Wrinkle formation of the skin is a consequence. Perioral Wrinkles: Histologic Differences Between Men and Women. Facial Surgery

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1 Facial Surgery INTERNATIONAL CONTRIBUTION Perioral Wrinkles: Histologic Differences Between Men and Women Emma C. Paes, MD; Hans J. L. J. M. Teepen, MD, PhD; Willemijn A. Koop, MD; and Moshe Kon, MD, PhD Wrinkle formation of the skin is a consequence of aging that is influenced by both intrinsic (biologic) and extrinsic (environmental) factors. 1-4 The number and depth of wrinkles are linearly related to a person s age. 5 Intrinsic aging is a largely genetically determined process that mimics the aging of all organs in the human body and results in cutaneous alterations. Those alterations include a flattening of the dermoepidermal junctions, atrophy of dermis and epidermis, and a reduction in the amount of collagen and reticuline, as well as the number of fibroblasts. 1,2 Dr. Paes and Dr. Kon are from the Department of Plastic, Reconstructive and Hand Surgery, University Medical Center, Utrecht, The Netherlands. Dr. Teepen is from the Department of Pathology, St. Elisabeth Hospital, Tilburg, The Netherlands. Dr. Koop is from the Department of Plastic, Reconstructive, and Hand Surgery, University Medical Center, Leeuwarden, The Netherlands. Dr. Kon is a member of the Dutch Society for Aesthetic Plastic Surgery. Background: Women tend to develop more and deeper wrinkles in the perioral region than men. Although much is known about the complex mechanisms involved in skin aging, previous studies have described histologic differences between men and women with respect to skin aging only incidentally and have not investigated the perioral region. Objective: The purpose of this study was to investigate gender-specific differences in the perioral skin. Methods: To determine wrinkle severity, skin surface replicas of the upper lip region in 10 male and 10 female fresh cadavers were analyzed by using the dermatop blue three-dimensional digitizing system (Breuckmann, Meersburg, Germany). In 30 fresh male and female cadavers, three full-thickness lip resections were investigated in a blinded fashion for specific histologic features. All results were statistically analyzed in a linear regression model with SPSS software (version 15.0; SPSS, Chicago, IL). Results: The female replicas showed more and deeper wrinkles than the male replicas (P <.01). Histologic analysis revealed that the perioral skin of men displayed a significantly higher number of sebaceous glands (P =.000; 95% confidence interval [CI] ), sweat glands (P =.002; 95% CI ), and a higher ratio between vessel area and connective tissue area in the dermis (P =.009; 95% CI ). The amount of hair follicles did not significantly differ between men and women, although the average number of sebaceous glands per hair follicle was greater in men (P =.002; 95% CI ). Conclusions: Women exhibit more and deeper wrinkles in the perioral region and their skin contains a significantly smaller number of appendages than men, which could be a feasible explanation for why women are more susceptible to development of perioral wrinkles. (Aesthet Surg J; 29: ) Skin that has been exposed to the sun, like the skin of the face and hands, develops permanent wrinkles, a process that is known as photoaging. 2,4,6 This process is the main contributor to extrinsic aging; as much as 80% of facial aging is caused by solar radiation. 1,4,6 The damaging effects of ultraviolet (UV) radiation lead to dermal elastosis, a reduction of collagen, and an initial thickening of the epidermis. 1,2 Clinical signs of photoaging include dryness, hyperpigmentation, telangectesia, and deep wrinkles. 3 These wrinkles do not disappear when the skin is stretched; this is in contrast to temporary wrinkles that arise during aging even on sun-protected skin. 7 Other environmental factors that contribute to the extrinsic aging process are smoking, 1,3,8 diet, 1 and hormone replacement therapy (HRT). 9,10 We can distinguish between several types of permanent facial wrinkles, one of which is linear wrinkles. Linear wrinkles arise at the site of expression lines on the face, such as frown lines on the forehead, crow s Aesthetic Surgery Journal Volume 29 Number 6 November/December

