.v,_1: y . .v.,._. ; ..... s .,...r..“ 130k4672 llllilillllltill!llllllllllllllllll 3 1293 00579 1532 This is to certify that the thesis entitled GUARDED RIGHT UTERINE HORN LAVAGE IN THE MARE FOR BACTERIOLOGY AND CYTOLOGY presented by Andrew Robert Schmidt has been accepted towards fulfillment of the requirements for m.s. degree in Large Animal Clinical Sciences @442 KM Major professor Date November , 1 9 8 8 0.7639 MS U is an Affirmative Action/Equal Opportunity Institution Lzzétblfi¢fiy {Mt}: higuflc Jtfitfi l University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before due due. DATE DUE DATE DUE DATE DUE £l¥ l l MSU Is An Affirmative Action/Equal Opportunity Institution GUARDED RIGHT UTERINE HORN LAVAGE IN THE MARE FOR BACTERIOLOGY AND CYTOLOGY BY Andrew Robert Schmidt A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Large Animal Clinical Sciences 1988 ABSTRACT GUARDED RIGHT UTERINE HORN LAVAGE IN THE MARE FOR BACTERIOLOGY AND CYTOLOGY BY Andrew Robert Schmidt Conventional methods for endometrial evaluation of subfertile mares include guarded swabs for culture and cytology. Efforts in this investigation were aimed at development of a new method which would improve diagnostic accuracy by sampling a larger endometrial surface area and by reducing the sample contamination. A guarded right uterine horn lavage (UHL) technique was evaluated in eight normal mares in estrus, diestrus, and anestrus. Uterine horn lavage for culture and cytology was accomplished by vaginal-rectal placement of a 10 Fr. silicone balloon catheter into the right horn. Aerobic cultures of centrifuged UHL fluid resulted' in significantly' more .no growths than the guarded endometrial swab technique. Mean ciliated cytoplasmic tuft scores were significantly higher in UHL cytology slides from mares in estrus. The results of this study indicate that UHL in the mare is a feasible technique that provides a .less contaminated sample than guarded swabs and additional cytologic information. ACKNOWLEDGMENTS I wish to express deep gratitude to my advisor, Dr. Carla L. Carleton, for her patience, enthusiasm, and guidance in preparation of this thesis. I am also indebted to my other graduate committee members: Drs. Christopher Brown, Robert D. Walker, and Edward C. Mather, for their expert assistance and counsel. Much of the research and manuscript preparation would not have been possible without the undaunted support and sacrifice of my wife, Shantelle, and son, Benjamin Carl. Inspiration gathered from my wife and committee members, Drs. Carleton and Brown, allowed completion of this thesis. iii Table of Contents LIST OF TABLES ....................................... v LIST OF FIGURES ..................................... vi INTRODUCTION....... .................. . .......... .....1 LITERATURE REVIEW .................................... 3 Endometrial Sampling Techniques: Women.......... .............. . .............. 3 Endometrial Sampling Techniques. Mares ......................................... 10 Interpretation of Endometrial Cytology: Women ....................................... 16 Interpretation of Endometrial Cytology: MareSOOOOOOOOOCO 0000000000000000 0.... 00000000 20 Interpretation of Endometrial Cultures: Women. ...... ....................... .. ...... 28 Interpretation of Endometrial Cultures: Mares ....... .... .. ..... ... ................. .32 Statement of Purpose ..... ..... ............ ...... 37 MATERIALS AND METHODS. .............................. 40 RESULTS... ...... ..... ............................... 59 DISCUSSION .......................................... 69 LIST OF REFERENCES ...... .... ......... ....... ....... .80 iv LIST OF TABLES Table ' Page 1. Shifts in endometrial bi0psy category . . 60 LIST OF FIGURES Guarded uterine horn lavage apparatus . Page 45 Position of the guarded UHL apparatus in the uterine body . . . . . . . . . . 48 Final placement and inflation of the cuff in the right uterine horn . . . . . . . 50 Percent recovery of UHL fluid . . . . . 62 Number of isolates from endometrial cultures . . . . . . . . . . . . 63 Isolation frequency from endometrial culture . . . . . . . . . . . . . 64 Percent intact epithelial cells from endometrial cytology . . . . . . . . 66 Tuft and red blood cell scores from endometrial cytology . . . . . . . . 