Increased Incidence of Telogen Effluvium Following Platelet-Rich Plasma Therapy in Patients with Elevated TNF-α Expression: A Prospective Cohort Study

Increased Incidence of Telogen Effluvium Following Platelet-Rich Plasma Therapy in Patients with Elevated TNF-α Expression: A Prospective Cohort Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Increased Incidence of Telogen Effluvium Following Platelet-Rich Plasma Therapy in Patients with Elevated TNF-α Expression: A Prospective Cohort Study Shabnam Feshangchian-Atashbar, Ali Ghamari, Sahel Jafarnejad-Komachali, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6905198/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Stimulation of hair regrowth with platelet-rich plasma (PRP) is a popular treatment for androgenic alopecia (AGA). However, a subset of patients experiences paradoxical telogen effluvium (TE) following treatment. To find the reason, we investigated the expression levels of inflammatory cytokines, dihydrotestosterone (DHT), its receptors, and related enzymes. In a prospective cohort study, we examined thirty male persons who complained of telogen effluvium following PRP administration in the last 12 months. The intervenors were tested with multiplex immunoassay for proinflammatory plasma cytokines, ELISA for Tissue DHT levels, western blot for androgen receptors (ARs), and RT-PCR for 5α-reductase isozymes and aromatase gene expression from the biopsy of dermal papilla cells (DPCs) of the vortex scalp. TE occurred in patients have twice-fold TNFα ( p ≤0.05) in their PRP, 40% higher levels of ARs ( p ≤0.05), and significant differences in 5α-reductase gene expression ( p =0.03) in their DPCs. There are significant quantitative differences in the down-expression levels of the aromatase in the outer root sheath of the HFs in these patients. We should consider that elevated TNF-α expression is a significant risk factor for the development of TE following PRP therapy. Biological sciences/Immunology Biological sciences/Molecular biology Alopecia PRP Telogen effluvium Teloptosis Tumor Necrosis Factor Figures Figure 1 Figure 2 Introduction Androgenetic alopecia (AGA) the most common alopecia, affects a large proportion of both men and women population ( 1 ). It is a chronic, age-related disorder specified by a reduction in hair length and diameter mainly located on the central scalp with varying baldness patterns ( 2 , 3 ). AGA is influenced by hormones, particularly dihydrotestosterone (DHT), a testosterone (T) metabolite that activates androgen receptors (ARs). In men, testosterone is converted to DHT by 5α-reductase, while in women, dehydroepiandrosterone (DHEA) and other androgens serve as DHT precursors ( 4 ). 5α-reductase expression is higher in the dermal papilla cells (DPCs) than in epithelial cells ( 5 ). Aromatase, which breaks the DHT structure, is expressed in the outer root sheath during the anagen phase ( 6 ). Hair follicles (HFs) in the vortex and frontotemporal scalp areas have increased ARs, making them more responsive to DHT, stimulating premature termination of the anagen growth phase in HFs, and leading to increased hair loss ( 7 ). Circulating DHT levels have less importance than T for optimizing the intracellular DHT concentrations because of local regulatory mechanisms that carefully regulate intracellular levels of androgens ( 8 ). The standard treatments for AGA involve a spectrum of medications including finasteride, dutasteride, ketoconazole, prostaglandin analogs, hormonal therapy, and topical minoxidil. However, these treatments may not work for all AGA-suffering individuals ( 9 ). These drugs need to be taken indefinitely, and treatment effectiveness is often hindered by patient compliance. Platelet-rich plasma (PRP) has emerged as an autologous, minimally invasive treatment for various dermatologic conditions, particularly alopecia. Its efficacy in stimulating hair regrowth has been attributed to the local delivery of growth factors such as PDGF, VEGF, IGF-1, and TGF-β, which promote follicular angiogenesis, stem cell activation, and dermal papilla survival ( 10 – 15 ). Overall, these proteins control cellular movements, adhesion, growth, specialization, and also accumulation of extracellular matrix (ECM) ( 16 ). By binding to the specific receptors on DPCs in the bulge area of HFs, the PRP growth factors stimulate hair regrowth when enter the anagen phase, creating the follicular unit and promoting hair regeneration ( 17 , 18 ). Then PRP induces the proliferation of DPCs by upregulating FGF7 and β-catenin, extracellular-signal–regulated kinase, and Akt signaling ( 19 ). However, an increasing number of clinicians report post-treatment telogen effluvium (TE), particularly in individuals with chronic or diffuse hair loss ( 20 , 21 ). Also, there have been patients who developed telogen effluvium with psoriasiform scalp dermatitis four weeks after being treated with PRP ( 22 ). While factors such as systemic illness, medications, or hormonal fluctuations are common triggers, recent studies have implicated inflammatory cytokines. This study explores the hypothesis that the levels of blood proinflammatory and inflammatory cytokines, DHT levels, its receptors activity, and enzymatic conversion may predispose patients to TE following PRP. Material and Methods Patients Criteria A prospective, single-center cohort study was conducted on 30 patients with TE. All patients (age range: 30–65) with male pattern hair loss provided written informed consent before participating in the intervention. All the experiments and interventions were placed in the Motahari Hospital, Stem Cell and Regenerative Innovation Center, Iran University of Medical Sciences (IUMS). All experimental protocols were approved by IUMS research ethics committee by approval code 1402.1080. All methods were performed in accordance with the relevant guidelines and regulations of Iranian Ministry of Health research and technology board. Thirty PRP-administrated patients in the last 12 months with telogen effluvium outcomes entered into the study (Group 1). Other twenty male persons with AGA and favored PRP results served as control (Group 2). All the selected patients have stage III to V patterns of hair loss according to the Norwood Hamilton classification. Patients, who had received topical minoxidil, prostaglandin, retinoids, and corticosteroids or systemic medications such as finasteride, dutasteride, and anti-androgens in the previous 12 months were excluded. Only patients with normal blood parameters in CBC were included in the study. PRP Preparation and administration To prepare PRP, blood was taken from a peripheral brachial vein using ACDA-1 (BD, UK) as an anticoagulant. The first step of centrifugation was done at 260 g for 8 min. After discarding RBC, the second step of centrifugation was performed in 1100 g for 4 minutes. The platelets were harvested and the growth factors are secreted once platelet activation begins with calcium gluconate. In addition, the numbers of platelets in PRP obtained from all participants were counted. The PRP was Intradermally injected on selected areas of the scalp at the amount of 0.1 mL/cm 2 using the nappage technique which multiple small injections in a linear pattern 1 cm apart to a depth of 1–2 mm. In our procedure, anesthesia is achieved with nerve block with lidocaine 1% with epinephrine. A portion of the PRP sample was subjected to immunoassay examination. Telogen Effluvium Evaluation criteria All patients were evaluated at the beginning of the study up to six months after the PRP administration. Assessment of the treatment was conducted in all patients with the standardized photo-trichograms in combination with the TrichoScan to measure hair count (number of hairs/0.65 cm 2 ), hair density (number of hairs/cm 2 ), hair diameter, anagen/telogen ratio, and vellus hair/terminal hair ratio. All hairs with a diameter larger than 40 µ m were categorized as terminal hairs, and those with lesser diameter were vellus hair. Multiplex Immunoassay of cytokines in PRP After the PRP preparation, one mL PRP was activated through a double freeze-thaw process from each patient. The cytokine concentration levels were evaluated with the MILLIPLEX Human cytokine magnetic bead-based panel kit (Millipore, Darmstadt, Germany). A total of 14 targeted proinflammatory cytokines and growth factors including the bFGF, EGF, HGF, interferon alpha 2 (IFNα2), IL-6, IL-8, IL-10, IL-17, IL-23, PDGF, TGF-β1, TNF-α, and VEGF were analyzed by the manufacturer’s instructions. Briefly, 20 µL buffer, 30 µL PRP, and 30 µL beads were added into a 96-well plate and were incubated for 12 hours at 4 ◦C, followed by washing twice with 200 µL washing buffer. An amount of 20 µL of antibody of each target was added into the wells, and the plates were incubated on a shaker plate for 1 h. Thirty µL of streptavidin-phycoerythrin solution was added into each well and incubated in the dark for 30 minutes. The wells contents were washed two times with 200 µL washing buffer. At the end 150 µL of sheath fluid was added into the wells, and were evaluated using the MAGPIX instrument. The median fluorescence intensities (MFI) of all cytokines were analyzed to calculate the final concentrations in each PRP sample. Hair Follicles Isolation and Processing All scalp specimens were processed as follows. Approximately ten hair follicles were obtained from the vortex area of a patient. All samples were taken in identical conditions by our hair transplant physician. Anagen hair follicles used for the processing show longer shafts with a consistent diameter, a rectangular shape, a slight distal angle, and darkly pigmented triangular or delta-shaped bulbs, featuring an angle to the hair shaft and an inner root sheath. The follicles were dissected and stored at -80°C until subjected to RNA extraction. RNA isolation from HFs RNA isolation was performed as described previously ( 23 ). The samples were homogenized in Trizol (Invitrogen, US). The samples were homogenized using a vortex to ensure complete lysis and release of RNA. Total RNA was isolated using chloroform (0.2 mL per 1 mL of lysis buffer) and then precipitated with isopropanol through centrifugation at 12000 g at 4°C. The upper aqueous phase contains RNA, the interphase contains DNA, and the organic phase contains proteins. The aqueous phase carefully transferred to a new tube. Then isopropanol (0.5 mL for every 1 mL of the aqueous phase) was added to precipitate the RNA. The RNA pellet underwent two washes with 75% ethanol and was then reconstituted in DEPC-treated water. DNase (Ambion) was added to eliminate any genomic DNA contamination. The quantity of RNA obtained was determined by spectrophotometer based on the A260/A280 ratio. The integrity of the isolated total RNA was confirmed through electrophoresis. Reverse transcription and quantitative real‑time PCR The cDNA was synthesized from 1µg of total RNA as described previously ( 24 ). Absolute quantification of mRNA of 5α-Reductase isozymes I, II, III, and aromatase in the hair follicles was evaluated by real-time RT-PCR using the Rotogen (Kiagen™, Germany) Real-time PCR system with SYBR Green PCR Master Mix (Promega). The final concentration used in the reactions was 0.5 µM for both the forward and reverse primers. Twenty microliters of the prepared PCR master mix were added to each well of the reaction plate. Three microliters of each reverse-transcribed RNA sample were added to the appropriate well to make a final reaction volume of 25 µl. The PCR protocol was as follows: denaturation at 94°C for 30s; annealing at 55°C for the 5α-Reductase I, 54°C for the 5α-Reductase I, 60°C for the 5α-Reductase III, 60°C for the aromatase gene for 30 s; and extension at 72°C for 30 s. The number of cycles was 35 in all cases. At the end of the amplification phase, a melting curve analysis was carried out. All reactions were run in triplicate and no cDNA was added to the negative reactions. The primer sequences are listed in Table 1 . Table 1 Primer sequences for 5α-Reductase isozymes and aromatase used in PCR amplification of harvested hair follicle samples. Gene Sequence (5′-3′) GenBank Access no. 5α-Reductase I -AGCCATTGTGCAGTGTATGC- -AGCCTCCCCTTGGTATTTTG- NM_001047.3 5α-Reductase II -TGAATACCCTGATGGGTGG- -CAAGCCACCTTGTGGAATC- NM_000348.3 5α-Reductase III -TCCTTCTTTGCCCAAACATC- -CTGATGCTCTCCCTTTACGC- NM_024592.4 Aromatase -TATTAGGGCCCTGTGTCTGC- -TGGGTTGGGACTTTTCCTCC- NM_000103.3 Western Blot Analysis DPCs were lysed in the lysis buffer supplemented with 1% protease inhibitor (Roche, Germany) and incubated on ice. Cell lysates were centrifuged at 5,000 g for 15 min at 4°C. Proteins (50 µg) in whole-cell lysates were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to the polyvinylidene difluoride membranes (Biocompare, US). After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with corresponding anti-AR primary antibody (1:500; Santa Cruz, USA), overnight at 4 ℃. On the next day, it was incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, UK) for 1 h at room temperature. The quantification of AR bands was conducted by densitometry using ImageJ software (US National Institutes of Health, USA). Enzyme linked immunosorbent Assay (ELISA) For the quantification of DHT levels in DPCs of both groups, the Human Dihydrotestosterone ELISA Kit (ab283979; Abcam, UK) was used, according to the supplier’s instruction. Patients DPCs were preserved at − 80°C, thawed, and centrifuged at 3500 g for 5 min. The supernatants were used for the ELISA assay. Briefly, 100 µL/well of a diluted sample was dispensed in each well and incubated 1 hour at room temperature. The samples were washed six times with washing buffer (300 µL/well). Then 100 µL/well of diluted enzyme-conjugated secondary antibody added to the solution and incubated for 1 hour in room temperature. It was washed 6 times with washing buffer and incubated with chromogen up to 15 minutes in the dark to develop the color. Finaly, the absorbance value at 450 nm was measured. DHT concentrations were measured from two groups of patients. Statistical analysis The non-parametric Mann–Whitney U test was used for comparisons. The Shapiro-Wilk test was used to analyze the compliance of the distribution of the analyzed variables with normal distribution. GraphPad Prism 7.0 software (San Diego, CA) was used for the statistical analysis. p ≤ 0.05 was considered significant. Results During the period from April 2022 to December 2023, thirty patients have complained of the previous PRP outcomes as the telogen effluvium (Group 1). The remaining patients were individuals who regularly underwent annual PRP treatments and showed enhancements in hair quality parameters. The median age of the patients included was 50.8 years (± 14.2 years). The blood counts of all participants in both groups are shown in Table 2 . The data shows a normal count for everyone. PRP was characterized according to platelet counts and growth factor concentrations. The median number of platelets was increased 4 to 5-fold, with a minimum of 1152.5 and a maximum of 2582 ×10 3 µL (Table 2 ). The age and platelet count in the PRP were not significantly different between the patients with successful and unsuccessful outcomes (Table 2 ). Age was not significantly correlated with any cytokine concentrations in both groups of patients. Table 2 Clinical parameters in the AGA patients’ blood from both groups were included in the study PRP (Group 1; n = 30) (Group 2; n = 20) p Age (Years Old) 46.5 ± 16.5 49.5 ± 14.5 0.643 Platelet count (×10 3 in µL) 1867 ± 715.48 1952 ± 720.25 ≤ 0.05** RBC (10 6 /µL) 4.97 ± 0.60 4.94 ± 0.41 0.002*** WBC (10 3 /µL) 6.19 ± 1.10 6.12 ± 1.55 ≤ 0.05** Neutrophils (10 3 /µL) 3.95 ± 0.89 3.77 ± 1.51 ≤ 0.05** Lymphocytes (10 3 /µL) 2.12 ± 0.55 2.32 ± 0.55 ≤ 0.05** Monocytes (10 3 /µL) 0.49 ± 0.22 0.48 ± 0.19 ≤ 0.05** Eosinophils (10 3 /µL) 0.14 ± 0.07 0.13 ± 0.09 ≤ 0.05** Basophils (10 3 /µL) 0.05 ± 0.01 0.04 ± 0.01 ≤ 0.05** RBC: Red blood cells; PLT: Platelets; WBC: White blood cells The cytokines and growth factors from 2 groups were measured. There was a variation between the expression levels of some agents in these 2 groups, with a mean coefficient of variability of 9.95% for bFGF, 5.89% for HGF, 22.64% for TGFβ and 5.8% for VEGF (Table 3 ). Also, the proinflammatory cytokines have significant differences between the two groups. There was variability of 13.41% for IFNα, 15.48% for IL-6, 13.74% for IL-10, 12.15% for IL-17, 25% for IL-23, and finally 188% for TNFα ( p ≤ 0.05** ) (Table 3 ). Among all the 14 targeted GFs and cytokines in PRPs, the level of TNF-α was dramatically higher in patients with unsuccessful outcomes ( p = 0.05** ). Table 3 Cytokine and Growth factor expression levels in both groups of patients PRPs included in the study. Patients PRP Group 1 (n = 30) Group 2 (n = 20) P value Cytokines (pg/mL) EGF 421.90 ± 336.10 413.12 ± 347.55 ≤ 0.5* bFGF 97.95 ± 24.89 108.77 ± 34.58 ≤ 0.05** HGF 483.12 ± 67.55 513.32 ± 48.55 ≤ 0.05** IFNα 16.49 ± 4.22 14.28 ± 3.19 ≤ 0.5* IL-6 12.28 ± 4.19 10.38 ± 5.49 ≤ 0.05** IL-8 818.35 ± 75.50 705.63 ± 68.60 ≤ 0.05** IL-10 12.72 ± 4.62 10.45 ± 3.82 ≤ 0.05** IL-17 9.22 ± 3.15 8.10 ± 3.55 ≤ 0.05** IL-23 14.88 ± 3.95 11.16 ± 4.10 ≤ 0.05** PDGF-A A 6511.20 ± 1661.25 6650.50 ± 1677.45 ≤ 0.5* PDGF-AB/BB 17700.00 ± 4200.50 16400.00 ± 4375.00 ≤0.05** TGF β1 56.28 ± 31.14 72.76 ± 30.16 ≤0.05** TNFα 64.47 ± 6.76 34.18 ± 8.15 = 0.05** VEGF 288.20 ± 38.5 305.33 ± 28.6 ≤0.05** EGF: epidermal growth factor; bFGF: basic fibroblast growth factor; HGF: Hepatocyte growth factor; IFNα2: interferon alpha 2; Il: interleukin; PDGF: platelet-derived growth factor; TNF-α: tumor necrosis factor-α; TGF-β: transforming growth factor-β; VEGF: vascular endothelial growth factor. The 5α-Reductase I and II mRNA levels were significantly higher in group 1 patients versus group 2 ( p = 0.003*** ) ( Fig. 1 A, B ) . Higher mean mRNA levels of 5α-Reductase III were also observed in this group, although the difference did not reach statistical significance ( Fig. 1 C ) . The aromatase mRNA levels ( Fig. 1 D ) significantly differ between the two groups. Nonetheless, there was notable variability observed in the results for aromatase expression among group 1 patients ( Fig. 1 D ) . The DHT concentrations in DPCs were elevated in 21 of 30 patients in group 1 ( Fig. 2 A ) , and 6 of the 20 patients in group 2 ( Fig. 2 A ). Differences in mean DHT concentration in DPCs of group 1 were statistically significant relative to group 2. Elevated levels of DHT in DPCs correlate with the progression of telogen effluvium following PRP. The expression of the androgen receptor was detected by Western blot ( Fig. 2 B ) . The levels of AR are standardized to the expression of GAPDH. Hair follicles from the vertex areas of the scalp express large quantities of AR especially in patients with teloptosis after PRP ( Fig. 2 B ) . After PRP therapy, all patients had mild effects in the form of transient erythema, immediately after injection. None of the patients experienced any serious adverse effects. All patients in two groups were followed up to six months for evaluation of outcomes. PRP efficacy was evaluated using various metrics such as photography, mean hair count and density, and anagen-to-telogen ratio. At the baseline, the patients in group 1 presented higher hair density in comparison with 6 months after PRP therapy (Table 4 ) and this difference was statistically significant ( P = 0.040 **). The results showed a decrease in anagen, an increase in telogen, and decreased anagen/telogen ratio ( p = 0.005*** ) (Table 4 ). These patients have nearly twice-fold TNFα ( p ≤ 0.05** ) concentrations in their PRP. Regarding hair count, the patients in group 2 showed a significant increase when the counts at baseline and 6 months after the PRP administration were compared ( P = 0.037** ). The improvement was observed in 60% of patients with an increase in anagen/telogen ratio (Table 4 ). Table 4 Clinical parameters in the PRP-treated AGA patients in both groups at baseline and six months. Group 1 Group 2 p baseline 6 months baseline 6 months Hair Diameter (mm) 0.048 ± 0.008 0.042 ± 0.005 0.049 ± 0.006 0.059 ± 0.008 0.05 Hair Count (n/cm2) 116.65 ± 11.95 101.35 ± 14.45 121.35 ± 15.45 168.80 ± 12.65 0.05 absolute anagen counts 71.15 ± 11.15 54.95 ± 08.35 91.55 ± 10.45 122.50 ± 11.75 0.05 Change from baseline 45.50 ± 0.80 46.40 ± 6.10 29.80 ± 5.00 46.30 ± 0.90 0.05 Discussion This study provides the first clinical evidence linking elevated TNF-α levels to increased risk of PRP-induced telogen effluvium. Platelet rich plasma therapy has been utilized for more than a decade in the treatment of AGA. Leukocyte rich PRP (L-PRP) is commonly employed in the treatment of dermatologic indications ( 25 ). Nevertheless, elevated levels of pro-inflammatory cytokines in L-PRP could trigger catabolic and inflammatory responses that may counteract the positive impact of growth factors on target tissue regeneration. In this study, we opted to prepare PRP using the manual double-spin technique which has a large quantity of leukocytes with the potential of producing proinflammatory cytokines like many clinics do (Table 2 ). Telogen effluvium can be a concerning issue, on the other hand, it's promising that the other 20 patients, who had regular annual PRP treatments, showed improvements in their hair parameters quality (Table 4 ). Undoubtedly the levels of growth factors and cytokines within PRP could impact the effectiveness of treatment and serve as a potential indicator of treatment success in clinical settings. The blood counts of all participants in our study, as shown in Table 2 , indicate normal values across the board. Our data also reveals that age and platelet count in the PRP did not show significant differences between patients with successful and unsuccessful outcomes. This suggests that while platelet count is important, there may be other factors at play influencing the efficacy of PRP treatment. We observed variations in the expression levels of some cytokines. The bFGF showed a mean coefficient of variability of 9.95%, and TGFβ at 22.64%. These differences could indicate distinct biological responses in each group. The variability in TGFβ plays a significant role between the two groups and their biological responses (Table 3 ). In pro-inflammatory cytokines expression, the variability was 13.41% for IFNα, 15.48% for IL-6, 13.74% for IL-10, 12.15% for IL-17, 25% for IL-23, and an astonishing 188% for TNFα (Table 3 ). The increased expression of IL-17, IL-23, and TNF-α, in PRPs of patients who develop telogen effluvium following PRP therapy was significant (p < 0.05 ). TNF-α is a powerful pro-inflammatory cytokine that plays a crucial role in coordinating the cytokine cascades in inflammations ( 26 ). Studies showed transient inflammations cause migration of mast cells and secretion of a variety of IL-4, IL-8, IL-13, IL-31, proteases (cathepsin S), prostaglandins, and platelet activating factor which can activate other cytokine cascades that affect HF cell cycle arrest ( 26 ). Some studies show that pro-inflammatory cytokines inhibit the growth of isolated HFs in in vitro cultures ( 27 ). The processes that control the hair growth cycles are situated within the HFs and are believed to be impacted by changes in the expression of specific regulatory molecules inside the follicles ( 28 ). Various studies have emphasized the importance of microinflammation within the microenvironment of HFs in the development of AGA ( 29 ). Microinflammation is a slow, subtle, and enduring process, contrasting with the rapid, inflammatory, and destructive process observed in classical inflammatory scarring alopecia ( 30 ). In 2000, Mahe and colleagues revealed that HFs adjacent keratinocytes, which express receptors for IL-1, engage the expression of IL-1 responsive genes which include mRNA coding for TNFα ( 30 ). Also, specific chemokine genes, such as IL-8, monocyte chemoattractant protein-1 (MCP-1), and MCP-3 are overexpressed, and their expression recruits’ neutrophils and macrophages ( 30 ) that strengthen production of TNFα. Moreover, neighboring fibroblasts are well-prepared to react to such pro-inflammatory signals. The increase in adhesion molecules on the capillary endothelium for circulating cells, along with the chemokine gradient, facilitates the movement of inflammatory cells, such as neutrophils via IL-8, T lymphocytes, and Langerhans cells partly due to MCP-1 action, across the endothelium. The outcome of these signals is the induction