ra ral disc A. Forabosco, MD,’ M. Criscuolo, IIJD,~ G. Coukos, MD,” E. Uccelli, MD,’ R. Weinstein, MD,a S. Spinato, MD,(’ A. Botticelli, IFID,~ A. Volpe, MD,” Modena and Cagliari, Italy UNIVERSITY

OF MODENA

MEDICAL

SCHOOL

AND

UNIVERSITY

OF CAGLIARI

MEDICAL

SCHOOL

The increase in incidence of oral discomfort among women in a menopause is probably due to hormone modifications. This study evaluated the efficacy of hormone replacement therapy in 27 postmenopausal patients, aged 48 to 58 years, with oral discomfort and no local irritants and in 47 postmenopausal women with no oral discomfort. Patients were treated with conjugated estrogens for 21 days and medroxyprogesterone acetate from day 12 through day 21. Hormone-replacement therapy had no effect on oral cytology in the 40 symptom-free postmenopausal women compared with a group of 47 postmenopausal women who had no oral symptoms and were not treated. Hormone-replacement therapy relieved symptoms and improved oral cytohormonal features in 15 of 27 patients with symptoms. Nuclear estrogen receptors were found by immunohistochemical assay in 8 of 10 randomly selected patients with symptoms who responded to hormone-replacement therapy, but not in 2 patients who did not benefit from hormone-replacement therapy. Estrogen receptors were also found in 6 of IO fertile women with no oral disease. Our results suggest that oral discomfort may be related to steroid hormone withdrawal only in some postmenopausal women and that replacement therapy may improve the clinical picture and cytologic features in this group of patients. lmmunohistochemical identification of estrogen receptors may help to identify patients for whom hormone-replacement therapy may be beneficial. (ORAL SURG ORAL Mm

ORAL PATHOL

1992;73:570-4)

ral discomfort (OD) represents a clinical condition characterized by oral dryness with a burning sensationand dysgeusia. Diffuse gingival atrophy and oral ulcerations may be part of the clinical picture. The incidence of OD increasesamong women past menopause, which suggests that steroid hormone withdrawal might be a cause.Some evidence that has been accumulated suggestsa positive relationship between ovarian hormone modifications and changes in the oral mucosa.1M3 Estrogens may stimulate proliferation of gingival fibroblasts4 and maturation of connective tissue, mainly through their influence on collagen turnover. In fact, estrogensseemto block degradation of collagen in rat skin5 Other studies have

failed to demonstrate such a relationship, recognizing that OD may have a number of different causes637 How ovarian hormones act on buccal mucosa is not known. Some authors8 have postulated that ovarian hormones act directly on gingiva, whereas others? have suggested that sex steroids act indirectly by modifying the inflammatory response of gingiva to local irritants. The aim of the present open study is to verify ( 1) the efficacy of hormone replacement therapy (HRT) on oral cytology and clinical symptoms, (2) the presence and localization of estrogen and progesterone receptors in the various layers of oral human mucosa, and (3) the possible association between steroid receptors and responseto HRT.

aDepartment bDepartment ‘Department l/13/29597

MATERIAL

570

of Dentistry, of Pathology, of Obstetrics

University of Modena. University of Modena. and Gynecology, University

of Cagliari.

AND METHODS

A total of I 14 postmenopausalpatients, aged 48 to 58 years, were evaluated. Eighty-seven of these

Hormone replacement therapy in postmenopausal women 571

Volume 73 Number 5

Table 1. Subjective symptoms in 27 postmenopausal No. of patients Before HRT 10 2 6 1 2 2 4 After HRT 3 4 2 2 1

1

dr$& + + + + +

(

Burning

Dysgeusia

+ +

+

Oral ulceration

Diffuse gingival atrophy

+ + + +

+ + +

women with oral discomfort before and after HRT

+ +

+

+

+ -I-

women had no symptoms of oral disease, and 2~7had various patterns of oral discomfort. None of the women had received any hormone treatment during the previous 6 months. Subjective symptoms were evaluated through the use of a questionnaire that mixed oral symptoms (dysgeusia, oral dryness, burning sensation, pain) with other systemic symptoms (malaise, insomnia, headaches, appetite modifications) and urogenital symptoms (urinary frequency, dysuria, vaginal dryness, itching, pain, irritation, discharge, dyspareunia). Severity of symptoms was estimated by patients along an arbitrary four-point scale. The questionnaire was completed by the patients both before and after the 3-month treatment period. Patients