67 vi INTRODUCTION Bacterial endometritis is a very significant cause of reproductive failure in mares (Couto and Hughes, 1984). The uterus of the mare is challenged by microorganisms and foreign material at breeding, foaling, during reproductive examinations, and as a result of poor perineal conformation. Chronic endometritis may develop in mares with decreased uterine defense mechanisms (Liu, 1988). Clinical signs of endometritis in mares are frequently subtle, and the only apparent abnormality may be infertility. Conventional diagnostic procedures used to evaluate the subfertile mare include endometrial swab culture, swab cytology, and endometrial biopsy. Results from swab culture techniques can be misleading due to contamination of the sample, and in other instances one may fail to obtain positive culture results from an infected uterus (Asbury, 1984a). Swab cytology is quick and easy, but has the disadvantage that many cellular artifacts may be present and it seldom provides an indication of causative factors (Couto and Hughes, 1984). Endometrial biopsy allows direct examination of the uterine lining, yet as in cytology, it seldom determines the etiology. The purpose of the present study was to develop and investigate a new technique, guarded uterine horn lavage (UHL), in endometrial evaluation. The UHL catheter system was developed with several goals in mind: (1) efficiently block the uterine horn for good recovery of fluid; (2) obtain an. uncontaminated sample; (3) obtain a high quality sample for culture and cytology from a large endometrial surface area; and (4) comparison of UHL results with conventional techniques. LITERATURE REVIEW This review will be limited to the literature involving outpatient endometrial sampling techniques and their cytologic and microbiologic interpretation in the human and equine species. Research has paralleled clinical efforts to optimize fertility in individuals of both species, and to diagnose endometrial carcinoma in women during early screening visits. Economic and anatomic factors have limited the use of sophisticated sampling techniques for individual cases in most other domestic species. Endometrial Sampling Techniques: Women The original Papanicolaou technique (Papanicolaou and Traut, 1941) described aspiration of cells from the posterior vaginal fornix using a hollow glass tube and a rubber bulb. Collected material was examined cytologically (x1 glass slide smears following wet fixation. In 1944, Ayre reported bulb aspiration of the external cervical as as more accurate than vaginal sampling for detecting cervical neoplasia. Endometrial cells were noted only when there was an abnormal discharge or endometrial bleeding. The use of cervical exfoliative cytology smears (Pap smear) has been very successful in screening for carcinoma of the cervix, yet has not been found to be accurate in detecting endometrial neoplasia. Only 1/3 to 1/2 of patients with uterine adenocarcinoma will have abnormal vaginal smear cytology (Creasman, 1986). Cary (1943) described an endometrial aspiration technique using a 28 cm 6 Fr. curved metal cannula with a bulbous tip. A 3 cc syringe provided negative pressure after the cannula was passed transcervically. Papanicolaou (1943) supported this technique in an accompanying paper and emphasized the need for simplicity and patient comfort in future instrument development as well. Romberg (1954) and Ferriera (1956) described aspiration techniques using larger syringes which made it possible to increase the force at the tip and apply a larger negative pressure.“ They were investigating the technique to accomplish endometrial dating by cytology. Koss, (1961) considered endometrial aspiration smears to be cumbersome and not always reliable compared to_ mechanical endometrial biopsy. This simple fundal aspiration technique missed many small carcinomas, and full dilatation and curettage under general anesthesia continued as the predominate diagnostic technique for endometrial cancer. The quest for a simple, accurate, and atraumatic outpatient endometrial sampling method continued. Techniques were developed using uterine lavage, endometrial washing, and more efficient aspiration. Barbaro (1969) reported a uterine lavage technique for assessment of endometrial cytology using a double lumen catheter that was coned at the tip to plug the cervix. A syringe attached to one lumen created negative pressure in utero, which drew fluid from a closed vial into the uterus and eventually into the syringe barrel. The author stated a 90% success rate in retrieving endometrial cells and accuracy approaching 85% in premalignant and malignant conditions of the endocervix and endometrium. The Gravlee jet washer was a more invasive vacuum-suction. method of ‘uterine lavage (Gravlee, 1969). .A straight double lumen metal cannula (5 mm diameter), with multiple ports in the uterine end, was inserted into the fundus and each cornu for sampling. As with Barbaro's technique, suction by syringe created fluid flow through the uterine lumen. Cytocentrifugation for cytology or a paraffin block technique for histology was suggested. Barbaro's and Gravlee's techniques are based on the siphon principle to prevent injection of fluid up the open Fallopian tubes of women and possible dissemination of malignant cells into the peritoneal cavity. Gravlee (1969) also reported results of a cytologic study of 