of teloptosis in the HFs cycles. In 1997, a study by Thein et al . surveyed the cytokine profiles of infiltrating T-lymphocytes from the margin of involved alopecia areata lesions ( 31 ). It was found that T-lymphocytes inhibited the proliferation of neonatal keratinocytes by releasing high amounts of IFN-γ and/or TNF-α ( 31 ). The Wnt/β-catenin signaling pathway plays a crucial role in both the development of HFs and the stimulation of hair growth ( 32 ). Subsequent studies aimed to uncover the specific functions of Wnt/β-catenin signaling in different cell types within the HFs. It was found that removing β-catenin in DPCs in mature HFs led to a decrease in the proliferation of stem cells responsible for generating the matrix keratinocytes in the anagen bulb. As a result, hair becomes thinner and shorter, and a premature transition from the anagen to catagen phase occurs ( 33 ). Other studies demonstrated that when Wnt ligand secretion was specifically blocked in the epidermal part of the HFs during the resting telogen phase, it impeded the transition to the anagen phase and caused the hair cycle to halt ( 34 ). On the other hand, ARs interact with β-catenin and use β-catenin as a co-factor to amplify the transcription of AR target genes ( 35 , 36 ). We noticed that hair follicles from the vertex areas of the scalp express large quantities of AR, particularly in patients with teloptosis after PRP treatment, as depicted in Fig. 2 B. This is an obvious link between AR expression and hair loss in these patients. It can be inferred that DHT is involved in the development of AGA by disrupting the hair follicle's β-catenin signaling pathway ( 37 , 38 ). This is supported by recent microarray gene expression data, which revealed a significant downregulation of Wnt/β-catenin signaling pathway genes in the bald frontal scalps compared to the haired occipital scalps of male patients with AGA ( 39 ). The results from our study indicate that DHT concentrations in dermal papilla cells (DPCs) were notably higher in a majority of patients in group 1 compared to group 2, as shown in Fig. 2 A. This is a potential association between elevated DHT levels in DPCs and the development of telogen effluvium following PRP treatment. Also, the T to DHT converter isozymes, the 5α-Reductase I and II mRNA levels were significantly higher in group 1 patients compared to group 2, with a p-value of 0.003 (Fig. 1 A, B). It suggests that there may be a dysregulation of the 5α-Reductase I and II genes in group 1 patients. We also found higher mean mRNA levels of 5α-Reductase III in group 1 patients (Fig. 1 C), although this difference was not statistically significant. It's possible that with a larger sample size, we may have been able to detect a significant difference in the expression of 5α-Reductase III as well. Dickkopf-related protein 1 (DKK1) is an inhibitor of the Wnt/β-catenin pathway that is overexpressed in bald areas of the scalp ( 40 ). The expression levels of DKK1 correlate with a reduction in anagen phase of HFs ( 41 ). In 2008, Kwack et al. , demonstrated that DHT induces the expression of DKK1 in DPCs, which results in apoptosis in follicular keratinocytes ( 40 ). Another factor in hair loss depends on the aromatase which is found in the outer root sheath that converts androstenedione into estrone and testosterone into estradiol, leading to the reduction in T and DHT levels ( 42 ). The aromatase mRNA levels in our study clearly show a significant decrease in group 1 patients, as indicated in Fig. 1 D. In vitro studies confirmed that TNF-α has a key role in the induction of HF apoptosis ( 43 ). In 2008, a study by Mikkol reported that HF apoptosis by TNF-α is a more important factor in hair loss than the interaction of DHT with AR ( 44 ). More studies have revealed that TNF-α causes vacuolation of matrix cells, and abnormal keratinization of the inner root sheath. This phenomenon disrupts follicular melanocytes in the DPs ( 45 ). The data from Hoffmann et al . experiments in cultures of HFs showed that TNF-α completely affects hair growth by induction of the formation of club-like HFs, similar to their morphology in the catagen phase ( 46 ). Our data also indicated a shift in hair growth phases, with a decrease in anagen and an increase in telogen phases, leading to a decreased anagen/telogen ratio ( P = 0.005 ) (Table 4 ). Additionally, the patients in this group exhibited nearly twice-fold TNFα concentrations in their PRP samples, which could be a potential factor affecting their hair growth outcomes. On the other hand, patients in group 2 showed a noticeable increase in hair count when comparing baseline levels to those at six months post-PRP treatment, with a significant difference (P = 0.037 ). Moreover, 60% of these patients demonstrated an improvement in their anagen-to-telogen ratio, indicating a positive response to the PRP therapy, as outlined in Table 4 . In contrast to our results, Teraki et al. reported that levels of TNF-α in alopecia areata patients are very high. They showed that the expression of TNF-α was significantly higher than those in patients with alopecia universalis ( 47 ). In 2001, Lis et al. found that TNF-α receptor type I was significantly elevated in patients with AA in comparison with the healthy control group ( 48 ). Later, in a 2002 study, Koubanova and Gadjlgoroeva examined the levels of TNF-α in patients with AA. The results showed no difference between this group of patients and the control group ( 49 ). However, despite the controversy in these results, our finding demonstrated the consistency between higher TNFα concentration and teloptosis. It can be asserted that the occurrence of telogen effluvium after PRP therapy is a multifaceted process, with numerous molecular mechanisms. Studies showed that Binding of TNFα to its cell surface receptor (TNFR) leads to the activation of IKK, which phosphorylates and degrades IkB, an inhibitor of NF-kB. NF-kB then translocate to the nucleus and activates Transcription ( 50 ). Androgen responsive cells are highly sensitive to TNF-α-induced arrest and apoptosis, where TNF-α did not induce phosphorylation of IkB ( 51 ). Long-term exposure of TNFα induces androgen hypersensitivity in cells, which was associated with decreased levels of total AR, but increased nuclear AR levels ( 50 ). Within this study, upregulation of AR expression, DHT levels, and 5a-reductase enzymes and downregulation of aromatase levels have been observed. These patients had elevated ranges of IL-17, IL-23, and TNF-α in their L-PRP. The potential exists to reduce this impact in these patients by substituting pure PRP with low leukocyte amounts instead of L-PRP. In conclusion, our current study revealed a novel finding: the concentration of TNF-α in PRP correlated with treatment effectiveness. Lower TNF-α levels could potentially predict improved therapeutic results of PRP for AGA treatment. The exclusion of leukocytes in PRP to create pure PRP is an endeavor to optimize therapeutic benefits for AGA patients with elevated TNF-α levels. Limitations The primary constraints of this study include a small sample size and the absence of a placebo control group. These factors can introduce several limitations, including limited generalizability, the risk of random variability, lack of precision and reliability, and limited exploration of heterogeneity. Also, this study was not a randomized, double-blind, controlled trial. The stability of cytokine levels in consecutive PRP treatments is still uncertain. We only included 14 types of cytokines in our analysis, and there may be additional cytokines that we have not yet studied that could also affect treatment outcomes. Future Directions Future studies should continue examining the role of inflammatory cytokines and hair-promoting mechanisms of PRP and amount of growth factors contained within PRP. Additionally, the hair-promoting effects of PRP that are unrelated to growth factor concentrations and cycles related to TNFα family should still be explored. Declarations Conflict of Interest: There is no conflict of interest. Funding: The study was supported by Iran University of Medical Sciences. Authors' Contributions: ASH and SFA visited, intervened, and followed up with the patients; SJ evaluated the criteria for telogen effluvium; ASH performed the biopsy; MA conducted the genetic tests; AG performed the multiplex immunoassay; SFA handled the preparation and administration of PRP; ASH designed the experiment, collected the final data, and wrote the manuscript. All authors have read and approved the final manuscript. Data Availability Statement: All data generated or analysed during this study are included in this manuscript. References Kuczara A, Waśkiel-Burnat A, Rakowska A, Olszewska M, Rudnicka L. Trichoscopy of Androgenetic Alopecia: A Systematic Review. J Clin Med. 2024;13(7):1962. doi: 10.3390/jcm13071962. Varothai S, Bergfeld WF: Androgenetic alopecia: an evidence-based treatment update. Am J Clin Dermatol 2014; 15:217-230. Alessandrini A., Starace M., D’Ovidio R., Villa L., Rossi A., Stan T.R., Calzavara-Pinton P., Piraccini B.M. Androgenetic alopecia in women and men: Italian guidelines adapted from European Dermatology Forum/European Academy of Dermatology and Venereology guidelines. G. Ital. Dermatol. Venereol. 2020; 155:622–631. doi: 10.23736/S0392-0488.19.06399-5. Owecka B, Tomaszewska A, Dobrzeniecki K, Owecki M. The Hormonal Background of Hair Loss in Non-Scarring Alopecias. Biomedicines. 2024;12(3):513. doi: 10.3390/biomedicines12030513. Asada Y, Sonoda T, Ojiro M, Kurata S, Sato T, Ezaki T, et al. 5 alpha-reductase type 2 is constitutively expressed in the dermal papilla and connective tissue sheath of the hair follicle in vivo but not during culture in vitro. J Clin Endocrinol Metab. 2001; 86:2875–80. doi: 10.1210/jc.86.6.2875. Sawaya ME, Penneys NS. Immunohistochemical distribution of aromatase and 3B-hydroxysteroid dehydrogenase in human hair follicle and sebaceous gland. J Cutan Pathol. 1992; 19:309–14. doi: 10.1111/j.1600-0560.1992.tb01367.x McElwee KJ, Shapiro JS: Promising therapies for treating and/or preventing androgenic alopecia. Skin Therapy Lett 2012; 17:1-4. Ronald S. Swerdloff, Robert E. Dudley, Stephanie T. Page, Christina Wang, Wael A. Salameh, Dihydrotestosterone: Biochemistry, Physiology, and Clinical Implications of Elevated Blood Levels. Endocrine Reviews 2017; 38(3):220–254. https://doi.org/10.1210/er.2016-1067. Cervantes J, Perper M, Wong LL, Eber AE, Villasante Fricke AC, Wikramanayake TC, Jimenez JJ. Effectiveness of Platelet-Rich Plasma for Androgenetic Alopecia: A Review of the Literature. Skin Appendage Disord. 2018;4(1):1-11. doi: 10.1159/000477671. Kourosh AS, Santiago Mangual KP, Farah RS, Rao M, Hordinsky MK, Arruda S, Sadick N. Platelet-Rich Plasma: Advances and Controversies in Hair Restoration and Skin Rejuvenation. Dermatol Surg. 2024; 50(5):446-452. doi: 10.1097/DSS.0000000000004115. Owczarczyk-Saczonek A, Wygonowska E, Budkiewicz M, Placek W. Serum sickness disease in a patient with alopecia areata and Meniere' disease after PRP procedure. Dermatol Ther. 2019;32(2):e12798. doi: 10.1111/dth.12798. Singhal P., Agarwal S., Dhot P., Sayal S. Efficacy of Platelet-Rich Plasma in Treatment of Androgenic Alopecia. Asian J. Transfus. Sci. 2015; 9:159. doi: 10.4103/0973-6247.162713. Khatu S., More Y., Gokhale N., Chavhan D., Bendsure N. Platelet-Rich Plasma in Androgenic Alopecia: Myth or an Effective Tool. J. Cutan. Aesthet. Surg. 2014; 7:107. doi: 10.4103/0974-2077.138352. Greco J., Brandt R. The Effects of Autologus Platelet Rich Plasma and Various Growth Factors on Non-Transplanted Miniaturized Hair. Int. Soc. Hair Restor. Surg. 2009; 19:49–50. doi: 10.33589/19.2.0049. Stevens J., Khetarpal S. Platelet-rich plasma for androgenetic alopecia: A review of the literature and proposed treatment protocol. Int. J. Women Dermatol. 