Urinary cytology in reproductive endocrinology

Urinary cytology in reproductive endocrinology

Urinary cytology in reproductive endocrinology P. J. O'MORCHOE, M.A., M.D. C. C. C. O'MORCHOE. M.A., M.D. Dublin, Ireland The direct effect of sex ho...

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Urinary cytology in reproductive endocrinology P. J. O'MORCHOE, M.A., M.D. C. C. C. O'MORCHOE. M.A., M.D. Dublin, Ireland

The direct effect of sex hormones on the urethral epithelium is reflected in the cells voided in the urine. Daily urinary smears from 5 women over 30 normal menstrual cycles were studied and correlated with simultaneous vaginal smears in 12 cycles and basal body temperature recordings in 15 cycles. The patte~n of the eosinophilic karyopyknotic (E.K.) index and of the individual cellular characteristics was similar in urinary and vaginal smears, but the E.K. index was higher throughout the cycle in vaginal smears. It is concluded that in reproductive endocrinology urinary cytology provides a valuable adjunct to vaginal cytology and is an important alternative when this is (Ontraindicated. Its value in the timing of ovulation is clearly demonstrated.

T H E L o w E R part of the urethra has an embryologic origin similar to that of the vagina1 • 2 and therefore cells exfoliated from the urethra and voided in the urine provide information regarding the comparative levels of at least two of the sex hormones (estrogen and progesterone). Under certain circumstance5, there would be advantages to the study of urinary cytology if it afforded information comparable to that of vaginal cytology. Del Castillo, Argonz, and GalliMainini3 in 1948, showed that the cytologic pictures of the vaginal smear and urinary sediment taken simultaneously from the same patient were similar. In 1948, Papanicolaou 4 suggested that significant cytologic changes might be noted even more readily in the urinary sediment than in vaginal secretions. In spite of this, the use of urinary cytology has been largely confined to research projects."-9 We believe that faulty techniques have been responsible for the failure to appreciate the advantages of urinary cytology in reproductive endocrinology. A technique10 was developed for the collection, storage, and processing of urine which consistently resulted in satisfactory smears. Using this technique, the urinary

cytology of the normal menstrual cycle was studied with special reference to simultaneous vaginal cytology and basal body temperature recordings. Materials and methods

Observations extended over 30 menstrual cycles in 5 normal women. In each cycle daily (approximately 3 per cent exceptions) smears were made from the urinary sediment and in 12 cycles vaginal smears were taken concurrently. In 15 of the cycles basal body temperature was recorded. Twenty milliliter samples of urine were collected directly into screw-top polythene test tubes containing 5 mi. of fixative (95 per cent alcohol with or without the addition of formalin) . Smears were made from the sediment obtained after centrifugation. Vaginal smears were taken from the posterior fornix (pool), immediately spread on the slide, and fixed (50: 50 ether and 95 per cent alcohol) . Smears were stained by a modified Shorr hematoxylin method 11 and examined for cell population, individual cell characteristics, and the differential cell count. For the differential cell count, cells were classified into the four basic types shown in Fig. 1 and expressed as a percentage of the total count (Index).

From The Department of Physiology and School of Anatomy, Trinity College, Dublin University.

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Fig. 2. T h <' urocytograph was constructed frolll th1 · E.K . index of :HI JJ u · nstru~l <' )Tit ·s and arranged so that the "peak" day (o) of •·ach cycle coiucid.,d . The teltlpernturc chart is also a compositt' one of recordings take n during I cycles. It was construc ted h" roin c idin <: lt'I JJ · peratures recordeJ on the urinary pea k days and s uc c~ssi\'o· da ys hdon· and ;..ft•· r tlu· [><'il k of pac h n •d e. The sha
Urinary cytology m reproductive endocrinology

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R·esults

A composite picture of the urinary eosinophil:ic karyopyknotic (E.K.) index of 30 cycles is shown in the lower part of Fig. 2, which was constructed by first coinciding the "peak" day of each cycle when the E.K . index was at its highest. It was completed by coinciding the days before and after the peak, respectively. The figure was not extended for more than 13 days on either side of the peak since many cycles were not longer than 27 days. The average value remain:; at about 4 per cent until 4 to 5 days before the peak when it starts to rise. Its original level is regained on the fifth day after the peak and is maintained until the end of the cycle. The secondary rise which has sometimes been described in vaginal cytology 12 did occur in a few of the cycles (as shown by the shaded area) but did not affect significantly the average value.

The average basophilic karyopyknotic (B.K.) index for the vaginal cycles remained at approximately 5 per cent whereas that of the urinary cycles was about 10 per cent. In occasional cycles, however, there was a slight increase around the time of ovulation and again toward the end of the cycle. Random variation of parabasal cells between 0 and 5 per cent occurred without any cyclic rhythm. Because of the relative constancy of the parabasal and B.K. indices the percentage of intermediate cells varied inversely with the E.K. index. Fig. 3 compares composite pictures of vaginal and urinary E.K. indices. The E.K. index is higher in vaginal smears and rarely falls to 0 per cent, at least in the first half of the cycle. Also in the vaginal cytograph the average value begins to rise several days earlier and the secondary peak is more apparent.