1481 women prior to conventional dilatation and curettage. All 53 endometrial adenocarcinomas were diagnosed by use of the jet washer and by histologic assessment of tissue obtained via dilatation and curettage. However, jet washings of 397 (27%) of these women produced unsatisfactory specimens. Multiple studies were published during the early 1970's describing results gathered using the Gravlee jet washer (Gravlee, 1975). Accuracy for carcinoma detection was high (90—100%) in the larger studies. Two studies report that cytological examination of the washings gave significantly more satisfactory samples for diagnosis than did the paraffin block technique for histology in postmenopausal women (Henderson et al., 1975; Girtanner et al., 1977). Slides for cytologic assessment were made by either sediment smear or ultrafiltration in these studies. Gravlee (1975) suggested the use of cytocentrifugation and albumen coated slides for cytologic analysis of poorly cellular fluids. Casey (1977) reported that of 201 asympatomatic women sampled with the jet washer 74.2% had slight or no discomfort. Moderate or severe discomfort was experienced by 41% of older women (60 - 69 years old), while only 19% of the 40 - 49 year old group communicated moderate or severe discomfort. Isaacs introduced a disposable guarded cell sampler as an endometrial aspiration technique in 1975. The cannula was made of malleable stainless steel (1.9 mm diameter) with 48 perforations in the 4 cm tip. The tip was covered with a sliding polyvinyl sheath that functioned to reduce contamination and plug the cervix. Aspiration with a 10 cc syringe drew in cells which were then expressed onto a slide and smeared with the cannula tip. In his office study of 250 postmenopausal patients, 88% of the samplings produced suitable endometrium for evaluation. The cannula could not be inserted in 17 women due to cervical or vaginal stenosis and no endometrial cells were obtained in 14 others after successful insertion. In a hospital study reported by Isaacs in the same paper, 6 of 7 cytologic reports indicating malignancy and 171 of 172 benign reports were substantiated histologically. Patients underwent Operative dilatation and currettage after sampling. A recent clinical study using the Isaacs cell sampler detected 11 of 11 malignancies and obtained suitable endometrium in 86% of patients (Polson et al., 1984). They believed that with instruction of the Operators this percentage would rise to 95%. A review of 3000 samplings obtained by a Swiss group of investigators using a pistol grip syringe aspirator, revealed suitable endometrium for cytology or histology in 93% of the patients (Bouchardy et al., 1987). Their pistol grip technique had the advantage of requiring only one hand to obtain the sample. Biopsy of the woman's endometrium can be accomplished mechanically or by suction. A disposable outpatient suction curette called the 'Vabra aspirator became popular in the 19705. At the base of the instrument near the suction release holes, in-line filters trapped the tissues for histologic analysis. Stainless steel instruments are also available for outpatient, mechanical or suction curettage. Examples include the multiple notched Novak curette (Wildhack et al., 1963) or the single notched Randall type. Obtaining tissue samples by biopsy of the cervix and endometrium has been suggested to be diagnostically superior to cytologic techniques alone (Creasman, 1986). The disposable Vabra aspirator is widely used today. However, Ellice et al. (1981) reported that from 100 women attending a menopausal clinic, adequate samples were obtained from only 61% of the patients using the Vabra suction curette compared to 91% for the Isaacs cell sampler. They also found that 23 of 30 women feund the Vabra aspirator moderately to severely uncomfortable and more painful than the cell sampler. Iversen and Segadal (1985) investigated three cell samplers (200 patients each) prior to dilatation and curettage. All cytology samplers were superior to curettage in obtaining adequate material for evaluation, Detection of histologically verified malignancies was accomplished in 65 of 66 cases by cytologic techniques. The Gravlee jet washer was criticized in this study due to its larger diameter and the necessity of handling fluid samples. Iversen and Segadal (1985) concluded that due to the small amount of tissue available ‘in postmenopausal women, cytological techniques were superior. Transcervical techniques reported for' procuring culture specimens from the endometria of women include uterine lavage with 10 cc normal saline (Wierdsma and Clayton, 1964), uterine lavage with 5 cc deoxygenated LRS (Gibbs et al., 1975), protected swab (Pezzloi et al., 1979), telescoping cannulas with a small nylon bristle brush (Knuppel et al., 1981), and guarded suction curettage (Hoyme et