2019; 5:46–51. doi: 10.1016/j.ijwd.2018.08.004. Akiyama M, Smith LT, Holbrook KA: Growth factor and growth factor receptor localization in the hair follicle bulge and associated tissue in human fetus. J Invest Dermatol 1996; 106:391-396. Trink A, Sorbellini E, Bezzola P, Rodella L, Rezzani R, Ramot Y, Rinaldi F: A randomized, double-blind, placebo- and active-controlled, half-head study to evaluate the effects of platelet-rich plasma on alopecia areata. Br J Dermatol 2013; 169:690-694. Gkini M-A, Kouskoukis A-E, Tripsianis G, Rigopoulos D, Kouskoukis K: Study of platelet-rich plasma injections in the treatment of androgenetic alopecia through a one-year period. J Cutan Aesthet Surg 2014; 7:213-219. Cervelli V, Garcovich S, Bielli A, Cervelli G, Curcio BC, Scioli MG, Orlandi A, Gentile P. The effect of autologous activated platelet rich plasma (AA-PRP) injection on pattern hair loss: clinical and histomorphometric evaluation. Biomed Res Int. 2014; 2014:760709. doi: 10.1155/2014/760709. Puig CJ, Reese R, Peters M. Double-blind, placebo-controlled pilot study on the use of platelet-rich plasma in women with female androgenetic alopecia. Dermatol Surg. 2016; 42:1243–7. Mapar MA, Shahriari S, Haghighizadeh MH. Efficacy of platelet-rich plasma in the treatment of androgenetic (male-patterned) alopecia: A pilot randomized controlled trial. J Cosmet Laser Ther. 2016; 18:452–5. De Sousa ICVD, Tosti A. New investigational drugs for androgenetic alopecia. Expert Opin Investig Drugs. 2013; 22: 573-589. Verdi J, Mortazavi-Tabatabaei SA, Sharif S, Verdi H, Shoae-Hassani A. Citalopram increases the differentiation efficacy of bone marrow mesenchymal stem cells into neuronal-like cells. Neural Regen Res. 2014;9(8):845-50. doi: 10.4103/1673-5374.131601. Shoae-Hassani A, Mortazavi-Tabatabaei SA, Sharif S, Madadi S, Rezaei-Khaligh H, Verdi J. Recombinant λ bacteriophage displaying nanobody towards third domain of HER-2 epitope inhibits proliferation of breast carcinoma SKBR-3 cell line. Arch Immunol Ther Exp (Warsz) . 2013; 61(1):75-83. doi: 10.1007/s00005-012-0206-x. Vladulescu D, Scurtu LG, Simionescu AA, Scurtu F, Popescu MI, Simionescu O. Platelet-Rich Plasma (PRP) in Dermatology: Cellular and Molecular Mechanisms of Action. Biomedicines 2024; 12, 7. https://doi.org/10.3390/biomedicines12010007 Karki R, Sharma BR, Tuladhar S, Williams EP, Zalduondo L, Samir P, Zheng M, Sundaram B, Banoth B, Malireddi RKS, Schreiner P, Neale G, Vogel P, Webby R, Jonsson CB, Kanneganti TD. Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes. Cell. 2021; 184(1):149-168.e17. doi: 10.1016/j.cell.2020.11.025. Philpott MP, Sander DA, Bowen J, Kealey T. Effects of interleukins, colony stimulating factor and tumour necrosis factor on human hair follicle growth in vitro: a possible role for interleukin-1 and tumour necrosis factor-a in alopecia areata. Br. J. Dermatol. 1996; 135, 942–948. Paus, R., 1997. Immunology of the hair follicle. In: Bos, J.D., (Ed.), Skin Immune System. Cutaneous Immunology and Clinical Immunodermatology, CRC Press, Boca Raton. Jaworsky C, Kligman AM, Murphy GF. Characterization of inflammatory infiltrates in male pattern alopecia: implication for pathogenesis. Br. J. Dermatol . 1992; 127, 239–246 Mahe´ YF, Michelet JF, Billoni N, Jarrousse F, Buan B, Commo S, Seint-Leger D, Bernard BA. Androgenetic alopecia and microinflammation. Int. J. Dermatol . 2000; 39, 576–584. Thein C, Strange P, Hansen ER, Baadsgaard O. Lesional alopecia areata T lymphocytes downregulate epithelial cell proliferation. Arch Dermatol Res . 1997; 289:384–8. Van Mater D, Kolligs FT, Dlugosz AA, Fearon ER. Transient activation of beta -catenin signaling in cutaneous keratinocytes is sufficient to trigger the active growth phase of the hair cycle in mice. Genes Dev. 2003; 17:1219–24. doi: 10.1101/gad.1076103. Enshell-Seijffers D, Lindon C, Kashiwagi M, Morgan BA. beta-catenin activity in the dermal papilla regulates morphogenesis and regeneration of hair. Dev Cell. 2010; 18:633–42. doi: 10.1016/j.devcel.2010.01.016. Myung PS, Takeo M, Ito M, Atit RP. Epithelial Wnt ligand secretion is required for adult hair follicle growth and regeneration. J Invest Dermatol. 2013; 133:31–41. doi: 10.1038/jid.2012.230 Yang F, Li X, Sharma M, Sasaki CY, Longo DL, Lim B, et al. Linking-catenin to androgen-signaling pathway*. J Biol Chem. 2002; 277:11336–44. doi: 10.1074/jbc.M111962200 Chesire DR, Isaacs WB. Ligand-dependent inhibition of beta-catenin/TCF signaling by androgen receptor. Oncogene. 2002; 21:8453–69. doi: 10.1038/sj.onc.120604 Kitagawa T, Matsuda K, Inui S, Takenaka H, Katoh N, Itami S, et al. Keratinocyte growth inhibition through the modification of Wnt signaling by androgen in balding dermal papilla cells. J Clin Endocrinol Metab. 2009; 94:1288–94. doi: 10.1210/jc.2008-1053 Kretzschmar K, Cottle DL, Schweiger PJ, Watt FM. The androgen receptor antagonizes wnt/β-catenin signaling in epidermal stem cells. J Invest Dermatol. 2015; 135:2753–63. doi: 10.1038/jid.2015.242 Premanand A, Rajkumari BR. In silico analysis of gene expression data from bald frontal and haired occipital scalp to identify candidate genes in male androgenetic alopecia. Arch Dermatol Res. 2019; 311:815–24. doi: 10.1007/s00403-019-01973-2 Kwack MH, Sung YK, Chung EJ, Im SU, Ahn JS, Kim MK, et al. Dihydrotestosterone-inducible dickkopf 1 from balding dermal papilla cells causes apoptosis in follicular keratinocytes. J Invest Dermatol . 2008; 128:262–9. doi: 10.1038/sj.jid.5700999 Mahmoud EA, Elgarhy LH, Hasby EA, Mohammad L. Dickkopf-1 expression in androgenetic alopecia and alopecia areata in male patients. Am J Dermatopathol . 2019; 41:122–7. doi: 10.1097/DAD.0000000000001266 Rossi A, Caro G, Magri F, Fortuna MC, Carlesimo M. Clinical aspect, pathogenesis and therapy options of alopecia induced by hormonal therapy for breast cancer. Explor Target Antitumor Ther. 2021;2(5):490-495. doi: 10.37349/etat.2021.00059 Botchkareva NV, Ahluwalia G, Shander D. Apoptosis in the hair follicle. J Invest Dermatol. 2006;126(2):258–64. Mikkola ML. TNF superfamily in skin appendage development. Cytokine Growth Factor Rev. 2008;19(3–4):219–30. Philpott MP, Sanders DA, Bowen J, Kealey T. Effects of interleukins, colony-stimulating factor and tumor necrosis factor-á in alopecia areata . Br J Dermatol. 1996; 135:942–8. Hoffmann R, Eicheler W, Huth A, Wenzel E, Happle R. Cytokines and growth factors influence hair growth in vitro: Possible implications for the pathogenesis and treatment of alopecia areata. Arch Dermatol Res . 1996; 288:153–6. Kasumagic-Halilovic E, Prohic A, Cavaljuga S. Tumor necrosis factor-alpha in patients with alopecia areata. Indian J Dermatol . 2011 Sep-Oct;56(5):494-6. doi: 10.4103/0019-5154.87124. Lis A, Pierzchala E, Brzezinska-Wcislo L. The role of cell-mediated immune response in pathogenesis of alopecia areata. Wiad Lek . 2001; 54:159–63. Koubanova A, Gadjlgoroeva A. EHRS Brussels, Conference abstracts. 2002. Jul 27, 29, On the problem of pathogenetic heterogeneity of alopecia areata; p. 13. Chopra DP, Menard RE, Januszewski J, Mattingly RR. 2004. TNF-alpha mediated apoptosis in normal human prostate epithelial cells and tumor cell lines. Cancer Lett 203:145–154. Harada S, Keller ET, Fujimoto N, Koshida K, Namiki M, et al. 2001. Long-term exposure of tumor necrosis factor alpha causes hypersensitivity to androgen and anti-androgen withdrawal phenomenon in LNCaP prostate cancer cells. Prostate 46:319–326. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6905198","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":502027225,"identity":"a347fc8b-74b7-42af-a871-34e33aaf08e0","order_by":0,"name":"Shabnam Feshangchian-Atashbar","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shabnam","middleName":"","lastName":"Feshangchian-Atashbar","suffix":""},{"id":502027228,"identity":"49fbdf71-9349-4c89-a29a-10e13d943ebb","order_by":1,"name":"Ali Ghamari","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Ghamari","suffix":""},{"id":502027230,"identity":"6e17630e-e0c1-4364-bebb-6e8c64d5643a","order_by":2,"name":"Sahel Jafarnejad-Komachali","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sahel","middleName":"","lastName":"Jafarnejad-Komachali","suffix":""},{"id":502027231,"identity":"8a9e6958-cbf3-4cd1-ac56-ef6dccedcddc","order_by":3,"name":"Mahsa Azhdari","email":"","orcid":"","institution":"Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Mahsa","middleName":"","lastName":"Azhdari","suffix":""},{"id":502027232,"identity":"dfe30f56-b550-4635-943a-85dfcf36f473","order_by":4,"name":"Alireza Shoae-Hassani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYDACdsYGMG0AIj4AMRs7IS3MSFoYZ4C0MBPUAqVBWph5kEVwAf5m5rYPH/7Y2ZuzNz98bPNrmzwfMwPjh485uLVIHGZsnjmDJzlxZ88xY+PcvtuGbcwMzJIzt+GxBqiFmUeCOcHgRoKZdG7PbUagFjZmXjxa5EFa/hjU2xvcSP/+27Lntj1BLQYgLQwJhxk33MgxY2b4cTuRoBZDoBbGngPHEzecOVMs2dtwO7mNmbEZr1/kjrc/Zvjxp9re4Hj7xg8//ty2nd/efPDDR3zeRwGMbWCygVj1IPCHFMWjYBSMglEwUgAAWWxPHnfm3a8AAAAASUVORK5CYII=","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Alireza","middleName":"","lastName":"Shoae-Hassani","suffix":""}],"badges":[],"createdAt":"2025-06-16 11:38:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6905198/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6905198/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89983890,"identity":"1cda926f-ec8f-4dec-9dbe-5d8edd3d9d67","added_by":"auto","created_at":"2025-08-27 06:35:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":120407,"visible":true,"origin":"","legend":"\u003cp\u003eThe levels of mRNA expression of isozymes 5α-Reductase I (A), 5α-Reductase II (B), 5α-Reductase III (C), and aromatase (D) in anagen hairs from both groups. The 5α-reductases mRNA levels were significantly higher in group 1 patients with telogen effluvium than in group 2 patients (\u003cem\u003ep = 0.003***\u003c/em\u003e). Higher mean mRNA levels of 5α-Reductase III were also observed in this group, although the difference did not reach statistical significance. The aromatase mRNA levels significantly differ between the two groups. Data are expressed as means ± SD.\u003c/p\u003e","description":"","filename":"PRPTNFManscriptImages1.png","url":"https://assets-eu.researchsquare.com/files/rs-6905198/v1/9cf6af48a5e98468ae0db95c.png"},{"id":89983892,"identity":"3d5353e7-d44b-4d1f-b9ba-843febecce64","added_by":"auto","created_at":"2025-08-27 06:35:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":140916,"visible":true,"origin":"","legend":"\u003cp\u003eThe concentrations of DHT levels in DPCs were analyzed by ELISA and found its levels in group 1 of patients exceed the majority of patients in group 2. The DHT concentrations in DPCs were elevated in 21 of 30 patients in group 1, and 6 of the 20 patients in group 2.\u003cstrong\u003e \u003c/strong\u003eElevated levels of DHT in DPCs correlate with the progression of telogen effluvium following PRP (A). Data are expressed as means ± SD (\u003cem\u003ep \u0026lt; 0.05**\u003c/em\u003e). The expression of the androgen receptor was detected by Western Blot, quantified and standardized to the expression of GAPDH. Hair follicles from the vertex areas of the scalp express large quantities of AR especially in patients with teloptosis after PRP (B). The western blot image of ARs in both group of patients and GAPDH as the control. The grouping of blots cropped from two parts of two gels (C).\u003c/p\u003e","description":"","filename":"PRPTNFManscriptImages2.png","url":"https://assets-eu.researchsquare.com/files/rs-6905198/v1/fdca65adccd90468e8947a01.png"},{"id":107303540,"identity":"23ef9d13-f725-4973-8ae8-0ec034ee26bb","added_by":"auto","created_at":"2026-04-20 07:57:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1384386,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6905198/v1/d0f95b06-f64d-44f5-80a9-43406d1a4601.pdf"},{"id":89983894,"identity":"c241c9c1-99ed-4c99-bffd-e114cb22896d","added_by":"auto","created_at":"2025-08-27 06:35:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":261378,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6905198/v1/74c9d5002501345ed1567d5f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Increased Incidence of Telogen Effluvium Following Platelet-Rich Plasma Therapy in Patients with Elevated TNF-α Expression: A Prospective Cohort Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAndrogenetic alopecia (AGA) the most common alopecia, affects a large proportion of both men and women population (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It is a chronic, age-related disorder specified by a reduction in hair length and diameter mainly located on the central scalp with varying baldness patterns (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). AGA is influenced by hormones, particularly dihydrotestosterone (DHT), a testosterone (T) metabolite that activates androgen receptors (ARs). In men, testosterone is converted to DHT by 5α-reductase, while in women, dehydroepiandrosterone (DHEA) and other androgens serve as DHT precursors (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). 