NUTRITION CLASSICS

THE JOURNAL OF THE AMERICAN

MEDICAL ASSOCIATION

VOLUME 226 1973 PP 1421-1424

MEGALOBLASTIC CHANGES IN THE CERVICAL EPITHELIUM Association with Oral Contraceptive Therapy and Reversal with Folic Acid

NANCY WHITEHEAD, FRANKLIN REYNER, MD, AND JOHN LINDENBALM, MD

EditOr’s Note: This paper by Whitehead et al. made a crucial observation with regard to the tissue-specific effects of hormone therapy (contraceptives) on folate-dependent fine- tion in cervical epithelium. Also, the observation that folate therapy, in the absence of systemic deficiency, could reverse cytologic changes has stimulated related studies of other epithelial organs that are of major potential importance to our understanding of the early stages of malignant cellular changes and the role of nutrients in chemoprevention.

It is well recognized that in severe folic acid or vitamin B,2 deficiency morphological abnormalities are not confined to hemopoietic cells. Reversible cytologic changes analo- gous to those found in the bone marrow have been observed in epithelial cells from a variety of organs with high cellular turnover rates. These changes have included morpho- logical abnormalities in cervical smears obtained from patients with pernicious anemia and megaloblastic anemia of pregnancy and the puerperium that have been demonstrated to return to normal after cyanocobalamin or folic acid therapy.

The cervical cytologic findings in patients with megaloblastic anemias due to folate deficiency were well described by Van Niekerk, (and confirmed by Klaus) to include nuclear enlargement, multinucleation, and cytoplasmic vacuolation. We recently ob- served similar cervical changes in several nonanemic patients taking oral contraceptive agents. In view of the reported increased incidence of megaloblastic anemia and other abnormalities of folate metabolism in users of oral contraceptives, we undertook a sys- tematic study of a group of women taking these agents to determine the incidence of cytologic abnormalities (using the criteria of folic acid deficiency reported by Van Nie- kerk), their relationship to other indices of folate depletion, and their response to vitamin therapy.

Methods

One hundred fifteen healthy ambulatory Caucasian patients from a middle class socio- economic background who were attending the offices of a private gynecologist were studied. They were receiving no medication other than oral contraceptive therapy at the time of study and had no clinical manifestations of disease. Fifty patients from a similar background who were not and had not been taking oral contraceptives in the past served as a control group.

Results

cytology

Cervicovaginal smears in which at least 10% of the nuclei of intermediate superficial cells were enlarged (greater than 10 p,m in diameter), often with an increase in nuclear- cytoplasmic ratio and frequent multinuclearity, were considered to be “megaloblastic, *’ i.e., demonstrating the changes as typically present in folate-deficiency megaloblastic anemia by Van Niekerk. Cytoplasmic vacuolation . . . was also commonly, but not in- variably, associated with these changes. By these criteria, the smears of 22 (19%) of the 115 patients surveyed were abnormal. The 22 patients with megaloblastic smears did not differ significantly from the remaining 93 normal subjects in mean age (32.5 vs. 31.7 years), mean duration of contraceptive therapy (42.0 vs. 34.4 months, respectively, P > .3), mean number of previous pregnancies or abortions, or the number of days since last menstrual period when the smear was taken. Megaloblastic abnormalities were seen in patients taking various types of oral contraceptive medication, including those taking oral progestational agents only. . . . Though a wide range of dosage levels for both the estro- gen and progestogen components were utilized by the group studied, there were no sig- nificant relationships between the presence of the cervicovaginal abnormalities and dos- age of either component. None of the smears from the 50 control patients not taking oral contraceptive steroids showed megaloblastic changes.

Hematologic Findings

The hematocrit reading and PMN lobe count were determined in 30 patients who were taking oral contraceptive steroids (1 2 with abnormal cervical smears and 18 with normal smears). None of the patients was anemic or had macrocytosis on blood smear. None had a PMN lobe count above the normal range (2.6 to 3.4 lobes) for this laboratory. The mean PMN lobe count of the patients with megaloblastic cervical smears was similar to that in the group with normal smears (2.92 k 0.24 vs. 3.01 k 0.19 lobes, respectively, P > .4). Serum folate concentrations were obtained in 37 patients. . . . The serum folate level was below the lower limit of normal for this laboratory (4 ng/ml) in 3 of 14 (21%) patients with megaloblastic smears and 4 of 23 (17%) with normal smears (P > .4). There was also no significant correlation between serum folate level and PMN lobe count in either of the groups. None of the 37 patients had a low serum vitamin Biz concentra- tion and mean serum vitamin B,, levels were similar in the abnormal and normal groups (635 f 195 vs. 537 f 131 pg/ml, respectively, P > .lo).