al., 1986). Rein and Mandell (1973) reported the bactericidal effect of using normal saline preparations in bronchial lavages for culture. Duff et al. (1983) published a comparative study of three transcervical methods and transfundal aspiration in 18 women during surgical tubal ligation. The telescoping brush technique or guarded double lumen uterine lavage were the most effective of the transcervical techniques in reducing cervicovaginal contamination. In this study, 10 11 (n? 18 transfundal aspirates obtained directly during surgery were sterile. Results from the transcervical brush culture agreed in four of" these cases and the uterine lavage culture in four. All single lumen unprotected lavages were contaminated, with. 13 of 18 cultures resulting in more than two different kinds of bacteria. Endometrial Sampling Techniques: Mares The mare's endometrium was originally sampled to determine if microorganisms were present. Endometrial swab culture today remains the most frequently used procedure in combination with endometrial cytology and endometrial biopsy to evaluate breeding soundness in the mare. Flushing, lavaging, and brushes are utilized much less commonly. The early work of Dimock and Edwards (1928) was an extensive report based on six years of reproductive observations in 1606 barren mares. Breeding evaluation of the mares consisted of passage of a vaginal speculum to visualize the vagina and cervix. Bacterial cultures were obtained with a 5 inch platinum needle attached to a holder, flamed, and passed via the speculum through the cervix and into the uterus 2 or 3 times. Based on 3,868 bacterial cultures done during estrus, 37% of the mares were determined to be infected. No mention is made of ll speculum sterilization between mares or perineal preparation prior to sampling. Knudsen (1964) described curettage in mares for endometrial cytology. In 151 mares with concurrent cytological and bacteriological examinations, 22 had polymorphonuclear leukocytes (PMN's) on cytology smears and pathogenic organisms upon culture. Three mares with positive cultures and endometritis were not detected by cytology. Cervical swabbing, however, was convenient and less traumatic and became more popular. An Irish clinician published a study of uterine infection in mares in response to the controversy around routine cervical swabbing of Thoroughbred mares prior to service (Collins, 1964). Cervical samples were taken from estrous mares for bacteriologic analysis only. His technique utilized a perineal wash, sterile speculum and a cotton-wool swab on a 30" wire sterilized in a glass tube with end plugs. Swabs of the cervix were taken through the speculum by advancing the glass tube into the cervix and exposing the swab. He found 26% of the 3,853 mares swabbed to have positive cultures and stressed continued "cervical" swabbing for bacteriological analysis. Collins (1964) suggested that the anterior vagina and cervix of normal mares was bacteriologically sterile and thus cervical swabs were accurate. Vaginal speculums and unguarded or partially guarded swabs (within open tubes) continued to 12 be used for bacteriology (Hughes et al., 1966; Peterson et al., 1969). A necropsy study of 100 mares showed decreasing colony counts and number of types of bacteria from the vagina to the uterus (Scott et al., 1971). Their results indicate that if positive cultures of the cervical 05 were used to diagnose uterine infection, false positives and false negatives were common. Newcombe (1978) compared cultures from the anterior vagina, cervix, and uterine lumen of a large number of mares of all breeds. He found that cervical and vaginal flora were similar and not representative of uterine culture results. The three cultures agreed well only when there was a purulent discharge from the uterus. The currently' accepted endometrial swabbing technique in our laboratory is inanual placement of a McCullough® double tube guarded swab1 through the cervix. The outer tube of this system has a sealed breakthrough cap. The manual technique described by Allen in 1979 had the advantages that no vaginal speculum was needed and it yielded more consistent samples. The use of vaginal speculums when swabbing the uterus has the disadvantages of introducing air and making cervical penetration more difficult (Brook, 1984). Blanchard et al. (1981) reported a comparison of endometrial sampling techniques using swabs within tubes; the first with an additional 13 outside guarding tube with a retractable plug and the second without. Both were placed manually and simultaneously into the uterus. Culture results indicated that the additional guard (tube with retractable plug) reduced cervicovaginal bacterial contamination. Combined cytologic and bacteriologic endometrial sampling of the mare was reported by numerous recent investigators as a, means to