5α-reductase expression is higher in the dermal papilla cells (DPCs) than in epithelial cells (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Aromatase, which breaks the DHT structure, is expressed in the outer root sheath during the anagen phase (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Hair follicles (HFs) in the vortex and frontotemporal scalp areas have increased ARs, making them more responsive to DHT, stimulating premature termination of the anagen growth phase in HFs, and leading to increased hair loss (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Circulating DHT levels have less importance than T for optimizing the intracellular DHT concentrations because of local regulatory mechanisms that carefully regulate intracellular levels of androgens (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe standard treatments for AGA involve a spectrum of medications including finasteride, dutasteride, ketoconazole, prostaglandin analogs, hormonal therapy, and topical minoxidil. However, these treatments may not work for all AGA-suffering individuals (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). These drugs need to be taken indefinitely, and treatment effectiveness is often hindered by patient compliance.\u003c/p\u003e\u003cp\u003ePlatelet-rich plasma (PRP) has emerged as an autologous, minimally invasive treatment for various dermatologic conditions, particularly alopecia. Its efficacy in stimulating hair regrowth has been attributed to the local delivery of growth factors such as PDGF, VEGF, IGF-1, and TGF-β, which promote follicular angiogenesis, stem cell activation, and dermal papilla survival (\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Overall, these proteins control cellular movements, adhesion, growth, specialization, and also accumulation of extracellular matrix (ECM) (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). By binding to the specific receptors on DPCs in the bulge area of HFs, the PRP growth factors stimulate hair regrowth when enter the anagen phase, creating the follicular unit and promoting hair regeneration (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Then PRP induces the proliferation of DPCs by upregulating FGF7 and β-catenin, extracellular-signal\u0026ndash;regulated kinase, and Akt signaling (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, an increasing number of clinicians report post-treatment telogen effluvium (TE), particularly in individuals with chronic or diffuse hair loss (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Also, there have been patients who developed telogen effluvium with psoriasiform scalp dermatitis four weeks after being treated with PRP (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). While factors such as systemic illness, medications, or hormonal fluctuations are common triggers, recent studies have implicated inflammatory cytokines. This study explores the hypothesis that the levels of blood proinflammatory and inflammatory cytokines, DHT levels, its receptors activity, and enzymatic conversion may predispose patients to TE following PRP.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cb\u003ePatients Criteria\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA prospective, single-center cohort study was conducted on 30 patients with TE. All patients (age range: 30\u0026ndash;65) with male pattern hair loss provided written informed consent before participating in the intervention. All the experiments and interventions were placed in the Motahari Hospital, Stem Cell and Regenerative Innovation Center, Iran University of Medical Sciences (IUMS). All experimental protocols were approved by IUMS research ethics committee by approval code 1402.1080. All methods were performed in accordance with the relevant guidelines and regulations of Iranian Ministry of Health research and technology board. Thirty PRP-administrated patients in the last 12 months with telogen effluvium outcomes entered into the study (Group 1). Other twenty male persons with AGA and favored PRP results served as control (Group 2). All the selected patients have stage III to V patterns of hair loss according to the Norwood Hamilton classification. Patients, who had received topical minoxidil, prostaglandin, retinoids, and corticosteroids or systemic medications such as finasteride, dutasteride, and anti-androgens in the previous 12 months were excluded. Only patients with normal blood parameters in CBC were included in the study.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePRP Preparation and administration\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo prepare PRP, blood was taken from a peripheral brachial vein using ACDA-1 (BD, UK) as an anticoagulant. The first step of centrifugation was done at 260 \u003cem\u003eg\u003c/em\u003e for 8 min. After discarding RBC, the second step of centrifugation was performed in 1100 \u003cem\u003eg\u003c/em\u003e for 4 minutes. The platelets were harvested and the growth factors are secreted once platelet activation begins with calcium gluconate. In addition, the numbers of platelets in PRP obtained from all participants were counted. The PRP was Intradermally injected on selected areas of the scalp at the amount of 0.1 mL/cm\u003csup\u003e2\u003c/sup\u003e using the nappage technique which multiple small injections in a linear pattern 1 cm apart to a depth of 1\u0026ndash;2 mm. In our procedure, anesthesia is achieved with nerve block with lidocaine 1% with epinephrine. A portion of the PRP sample was subjected to immunoassay examination.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTelogen Effluvium Evaluation criteria\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll patients were evaluated at the beginning of the study up to six months after the PRP administration. Assessment of the treatment was conducted in all patients with the standardized photo-trichograms in combination with the TrichoScan to measure hair count (number of hairs/0.65 cm\u003csup\u003e2\u003c/sup\u003e), hair density (number of hairs/cm\u003csup\u003e2\u003c/sup\u003e), hair diameter, anagen/telogen ratio, and vellus hair/terminal hair ratio. All hairs with a diameter larger than 40 \u003cem\u003e\u0026micro;\u003c/em\u003em were categorized as terminal hairs, and those with lesser diameter were vellus hair.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMultiplex Immunoassay of cytokines in PRP\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter the PRP preparation, one mL PRP was activated through a double freeze-thaw process from each patient. The cytokine concentration levels were evaluated with the MILLIPLEX Human cytokine magnetic bead-based panel kit (Millipore, Darmstadt, Germany). A total of 14 targeted proinflammatory cytokines and growth factors including the bFGF, EGF, HGF, interferon alpha 2 (IFNα2), IL-6, IL-8, IL-10, IL-17, IL-23, PDGF, TGF-β1, TNF-α, and VEGF were analyzed by the manufacturer\u0026rsquo;s instructions. Briefly, 20 \u0026micro;L buffer, 30 \u0026micro;L PRP, and 30 \u0026micro;L beads were added into a 96-well plate and were incubated for 12 hours at 4 ◦C, followed by washing twice with 200 \u0026micro;L washing buffer. An amount of 20 \u0026micro;L of antibody of each target was added into the wells, and the plates were incubated on a shaker plate for 1 h. Thirty \u0026micro;L of streptavidin-phycoerythrin solution was added into each well and incubated in the dark for 30 minutes. The wells contents were washed two times with 200 \u0026micro;L washing buffer. At the end 150 \u0026micro;L of sheath fluid was added into the wells, and were evaluated using the MAGPIX instrument. The median fluorescence intensities (MFI) of all cytokines were analyzed to calculate the final concentrations in each PRP sample.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHair Follicles Isolation and Processing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll scalp specimens were processed as follows. Approximately ten hair follicles were obtained from the vortex area of a patient. All samples were taken in identical conditions by our hair transplant physician. Anagen hair follicles used for the processing show longer shafts with a consistent diameter, a rectangular shape, a slight distal angle, and darkly pigmented triangular or delta-shaped bulbs, featuring an angle to the hair shaft and an inner root sheath. The follicles were dissected and stored at -80\u0026deg;C until subjected to RNA extraction.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA isolation from HFs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRNA isolation was performed as described previously (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The samples were homogenized in Trizol (Invitrogen, US). The samples were homogenized using a vortex to ensure complete lysis and release of RNA. Total RNA was isolated using chloroform (0.2 mL per 1 mL of lysis buffer) and then precipitated with isopropanol through centrifugation at 12000 \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C. The upper aqueous phase contains RNA, the interphase contains DNA, and the organic phase contains proteins. The aqueous phase carefully transferred to a new tube. Then isopropanol (0.5 mL for every 1 mL of the aqueous phase) was added to precipitate the RNA. The RNA pellet underwent two washes with 75% ethanol and was then reconstituted in DEPC-treated water. DNase (Ambion) was added to eliminate any genomic DNA contamination. The quantity of RNA obtained was determined by spectrophotometer based on the A260/A280 ratio. The integrity of the isolated total RNA was confirmed through electrophoresis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReverse transcription and quantitative real‑time PCR\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe cDNA was synthesized from 1\u0026micro;g of total RNA as described previously (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Absolute quantification of mRNA of 5α-Reductase isozymes I, II, III, and aromatase in the hair follicles was evaluated by real-time RT-PCR using the Rotogen (Kiagen\u0026trade;, Germany) Real-time PCR system with SYBR Green PCR Master Mix (Promega). The final concentration used in the reactions was 0.5 \u0026micro;M for both the forward and reverse primers. Twenty microliters of the prepared PCR master mix were added to each well of the reaction plate. Three microliters of each reverse-transcribed RNA sample were added to the appropriate well to make a final reaction volume of 25 \u0026micro;l. The PCR protocol was as follows: denaturation at 94\u0026deg;C for 30s; annealing at 55\u0026deg;C for the 5α-Reductase I, 54\u0026deg;C for the 5α-Reductase I, 60\u0026deg;C for the 5α-Reductase III, 60\u0026deg;C for the aromatase gene for 30 s; and extension at 72\u0026deg;C for 30 s. The number of cycles was 35 in all cases. At the end of the amplification phase, a melting curve analysis was carried out. All reactions were run in triplicate and no cDNA was added to the negative reactions. The primer sequences are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer sequences for 5α-Reductase isozymes and aromatase used in PCR amplification of harvested hair follicle samples.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSequence (5\u0026prime;-3\u0026prime;)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGenBank Access no.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5α-Reductase I\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-AGCCATTGTGCAGTGTATGC-\u003c/p\u003e\u003cp\u003e-AGCCTCCCCTTGGTATTTTG-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNM_001047.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5α-Reductase II\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-TGAATACCCTGATGGGTGG-\u003c/p\u003e\u003cp\u003e-CAAGCCACCTTGTGGAATC-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNM_000348.