2 Figure 1. Marked silicone skin surface replica of the perioral region indicating the location of three set sites (G, C, and D) that were used for the measurements. Figure 2. Measuring the skin surface replica. Orientation of the replica during the measurement using a high-resolution camera. In this figure, site C is seen. Three-dimensional topography was then extracted from each 12-mm 20-mm site. feet at the lateral canthus of the eye, the nasolabial crease, and wrinkles at the skin of the perioral site. 7 This last type seems to be observed more often in women. 11,12 Most requests for the correction of perioral wrinkles are made by women. While this may be explained in part by a greater concern among women about their appearance, it also appears that distinctive vertical wrinkles at the perioral region develop more frequently in women than in men. The growing demand for correction of these wrinkles has led to an expansion of treatment choices. While dermabrasion and peeling 13 were previously the most commonly used methods, laser treatment, botulinum injections, and injectable or implantable wrinkle fillers are the latest options. 12,14 Nevertheless, the treatment of wrinkles at the perioral region still remains a difficult matter. Although some papers have described the complex mechanism of aging of facial skin 2,5,7,13-15 and the associated histologic changes, 3,5,11,15 little is known about specific differences in facial skin aging between men and women, particularly with respect to the perioral region. Could there be a histologic explanation for why women are more susceptible than men to development of wrinkles in that region? In this study, the perioral skin of men and women was investigated in an effort to resolve this issue, in the hope that our results might contribute to the understanding of facial skin aging and ultimately lead to the development of better strategies for the prevention and treatment of perioral wrinkles. METHODS To define wrinkle severity (amount and depth), a skin surface replica was made of the perioral region in 10 white male and 10 white female fresh cadavers (age range years). With the exceptions of date of birth and exterior appearance, nothing was known about their medical history. Subsequently, three full-thickness lip resections were taken for histologic analysis. To provide additional histologic data, lip resections were also taken from another five white male and five white female fresh cadavers with the same characteristics. Skin Surface Replica The perioral skin was replicated by imprinting it on a silicone elastomer. The elastomer was prepared by mixing a monomer (Xantopren L blue; Heraeus Kulzer, South Bend, IN) with a catalyst (Optosol-Xantopren; Heraeus Kulzer). This suspension was applied with light pressure to the skin of the upper perioral region. The low viscosity and hydrophobic properties of the suspension promoted its penetration into all the irregularities of the skin. After several minutes, the catalyst converted the silicone monomer into a harder polymer, producing a high-resolution, permanent negative replica of the skin surface (Figure 1). The replicas were analyzed at three set sites, using the dermatop blue three-dimensional (3-D) digitizing system (Breuckmann, Meersburg, Germany). The replicas were oriented so that the fine perioral wrinkles were horizontal and the region of interest was centered in the field of view of the 3-D sensor (Figure 2). Three-dimensional topo- 468 Volume 29 Number 6 November/December 2009 Aesthetic Surgery Journal

3 A Figure 3. Three-dimensional wrinkle topography and visualization at a fixed scale of the upper perioral region. A, Three-dimensional topographic image of male perioral skin. The colors indicate the height difference measured, with red being the highest measured point and green being the lowest. B, Three-dimensional topographic image of female perioral skin. Note the deepness of the wrinkles, recognizable by the red lines. A Figure 4. Wrinkle depth set out in measurable two-dimensional (2-D) parameters. A, Wrinkle depth was scanned in all the topographies, which resulted in 50 single profiles. B, Calculation of the roughness parameters Ra, Rz, and Lr for each single profile using the Gaussian 2-D filtering standard method. graphic images measuring 12 mm 20 mm were extracted (Optocat and dermatop blue 2.0) and an image from the topography at fixed Z-scale was recorded. The fixed Z-scale allowed comparison of the replicas and performance of a qualitative analysis (Figure 3, A and B). Three-dimensional surface roughness parameters (Sa, Sq, St, and Sr) were calculated out of the topographies. Subsequently, the topographies were loaded and 50 profiles were extracted in a direction perpendicular to the wrinkles. Three roughness parameters (Ra, Rz, and Lr) were calculated for each two-dimensional (2-D) profile, using the Gaussian filtering standard method (Figure 4, A and B). The analysis of the parameters led to an interpretation of wrinkle depth and the number of wrinkles. These