improve diagnostic accuracy (Digby,1978; Woolcock, 1980; Knudsen, 1982; Brook, 1984; 1aCour and Sprinkle, 1985; Crickman and Pugh, 1986). All utilized swabs, except for Knudsen (1982) who used an instrument with a swab for bacteriology behind a grooved plastic tip fer cytology. The others used two separate swabs or a single swab for making smears and inoculating agar plates. Other methods reported for cytologic or bacteriologic endometrial sampling, or both, in mares are uterine flushing (Varadin, 1975; Asbury, 1984a; Slusher et al., 1984; Ball at al., 1988), uterine tampon (Stratton et al., 1979), and guarded biopsy (Brook,- 1984). Flushing techniques for mares were originally reported by Zavy et al., 1978. Eighty mls of saline were infused and aspirated through a single lumen metal 16 g cannula with a Silastic tip. This was inserted through a previously placed 26 - 30 Fr. Foley catheter with a 60 14 cc cuff positioned cranial to the cervix. The average volume of fluid recovered was 85 mls (106.3%, N :- 42). The uterus was manipulated transrectally during collection of fluid. Modifications of this technique have been used to flush equine uteri for analysis of immunoglobulins (Mitchell et al., 1982), total protein content (Strzemienski & Kenney, 1984), prostaglandins (Watson et al., 1987), and neutrophils (Asbury et al., 1980; Liu et al., 1985). Cervicovaginal cells and bacteria undoubtedly contaminated these flushes as efficient guarding systems were not described. Ball at al. (1988) used a steel tube to guard the entrance of a 20 Fr. Gibbons balloon catheter through the cervix. Once inflated with 60 mls of air, the balloon blocked the internal cervical os. Sixty mls of phosphate buffered saline solution (PBSS) were infused and the uterus manipulated transrectally to distribute the fluid and help retrieve it. Approximately 55 - 60 percent of the fluid was consistently recovered. A 15 ml aliquot was centrifuged (15,000 G x 15 min.) for culture, and another centrifuged (1,000 to 1,500 rpm x 5 min) for cytologic smears. Fluid cell counts were not reported. Culture results from the flush solution in this study were significantly correlated to clitoral sinus cultures suggesting bacterial flora contamination. Ball's results indicated that a single-procedure cytologic and 15 bacteriologic technique for the evaluation of the mare's endometrium was suitable in a clinical situation, and possibly' more sensitive than. previously described methods. Endometrial biopsy currently remains in the mare, as it does in women, the primary diagnostic and prognostic tool for endometrial evaluation. Tobler introduced a bullet-shaped equine endometrial biopsy instrument in 1966. While in the uterus, the tip of the instrument is moved forward to expose a stainless steel cutting edge. As the instrument closes, a sample of the endometrium is pinched off. Transrectal manipulation was not required, and sample size ranged from 1 mm to 2 cm (Brandt and Manning, 1969). This group also reported endometrial biopsy with a small forceps through a fiberoptic scope. The sample obtained was small, yet the site biopsied could be visualized. In the mid 1970's, three studies reported evaluation of endometrial biopsies taken with an alligator jawed punch instrument. Transrectal uterine manipulation was used after digital placement of the tip of the instrument through the cervix (Ricketts, 1975; Kenney, 1978; Gordon and Sartin, 1978). All of these investigators developed numerical systems to categorize endometrial biopsies, based on inflammation and glandular abnormalities, for prediction of fertility in a l6 particular mare. Two used Bouin's solution as the initial fixative to facilitate trimming and provide better cellular detail. Endometrial punch biopsy causes an acute transient endometritis similar to saline infusion (Bennett et al., 1980) and may shorten the return to estrus when used in diestrual mares (Hurtgen and Ganjam, 1979). The endometrial punch biopsy, together with the category classification system, is currently a routine component of the mare's breeding soundness examination (BSE). Interpretation of Endometrial Cytology: Women Papanicolaou in 1941 stated that 26,000 deaths per year were attributable to uterine cancer in women. He stated that early diagnosis would result in a higher percentage of cures. He developed alcohol based stains, that bear his name, to determine better the structure of cervical and endometrial cells in the presence of mucus and blood (Papanicolaou, 1942). The incidence of endometrial cancer is increasing and replacement therapy in menopausal women with exogenous conjugated estrogens has been incriminated (Bouchardy et al., 1987). A decrease in deaths due to cervical malignancy is ascribed to widespread screening (Pap smear), leading to early diagnosis. l7 Cytologic techniques have gained preference