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B

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Fig. 3. A is a composite picture constructed from the E.K. index of 12 vaginal cycles arranged so that the "peak" day ( o) of each cycle coincided. B is a urocytograph constructed in a similar way from the E.K. index of the same 12 cycles. The urocytograph in C was constructed frcm the E.K. index of the remaining 18 cycles studied.

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Table I. Correlation of urinary and vaginal peak days with the change m basal body temperature j

Cytology peak (days before first day of next cyclej

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Subject

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10.5

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6

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4

1

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97.57

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97.91

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98.03

0.49

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97.64

98.32

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98.16

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Total average

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30

12

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12.33

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Table I correlates the urinary and vaginal peak days and the change in basai body temperature. The figures in columns 2, 3, 5, ~"'1d 6 \Vere obtained by counting from the peak day to the last day of the cycle inclusive. The total averages are shown in the bottom line of Table I. If, however, the 12 cycles in which simultaneous urinary and vaginal smears were taken are considered alone, then on an average the urinary peak occurred 12.83 days and the vaginal peak 12.33 days before the first day of the next cycle. The pattern of the temperature rise was found to follow one of two forms. In the first type (Fig. 4, A) the rise was completed within 24 hours. For correlation of these cycles with cytology the day of rise was taken as the first day on which the temperature approximately equaled the average of the second part of the cycle. This day was chosen because it was the first day on which the subject was aware of the change. The second type of temperature rise occurred over 2 to 3 days (Fig. 4, B). The day of rise was taken as the first day on which the temperature was higher than all the preceding temperatures of that cycle (excluding abnormal values) . Occasionally,

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2

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this day could be chosen only in retrospect. When the temperature rise was compieted within 24 hours (9 cycles) the day of rise occurred either 1 or 2 days after the urinary peak. On the other hand when the tempera· ture rise was gradual ( 6 cycles) the day of rise occurred between 3 days before and 1 day after the urinary peak. Fig. 2 correlates the basal body temperature with the composite urocytograph. The average temperature line has three segments. The middle segment slopes upward for approximately 0.5° F. over 2 days, from the morning of the day before the peak to the morning of the day after the peak. The first and third segments are comparatively level at about 97.5° F. and about 98.2° F., respectively. Comment

The features of vaginal smears which characterize the different stages of the menstrual cycle12 - 14 are dosely paralleled in urinary smears. Urinary smears (Figs. 5, 6, and 7) taken 5 days before the peak, on the peak day, and 5 days after the peak show the cellular characteristics- of the main phases of the cycle. Subdivision of these phases16 was found to be of little practical

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value since the finer features of smears vary from day to day, from cycle to cycle, and from subject to subject, so that it may even be di:fficult to decide whether a smear is pre- or postovulatory unless other smears from the same cycle are available for comparison. As with vaginal cytology, postovulatory urinary smears are characterized principally by an angularity of the cell outlines (Fig. 7) due at least in part to infolding of the cytoplasmic borders and by a tendency of the cells to occur in groups. Preovulatory smears have a higher incidence of discrete, rounded, and flat intermediate cells (Fig. 5). This contrast is attributed to the effect of hig:h progesterone levels in the second part of the cycle. An increasing number of leukocytes and a tendency to "dirty" smears, sometimes seen in vaginal cytology after ovulation, have also been attributed to the effect of progesterone, but these signs are not seen in urinary smears. Except where there is excess sediment, urinary smears remain "clean" throughout the cycle.

At about the middle of the cycle (Fig. 6) the urinary smear clearly resembles the vaginal smear in that there is a high E.K. index and all the cells tend to be flat, rounded, and discrete. When subsequent smears exhibit a progesterone effect, it is generally accepted th-.t ovulation has occurred at or shortly before the peak of the E.K. index. Hence, cytologic examination has been used in the timing of ovulation as an adjunct to fertilization.H If, however, ovulation occurs before the peak and the peak is not recognized for a number of hours, at least, it may then be too late for fertilization to take place. The temperature chart also fails to give an early indication of ovulation; the average values (Fig. 3) show that the rise in temperature and the urinary peak occur on the same day. One method designed to overcome this difficulty is to study vaginal cytoiogy in each patient for two or three cycles. With regular cycles and reproducible cytology the imminence of the peak can be gauged by reference to previous cycles.

484 O'Morchoe and O'Morchoe

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B Fig. 5. Photomicrographs of a urinary smear, made 5 days bdore the "peak" day, in which the cells are predominantly disc rete, flat, and rounded (compa re with Fig. 7) . (A. x !OO: B, >< 250.)

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Urinary cytology rn reproductive endocrinology

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F:ig. 6. Photomicrographs of a urinary "peak day" superficial in type. (A, x JOO ; B, x 250. )

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B Fig. 7. Photomicrographs of a urinary smear, made 5 days after the "peak" day, in which the cells are predominantly angular in outline (compare with Fig. 6). (A, x!OO; B, x250.)

