**4. Discussion**

AGA is a challenging disorder for both healthcare providers and patients. Current therapeutic options for AGA may generate biochemical abnormalities and clinical adverse effects. Therefore, new personalized therapies are necessary according to the characteristics of the patients, with fewer side effects and satisfactory results [46–49]. An increasing body of evidence emphasizes an important dermal and follicular response to certain growth factors such as platelet-derived growth factor, transforming growth factor beta, etc. [17,50,51]. In line with this, recent research has focused on evaluating the effectiveness of PRP in AGA.

The present meta-analysis succeeded in retrieving a sufficiently large number of clinical trials including PRP treatments for AGA in male, female, or mixed gender groups to allow pertinent quantitative analysis of results. By comparison, a relatively recent similar meta-analysis, Giordano, Romeo and Lankinen (2017) [3], found only 6 out of 16 clinical trials of PRP in AGA accurately reporting changes in hair density following treatment. A similar attempt of meta-analysis, Kramer & Keaney (2018) [52], focusing on technical details of PRP preparation, concluded that only 21% of the 19 studies selected for analysis provided complete quantitative assessment of initial and final cellular composition of PRP preparations. Data reporting seems to have improved in recent clinical trials [17,53], so we were able to retrieve accurate quantitative estimates of changes in hair density obtained with reliable standardized methods in 14 out of 15 studies (Table 1), as well as sufficient details concerning PRP preparation or delivery protocols.

The amount of PRP administered per treatment varied between 0.9 and 10 mL (Tables 1 and S2), and it was distributed at multiple subcutaneous injection points, each receiving 100–150 µL. Interestingly, some studies reported significant increases in hair density even for the placebo treatment (e.g., from 151.04 ± 41.99 to 166.72 ± 37.13 hairs/cm<sup>2</sup> , mean ± SD [43]). Although the authors explained such effects as resulting from a possible diffusion of the growth factors from the PRP-injected to the placebo-injected area, it is also plausible that microstimulation by needle puncture itself exerts beneficial effects on hair growth; this principle is currently applied in microneedling, another novel therapy used for AGA [54,55].

Among the 15 clinical trials selected for this meta-analysis, only two included histopathological and/or immunohistochemical assessment of PRP treatment effects. Gentile et al. (2015) showed significant increases in epidermal thickness and number of hair follicles after PRP injection on hematoxylin-eosin-stained scalp biopsies; they also assessed Ki67 proliferation index of hair follicle bulge cells and basal cells of epidermis [37]. Pakhomova and Smirnova (2020) proved by immunohistochemistry post-PRP treatment increases in areas of CD34 and β-catenin reactivity, as well as almost doubling of Ki67 proliferative index [40]. Rodrigues et al. (2018) used the multiplex method (Luminex®) to assess VEGF, PDGF and EGF concentrations in PRP preparations and correlate with their platelet counts, but also showed a lack of correlation with clinical effects on hair density [42].

Although there is no consensus concerning the molecular mechanisms and signaling pathways involved in PRP therapy, histological examination of biopsy samples from treated vs. control areas and immunohistochemical markers suggest multiple changes such as increased epidermal cell proliferation, particularly at hair follicles dermal papillae, augmentation of blood capillary network, and strengthening of the basal dermal extracellular matrix [43]. Platelet-secreted factors present in PRP preparations seem to enhance hair growth by stimulating multiple signaling pathways involved in the hair follicle cell cycle, such as the protein kinase B (Akt) pathway [18]. Upon Akt phosphorylation, subsequent Wnt/β-catenin transcription upregulation induces stem cell differentiation into hair follicle cells [56]. In vitro studies also showed that PRP causes papillary cells of the skin to grow by activating the extracellular signal-regulated kinases (ERK) [49]. Another important effect of PRP is reduction of hair follicle microinflammation associated with hair loss during AGA [1,57]. PRP growth and transcription factors signal the follicle to enter the anagen versus catagen phase, playing a role in regeneration and renewal; these effects partly overlap those of other effective AGA therapies such as minoxidil [58] or finasteride [59], justifying combined therapy approaches [44].

Due to these remarkable properties and activation of multiple signaling pathways by platelet-secreted factors, PRP raised a lot of interest as a potentially beneficial therapy in various medical fields, such as dermatology and cosmetology, ophthalmology, neurology, sports medicine, stomatology, gynaecology and reproductive medicine. A similar remedy is PRF, which is even easier to prepare in either leukocyte-rich or leukocyte-poor varieties, and includes cytokines and growth factors that are released for up to several weeks, resulting in enhanced wound healing [60,61].

Our study presents a number of limitations, such as those related to the extent of literature search, as well as the lack of accurate reporting of quantitative assessment variables and details concerning parameters of study groups, particularly in earlier published clinical trials. There is also a high degree of variability concerning PRP preparation and administration protocols, selection of experimental groups and general design of the trials, objective assessment methods. Possibly standardization requirements and larger patient samples will facilitate comparisons between objective outcomes of future studies.