Three groups of postmenopausal women were studied. The first group included 27 postmenopausal women, aged 48 to 58 years, with .variable patterns of OD that included oral dryness, burning sensation, and dysgeusia. Seven patients had OD associated with oral ulcerations (1 patient) or diffuse atrophy of oral mucosa (6 patients) (Table I). All 27 patients also gave detailed information concerning systemi’c diseases, dietary regimen, and oral hygiene habits. Moreover, all had gynecologic examinations as well as comprehensive oral exfoliative cytology and oral examination on admission to the study to rule out local irritants such as excessive calculus and inadequate protheses. Patients included in the study were given conjugated estrogens (Premarin), 0.625 mg/day for 2 1 days, plus medroxyprogesterone acetate (Farlutal), 10 mg/day from. day 12 through day 21 of the treatment cycle, for three consecutive 21-day cycles. Each new cycle was started 7 days after completion of the previous one. Oral examination and exfoliative

+

cytology were repeated at the end of the 3-month treatment period. The second group included 47 postmenopausal women, aged 48 to 58 years, with no oral symptoms, These patients were given the same dosage of conjugated estrogens plus medroxyprogesterone acetate as group 1. A third group of 40 postmenopausal patients with no oral symptoms did not receive any treatment. Oral examination and exfoliative cytology were performed on groups 2 and 3. Women in group 2 were examined both before and after the 3-month treatment period. Oral exfoliative cytology

Oral exfoliative cytology was performed with sterile kits for Papanicolaou smear preparation. An Ayre’s spatula was grazed against oral mucosa from an area near Stensen’s duct through the labial commissure. Material was used to prepare smears according to Papanicolaou’s method. Cytohormonal evaluation was conducted by visual interpretation of smears by one of us, without knowledge of the patient’s history. Cytologic criteria that applied to vaginal exfoliative cytology were used. For instance, cells were classified as three types: superficial (polygonal, flat cells with pycnotic nucleus and large eosinophilic or cyanophilic cytoplasm), intermediate (polygonal, flat cells with vesicular nucleus showing the chromatic structure and cyanophilic cytoplasm), and parabasal (round, three-dimensional cells with large vesicular nucleus and smaller cyanophilic cytoplasm). Maturation of epithelium was estimated through differential cell count by consideration of these three cell types and by evaluation of maturation index and maturation value. The maturation index was the direct expression of percentages of the three cell types: % parabasal, %

572

Forabosco

et al.

&AL

SLRG&AL

MIED ORAL PATHOL May 1992

I

i i-

n=2 Before

HRT

After

HRT

After HRT

Before

HRT

After

HRT

Fig. 1. Oral exfoliative cytology maturation value in 27 postmenopausal patients with oral discomfort before (first bar] and after hormonal-replacement therapy (HRT). The second bar represents the 12patientsof the groupin whom HRT relieved orai symptomspartially or not at all. The third bar representsthe 15 patientsin whom HRT completely relievedsymptoms.Maturation value isexpressed as percentage.Resultsare expressedas mean + 1SD (*P < 0.05).

Fig. 2. Oral exfoliative cytology maturation value in 47 postmenopausal womenwith no oral discomfortbeforeand after HRT andin 40 symptom-freepostmenopausal women who receivedno treatment (hatched bar).

intermediate, and % superficial. The maturation value was the final value, expressedin percentages, derived from the addition of the percentages of single cell classesand multiplied by a corresponding conventional numeric factor (superficial eosinophilic = 1.O; superficial cyanophilic = 0.8; large intermediate = 0.6; small intermediate = 0.5; parabasal = 0.0).

RESULTS Oral cytology and clinical appearance in postmenopausal patients

lmmunohistochemical assay of steroid hormone receptors The expression of nuclear estrogen and progesterone receptor protein in oral mucosa was investigated in 10 women randomly selected from 27 postmenopausal patients with symptoms. Among them, 3 had macroscopic lesionsof oral mucosa, whereas7 had no macroscopic evidence of oral mucosa lesions. Ten symptom-free fertile women, aged 38 to 44 years, were also evaluated ascontrols. With the patient’s informed consent and approval of the hospital ethics committee, gingival tissue specimenswere removed surgically before HRT was started; specimenswere immediately frozen in liquid nitrogen and sent to the pathology department. Cryostat sections were incubated with one to two drops of monoclonal antibodies raised against the estrogen and progesteronereceptor proteins (ER-ICA and PR-ICA, respectively, Abbo H, IJ.S.A.),‘u and an immunohistochemical study was performed to identify thesereceptors. The pathologist who performed the immunohistochemical study was not aware of patient age and history.