Volume ~9 Number •I

However, problems, including a high incidence of vaginitis, are created when vaginal cytology is followed for such a length of time. Urinary cytology, on the other hand, may be followed daily, if necessary, without risk of infection. A further advantage in the use of urinary cytology can be seen from Figs. ~! and 3. In Fig. 3, A the line representing the average E.K. index rises gradually for as much as ten days before the peak. In Fig. 2 the first sign of a rising E.K. index occurs only 4 days before the peak. If the urocytograph of a patient's E.K. index corresponds with the average values shown in Fig. 2, then it may be assumed that if ovulation occurs it will do so in the 3 to 4 day interval between the beginning of the rise and the peak day. Therefore, insemination during this time will have a greater chance of success than if carried out after the peak. The use of vaginal cytology in the diagnosis and the treatment of endocrine disorders may oecasionally be unsatisfactory or even contraindicated. Reference has already been made to the risk of inducing vaginitis, especially when repeated smears are required, and the existence of vaginitis, whatever the cause, renders vaginal smears difficult or impossible to assess. For psychological reasons, the taking of vaginal smears in young girls is to be avoided if possible and even adults may dislike making their own smears at home. If it is considered that vaginal smears must be taken from the lateral wall with the aid of a speculum, then attendance at an outpatient department is a necessity. In all these instances, urinary cytology offers a simple and effective alternative; as well as providing a procedure for use in endocrine diagnosis in the male.

Urinary cytology in reproductive endocrinology 487

Although in urinary cytology individual variation makes it difficult to define the normal average and limits of the E.K. index, the composite urocytograph in Fig. 2 may be used as a guide. By superimposing any urocytograph over that of Fig. 2, it can be seen whether it fits within the limits and how closely it corresponds with the average. It is of interest that on no occasion did the E.K. index rise above 21 outside the 11 day period which had the peak day at its center. In contradistinction, the lowest value recorded for the peak E.K. index was 20. The consistently lower E.K. index in urinary as compared with vaginal smears may be due to frequent washing away of superficial cells from the urethral epithelium. It must be emphasized that, as with vaginal cytology, a normal E.K. index is not in itself evidence of a "normal" cycle. For a cycle to be accepted as "normal," it is necessary to take into account the over-all cellular pattern and composition, especially with regard to those findings associated with the effect of progesterone. This study, we believe, has shown that urinary cytology can be accepted as a simple and reliable method for the study of hormone levels during the menstrual cycle. Its simplicity makes it especially suitable for use by the patient at home, thus making provision for daily recordings on an outpatient basis with the minimum of inconvenience. Its close correlation with vaginal cytology also renders it a suitable alternative when the latter is contraindicated or inadvisable. We wish to express our thanks to C. Foran for his excellent technical assistance.

REFERENCES

l. Zuckerman, S.: Lancet 1: 135, 1938.

2. Zuckerman, S.: Bioi. Rev. 15: 15, 231, 1940. 3. Del Castillo, E. B., Argonz, J., and GalliMadnini, C.: J. Clin. Endocrinol. 8: 76, 1948. 4. Papanicolaou, G. N.: Proc. Soc. Exper. Bioi. & Med. 67: 247, 1948. 5. Sera, P.: Acta cyto1. 3: 305, 1959. 6. Castellanos, H., and Taymor, M. L.: Fertil. & Steril. 13: 314·, 1962.

7. Castellanos, H., Fairgrieve, J., O'Morchoe, P. ]., and Moore, F. D.: J. A. M. A. 184: 295, 1963. 8. Castellanos, H., and Sturgis, S. H.: Progress in Gynecology IV, New York, 1963, Grune & Stratton, Inc. 9. Haour, P., and Cardon, J. P.: Proc. of the Second International Congress of Exfoliative Cytology, 1965, p. 117.

\ lttob<·t l'>. 1'167

488 O'Morchoe and O'Morchoe

10. O'Morchoe, P. J., and O'Morchoe, C. C. C.: Acta cytol. 11: 145, 196i. II. Murray, M.: Personal communication. 12. Smolka, H., and Soost, H-J.: An Outline and Atlas of Gynaecological Cytodiagnosis, ed. 2. Baltimore, 1965, The Williams & Wilkins Company. 13. Papanicolaou, G. N .. Traut, H. F., and

\w

14. 15. 16. 17.

J t >hq _ & r;,.nt·•

Marchetti, A. A.: The Epithelia of Woman'; Reproductive Organs; New York. 1948. Rauscher, H.: Acta cytol. 4: 81, 1960. Wachtel, E.: Acta cytol. 4: 82, 1960. de Allende, I. L. C., and Orias, 0.: Cytology of the Human Vagina, U. S. A., 1950. Gaudefroy, M., Rakoff, A. E., Rauscher, H., and Wachtel, E.: Acta eytol. 4: 80, 1960.