All the study procedures were carried out according to World Medical Association’s (WMA) Helsinki Declaration and its amendments (Ethical Principles for Medical Research Involving Human Subjects, adopted by the 18th WMA General Assembly Helsinki, Finland, June 1964 and amendments). To participate in the study, each participant was fully informed on study risks and benefits, aims, and procedures. An informed consent form and a consent release for publication of photos were signed by the subject prior to participating in the study.
Subjects and Study Design
Healthy male volunteers suffering from alopecia grade II to IV according to the Hamilton–Norwood scale  (Fig. 1) were enrolled in the study. Subjects were enrolled in the study by a certified dermatologist if they fulfilled the inclusion and exclusion criteria laid down in the study design (Table 1) were applicable. Clinical examination was carried out in order to evaluate the degree and pattern of hair loss, and hair (length, diameter, and breakage) and scalp (inflammation, erythema and, scaling) conditions. Active and placebo treatments were then allocated by means of the Efron’s biased coin algorithm using PASS 11 (version 11.0.8 for Windows; PASS, LLC., Kaysville, UT, USA). The tested and the placebo products were used for 4 months according to the following procedure: apply one vial (5 mL) of the product on clean and dry scalp, line by line, concentrating on the areas where thinning is more evident; massage gently to aid penetration; apply every day for 5 consecutive days, stop the treatment for 2 days and then continue the application.
The tested product is a commercially available cosmetic lotion named Crescina® HFSC. The ingredients of the tested product and placebo are listed in Table 2.
A transitional area of hair loss between normal hair and the balding area of 1.8 cm2 was defined using a stencil template and chosen for clipping. The clipped hairs within the target area were dyed for gray or fair hairs with a commercially available solution (RefectoCil®, GW Cosmetics GmbH, Leopoldsdorf, Germany) in order to enhance their contrast. Thereafter, the dyed hairs were cleansed using an alcoholic solution and digital images were taken while the area was still wet with a digital close-up camera. Images were taken at day 0, immediately after clipping, and 2 days after clipping. These two photographs were then examined by a software system that is able to recognize individual hair fibers in the photographs. By comparing the two photographs, the computer can determine which hairs are growing (anagen hairs) and which are not (telogen hairs). The same procedure was carried out after 2 and 4 months of treatment.
Gentle traction was exerted on a cluster of hairs (approximately 60 hairs) on at least three different areas of the scalp, and the number of extracted hairs was counted. Normally, less than three telogen-phase hairs should come out with each pull . If at least three hairs were obtained with each pull or if more than ten hairs total were obtained, the pull test was considered positive and suggestive of telogen effluvium.
Global Photographic Assessment
Patients were asked to maintain the same hair style, color, and length throughout the study. Standardized global photographs of the frontal/parietal region were taken using a digital camera equipped with a macro lens.
An intention to treat statistical analysis was performed using NCSS 8 (version 8.0.4 for Windows; NCCS, LLC., Kaysville, UT, USA). Data were checked for normality using either the Shapiro–Wilk W, Kolmogorov–Smirnov, and D’Agostino omnibus normality tests. If the data were normal, the repeated measure analysis of variance (RM-ANOVA) followed by Tukey–Kramer multiple comparison test was performed both for intra- and inter-group comparisons. If data were not normal, the Wilcoxon signed-rank test was performed for intra-group comparisons, whereas the Mann–Whitney U test was performed for inter-group comparisons. Values are expressed as arithmetic mean ± standard deviation (SD). P < 0.05 was considered significant.