) + 0.1A(c) D(c). The range of the total score is 0 to 24. Area (A) is scored as – 0 = absent, 1 = < 10%, 2 = 10–29%, 3 = 30–49%, 4 = 50–69%, 5 = 70–89% and 6 = 90–100%. Darkness (D) is scored as – 0 = absent, 1 = slight, 2 = mild, 3 = marked and 4 = severe.
A total of 112 patients, both women and men, suffering from melasma were included in this study and divided in two groups. Patients belonging to Group A were treated with autologous PRP with microinjections along with topical 4% hydroquinone while Group B patients received topical 4% hydroquinone only.
PRP preparation
Platelet-rich plasma was prepared by the double spin centrifugation method using a centrifuge machine Remi R-8CTM (Remiworld, India) [10]. Under aseptic precautions 10 ml blood was taken from each patient and put into sterile conical tubes (15 ml), having 2 drops of anticoagulant citrate dextrose solution, Solution A (ACD-A). To isolate plasma, the tube was centrifuged at 1500 rotations for 10 min. RBCs precipitated at the bottom of the tube and the plasma-containing platelets at the rest of the tube. The plasma was then gently transferred to an empty tube and centrifuged again at 3000 rotations for 10 min. Two layers thus obtained were platelet-rich plasma (PRP – the lower one-third) and the platelet-poor plasma (PPP – the remaining upper portion). PPP was discarded and PRP was transferred to 31G insulin syringes.
PRP injection
Prior to each session the patient was asked to wash face with soap and water. Topical anaesthetic cream EMLA (2.5% lidocaine + 2.5% prilocaine) was applied under occlusive dressing for 1 h and then washed off.
About 0.1 ml of PRP was injected intradermally in lesional skin using 31G insulin syringe. 1 cm distance was kept between each injection.
After the procedure, the patients were advised to use a sunscreen with a sun protection factor of 50 after the therapy, twice daily and on a regular basis along with 4% hydroquinone at bed time throughout the treatment course.
Patients were treated using intradermal injections of PRP for a maximum of 5 sessions of PRP at an interval at an interval of 2 weeks i.e. week 2, 4, 6, 8 and 10.
Group B patients were prescribed topical 4% hydroquinone at bed time, along with a sunscreen with a sun protection factor of 50, twice during day time (morning and afternoon) and on a regular basis throughout the treatment course.
At the end of the treatment course, only a sunscreen was prescribed to all patients as maintenance therapy.
Follow-up
All the patients were followed up every 2 weeks for a maximum of 6 months. Patients were photographed immediately before each session of intradermal PRP injections while after the last treatment session they were photographed every month for 3 months. Assessment of improvement was done directly on patients before each session of intradermal PRP injection. (Note: Improvement from the last treatment session was assessed just before intradermal PRP injections of the next session). During each visit patients were asked to report any side-effects of the treatment, for example ecchymosis, post-treatment dyschromia, severe erythema, oozing, crusting etc.
Patients were asked to rate their overall satisfaction with the treatment, 4 weeks after its completion as Not satisfied (< 25% satisfaction), Slight satisfied (25–50% satisfaction), Satisfied (50–75% satisfaction) and Very satisfied (≥ 75% satisfaction).
Objective evaluation was done by calculating mMASI score at baseline and before each session for up to 3 months after the end of treatment. The grades of improvement were calculated as mild (0–25% reduction in mMASI score), fair (25–50% reduction in mMASI score), good (50–75% reduction in mMASI score) and excellent (> 75% reduction in mMASI score).
Statistical analysis
All the data was tabulated and analyzed using IBM SPSSTM software version 20.0. Qualitative data was described using numbers and percentages. Quantitative data was described using means and standard deviation (SD). Chi-square test, Fisher exact test, independent ‘t’ test and Mann-Whitney U test were used to compare data between the groups. P-values < 0.05 were considered significant.
