Efficacy of Botulinum Toxin A Versus Nanofat in the Treatment of Androgenetic Alopecia: A Comparative Clinical Trial

This randomized prospective comparative clinical trial will compare the efficacy and safety of intramuscular botulinum toxin type A injection with intradermal autologous nanofat injection for the treatment of male androgenetic alopecia. The study will be conducted at Assiut University Hospital and will include 40 male participants, with 20 participants allocated to each treatment group. The planned allocation ratio is 1:1.

Eligible participants will be men aged 18 to 40 years with a clinical diagnosis of androgenetic alopecia confirmed by history, scalp examination, and dermoscopy. Participants must have Norwood Hamilton grade II or III androgenetic alopecia and visible hair follicles within the target scalp area on dermoscopic examination.

Participants will be excluded if they have significant systemic conditions that could interfere with safety, wound healing, or hair growth, including neuromuscular disorders, uncontrolled diabetes mellitus, autoimmune disease, hematological disease, active malignancy, or recent chemotherapy. Other exclusion criteria include active scalp infection or inflammation, dermatitis, wounds, scarring, or other lesions within the proposed treatment area. Participants with other causes of hair loss, including alopecia areata, telogen effluvium, traction alopecia, cicatricial alopecia, thyroid-related hair loss, iron deficiency-related hair loss, or drug-induced alopecia, will also be excluded.

Additional exclusion criteria include previous topical treatment for androgenetic alopecia within the preceding 1 to 2 months, systemic corticosteroids, JAK inhibitors, immunosuppressive therapy, or other medications that may affect hair growth within the preceding 3 months. Participants with known hypersensitivity to botulinum toxin, albumin, local anesthetics, or any component used during the study procedures will not be eligible. Bleeding disorders, thrombocytopenia, current anticoagulant or antiplatelet therapy, and inadequate or unsuitable donor fat sites, including infection or scarring at the proposed harvesting site, will also constitute exclusion criteria.

Before treatment, all participants will undergo a standardized baseline evaluation. This will include complete history taking, including personal history, history of the present illness, medication history, and family history. Clinical examination will include general assessment, body mass index measurement, dermatological examination, and grading of alopecia according to the Norwood Hamilton classification.

A standardized dermoscopic evaluation of the scalp and areas affected by hair loss will also be performed. Two reference points will be identified along the midline of the scalp. The first will be located in the frontal region, approximately 18 cm from the tip of the nose, and the second in the vertex region, approximately 24 cm from the tip of the nose. Dermoscopic examination will also be performed on the right and left sides, approximately 5 cm from Kang's point, defined in the protocol as the point where the midsagittal line intersects the coronal line connecting the tips of the tragi. These predefined locations are intended to improve consistency of serial dermoscopic assessment during follow-up.

Standardized digital photographs will be obtained at baseline and repeated at 3 months and 6 months. Photographs will be taken using consistent photographic settings, lighting conditions, participant positioning, and camera-to-scalp distance to facilitate longitudinal comparison.

After providing informed consent, participants will be randomly assigned to one of two treatment groups.

Group 1, Botulinum toxin type A Participants assigned to the botulinum toxin group will receive a single treatment session at baseline. Botulinum toxin type A will be prepared by dilution of 100 units in 2 mL of normal saline. The preparation will be injected intramuscularly at 30 injection points, spaced approximately 2 cm apart, at a depth of approximately 4 mm. Injection points will be distributed across the frontal hairline, temporalis, periauricular, and occipitalis muscle regions. Each injection point will receive approximately 3.3 units of botulinum toxin type A.

No repeated botulinum toxin treatment session is planned within the 6-month follow-up period according to the study protocol.

Group 2, Autologous nanofat Participants assigned to the nanofat group will also receive a single treatment session at baseline. Autologous adipose tissue will be harvested under sterile conditions from an appropriate donor site, such as the lower abdomen, flank, or thigh, using small-volume liposuction under local anesthesia.

The harvested adipose tissue will be washed using sterile saline or Ringer solution. It will then undergo mechanical emulsification through repeated transfer between syringes using Luer-lock connectors. The processed fat will subsequently be filtered to produce a smooth injectable nanofat suspension.

The resulting nanofat preparation will be injected into the affected scalp using an intradermal microbolus technique. Injection points will be distributed approximately 1 cm apart throughout the treatment area. The total number of injection points will depend on the surface area of the affected scalp being treated. Approximately 0.1 mL of nanofat will be administered at each injection point.

Follow-up and efficacy assessment

All participants will be followed for 6 months. Assessments will be conducted at three predefined time points:

• Baseline, before treatment
• 3 months after treatment
• 6 months after treatment Clinical and dermoscopic evaluations will be performed at these visits. According to the protocol, participants will be assessed by two blinded dermatologists at baseline, 3 months, and 6 months. Standardized photographs will also be obtained at these time points.

The primary efficacy outcome will be the change in total hair count from baseline to 6 months after treatment. This outcome will be used as the principal measure for comparison between the botulinum toxin A and nanofat groups.

Secondary efficacy assessments will evaluate several dermoscopic characteristics associated with hair growth and follicular response, including:

• Change in terminal hair count
• Change in vellus hair count
• Change in the terminal-to-vellus hair ratio
• Change in follicular unit pattern
• Change in hair shaft diameter diversity
• Change in yellow dots
• Change in the peripilar sign These measurements will allow comparison not only of the total number of hairs but also of changes in hair type, follicular structure, and dermoscopic features during the 6-month follow-up period.

Safety assessment Safety will be evaluated throughout follow-up by documenting adverse reactions reported by participants. Prespecified adverse effects listed in the study protocol include edema, erythema, pruritus, and headache. Other clinical complications identified during follow-up may also be documented as part of the safety evaluation.

Because the interventions involve different procedures, safety monitoring will include the clinical consequences of both the scalp treatment and, in the nanofat group, the fat-harvesting procedure.

Participant satisfaction

Participant satisfaction will be assessed during follow-up. Participants will be asked to evaluate improvement in their hair condition compared with the start of the study. Responses will be classified as:

• Satisfied
• Neutral
• Dissatisfied Application form Application form Statistical analysis will be performed using IBM SPSS Statistics version 26. Quantitative variables will be summarized using mean and standard deviation. Comparisons of quantitative outcomes between the two treatment groups will be performed using the independent-samples t-test, while changes over time will be evaluated using repeated-measures analysis of variance (ANOVA). Categorical variables will be compared using the Chi-square test or Fisher's exact test, as appropriate. Statistical significance will be defined as p < 0.05.

The planned sample size is 40 participants, 20 per treatment group. The sample-size calculation was performed using G*Power version 3.1.9.7 and was based on comparison of the change in total hair count from baseline to 6 months between the two independent treatment groups. The calculation assumed a standardized effect size of Cohen's d = 0.81, a one-sided significance level of 5%, statistical power of 80%, and equal allocation between groups.

Ethical considerations Participation will occur only after informed consent is obtained. Participants will receive information regarding the study procedures, the investigational nature of the treatments, potential risks and benefits, available treatment alternatives, and their right to withdraw from the study without affecting the healthcare they receive.

Participant information and study data will be kept confidential and used only for research purposes by qualified personnel. Application form The overall study period for each participant will extend from baseline treatment through the final 6-month assessment, allowing direct comparison of the efficacy, dermoscopic response, safety, and participant satisfaction associated with a single session of intramuscular botulinum toxin type A versus a single session of intradermal autologous nanofat injection.