STATISTICAL ANALYSlS Statistical analysis of data regarding maturation value was performed with the Student t test.

Hormone-replacement therapy relieved oral drynessin 12 of 21 patients, burning sensation in 10 of 16 patients, and dysgeusia in all patients (22 before HRT). Moreover, HRT reversed atrophic changesin oral mucosa in 3 of 6 patients and oral ulceration in 1 patient. In all, HRT completely relieved symptoms in 4 of 7 patients with macroscopic changes of oral mucosa before therapy and in 11 of 20 patients with oral discomfort and no macroscopic lesionsof the oral mucosa (Table I). Cytohormonal evaluation showed maturation of oral epithelium in the 15 patients who benefited from HRT; no significant changeswere observed in the 12 patients in whom QD persisted after therapy. In particular, the maturation index estimation showed an increase in the percentage of superficial cells and the maturation value increased significantly in these 15 patients (Fig. 1). The maturation index and maturation value were similar in groups 2 and 3 (Fig. 2). Steroid hormone receptors Immunostaining of estrogen receptor protein was evident in 8 of 10 patients with OD and 6 of 10 fertile women (Fig. 3). Progesterone receptor protein was evident in 3 patients with OD and in 3 fertile women (Fig. 4). All 8 patients with evident estrogen receptors responded to HRT; the 2 negative specimenswere

Volume 73

Hormone replacement therapy in postmenopausal women 573

Number5

Fig. 3. Immunohistochemical evidenceof estrogenreceptor proteinin oral mucosaspecimenfrom a patient with oral discomfortwho respondedto replacementtherapy. Ihtrogen receptor-positivenuclei within epitheliumare marked with squares.(Original magnification,X125.) from 2 patients who did not respond satisfactorily to HRT. Immunostaining for estrogen receptor was stronger in the epithelial layer of the oral mucosa; staining for progesterone receptor was stronger in the stroma (Figs. 3 and 4). DISCUSSION Oral discomfort represents an assemblageof subjective symptoms that may be accompanied by macroscopic evidence of oral lesions.In many cases,however, such evidence is lacking. All of the patients with symptoms but no oral macroscopic lesionshad microscopic evidence of altered mucosal trophism, but postmenopausalwomen with no symptoms had a normal pattern of oral cytology. These results suggesta relationship between oral symptoms and microscopic changes in oral mucosa. Oral mucosa is similar to vaginal mucosa in some respects. In fact, both mucosaepresent a pluristratified epithelium and a desquamative growth pattern. However, it is still deblated whether oral mucosa exhibits properties similar to those of vaginal mucosain relation to sensitivity to ovarian steroids and whether oral mucosa is an index of hormonal milieu in menopause. Bercovici et al. I1 did not find any correlation between estrogen deficiency and oral cytology, concluding that oral discomfort in menopausalwomen is due to local irritants. Wardrop et aILl found a significantly higher prevalence of oral discomfort in perimenopausaland postmenopausalwomen than in premenopausalwomen, and they later reported13 that in approximately two thirds of the menopausal women with oral discomfort but normal-appearing oral mucosa symptoms were relieved by appropriate hormone-replacement therapy. Some evidence in animals

Fig. 4. Immunohistochemicalevidence of progesterone receptor(PR) protein in oral mucosaspecimenfrom a patient with oral discomfortwho respondedto replacement therapy. An intensivelypositivePR reactionof nucleiis observedin chorionmucosae.PRs are marked with squares. (Original magnification,X 125.)