Results
A total of 112 patients participated in the study. Fifty-six patients were allocated to Group A, and they were treated with intradermal autologous PRP injections along with hydroquinone 4%. Group B also included 56 patients, who were treated with hydroquinone 4% alone. There were six dropouts (4 patients from Group A and 2 patients from Group B), so 106 patients completed the study. There were 32 (28.57%) males and 80 (71.42%) females in the study. The highest number of patients belonged to the age group 21–30 years (76.7%). Mean age was calculated as 26.2 years. Majority of the patients in our study were from urban areas (68.75%). Majority of patients in our study were housewives (46.4%), followed by students (27.6%). Majority of the patients had a malar pattern of melasma (51.7%), followed by a centrofacial pattern (45.5%). Majority of the patients had epidermal melasma (76.7%), followed by mixed type (20.5%). Most of the patients had disease for 1–3 years (45.5%) (table 1). At the end of the study, Group A patients responded better than Group B. The mean mMASI score decreased from 4.04 ±1.54 to 1.53 ±0.72 in Group A (PRP + HQ 4%) whereas the score decreased from 3.89 ±1.16 to 2.26 ±0.98 in Group B (HQ 4%). Patients with epidermal melasma responded better than mixed and dermal subtype in both the Groups. At the end of the study, Group A patients were more satisfied with the treatment than Group B patients. In Group A, the most common adverse effect was procedural pain (60.7%) whereas erythema (12.5%) was the most common adverse effect in Group B (figs. 1–22).
Discussion
PRP has recently gained popularity in the field of aesthetic medicine. It contains more than 30 growth factors. Two of these growth factors. TGF-β and EGF, have shown an important role in reducing hyperpigmentation [7].
Our understanding of PRP’s efficacy in treating melasma is currently limited.
The age of the patients in our study ranged between 15 to 35 years. The majority of the patients were in age group of 21–30 years (76.8%), with the mean age of 26.3 years. This is consistent with 27.54 years in a previous study conducted by Yalda et al. [11]. It was observed that younger patients are predominantly affected. This may be because younger patients are more cosmetically concerned.
In our study there were more women (71.4%) than men (28.5%). The same was observed by KrupaShankar et al. [12], Achar et al. [2], and Pawar et al. [13] where 75.6%, 80.1% and 74.2% of female patients had melasma. The greater predilection of melasma in women is attributed to the role of female sex hormones in its pathophysiology [14].
In our study, majority of the patients were from urban background (68.7%) compared to rural areas (31.2%). Similarly, Qazi et al. also found that 64.3% and 35.7% of the patients came from rural and urban areas respectively [15]. This may be due to location of hospital in the urban area leading to hospital bias. Also, people living in the urban areas have better access to facilities like transport, better access of information and these people are more self-conscious.
In our study, most of the patients seeking treatment were housewives (46.4%). followed by students (27.6%). Also in the study by Raju et al. [16] and Ali et al. [17], most of participants were housewives, 66% and 56% respectively. More than 25% of patients were students, which may be because our study was conducted in a university hospital where students form a considerable portion of outpatients.
We observed that half of our patients were suffering from the malar pattern of melasma (51.7%) followed by the centrofacial pattern (45.5%). These findings are consistent with those of Handa et al. [18] who observed a malar pattern in 52% of patients. Kumar et al. [19] and Mahmood et al. [20] also found malar melasma as the most common pattern in their study.
In our study we found epidermal melasma (76.7%) as the most common type followed by mixed (20.5%) and dermal type (2.6%) on Wood’s lamp examination. Likewise, in a study by Bhattarai et al. [21], 65.2% of patients had epidermal melasma. Pawar et al. [13], Ali et al. [17] and Nanjundaswamy et al. [22] also found epidermal melasma as the most common pattern in their studies.
Our study found that in most of the patients (45.5%) the duration of disease was between 1 to 3 years, with a mean duration of 3.1 years. Similarly Handa et al. [18] and Bhattarai et al. [21] also reported mean duration of disease as 3.25 years and 3.26 years, respectively.
In our study, the percentage of clinical improvement seen after the 5th (last) treatment session (after 10 weeks) (fig. 3) and 1 month after the last treatment session (after 14 weeks) (fig. 4) was better in Group A. The difference in response between both the groups was found to be statistically significant (p = 0.043 and p = 0.048, respectively).
In our study, the percentage of clinical improvement seen 3 months after the last treatment session (after 22 weeks) was better in Group A. Excellent response (more than 75%) was seen in 32.1% of patients in Group A as compared to 12.5% in Group B. Good response (50–75%) was seen in 46.4% of patients in Group A as compared to 41.1% in Group B. Fair response (25–50%) was seen in 12.5% of patients in Group A as compared to 26.7% in Group B. Poor response (less than 25%) was seen 1.8% of patients in Group A as compared to 16.1% of patients in Group B. The difference in response between both the groups was found to be statistically significant (p = 0.0025) (fig. 5). A similar study done by Hofny et al. [23] found excellent response in 13%, good response in 34.8%, fair response in 39.2% and poor response in 13% of patients. Similarly, a study done by Boparai et al. [24], found 10%, 30% and 60% of patients showing < 25%, 25–50% and 50–75% improvement, respectively. Higher percentage of improvement in our study could be because of additional depigmentary effects of hydroquinone 4%, which is the established gold standard treatment.