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5α-Reductase III\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-TCCTTCTTTGCCCAAACATC-\u003c/p\u003e\u003cp\u003e-CTGATGCTCTCCCTTTACGC-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNM_024592.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAromatase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-TATTAGGGCCCTGTGTCTGC-\u003c/p\u003e\u003cp\u003e-TGGGTTGGGACTTTTCCTCC-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNM_000103.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern Blot Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDPCs were lysed in the lysis buffer supplemented with 1% protease inhibitor (Roche, Germany) and incubated on ice. Cell lysates were centrifuged at 5,000 \u003cem\u003eg\u003c/em\u003e for 15 min at 4\u0026deg;C. Proteins (50 \u0026micro;g) in whole-cell lysates were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to the polyvinylidene difluoride membranes (Biocompare, US). After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with corresponding anti-AR primary antibody (1:500; Santa Cruz, USA), overnight at 4 ℃. On the next day, it was incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, UK) for 1 h at room temperature. The quantification of AR bands was conducted by densitometry using ImageJ software (US National Institutes of Health, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnzyme linked immunosorbent Assay (ELISA)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor the quantification of DHT levels in DPCs of both groups, the Human Dihydrotestosterone ELISA Kit (ab283979; Abcam, UK) was used, according to the supplier\u0026rsquo;s instruction. Patients DPCs were preserved at \u0026minus;\u0026thinsp;80\u0026deg;C, thawed, and centrifuged at 3500 g for 5 min. The supernatants were used for the ELISA assay. Briefly, 100 \u0026micro;L/well of a diluted sample was dispensed in each well and incubated 1 hour at room temperature. The samples were washed six times with washing buffer (300 \u0026micro;L/well). Then 100 \u0026micro;L/well of diluted enzyme-conjugated secondary antibody added to the solution and incubated for 1 hour in room temperature. It was washed 6 times with washing buffer and incubated with chromogen up to 15 minutes in the dark to develop the color. Finaly, the absorbance value at 450 nm was measured. DHT concentrations were measured from two groups of patients.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe non-parametric Mann\u0026ndash;Whitney U test was used for comparisons. The Shapiro-Wilk test was used to analyze the compliance of the distribution of the analyzed variables with normal distribution. GraphPad Prism 7.0 software (San Diego, CA) was used for the statistical analysis. p\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eDuring the period from April 2022 to December 2023, thirty patients have complained of the previous PRP outcomes as the telogen effluvium (Group 1). The remaining patients were individuals who regularly underwent annual PRP treatments and showed enhancements in hair quality parameters. The median age of the patients included was 50.8 years (\u0026plusmn;\u0026thinsp;14.2 years).\u003c/p\u003e\u003cp\u003eThe blood counts of all participants in both groups are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The data shows a normal count for everyone. PRP was characterized according to platelet counts and growth factor concentrations. The median number of platelets was increased 4 to 5-fold, with a minimum of 1152.5 and a maximum of 2582 \u0026times;10\u003csup\u003e3\u003c/sup\u003e \u0026micro;L (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The age and platelet count in the PRP were not significantly different between the patients with successful and unsuccessful outcomes (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Age was not significantly correlated with any cytokine concentrations in both groups of patients.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eClinical parameters in the AGA patients\u0026rsquo; blood from both groups were included in the study\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePRP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(Group 1; n\u0026thinsp;=\u0026thinsp;30)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(Group 2; n\u0026thinsp;=\u0026thinsp;20)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge (Years Old)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e46.5\u0026thinsp;\u0026plusmn;\u0026thinsp;16.5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e49.5\u0026thinsp;\u0026plusmn;\u0026thinsp;14.5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.643\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlatelet count (\u0026times;10\u003csup\u003e3\u003c/sup\u003e in \u0026micro;L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1867\u0026thinsp;\u0026plusmn;\u0026thinsp;715.48\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1952\u0026thinsp;\u0026plusmn;\u0026thinsp;720.25\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003e\u0026le;\u0026thinsp;0.05**\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRBC (10\u003c/b\u003e\u003csup\u003e\u003cb\u003e6\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e/\u0026micro;L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e4.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e4.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.002***\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eWBC (10\u003c/b\u003e\u003csup\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e/\u0026micro;L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e6.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e6.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNeutrophils (10\u003c/b\u003e\u003csup\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e/\u0026micro;L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e3.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e3.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLymphocytes (10\u003c/b\u003e\u003csup\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e/\u0026micro;L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e2.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e2.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMonocytes (10\u003c/b\u003e\u003csup\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e/\u0026micro;L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eEosinophils (10\u003c/b\u003e\u003csup\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e/\u0026micro;L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBasophils (10\u003c/b\u003e\u003csup\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e/\u0026micro;L)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cb\u003eRBC: Red blood cells; PLT: Platelets; WBC: White blood cells\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe cytokines and growth factors from 2 groups were measured. There was a variation between the expression levels of some agents in these 2 groups, with a mean coefficient of variability of 9.95% for bFGF, 5.89% for HGF, 22.64% for TGFβ and 5.8% for VEGF (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Also, the proinflammatory cytokines have significant differences between the two groups. There was variability of 13.41% for IFNα, 15.48% for IL-6, 13.74% for IL-10, 12.15% for IL-17, 25% for IL-23, and finally 188% for TNFα (\u003cem\u003ep\u0026thinsp;\u0026le;\u0026thinsp;0.05**\u003c/em\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Among all the 14 targeted GFs and cytokines in PRPs, the level of TNF-α was dramatically higher in patients with unsuccessful outcomes (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.05**\u003c/em\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCytokine and Growth factor expression levels in both groups of patients PRPs included in the study.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePatients PRP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroup 1 (n\u0026thinsp;=\u0026thinsp;30)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGroup 2 (n\u0026thinsp;=\u0026thinsp;20)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCytokines (pg/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEGF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e421.90\u0026thinsp;\u0026plusmn;\u0026thinsp;336.10\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e413.12\u0026thinsp;\u0026plusmn;\u0026thinsp;347.55\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cem\u003e\u0026le;\u0026thinsp;0.5*\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ebFGF\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e97.95\u0026thinsp;\u0026plusmn;\u0026thinsp;24.89\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e108.77\u0026thinsp;\u0026plusmn;\u0026thinsp;34.58\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHGF\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e483.12\u0026thinsp;\u0026plusmn;\u0026thinsp;67.55\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e513.32\u0026thinsp;\u0026plusmn;\u0026thinsp;48.55\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIFNα\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e16.49\u0026thinsp;\u0026plusmn;\u0026thinsp;4.22\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e14.28\u0026thinsp;\u0026plusmn;\u0026thinsp;3.19\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.5*\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIL-6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e12.28\u0026thinsp;\u0026plusmn;\u0026thinsp;4.19\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e10.38\u0026thinsp;\u0026plusmn;\u0026thinsp;5.49\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIL-8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e818.35\u0026thinsp;\u0026plusmn;\u0026thinsp;75.50\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e705.63\u0026thinsp;\u0026plusmn;\u0026thinsp;68.60\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIL-10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e12.72\u0026thinsp;\u0026plusmn;\u0026thinsp;4.62\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e10.45\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIL-17\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e9.22\u0026thinsp;\u0026plusmn;\u0026thinsp;3.15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e8.10\u0026thinsp;\u0026plusmn;\u0026thinsp;3.55\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIL-23\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e14.88\u0026thinsp;\u0026plusmn;\u0026thinsp;3.95\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e11.16\u0026thinsp;\u0026plusmn;\u0026thinsp;4.10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePDGF-A A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e6511.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1661.25\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e6650.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1677.45\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;\u0026thinsp;0.5*\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePDGF-AB/BB\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e17700.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4200.50\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e16400.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4375.00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTGF β1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e56.28\u0026thinsp;\u0026plusmn;\u0026thinsp;31.14\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e72.76\u0026thinsp;\u0026plusmn;\u0026thinsp;30.16\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTNFα\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e64.47\u0026thinsp;\u0026plusmn;\u0026thinsp;6.76\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e34.18\u0026thinsp;\u0026plusmn;\u0026thinsp;8.15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e= 0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eVEGF\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e288.20\u0026thinsp;\u0026plusmn;\u0026thinsp;38.5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e305.33\u0026thinsp;\u0026plusmn;\u0026thinsp;28.