also suggests a relationship between ovarian hormones and structural modifications of oral mucosa. Estrogens affect cellular proliferation, differentiation, and keratinization of the gingival epithelium in the rat14 and stimulate the proliferation of gingival fibroblasts in mice.15 In the present open study, approximately one half of the patients complaining of oral discomfort showed a consistent decrease in subjective symptoms after HRT. Moreover, maturation aspects of exfoliative cytology significantly improved only in those patients in whom HRT completely relieved oral symptoms. This closecorrelation suggeststhat HRT may relieve symptoms through amelioration of oral mucosain responsive patients. A small number of patients reported partial amelioration of oral symptoms without cytohormonal improvement. In these patients a placebo effect of HRT may- not be excluded. The evidence that estrogen receptors were not detected in 2 of 10 randomly selected patients with symptoms may explain why somepatients with symptoms do not benefit from HRT. In fact, all eight patients who displayed oral mucosa estrogen receptors responded to HRT; the two remaining patients with no estrogen receptors did not respond to HRT. Estrogen receptors were identified in only six of 10 fertile women, suggesting that oral mucosa is sensitive to hormonal milieu in only some subjects during the reproductive period of life. These data suggestthat modifications of oral mucosa and symptoms may be related to steroid hormone withdrawal in only some postmenopausal patients with OD and may explain discordant results among authors.

574

Forabosco

et al.

Conclusions

Our data show that patients with oral discomfort displayed microscopic changes in oral mucosa. Not all patients, however, greatly benefited from HRT. A close correlation between the benefits of HRT and the presence of estrogen receptors in the oral mucosa may explain the diversity of response among postmenopausal patients and the discordant results among authors. The evidence that not all fertile patients examined displayed estrogen receptors in the oral mucosa suggests that the hormonal milieu affects oral mucosa in only some women. Identification of estrogen receptors in oral mucosa by an immunohistochemical technique may be appropriate to identify those patients for whom hormonal therapy could be beneficial.

ORAL

S~JRGORAL MED ORAL PATW~L May 1992

5. Skosey JL, Damgaard E. Effects of estradiol benzoate on the degradation of insoluble collagen of rat skin. Endocrinoloevu, 1973;93:311-6. 6 7.

8. 9.

10. 11. 12. 13.

Trott JR. A desquamative cytological study of healthy oral mucosa. J Periodontol 1958;29:213-20. Silverman Jr S, Shouse C. Estrogen effects on human oral epithelium: cytologic, histologic, and clinical comparisons. Oral Ther Pharm 1966;3:87-93. Turesky S, Fisher B, Glickman I. A histochemical study of the attached gingiva in pregnancy. J Dent Res 1958;37: 1115-9. Loe H, Silness J. Periodontal disease in pregnancy. I. Prevalence and severity. Acta Odontol Stand 1963;21:533-551. Greene CL, Nolan C, Engler JP, Jensen EV. Monoclonal antibodies to human estrogen receptor. Proc Nat1 Acad Sci U S A 1980;5115-9. Bercovici B, Gran S, Pisanty S. Vaginal and oral cytology of the menopause. Acta Cytol 1985;29:805-9. Wardrop RW, Reade PC, Hailes J, Burger HG. Oral discomfort during the menopause [Abstract]. Maturitas 1984;6:206. Wardrop RW, Hailes J, Burger H, Reade PC. Oral discomfort at menopause. ORAL SURG ORAL MED ORAL PATH~L 1989; 67:535-40.

REFERENCES 1. Loe H. Periodontal changes in pregnancy. J Periodontal 1965;36:209-17. 2. Wingrove FA. Influence of ovarian hormone situation on atrophy, hypertrophy, and/or desquamation of human gingiva in premenopausal and postmenopausal women. J Periodontol 1979;50:445-8. 3. Volpe A, Lucenti V, Forabosco A, et al. Oral discomfort and hormone replacement therapy in postmenopausal period. Maturitas 1990;13:1-5. 4. Fukupa H. Experimental studies on the effect of sex hormones on the proliferation of cells derived from the gingival tissues in tissue culture. Shikwa Gaku Ho 1973;71:1214-9.

14. Vittek J, Altman K, Rappaport SC, Gordon GC, Hagedoorn J, Southren AL. Effect of progestins on androgen 3-4-ketosteroid-5 A-ring reductase system in rat oral mucosa. J Dent Res, 1978;57:51 l-5. 15. Beagrie GS. Observations on cell biology of gingival tissues of mice. Br Dent J, 1966;121:417-20. Reprint

requests:

Annibale Volpe, MD, Clinica Ostetrica e Ginecologica University of Cagliari Medical School Osoedale S. Giovanni di Dio Via Ospedale 46 09 1OO-Cagliari, Italy