were used as a quantifiable measurement for wrinkle severity. The B B roughness parameters were statistically analyzed in a linear regression model using SPSS software (version 15.0; SPSS, Chicago, IL). Histology Full-thickness lip resections were taken at three different sites in the perioral region (Figure 5). The tissue was excised within 24 hours postmortem in fresh cadavers to prevent distortion of the morphology of the skin. After fixation in formalin 4% solution and dehydration, paraffin slides were made. After the slides were stained (hematoxylin eosin, Elastica van Gieson, and Masson trichrome stains) and marked with CD31 and an estrogen receptor marker, a histologic pilot study was performed. The slides were reviewed in a blinded fashion for characteristics of the epider- Perioral Wrinkles: Histologic Differences Between Men and Women Volume 29 Number 6 November/December

4 Figure 5. Full-thickness lip resection. The resections were taken at three different sites of the perioral region, marked in this figure. mis, dermis, and appendages. The collected data were analyzed and set out in a linear regression model using SPSS software (version 15.0). RESULTS Skin Surface Replica Women seemed to express more severe wrinkles in the perioral region than did men. In general, more and deeper wrinkles were seen on the 3-D topog raphies (Figure 3). In addition, two of the four 3-D surface roughness parameters calculated from the topographies showed significant differences between men and women (Table 1). All extracted average 2-D roughness parameters showed significantly more and deeper wrinkling in women than in men (Table 1). Histology There were several significant differences encountered between men and women. The perioral skin of men displayed a significantly higher number of sebaceous glands (P =.000; 95% CI ) and sweat glands (P =.002; 95% CI ). Moreover, the average number of sebaceous glands per hair follicle was larger in men than in women (P =.002; 95% CI ), although the number of hair follicles did not significantly differ. There was also a higher ratio between the vessel area and connective tissue area in the dermis in men compared to women (P =.009; 95% CI ). Finally, the distance between the dermis and the orbi - cularis oris muscle was significantly larger in men (P =.004; 95% CI ; Table 2). DISCUSSION Five out of seven calculated parameters of the replicas expressed significant differences between men and women (P <.01). Women exhibited significantly more and deeper wrinkles in the perioral region than men. The skin in this region also contained a significantly smaller number of appendages in women than in men. Many studies have described the complex mechanism of wrinkle formation and the factors that influence this process. However, gender differences were not addressed in these previous studies, although such differences could play an important role in the etiology of perioral wrinkles because perioral wrinkles seem to develop more in women than in men. This might be caused by greater exposure to extrinsic factors, of which solar radiation is the most important. However, it might be assumed that men and women are exposed to the same amount of UV radiation, as members of both genders do not readily cover up their face and hence solar exposition is approximately similar in both genders. Intrinsic factors are, of course, very variable; they were not considered in this study because they are not gender-specific. The results of this study clarify the differences between men and women with respect to several histologic issues, in addition to intrinsic and extrinsic factors. Similar results are described only incidentally in the literature, do not address the perioral region specifically, 6 and state that sebum levels tend to drop more than 40% in postmenopausal women, whereas no major changes appear in men. 16 These studies discuss differences between men and women in the number of perioral wrinkles; their conclusions are pertinent to the results of this study because male facial skin contains a significantly higher number of sebaceous glands than does female facial skin (Table 2). The postmenopausal decrease in sebum levels among women might be a feasible explanation for why perioral wrinkles generally develop in women with age, while they do not develop at all in a large percentage of men. Although the number of hair follicles in the perioral skin of men and women did not differ significantly, male follicles contained a significantly higher average number of sebaceous glands per hair. Comparable results are not found elsewhere in the literature. Epidermal thickness has been found to be greater in men than in women 10,17,18 ; it was also observed in this study, although the differences were not significant (Table 2). In addition, Contet-Audonneau et al 2 reported that, during aging, the stratum corneum is thickened in wrinkles and the incidence of dyselastosis is greater. However, they did not investigate gender-specific differences. In this study, we confirmed that women have a thicker stratum corneum and a significantly higher incidence of dyselastosis than men (Table 2). Capillary blood flow velocity has been shown to decrease in postmenopausal women. 