VAGINAL CURETTAGE AS A MEANS O F DIAGNOSING PREGNAKCY IN THE GUINEA-PIG' G . LOMBARD KELLY Departnient of Anatomy, Medical Department, University of Georgia, Augusta OKE TEXT FIGURE A N D ONE EIELIOTYPE PLATE (FOUR FIGURES) INTRODUCTION The fact that the vaginal epithelium of the guinea-pig, like that of several other rodents, undergoes a very marked transformation during pregnancy has been known f o r many years. Lataste(4) seems to have been the first investigator to make a note of this phenomenon ( '87), and under his direction Xorau(5, 6) published two papers bearing directly on the subject ( '88, '89). Following these writers, articles appeared by Salvioli(8) and de Retterer(7), in 1890 and 1892. Since then this subject has been treated extensively in the literature. Recently, the present writer(1) has taken up the investigation and followed it from a number of angles, working out the vaginal epithelial changes in the guinea-pig under a wide variety of conditions. This paper cites most of the literature in this particular field up to the present. As stated in this article, the changes that occur in the vaginal epithelium are quite marked. The stratified squamous epithelium of estrus is transformed gradually to a high columnar, mucous epithelium (figs. 2 and 3 ) . The writer has shown (see reference 1) that this change begins about 'Contribution no. 3, series B, from the Department of Anatomy, Medical Department of the University of Georgia. This work has been aided by a grant from .the National Research Council through its Committee for Research on Problems of Sex. 373 T H E ANATOMlC.41, RECORD, VOL. 40, NO. 3 374 G . LOMBARD KELLY the second week of pregnancy and is well developed by the end of the third week. From this time on the changes that occur are due in large part to the hypertrophy of the vagina, though the mueous nature of the cells becomes enhanced as pregnancy continues. Between estrous periods the vaginal mucosa assumes a traiisitiorial form; it is very thin and cannot be describcd a s strictly stratified syuamous or simple columnar. For illustrations of this form see reference l. Since no mucous cells are present in this epithelium, curettage at this interval will give negative scrapings. METHODS The extent of this epithelial transformation first came to my attention while working on the mechanism of the opening and closing of the vaginal orifice in the guinea-pig(2), a female at term having been sacrificed during this investigation. It occurred to me then that, if these cells could be scraped from the vagina, they would serve as a means of diagnosing pregnaiicy in this animal. Some time later, I happened t o be examining vaginal smears from guinea-pigs daily, and in many of them I noticed the large mucous cells that I have since found are present in large numbers after abortions o r deliveries (figs. 4 and 5). Their presence in such smears is due to their desquamation after the termination of the pregnant state. The appearance of the cells in these two figures is the same as when they are obtained b y the method described below, that is, by vaginal curettage. These cells are characteristic in appearance, being x7er.Jlarge and reticulated, and having a small, dark nucleus. They are as a whole basophilic and take a pale blue or lavender stain. Usually they are seen in clumps, as shown in the illustrations ; very seldom singly. By sectioning vaginas of animals that had been pregnant from seven to nine weeks, it was easy to identify these cells as the high columnar, mucous epithelial cells of pregnancy (compare figs. 3 and 5). DIAGNOSING PREGNANCY IN THE GUINEA-PIG 375 Since it has been found that very few of these mucous cells desquamate before delivery, the vaginal smear as first described by Stockard and Papanicolaou(9) is not satisfactory for obtaining cells from the vaginal wall as a diagnostic method. It is true, however, that many smears from pregnant animals will show quite a number of these cells; but all smears do not. Consequently, curettage seemed to be the best avenue of approach. Fig. 1 Thr rdurettr. Some difficulty was experienced in devising a curette that would work satisfactorily. The epithelium was so smooth that the edge of the curette did not take hold easily, Finally, the type illustrated (fig. 1) was found to accomplish the purpose. It was made from a narrow, flat piece of steel by bending one end back to less than a right angle with the shaft and then sharpening the bent-over end to a fine edge. The curettage is performed as follows: With the aid of an assistaiit, the animal is wrapped in a towel with only its 376 G . LOMBARD KELLY head out, in order to prevent struggling, and given just sufficient ether to get it asleep. The vulvar site is cleaned with a sponge saturated with alcohol and the vulvar lips torn open. The curette is now introduced and the curettage performed, pressing the instrument firmly against the surface scraped (usually the ventral wall of the vagina). With the end of a clean pair of forceps the curetted