Folute Therapy

Eight patients with megaloblastic cervicovaginal smears on initial screening continued taking oral contraceptive therapy and were restudied after six months. There was no significant change in any patient in the mean nuclear diameter based on measurements of 50 cells ( 8 . 2 6 ~ f 3 . 1 1 ~ vs. 8 . 2 7 ~ 2 2.94p), nor were there significant variations in numbers of cells with enlarged nuclei . . . multiple nuclei, or cytoplasmic vacuolation (Table 2). Each patient then agreed to take two 5-mg pteroylglutamic acid tablets daily for three weeks while continuing the oral contraceptive medication. Cervicovaginal smears obtained on the 21st day of folic acid therapy showed a striking reversion of the megaloblastic changes towards normal. The mean nuclear diameter fell to 6 . 6 9 ~ 2 2 . 0 2 ~ (P < .001), reflecting a marked reduction in the number of cells with abnormally large nuclei. . . . When paired data from each patient were analyzed, there was also a

TABLE 2 Number of Cells per 200 Cells Counted in Eight Patients Before and After Folic

Acid (FA) Therapy

Multinucleated CellsNacudated CeUs

Patient 1 2 3 4 5 6 7 8

Initial testing 1014 1411 1010 18/27 23/18 3019 810 1018 Immediately before FA therapy 1513 1410 810 23/29 21/16 3718 910 717 After 21 days of FA therapy 010 010 010 6/5 515 514 010 010

significant fall in the numbers of multinucleated cells (Table 2, P < -001) and a trend towards disappearance of vacuolated cells (Table 2, . 10 > P > .05). Serum folate con- centrations, which were low in three of the eight patients before therapy, rose to high normal or elevated levels during therapy but no significant change in PMN lobe average was observed in any patient.

Comment

In this study, cervical cytologic abnormalities similar to those found in patients with megaloblastic anemia due to folic acid or vitamin B,, deficiency were present in nearly one fifth of a group of middle class Caucasian women taking oral contraceptive agents. Though the morphologic changes observed were qualitatively identical to those described by Van Niekerk in puerperal megaloblastic anemias, in most of our patients they were less severe quantitatively; grossly enlarged nuclei with diameters in the 19p to 25p range were seen only rarely in our subjects, though in an occasional patient the degree of abnormality was equal to that reported in the average patient with a severe megaloblastic anemia. Even in patients with more subtle changes, however, reversal of the abnomali- ties or improvement towards normal was observed after folic acid therapy. . . .

It has been repeatedly observed that the cytologic findings on cervical smears from patients with vitamin B,, or folate deficiency may be mistaken for dysplastic or prema- lignant changes. Some of the abnormalities that were shown to be responsive to folic acid therapy in this study may have been previously noted by others in patients taking oral contraceptive medication and interpreted as evidence of dyskaryosis consistent with dys- plasia or the presence of premalignant change.

Recently megaloblastic anemia secondary to folate deficiency has been increasingly reported as an occasional complication of oral contraceptive therapy. In addition, a high incidence of subnormal serum folate levels and increased urinary excretion of formimin- oglutamic acid have been reported by some observers in women taking oral contracep- tives, and denied by others. In our study 17% to 21% of the subjects studied had de- creased serum folate concentrations, but the incidence of low levels was similar in the patients with normal and abnormal cytologic findings. That systemic folate deficiency was generally not present in the women with “megaloblastic” cervical changes is further supported by the absence of anemia, macmytosis, or increased PMN lobe segmentation.

Increasing evidence in man and experimental animals suggests strong interrelations between sex hormones and folate coenzymes. Studies in chicks and monkeys indicate that folk acid is required for estrogenic action on target organs. In the chick oviduct, folate was shown to be essential to the mechanism of action of estrogen in promoting tissue proliferation at the level of the end organ itself. Recent work has demonstrated that both oral doses of folate and a number of sex steroids induced increases in human small- intestinal glycolytic enzyme activities. In addition, oral doses of sex hormones produce

adaptive changes in the activities of several jejunal folate-metabolizing enzymes in nor- mal and castrated rats. Sex hormones appear to act on target organs via regulation of protein synthesis, and folate coenzymes function as important one-carbon donors in pro- tein and DNA synthesis. It has been suggested that sex hormones may increase the rate of metabolism of folate compounds. This effect would be predicted to be most striking at the end organ level, and this may explain why the women with “megaloblastic” cervical cytologic abnormalities in this study had no evidence of systemic folate deficiency. The rare patients taking oral contraceptives who develop megaloblastic anemia may be ex- treme instances of an effect on folate metabolism manifested much more commonly by localized morphological changes in the reproductive system.

Abridged and reprinted with permission from The Journal of the American Medical Association. 0 1973, American Medical Association.