2,16,19 This result is in line with our findings; an almost three times higher ratio between the area containing blood vessels and the area of the connective tissue measured in a set surface in the dermis of the perioral skin was found in men (Table 2). The perioral skin of men therefore contains more blood vessels than perioral skin in women. Better vascularization might have a decelerating effect on the development of wrinkles in the perioral skin. Nothing was previously described concerning the relationship 470 Volume 29 Number 6 November/December 2009 Aesthetic Surgery Journal

5 Table 1. Roughness statistics of the skin surface replicas of the perioral region in 10 male and 10 female fresh cadavers Mean (SD) Variable Male Female Difference (95% CI) P* Three-dimensional surface roughness parameters Sa (mm) (.00904) (.01513) ( ).000 Sq (mm) (.01197) (.01906) ( ).001 St (mm) (.78157) (.29030) ( ).781 Sr (.02935) (.02795) ( ).578 Average two-dimensional roughness parameters Ra (mm) (.00484) (.00731) ( ).000 Rz (mm) (.01817) (.02610) ( ).010 Lr (.01265) (.01086) ( ).003 CI, confidence interval; LR, ratio of developed line to the profile length; Ra, arithmetic average of absolute values of roughness profile ordinates (Z), where Z is the sum of the heights of the highest peaks and the lowest valley depth within a sampling length; Rz, arithmetic mean value of the single roughness depths of consecutive sampling lengths; Sa, linear average surface roughness; SD, standard deviation; Sq, quadratic average surface roughness; Sr, ratio of developed area to target area; St, maximum surface height difference (peak to peak value). *P <.05 was considered statistically significant. Table 2. Average statistical results of the histologic analysis of 90 biopsies taken from the perioral region in 15 male and 15 female fresh cadavers Mean (SD) Variable Male Female Difference (95% CI) P* Maximum epidermal thickness (μm) 271 (164) 205 (60) 66 ( ).158 Maximum corneum thickness (μm) 19 (9) 21 (15) 28 ( 3 59).075 Dyselastosis dermis 1.93 (.59) 3.0 (.66) 0.92 ( ).002 Ratio distribution of collagen to reticulin 1.05 (.40).99 (.70) 0.05 ( ).835 No. of hair follicles in coupe 42.0 (14.5) 36.0 (12.5) 4.1 ( ).426 No. of (interspace) hair follicles/mm.93 (.23) 1.03 (.29) ( ).661 No. of (interspace) sebaceous glands in coupe 84.5 (25.1) 45.9 (21.4) 38.4 ( ).000 No. of sebaceous glands/hair (interspace) follicle 2.08 (.45) 1.29 (.67) 0.80 ( ).002 Percent of sebaceous glands with estrogen receptors 59.5 (28.8) 52.7 (30.7) 9.7 ( ).318 No. of sweat glands 13.1 (3.7) 8.4 (4.0) 5.0 ( ).002 Ratio of vessel area to connective tissue area (μm).0346 (.0290).0117 (.0095).0120 ( ).009 Maximum distance from orbicularis oris (3144.6) (2200.3) ( ).004 to the epidermis (μm) CI, confidence interval; SD, standard deviation. *P <.05 was considered statistically significant. between gender and the presence of sweat glands in the perioral skin. Our study shows that the perioral skin of men contains significantly more sweat glands than that of women (Table 2). It has been reported that women who receive HRT have lower facial wrinkle scores than women who are not treated with this therapy. 9,10 In addition, mean levels of epidermal skin moisture, elasticity, and skin thickness are improved with HRT, 10 although this has never been shown with respect to the perioral region and the results of this study showed no difference between men and women regarding the presence of estrogen-positive sebaceous glands in this area. This suggests that other factors may have a greater influence on the development of perioral wrinkles. As described, the perioral skin of men contains more appendages than that of women, which could influence, in some way, the natural filling of the dermis. Because the dermis of the perioral skin in men includes considerably more sebaceous glands, sweat glands, and blood vessels, one could imagine that the formation of wrinkles in that region is more difficult in men. Another interesting observation was that the orbicularis oris muscle, which surrounds the lips, is anchored 1.5 times closer to the dermis in women than in men (Table 2). This difference has not been previously reported Perioral Wrinkles: Histologic Differences Between Men and Women Volume 29 Number 6 November/December