In our study we found that mean mMASI scores in Group A were reduced from 4.04 ±1.54 at baseline to 1.53 ±0.72 at the end of 22 weeks (62.1% improvement). On the other hand, scores were reduced from 3.89 ±1.16 at baseline to 2.26 ±0.98 at the end of
22 weeks (41.9% improvement) in Group B. It is only at the 5th treatment session and later that Group A mMASI scores improved as compared to Group B. However, the overall decrease of mMASI scores was higher in Group A as compared to Group B (fig. 6). Similarly, Tuknayat et al. [25] and Boparai et al. [24] reported 54.5% and 52.16% improvement in mMASI scores with intralesional PRP in their study, respectively. Higher percentage of improvement in mMASI scores in our study could be because of additional effects of hydroquinone 4%.
In our study the percentage of clinical improvement seen after the end of treatment was found to be better in epidermal melasma as compared to mixed and dermal subtypes.
The mean mMASI score decreased from 4.07 ±1.48 at baseline to 1.47 ±0.71 at the end of 22 weeks in Group A while it decreased from 3.89 ±1.18 at baseline to 2.21 ±1.01 at the end of 22 weeks in Group B, in epidermal melasma patients. Also, there was a steady decrease in mMASI scores from baseline to the end of treatment within the each group (fig. 7).
A similar decrease in mMASI scores was also found in mixed melasma (fig. 8). Since dermal melasma is comparatively more resistant to treatment, a decrease in mMASI score was little. At 1-month follow-up, mMASI scores went back to pre-treatment levels, even at 3 months a very little decrease was seen (fig. 9).
We also compared the mean mMASI scores between both groups in terms of duration of melasma. We found a steady decrease in mean mMASI scores from baseline to the end of treatment, within each group (figs. 18–22).
A total of 6 patients did not turn up for follow up (4 in Group A and 2 in Group B). Despite a comparatively better response, more dropout in Group A could be explained by the interventional nature of the study and pain during the procedure.
At the end of our study, we found that Group A patients had a higher percentage of satisfaction as compared to Group B. In Group A, 17 (30.3%) patients showed a satisfaction rate of more than 75% (very satisfied), 13 (23.2%) patients showed a satisfaction rate of more than 50% (satisfied), 14 (25.0%) patients showed a satisfaction rate of 25-50% (slightly satisfied) and 8 (14.2%) patients showed a satisfaction rate of less than 25% (unsatisfied). In group B, 7 (12.5%) patients showed a satisfaction rate of more than 75%, 11 (19.6%) patients showed a satisfaction rate of more than 50%, 17 (30.3%) patients showed a satisfaction rate of 25–50% and 19 (33.9%) patients showed a satisfaction rate of less than 25% (fig. 10). Similar findings were reported by Hofny et al. [23], Gamea et al. [26] and Sirithanabadeekul et al. [27].
In our study the adverse effects noted were few and mild. Procedural pain (61.5%) was the most common adverse effect in Group A while erythema (12.9%) was most common in Group B (fig. 11). Most of the side effects noted were mild and self-limiting. No ochronosis after hydroquinone 4% was seen in our study. No severe and long-term side effect was seen in our study.
Limitations. We did not include a control group comparing intradermal injections of PRP + hydroquinone vs. intradermal injections of saline + hydroquinone. Since, the needling component of PRP alone would cause some improvement in melasma. Hormonal evaluation, quality of life assessment and effect of family history of melasma on the outcome of the treatment, was not done in our study.
Conclusions
For numerous years, autologous platelet-rich plasma has been utilised as an adjunctive therapy for hyperpigmentation. Hydroquinone is the gold standard treatment for melasma. Hence combining with PRP seems logical. The use of this combination is seldom reported. As a result, this study was novel in its approach.
Autologous PRP may be a safe and effective therapeutic option in the treatment of melasma. This can also be an adjuvant technique to existing conventional modalities. More studies with a larger sample size are required to fully establish its therapeutic effectiveness.
Funding
No external funding.
Ethical approval
Approval number: 85/FM/IEC.
Conflict of interest
The authors declare no conflict of interest.
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