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026le;0.05**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003eEGF: epidermal growth factor; bFGF: basic fibroblast growth factor; HGF: Hepatocyte growth factor; IFNα2: interferon alpha 2; Il: interleukin; PDGF: platelet-derived growth factor; TNF-α: tumor necrosis factor-α; TGF-β: transforming growth factor-β; VEGF: vascular endothelial growth factor.\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe 5α-Reductase I and II mRNA levels were significantly higher in group 1 patients versus group 2 (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.003***\u003c/em\u003e) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e. Higher mean mRNA levels of 5α-Reductase III were also observed in this group, although the difference did not reach statistical significance \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. The aromatase mRNA levels \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e significantly differ between the two groups. Nonetheless, there was notable variability observed in the results for aromatase expression among group 1 patients \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe DHT concentrations in DPCs were elevated in 21 of 30 patients in group 1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, and 6 of the 20 patients in group 2 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e Differences in mean DHT concentration in DPCs of group 1 were statistically significant relative to group 2. Elevated levels of DHT in DPCs correlate with the progression of telogen effluvium following PRP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe expression of the androgen receptor was detected by Western blot \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The levels of AR are standardized to the expression of GAPDH. Hair follicles from the vertex areas of the scalp express large quantities of AR especially in patients with teloptosis after PRP \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eAfter PRP therapy, all patients had mild effects in the form of transient erythema, immediately after injection. None of the patients experienced any serious adverse effects. All patients in two groups were followed up to six months for evaluation of outcomes. PRP efficacy was evaluated using various metrics such as photography, mean hair count and density, and anagen-to-telogen ratio. At the baseline, the patients in group 1 presented higher hair density in comparison with 6 months after PRP therapy (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and this difference was statistically significant (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.040\u003c/em\u003e**). The results showed a decrease in anagen, an increase in telogen, and decreased anagen/telogen ratio (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.005***\u003c/em\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These patients have nearly twice-fold TNFα (\u003cem\u003ep\u0026thinsp;\u0026le;\u0026thinsp;0.05**\u003c/em\u003e) concentrations in their PRP. Regarding hair count, the patients in group 2 showed a significant increase when the counts at baseline and 6 months after the PRP administration were compared (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.037**\u003c/em\u003e). The improvement was observed in 60% of patients with an increase in anagen/telogen ratio (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eClinical parameters in the PRP-treated AGA patients in both groups at baseline and six months.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eGroup 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eGroup 2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ebaseline\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6 months\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ebaseline\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6 months\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHair Diameter (mm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.048\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.042\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.049\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.059\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHair Count (n/cm2)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e116.65\u0026thinsp;\u0026plusmn;\u0026thinsp;11.95\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e101.35\u0026thinsp;\u0026plusmn;\u0026thinsp;14.45\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e121.35\u0026thinsp;\u0026plusmn;\u0026thinsp;15.45\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e168.80\u0026thinsp;\u0026plusmn;\u0026thinsp;12.65\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.05\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eabsolute anagen counts\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e71.15\u0026thinsp;\u0026plusmn;\u0026thinsp;11.15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e54.95\u0026thinsp;\u0026plusmn;\u0026thinsp;08.35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e91.55\u0026thinsp;\u0026plusmn;\u0026thinsp;10.45\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e122.50\u0026thinsp;\u0026plusmn;\u0026thinsp;11.75\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.05\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eChange from baseline\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e45.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e46.40\u0026thinsp;\u0026plusmn;\u0026thinsp;6.10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e29.80\u0026thinsp;\u0026plusmn;\u0026thinsp;5.00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e46.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.05\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides the first clinical evidence linking elevated TNF-α levels to increased risk of PRP-induced telogen effluvium. Platelet rich plasma therapy has been utilized for more than a decade in the treatment of AGA. Leukocyte rich PRP (L-PRP) is commonly employed in the treatment of dermatologic indications (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Nevertheless, elevated levels of pro-inflammatory cytokines in L-PRP could trigger catabolic and inflammatory responses that may counteract the positive impact of growth factors on target tissue regeneration.\u003c/p\u003e\u003cp\u003eIn this study, we opted to prepare PRP using the manual double-spin technique which has a large quantity of leukocytes with the potential of producing proinflammatory cytokines like many clinics do (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Telogen effluvium can be a concerning issue, on the other hand, it's promising that the other 20 patients, who had regular annual PRP treatments, showed improvements in their hair parameters quality (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Undoubtedly the levels of growth factors and cytokines within PRP could impact the effectiveness of treatment and serve as a potential indicator of treatment success in clinical settings. The blood counts of all participants in our study, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, indicate normal values across the board. Our data also reveals that age and platelet count in the PRP did not show significant differences between patients with successful and unsuccessful outcomes. This suggests that while platelet count is important, there may be other factors at play influencing the efficacy of PRP treatment. We observed variations in the expression levels of some cytokines. The bFGF showed a mean coefficient of variability of 9.95%, and TGFβ at 22.64%. These differences could indicate distinct biological responses in each group. The variability in TGFβ plays a significant role between the two groups and their biological responses (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In pro-inflammatory cytokines expression, the variability was 13.41% for IFNα, 15.48% for IL-6, 13.74% for IL-10, 12.15% for IL-17, 25% for IL-23, and an astonishing 188% for TNFα (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The increased expression of IL-17, IL-23, and TNF-α, in PRPs of patients who develop telogen effluvium following PRP therapy was significant \u003cem\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e\u003cp\u003eTNF-α is a powerful pro-inflammatory cytokine that plays a crucial role in coordinating the cytokine cascades in inflammations (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Studies showed transient inflammations cause migration of mast cells and secretion of a variety of IL-4, IL-8, IL-13, IL-31, proteases (cathepsin S), prostaglandins, and platelet activating factor which can activate other cytokine cascades that affect HF cell cycle arrest (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Some studies show that pro-inflammatory cytokines inhibit the growth of isolated HFs in \u003cem\u003ein vitro\u003c/em\u003e cultures (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The processes that control the hair growth cycles are situated within the HFs and are believed to be impacted by changes in the expression of specific regulatory molecules inside the follicles (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Various studies have emphasized the importance of microinflammation within the microenvironment of HFs in the development of AGA (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Microinflammation is a slow, subtle, and enduring process, contrasting with the rapid, inflammatory, and destructive process observed in classical inflammatory scarring alopecia (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In 2000, Mahe and colleagues revealed that HFs adjacent keratinocytes, which express receptors for IL-1, engage the expression of IL-1 responsive genes which include mRNA coding for TNFα (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Also, specific chemokine genes, such as IL-8, monocyte chemoattractant protein-1 (MCP-1), and MCP-3 are overexpressed, and their expression recruits\u0026rsquo; neutrophils and macrophages (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) that strengthen production of TNFα. Moreover, neighboring fibroblasts are well-prepared to react to such pro-inflammatory signals. The increase in adhesion molecules on the capillary endothelium for circulating cells, along with the chemokine gradient, facilitates the movement of inflammatory cells, such as neutrophils via IL-8, T lymphocytes, and Langerhans cells partly due to MCP-1 action, across the endothelium. The outcome of these signals is the induction of teloptosis in the HFs cycles. In 1997, a study by Thein \u003cem\u003eet al\u003c/em\u003e. surveyed the cytokine profiles of infiltrating T-lymphocytes from the margin of involved alopecia areata lesions (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). It was found that T-lymphocytes inhibited the proliferation of neonatal keratinocytes by releasing high amounts of IFN-γ and/or TNF-α (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe Wnt/β-catenin signaling pathway plays a crucial role in both the development of HFs and the stimulation of hair growth (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Subsequent studies aimed to uncover the specific functions of Wnt/β-catenin signaling in different cell types within the HFs. It was found that removing β-catenin in DPCs in mature HFs led to a decrease in the proliferation of stem cells responsible for generating the matrix keratinocytes in the anagen bulb. As a result, hair becomes thinner and shorter, and a premature transition from the anagen to catagen phase occurs (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Other studies demonstrated that when Wnt ligand secretion was specifically blocked in the epidermal part of the HFs during the resting telogen phase, it impeded the transition to the anagen phase and caused the hair cycle to halt (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). On the other hand, ARs interact with β-catenin and use β-catenin as a co-factor to amplify the transcription of AR target genes (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). We noticed that hair follicles from the vertex areas of the scalp express large quantities of AR, particularly in patients with teloptosis after PRP treatment, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. This is an