6 in the literature. It might play a role in wrinkle formation because fibrous connections between the muscle and the dermis can cause an inward traction, thereby creating deeper wrinkles. It should be noted that during data collection, the assumption was made that the cadavers had a roughly equal exposure to UV radiation during their lives. We also cannot exclude the possibility that other external factors that influence the aging process such as smoking, hormones, and diet may have been operative to varying extents in the cadavers. CONCLUSIONS The treatment of perioral wrinkles remains a difficult matter. To our knowledge, ours is the first study to investigate skin surface and specific histologic differences between the perioral skin of men and women and their possible relationship to wrinkle formation. We found that, in comparison to men, women exhibit more and deeper wrinkles in the perioral region and that women s perioral skin contains significantly fewer appendages. These findings provide a feasible explanation for why women are more susceptible to development of perioral wrinkles and contribute to our current understanding of wrinkle formation. ACKNOWLEDGMENTS The authors thank Mr. Simon Plomp and Mr. Willem van Wolferen from the anatomy department of the University Medical Centre Utrecht for their assistance in collecting the biopsies from the cadavers. DISCLOSURES The authors have no financial interest in and received no compensation from manufacturers of products mentioned in this article. REFERENCES 1. Baumann L. Skin ageing and its treatment. J Pathol 2007;211: Contet-Audonneau JL, Jeanmaire C, Pauly G. A histological study of human wrinkle structures: comparison between sun-exposed areas of the face, with or without wrinkles, and sun-protected areas. Br J Dermatol 1999;140: Gilchrest BA. A review of skin ageing and its medical therapy. Br J Dermatol 1996;135: Jenkins G. Molecular mechanisms of skin ageing. Mech Ageing Dev 2002;123: Leveque JL, Goubanova E. Influence of age on the lips and perioral skin. Dermatology 2004;208: Zouboulis CC, Boschnakow A. Chronological ageing and photoageing of the human sebaceous gland. Clin Exp Dermatol 2001;26: Hatzis J. The wrinkle and its measurement a skin surface Profilometric method. Micron 2004;35: El-Domyati M, Attia S, Saleh F, et al. Intrinsic aging vs. photoaging: a comparative histopathological, immunohistochemical, and ultrastructural study of skin. Exp Dermatol 2002;11: Castelo-Branco C, Figueras F, Martínez de Osaba MJ, Vanrell JA. Facial wrinkling in postmenopausal women. Effects of smoking status and hormone replacement therapy. Maturitas 1998;29: Sator PG, Schmidt JB, Sator MO, Huber JC, Honigsmann H. The influence of hormone replacement therapy on skin ageing: a pilot study. Maturitas 2001;39: Wojnarowska F. Clinical aspects of ageing skin. In: Fry L, editor. Skin problems in the elderly, 2nd ed. Edinburgh: Churchill Livingstone; 1985, pp Monhian N. Injectable implantable materials for facial wrinkles. Aesthetic Facial Surgery ed. 2008, pp Holmkvist KA, Rogers GS. Treatment of perioral rhytides: a comparison of dermabrasion and superpulsed carbon dioxide laser. Arch Dermatol 2000;136: Semchyshyn N, Sengelmann RD. Botulinum toxin A treatment of perioral rhytides. Dermatol Surg 2003;29: Piérard GE, Uhoda I, Piérard-Franchimont C. Update on the histological presentation of facial wrinkles. Eur J Dermatol 2002;12:XIII XXIV. 16. Sandby-Moller J, Poulsen T, Wulf HC. Epidermal thickness at different body sites: relationship to age, gender, pigmentation, blood content, skin type and smoking habits. Acta Derm Venereol 2003;83: Castelo-Branco C, Duran M, Gonzalez-Merlo J. Skin collagen changes related to age and hormone replacement therapy. Maturitas 1992;15: Vaillant L, Callens A. Hormone replacement treatment and skin aging. Therapie 1996;51: Raine-Fenning NJ, Brincat MP, Muscat-Baron Y. Skin aging and menopause: implications for treatment. Am J Clin Dermatol 2003;4: Accepted for publication May 22, Presented at the 20th Annual Meeting of the European Association of Plastic Surgeons, May 28 30, 2009, Barcelona, Spain. Reprint requests: Moshe Kon, MD, PhD, University Medical Center Utrecht, Department of Plastic, Reconstructive and Hand Surgery, PO Box 85500, 3508 GA Utrecht, the Netherlands. M.Kon@umcutrecht.nl. Copyright 2009 by The American Society for Aesthetic Plastic Surgery, Inc X/$36.00 doi: /j.asj Volume 29 Number 6 November/December 2009 Aesthetic Surgery Journal