material is pushed from the angle of the curette to a glass slide and smeared there. Fixation is with alcohol-ether, equal parts ; staining, with liaematin and eosin. The entire procedure up to fixation requires but a few minutes, and the animal is usually awake by the time the slide is placed in the alcohol-ether. In giving guinea-pigs ether it is advisable to use an indirect method, that is, to pump it from a bottle with a rubber hulh. The gas is thus delivered to the animal’s nostrils through a rubber tube, on the end of which the lialf of an old rubber bulb with two or three air holes in its sides can he attached as a nose-piece. If ether is applied directly, postoperative pneumonia is apt to be frequent. Incidentally, we have never had any accidents whenever these methods have beeii used. OBSERVATIONS At times it is very desirable t o know whether a female guinea-pig is pregnant or not. The long gestation period (nine weeks) tends to make this knowledge even more desirable if non-pregnant animals are needed within a short time f o r experimental work. On January 18, 1928, we received a shipment of ten female guinea-pigs. These animals had been ordered for a particular experiment that required non-gravid animals. On January 23rcl, vaginal curettage was undertaken, with the results shomm in table 1. As will be noted, two of these females had abortions the day following arrival-a. not uncommon accident after long transportation to new surroundings. Of course, the scrapings from these animals were positive, though they were not then pregnant. A third animal was in stage 1 of estrns 377 DIAGNOSING PREGNANCY IX THE GUINEA-PIG the day after arrival. Naturally, it had a negative scraping three days later; but in examining the slides no identifying marks revealed from which animal they came, each diagnosis being made on the presence or absence of the characteristic cells. Since the vaginal epithelium of the high columnar, mucous type does not develop until nearly three weeks after copulation, it can readily be seen that two curettages, three weeks apart, are necessary in order to rule out pregnancy. A curetTABLE 1 This shows the results obtained in curetting a new shipment of ten animals - -. NO. - 1051 1052 1053l 1054 1055 1056a 1057 1058 1059' * . . ~ FIRST CURETTAGE L SECOND CURETTAGE ~ FINAL DIAGNOSIS CORRECT -- ~- Yes Yes 1/23/28, negative 1/23/28, doubtful 2/24/28, positive 2/14/28, positive Positive Positive ...... .... 1/23/28, 1/23/28, 1/23/28, 1/23/28, 1/23/28, None None Nolie 2/14/28, positive None Positive Positive Negative Positive Positive Yes Yes Yes Yes Yes ...... positive positive negative negative positive ...... ...... ...... None -~~ __ ...... Negative .... Yes ~ Animal 1060 returiied to estrus before time f o r the second curettage. tage once a week for three weeks would be better. This explains why three of the animals referred to were first diagnosed as non-pregnant, though a subsequent examination revealed positive findings. They had been pregnant less than three weeks when they arrived. It is worthy of note that, with this precaution, the final diagnoses were all correct (omitting the two animals that were known just to have aborted). I n doing this work, especial care was taken to avoid bias. No palpations were made and none of the animals were big with young when they came. Finally, the slides were stained and mounted by an assistant, anct the eliamiiier did not know one slide from aiiotlier, but hased his diagnosis only on the histological appearance. In the spring of 1928, we were doing some experiments on artificial insemination of the guinea-pig by way of the ovarian hursa(3) ailti were very desirous of knowing as soon as possible whether a particular animal was impregnated or not. Of course, those that returned to estrus a t the expected time WIT not pregnant, but about the others we could not be absolutely sure, since these animals may occasionally have estrus so weakly expressed that the vaginal orifice does not opcn. So in each of this group a curettage was done three weeks after the operation. Sever1 animals were operate(! upon. Three came hack into estrus and the remaining four were cnrctted. All four of the latter showed positive scrapi n g ~and subsequently were found actually to be pregnant. Tn tr'ying out this method in tlie heginning a large nnmbcr' of c u r d tages were done on animals whose state was kiiowii, h u t the scrapings were examined without knowledge of their source. It was soon found that it was not at all a difficult mattor to tell wlietlicii* a given slide was from a pregnaiit or 11 oil-pregnaii t animal. DISCUSSION making such diagnoses three limitations must he boriie miacl : First, the vagirial epithelium of immature females is of the high columnar, mucous type and practically identical with that of advanced pregnancy. Consequently, curettage 0 1 1 ariimals that have iiever come into estrus woalcl show the mucous cells. The cells, howcver, arc smaller than those of pregnancy, though the difference in size is not