Relationship between collagen autofluorescence of the human cervix and menopausal status

Photochemistry and Photobiology, Jun 2003 by Gill, Erin M, Malpica, Anais, Alford, Raphael E, Nath, Audrey R, Et al

ABSTRACT

The goal of this study was to evaluate the effect of different menopausal states (pre- and post-) on the endogenous fluorescence of normal cervical tissues. In particular, the average fluorescence as well as the interpatient and intrasample variability in the average fluorescence of the epithelium and stroma were evaluated as a function of pre- and postmenopausal states. High-resolution fluorescence images at excitation-emission wavelengths of 440, 520 nm and 365, 465 nm were obtained from epithelia and stroma of freeze-trapped cervical tissue blocks maintained at -196[degrees]C. The fluorescence images were recorded using a low temperature optical scanner. Fluorescence images from a normal sample population (n = 27) were quantitatively analyzed, and the average epithelial and stromal fluorescence intensities were obtained. Data grouped according to menopausal status (pre- vs post-) showed statistically significant differences (P

INTRODUCTION

It has been estimated that in the United States, 12 200 cases of invasive cervical cancer will be diagnosed and 4100 women will die from this disease in 2003 (1). Risk factors include human papilloma virus (HPV) infection, increasing age, no previous Papanicolaou (Pap) smear, multiple sexual partners or high-risk male partners, first intercourse at an early age, sexually transmitted diseases, smoking and using nonbarrier birth control methods (2). Because symptoms are present only in advanced stages, effective screening and diagnostic techniques are needed. Current screening and diagnostic methods for cervical cancer include the Pap smear and colposcopy. Although Pap smear screening and diagnostic colposcopy have dramatically reduced the number of cervical cancer deaths over the past 50 years, there is still a critical need for promising new technologies that can potentially improve the accuracy and efficacy of these screening and diagnostic programs.

Optical methods offer an alternative for early diagnosis of cervical cancer. Optical methods are noninvasive, fast and relatively inexpensive technologies. A promising optical diagnostic technique for cervical squamous intraepithelial lesions (SIL) under development is fluorescence spectroscopy. Fluorescence spectroscopy has been demonstrated to successfully detect cervical SIL in women undergoing colposcopy (3-10). In a recent study, fluorescence excitation-emission matrices were measured from 351 sites from the cervices of 146 patients undergoing colposcopy. The unbiased sensitivity and specificity for discriminating between squamous normal tissue and high-grade SIL were 71 and 77%, respectively, using fluorescence spectra at three excitation wavelengths, 330-340, 350-380 and 400-450 nm (10).

Fluorescence spectroscopy of cervical cell suspensions and tissue cultures has revealed excitation-emission maxima that are associated with the known endogenous fluorophores: tryptophan, reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and collagen (11). Fluorescence spectroscopy of human cervical tissue at these excitation-emission wavelengths can probe the concentration and distribution of these endogenous fluorophores in vivo. NADH and FAD are found in epithelial cells and are associated with cellular metabolism (12), and collagen is the structural component of the extracellular matrix (12). Differences in the fluorescence spectra of the neoplastic and nonneoplastic cervix have been attributed to NADH and collagen; in particular, an increase in epithelial NADH fluorescence and a decrease in stromal collagen fluorescence have been noted in dysplastic cervical tissues (13).

It is important to note that the biochemistry and morphology of healthy cervical tissue are complex and may be influenced by multiple factors such as menstrual-cycle phase, menopausal status and pregnancy. Therefore, it is essential to understand the variability in normal cervical tissue fluorescence to properly interpret differences between neoplastic and nonneoplastic tissues.

The cervix is composed of a mixture of fibrous, muscular and elastic tissue and is lined by squamous and columnar epithelia. Hormone levels have a significant influence on the epithelial and stromal layers of the cervix. In general, estrogen stimulates epithelial proliferation, maturation and desquamation, whereas progesterone inhibits maturation. In postmenopausal women, the squamous epithelium is atrophic because of the ceasing of epithelial maturation (14). During pregnancy, elevated gestational hormones (estrogen, progesterone) stimulate the enlargement and softening of the cervix. The enlargement is caused by increased vascularity and edema in the stroma. Cervical softening is due to the rearrangement of collagen fibers and accumulation of extracellular ground substance within the stroma (15). The cervical softening, together with enlargement, facilitates dilation of the cervix during labor (14).

Brookner et al. (11) characterized the endogenous fluorophores within normal human cervical tissue and evaluated the changes in the endogenous cervical fluorescence with age and menopause. Using a novel technique, they prepared short-term "live" tissue cultures of normal cervical biopsies and imaged the endogenous fluorescence from the epithelial and stromal layers using epifluorescence microscopy at excitation wavelengths of 380 and 460 nm. NADH is fluorescent at 380 nm excitation, whereas FAD is fluorescent at 460 nm excitation. Collagen fluoresces at both excitation wavelengths. The fluorescence images were stratified into three statistically distinct age groups with average ages of 31, 38 and 49 years. The endogenous epithelial NADH and FAD fluorescence did not display statistically significant differences between the three age groups. However, an increase in the stromal collagen fluorescence was observed in the 38 and 49 year age groups relative to that in the 31 year age group, suggesting an increase in collagen cross-linking with age (16). Because there was a relative increase in the number of postmenopausal patients with age, these results indicate that there is an increase in stromal collagen fluorescence in postmenopausal women, relative to that in premenopausal women.