IMMUNOHISTOCHEMICAL STUDY OF ESTROGEN AND PROGESTERONE RECEPTOR EXPRESSION IN MELASMA PROTOCOL OF THESIS SUBMITTED FOR THE PARTIAL FULFILLMENT OF MASTER DEGREE IN DERMATOLOGY, VENEREOLOGY AND ANDROLOGY BY EMAN MOHAMMED AHMED ALIAN (M.B, B. CH.) FACULTY OF MEDICINE – AIN SHAMS UNIVERSITY SUPERVISED BY DR. MAHIRA HAMDY EL-SAYED PROFESSOR OF DERMATOLOGY, VENEREOLOGY AND ANDROLOGY FACULTY OF MEDICINE – AIN SHAMS UNIVERSITY DR. AZZA ESMAT MOSTAFA LECTURER OF DERMATOLOGY, VENEREOLOGY AND ANDROLOGY FACULTY OF MEDICINE – AIN SHAMS UNIVERSITY FACULTY OF MEDICINE AIN SHAMS UNIVERSITY 2010 INTRODUCTION Melasma is a common acquired symmetric hypermelanosis characterized by irregular light-brown to gray-brown macules and patches involving sun-exposed areas most commonly the face (1). Although the majority of melasma cases occur in women, 10% of cases have been reported in males (2). Clinically, melasma usually occurs in one of three patterns. The most common is the centrofacial pattern with involvement of the lateral forehead, cheeks, nose, upper lip and chin. The less common patterns include the malar and the mandibular types. Lesions may occasionally occur in other sun-exposed areas including the forearms and the mid upper chest (3). Although histologically, melasma was formerly classified as epidermal, dermal and mixed type by the locations of pigments. Kang et al., 2002 reported that there is no true dermal type of melasma (4). In addition, it has been demonstrated that the melanocytes within affected skin are larger, intensely stained with prominent dendrites and contain more melanosomes than melanocytes of unaffected skin, suggesting that these cells may be hyperfunctional in melasma (5). The exact etiopathogenesis of melasma is unknown, although multiple factors have been implicated. These include genetic predisposition; medications, such as anticonvulsants; ultraviolet light exposure (6); and, most notably, hormonal influences (7). Many cases appear to be related to excess estrogen, either produced endogenously during pregnancy or delivered exogenously through the use of oral contraceptive pills and hormone replacement therapy. In vitro studies have shown that estradiol increases levels of tyrosinase, tyrosinase-related-protein 1 and tyrosinase-related-protein 2, enzymes involved in human eumelanogenesis, within normal human melanocytes (8). On the other hand, the results of studies on the effects of progesterone on skin pigmentation have been inconsistent. It has been reported that progesterone increased cell numbers and tyrosinase activity in melanocytes. The observation that postmenopausal woman who are given progesterone develop melasma, while those who are given only estrogen do not, implicates progesterone as playing a critical role in the development of melasma (9). In contrast, significant inhibitory effect of progesterone on the proliferation of melanocytes in cell culture was also reported (10). However, estrogen receptor and progesterone receptor expression in melasma affected skin has not been investigated to date, except for two reports in the literature (2, 11). Such information is a prerequisite for the understanding the pathogenesis of melasma in relation to female sex hormones such as estrogen and progesterone, perhaps then a treatment protocol can be constructed probably implementing topical anti-estrogen therapy. AIM OF THE WORK The aim of this thesis is to investigate the immunohistochemical differences of the estrogen receptor and progesterone receptor expression between melasma- affected skin and adjacent-unaffected control skin. SUBJECTS AND METHODS This thesis is designed to be prospective case-control study. It will be carried on thirty(30) patients with melasma presenting to the Dermatology outpatient clinic of Ain Shams University Hospital, Faculty of Medicine, Ain Shams University. Exclusion criteria: Patients with other diseases leading to hyperpigmentation will be excluded. Each patient will be subjected to: 1- History taking: including full information about the medical history of the patients and history of the disease. 2- Full dermatologic examination. 3- Skin biopsy: i. A 3mm punch biopsy will be taken from an area of melasma affected skin. ii. Another 3mm punch biopsy will be taken from unaffected adjacent skin as a control. iii. The tissues will be fixed in 10% buffered formalin and embedded in paraffin. iv. From the paraffin block, 5 micron thick serial sections will be cut and prepared for routine hematoxylin and eosin (H&E) stain for histopathological examination by light microscopy. v. Sectioned will be examined for: a. The extend and the distribution of melanin pigment. b. The histologic type of melasma. c. Other relevant histologic findings will be reported. vi. Immunohistochemical staining will be performed using the following stains: a) Staining for estrogen receptors (ER–β) in lesional and perilesional skin. b) Staining for progesterone receptors (PR) in lesional and perilesional skin. vii. All slides were evaluated by two observers in a blinded fashion. Statistical methodology Analysis of the data will be done using SPSS (Statistical Program for Social Science) computer program. THE THESIS WILL INCLUDE: Introduction and aim of the work. Review of literature. Subjects and methods. Results. Discussion. Conclusion Recommendation Summary References Arabic summary REFERENCES (1) Sanchez NP, Pathak MA, Sato S, Fitzpatrick TB, Sanchez JL and Mihm MC Jr (1981): Melasma: a clinical, light microscopic, ultrastructural, and immunofluorescence study. J Am Acad Dermatol; 4: 698-710. (2) Lieberman R and Moy L (2008): Estrogen receptor expression in melasma: results from facial skin of affected patients. J Drugs Dermatol; 7(5): 463-465. (3) Johnston GA, Sviland L and Mc Lelland J (1998): Melasma of the arms associated with hormone replacement therapy. Br J Dermatol; 139:932. (4) Kang WH, Yoon KH, Lee ES, Kim J, Lee KB and Yim H (2002): Melasma: histopathological characteristics in 56 Korean patients. Br J Dermatol; 146: 228-237. (5) Grimes PE, Yamada N and Bhawan J (2005). Light microscopic, immunohistochemical, and ultrastructural alterations in patients with melasma. Am J Dermatopathol; 27: 96-101. (6) Ortonne JP, Arellano I, Berneburg M, Cestari T, Chan H, Grimes P, Hexsel D, Im S, Lim J, Lui H, Pandya A, Picardo M, Rendon M, Taylor S, Van Der Veen JPW and esterhof WW (2009): Global survey of the role of ultraviolet radiation and hormonal influences in the development of melasma. J Europ Acad Dermatol Venereol; 23, 1254-1262. (7) Hassan I, Kaur I, Sialy R and Dash RJ (1998): Hormonal milieu in the maintenance of melasma in fertile women. J Dermatol; 25: 510-512. (8) Kippenherger S, Loitsch S, Solano F, Bernd A and Kaufmann R. (1998): Quantification of tyrosinase, TRP-1, and TRP-2 transcripts in human melanocytes by reverse transcriptase-competitive multiplex PCR-regulation by steroid hormones. J Invest Dermatol; 110: 364-367. (9) Bolanca I, Bolanca Z, Kuna K, Vuković A, Tuckar N, Herman R and Grubisić G (2008): Chloasma: the mask of pregnancy. Coll Antropol; 32 (S2): 139-141. (10) Wiedemann C, Nägele U, Schramm G and Berking C (2009): Inhibitory effects of progestogens on the estrogen stimulation of melanocytes in vitro. Contraception; 80: 292-298. (11) Jang, YH, Lee YJ, Kang HY, Lee E-S and Kim YC (2010): Oestrogen and progesterone receptor expression in melasma: an immunohistochemical analysis. J Europ Acad Dermatol Venereol; 24: 1312-1316.