very great. These animals are very small and their sexual immaturity ciiii usually be inferred from their size. While the majority of them have their first estrus at about three months of age, we have had quite a number that were only a little over two months old when the first stage-1 smear was obtained. Secondly, curettage of animals within one or two weeks after abortion or tldivery will probahly reveal mucous cells 111 iii DIAGNOSING PREGNANCY I N T H E GUINEA-PIG 379 in the scrapings, since all the cells do not desquamate a t the delivery estrus. Occasionally, the mucous cells may persist until the estrus following the delivery estrus, but not thereafter in a scraping will they be abundant, as from a pregnant animal, though in smears a few may be seen for two or three weeks longer. I n a vaginal scraping from a pregnant animal the domiiiancy of the mucous cells is easily apparent. A third limitation has already hecn mentioned, viz., the animal must be pregnant three weeks before the test is effective. For those experienced enough to determine early pregnancy in the guinea-pig by palpation, this method may not hold much of value. It may provide a method of differential diagnosis in case of abdominal tumors, but this is doubtful. Still, many persons can not by palpation of this aiiimaI determine pregnancy until it is well advanced. For them vaginal curettage should solve the question. We have already found it practicable within its limitations and often very useful. I t s theoretical value is not without interest. Finally, the question arises as to the diagnostic value of this procedure in other species. We have not tried vaginal curettage in other animals, with one exception, and can say nothing about it. A student became interested in the method and tried it on white rats known to be pregnant, but without success. That is, he found no characteristic mucous cells. This x7ork was not supervised and possibly the curettage was not properly done. It requires firm pressure to scrape the mucous cells off. Of course, in animals with a persistent form of epithelium throughout pregnancy this method would have no value. For this reason it has no application in the human (with our present knowledge of the histology of the human vagina). 380 G . LOMBARD KELLY CONCLUSIONS 1. By vaginal curettage pregnancy can be diagnosed in guinea-pigs three weeks or more after copulation. 2. Positive scrapings can be obtained from sexually immature females and from females within two or three weeks after abortion or miscarriage; these are limitations of the method. 3. The method has no valne, so f a r as can be told a t present, in animals that do not have a marked transition in the vaginal epithelium during pregnancy, such as is seen in the guinea-pig. LITERATURE CITED The histological transformations in the vaginal epithelium of the guinea-pig. (To be published.) KELLY,G. L., AND PAPANICOLAOU 1927 The mechanism of the periodical opening and closing of the vaginal orifice in the guinea-pig. Am. Jour. Anat., vol. 40, no. 2. KELLY,G . L., Err AL. Artificial insemination by way of the ovarian bursa in the guinea-pig. (To be published.) LATASTE,F. 1887 Rccherches de xo66thique. Actes de la Soe. Linnienne de Bordeaux, T. 40, pp. 317, 360, and 500. MORAU, 13. 1888 Des transformations pgriodiques de l’epith6lium de la muqueuse de quelques rongeurs. Comptes Rendus de Biol., Paris, 85, T. 5, p. 831. 1889 Des transformations epith6liales de la muqueuse du vagiii de quelques rongeurs. Jour. de 1’Anat. et Phys., Paris, T. 2.5, pp. 277-297. DE RETTEEER,ED. 1892 Sur la morphologie et l’bvolution de I’epith6lium du vagin de mammif6res. Comptes Rendus, SOC. de Biol., P a r k , 95, T. 4, pt. 2, pp. 101-107. SALVIOLI, I. 1890 Contribution B la physiologie de l’epith6lium. De la structure de l’epith6lium vaginal de la lapine et des modifications qu’il suhit pendant la gestation. Translated from Atti d. r. Acad. d. Sv. di Torino, svi. STOCKARD AND PAPANICOLAOU 1917 The existence of a typical oestrous cycle in the guinea-pig with a description of its histological and physiological changes. Am. dour. Anat., vol. 22, no. 2. 1 KELLY,G. L. 2 3 4 5 6 7 8 9 PLATE 381 I’LSTM 1 EXPLANATION OF FIGURES 9 Low-pnww niicropliotograpl~of v a g i n a of a guinea-pig 11he weeks ylrgn;tnt, ~howingthe. high c.oluinnar rnuvous epithelium. X 130. 3 High-powrr view of the same. Comp;ire tliesc cells w i t h those in figure 3. Soticr the diffrrence i n color and sizr of tho nurlri in the two figurt,s. X 360. 4 Ilow-powrr mierophotogrnph of mucous cells :IS they :tppe:ir in :t vugiiial scraping. The collwtion in e l u s t t ~ sis characteristic. X 150. ?. High-power view of the same. Compare the size of thc crlls with t h a t of the war-by leiirocytes. Note that the nuclei appear to be undergoing caryolysis. Thcs rrtieular structure of t h r cells is well shown here. X 360. 3IAGNOSING PREGXAKCY I N THE GUINEA-PIG 0. I,ODIBABD KBIiIiY 383 THE ANATOMICAL RRCORD, VOL. 40, NO. 3 PLATE 1
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