obvious link between AR expression and hair loss in these patients. It can be inferred that DHT is involved in the development of AGA by disrupting the hair follicle's β-catenin signaling pathway (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). This is supported by recent microarray gene expression data, which revealed a significant downregulation of Wnt/β-catenin signaling pathway genes in the bald frontal scalps compared to the haired occipital scalps of male patients with AGA (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). The results from our study indicate that DHT concentrations in dermal papilla cells (DPCs) were notably higher in a majority of patients in group 1 compared to group 2, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. This is a potential association between elevated DHT levels in DPCs and the development of telogen effluvium following PRP treatment. Also, the T to DHT converter isozymes, the 5α-Reductase I and II mRNA levels were significantly higher in group 1 patients compared to group 2, with a p-value of 0.003 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). It suggests that there may be a dysregulation of the 5α-Reductase I and II genes in group 1 patients. We also found higher mean mRNA levels of 5α-Reductase III in group 1 patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), although this difference was not statistically significant. It's possible that with a larger sample size, we may have been able to detect a significant difference in the expression of 5α-Reductase III as well. Dickkopf-related protein 1 (DKK1) is an inhibitor of the Wnt/β-catenin pathway that is overexpressed in bald areas of the scalp (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). The expression levels of DKK1 correlate with a reduction in anagen phase of HFs (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). In 2008, Kwack \u003cem\u003eet al.\u003c/em\u003e, demonstrated that DHT induces the expression of DKK1 in DPCs, which results in apoptosis in follicular keratinocytes (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAnother factor in hair loss depends on the aromatase which is found in the outer root sheath that converts androstenedione into estrone and testosterone into estradiol, leading to the reduction in T and DHT levels (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). The aromatase mRNA levels in our study clearly show a significant decrease in group 1 patients, as indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e studies confirmed that TNF-α has a key role in the induction of HF apoptosis (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). In 2008, a study by Mikkol reported that HF apoptosis by TNF-α is a more important factor in hair loss than the interaction of DHT with AR (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). More studies have revealed that TNF-α causes vacuolation of matrix cells, and abnormal keratinization of the inner root sheath. This phenomenon disrupts follicular melanocytes in the DPs (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). The data from Hoffmann \u003cem\u003eet al\u003c/em\u003e. experiments in cultures of HFs showed that TNF-α completely affects hair growth by induction of the formation of club-like HFs, similar to their morphology in the catagen phase (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur data also indicated a shift in hair growth phases, with a decrease in anagen and an increase in telogen phases, leading to a decreased anagen/telogen ratio (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.005\u003c/em\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Additionally, the patients in this group exhibited nearly twice-fold TNFα concentrations in their PRP samples, which could be a potential factor affecting their hair growth outcomes. On the other hand, patients in group 2 showed a noticeable increase in hair count when comparing baseline levels to those at six months post-PRP treatment, with a significant difference \u003cem\u003e(P\u0026thinsp;=\u0026thinsp;0.037\u003c/em\u003e). Moreover, 60% of these patients demonstrated an improvement in their anagen-to-telogen ratio, indicating a positive response to the PRP therapy, as outlined in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. In contrast to our results, Teraki \u003cem\u003eet al.\u003c/em\u003e reported that levels of TNF-α in alopecia areata patients are very high. They showed that the expression of TNF-α was significantly higher than those in patients with alopecia universalis (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). In 2001, Lis \u003cem\u003eet al.\u003c/em\u003e found that TNF-α receptor type I was significantly elevated in patients with AA in comparison with the healthy control group (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Later, in a 2002 study, Koubanova and Gadjlgoroeva examined the levels of TNF-α in patients with AA. The results showed no difference between this group of patients and the control group (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). However, despite the controversy in these results, our finding demonstrated the consistency between higher TNFα concentration and teloptosis. It can be asserted that the occurrence of telogen effluvium after PRP therapy is a multifaceted process, with numerous molecular mechanisms. Studies showed that Binding of TNFα to its cell surface receptor (TNFR) leads to the activation of IKK, which phosphorylates and degrades IkB, an inhibitor of NF-kB. NF-kB then translocate to the nucleus and activates Transcription (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Androgen responsive cells are highly sensitive to TNF-α-induced arrest and apoptosis, where TNF-α did not induce phosphorylation of IkB (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Long-term exposure of TNFα induces androgen hypersensitivity in cells, which was associated with decreased levels of total AR, but\u003c/p\u003e\u003cp\u003eincreased nuclear AR levels (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Within this study, upregulation of AR expression, DHT levels, and 5a-reductase enzymes and downregulation of aromatase levels have been observed. These patients had elevated ranges of IL-17, IL-23, and TNF-α in their L-PRP. The potential exists to reduce this impact in these patients by substituting pure PRP with low leukocyte amounts instead of L-PRP.\u003c/p\u003e\u003cp\u003eIn conclusion, our current study revealed a novel finding: the concentration of TNF-α in PRP correlated with treatment effectiveness. Lower TNF-α levels could potentially predict improved therapeutic results of PRP for AGA treatment. The exclusion of leukocytes in PRP to create pure PRP is an endeavor to optimize therapeutic benefits for AGA patients with elevated TNF-α levels.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLimitations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe primary constraints of this study include a small sample size and the absence of a placebo control group. These factors can introduce several limitations, including limited generalizability, the risk of random variability, lack of precision and reliability, and limited exploration of heterogeneity. Also, this study was not a randomized, double-blind, controlled trial. The stability of cytokine levels in consecutive PRP treatments is still uncertain. We only included 14 types of cytokines in our analysis, and there may be additional cytokines that we have not yet studied that could also affect treatment outcomes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFuture Directions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFuture studies should continue examining the role of inflammatory cytokines and hair-promoting mechanisms of PRP and amount of growth factors contained within PRP. Additionally, the hair-promoting effects of PRP that are unrelated to growth factor concentrations and cycles related to TNFα family should still be explored.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eConflict of Interest:\u003cstrong\u003e\u0026nbsp;There is no conflict of interest.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The study was supported by Iran University of Medical Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' Contributions:\u003c/strong\u003e ASH and SFA visited, intervened, and followed up with the patients; SJ evaluated the criteria for telogen effluvium; ASH performed the biopsy; MA conducted the genetic tests; AG performed the multiplex immunoassay; SFA handled the preparation and administration of PRP; ASH designed the experiment, collected the final data, and wrote the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eAll data generated or analysed during this study are included in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKuczara A, Waśkiel-Burnat A, Rakowska A, Olszewska M, Rudnicka L. Trichoscopy of Androgenetic Alopecia: A Systematic Review. \u003cstrong\u003e\u003cem\u003eJ Clin Med.\u003c/em\u003e\u003c/strong\u003e 2024;13(7):1962. doi: 10.3390/jcm13071962.\u003c/li\u003e\n\u003cli\u003eVarothai S, Bergfeld WF: Androgenetic alopecia: an evidence-based treatment update. Am \u003cstrong\u003e\u003cem\u003eJ Clin Dermatol\u003c/em\u003e\u003c/strong\u003e 2014; 15:217-230.\u003c/li\u003e\n\u003cli\u003eAlessandrini A., Starace M., D\u0026rsquo;Ovidio R., Villa L., Rossi A., Stan T.R., Calzavara-Pinton P., Piraccini B.M. 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Long-term exposure of tumor necrosis factor alpha causes hypersensitivity to androgen and anti-androgen withdrawal phenomenon in LNCaP prostate cancer cells. \u003cstrong\u003e\u003cem\u003eProstate\u003c/em\u003e\u003c/strong\u003e 46:319\u0026ndash;326.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alopecia, PRP, Telogen effluvium, Teloptosis, Tumor Necrosis Factor","lastPublishedDoi":"10.21203/rs.3.rs-6905198/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6905198/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStimulation of hair regrowth with platelet-rich plasma (PRP) is a popular treatment for androgenic alopecia (AGA). However, a subset of patients experiences paradoxical telogen effluvium (TE) following treatment. To find the reason, we investigated the expression levels of inflammatory cytokines, dihydrotestosterone (DHT), its receptors, and related enzymes.\u003c/p\u003e\n\u003cp\u003eIn a prospective cohort study, we examined thirty male persons who complained of telogen effluvium following PRP administration in the last 12 months. The intervenors were tested with multiplex immunoassay for proinflammatory plasma cytokines, ELISA for Tissue DHT levels, western blot for androgen receptors (ARs), and RT-PCR for 5α-reductase isozymes and aromatase gene expression from the biopsy of dermal papilla cells (DPCs) of the vortex scalp.\u003c/p\u003e\n\u003cp\u003eTE occurred in patients have twice-fold TNFα (\u003cem\u003ep \u003c/em\u003e≤0.05) in their PRP, 40% higher levels of ARs (\u003cem\u003ep \u003c/em\u003e≤0.05), and significant differences in 5α-reductase gene expression (\u003cem\u003ep\u003c/em\u003e =0.03) in their DPCs. There are significant quantitative differences in the down-expression levels of the aromatase in the outer root sheath of the HFs in these patients.\u003c/p\u003e\n\u003cp\u003eWe should consider that elevated TNF-α expression is a significant risk factor for the development of TE following PRP therapy.\u003c/p\u003e","manuscriptTitle":"Increased Incidence of Telogen Effluvium Following Platelet-Rich Plasma Therapy in Patients with Elevated TNF-α Expression: A Prospective Cohort Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 06:34:55","doi":"10.21203/rs.3.rs-6905198/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fd9a2243-1ae7-470e-a5f0-1aa845b98b13","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53321774,"name":"Biological sciences/Immunology"},{"id":53321775,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-04-20T07:57:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-27 06:34:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6905198","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6905198","identity":"rs-6905198","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}