over histology through the years in an effort to avoid sharp biopsy, anesthetic risk, and outpatient pain during screening of patients for endometrial malignancy (Hutton et al., 1978; Morse et al., 1981). Iversen and Segadal (1985) concluded that the general opinion was that cytologic techniques were as accurate as histologic ones for the diagnosis of malignancy of the endometrium. Others claimed that cytology was not valuable for endometrial evaluation (Studd et al., 1979). Currently routine screening of asymptomatic women for endometrial cancer is not widespread. Indications for evaluation include menstrual irregularities, abnormal premenopausal bleeding, and postmenopausal bleeding. High risk women include those with increasing age, obesity, and a history of estrogen therapy without progesterone (Anderson, 1986). Creasman (1986) stated that 20% of women with postmenopausal uterine bleeding will have genital malignancy. Cytologic techniques necessitate intensive laboratory skills for some procedures and a skilled and experienced cytopathologist for the interpretations (Isaacs, 1975). Anderson (1986) considered that because all laboratories could not provide this level of service, a negative or normal cytologic result was no guarantee that the uterus was healthy. The difficulty in cytologic 18 diagnosis of endometrial malignancy may be due in part to the small size of the endometrial epithelial cell, and the observation that cell changes associated with adenocarcinoma are not as obvious in malignant endometrial cells as in.1nalignant, cervical. cells (von Haam, 1962). Endometrial smears contain cells from several parts of a complex tissue that is changing constantly in response to ovarian hormones (Morse et al., 1981). Several 'authors, however, have described in detail the morphologic changes in endometrial adenocarcinoma and its precursors, and have included classification systems to compare their cytologic diagnosis to histology (K035 and Durfee, 1962; Ellice et al., 1981; Morse et al., 1981). Examples of a cytologic classification would be normal-premenopausal, normal- postmenopausal, cystic hyperplasia, adenomatous hyperplasia, cystic and adenomatous hyperplasia, and adenocarcinoma. Currently, symptomatic women with cytology results indicating hyperplasia are either monitored, or treated by altering or adding hormone therapy, or both. Those with normal or negative results that remain symptomatic usually are hospitalized and undergo dilatation and curettage. Cytologic endometrial dating (interpreting smears for detection of ovulation) in .normal. women. or infertility cases has not received as much attention as 19 cytologic screening for cancer. Greater effort has been invested in the study of endometrial biopsy and curettage for dating. Historically, however, the ease of the cytologic technique has permitted frequent sampling. Using aspiration cytology, Romberg (1954) was able to evaluate endometrial smears nearly every other day throughout one cycle in 10 women. Two normal cycling women were evaluated daily throughout their 28 day cycles. His detailed descriptions and those of Ferreira (1956) present the prominent stromal and epithelial changes that occur in the cycle. Ferreira separates the phases into early proliferative (onset of menstruation to day 7), late proliferative (day 8 to day 14), early secretory (day 14 to 21), and late secretory (day 25 on). From early to late proliferative, the glandular epithelial cells changed from round cells with dark eccentric nuclei, to "rose petal" in form with basal nuclei. stromal changes were not remarkable. The glandular epithelial cells of the early and late secretory phase (after ovulation) developed vacuoles, were larger, and underwent nuclear lightening and shrinkage. On the day prior to the onset of the next menstruation the glandular cells collapsed and necrosis took place (Romberg, 1954). Abnormal cytologic patterns seen by these investigators included: "fixed endometrium with cells characteristic of both proliferative and 20 secretory phases, insufficient secretory endometrium, and a persistence of proliferative morphology, characteristic of anovulation. Inflammatory cells are infrequently mentioned in the human studies, due to the fact that chronic endometritis is not a significant problem in women. Koss (1961) noted that a variety of leukocytes may be present in endometrial . smears (mostly lymphocytes and polymorphonuclear leukocyteS) in the absence of endometritis, yet that the presence of plasma cells indicated a chronic endometritis. The physiologic inflammatory infiltrate of the premenstrual phase: may mask a pathologic inflammation, making the timing of biopsy important (Czernobilsky, 1978). Nonspecific chronic endometritis was described histologically in 9% of 899 patients undergoing evaluation for neoplasia (Greenwood and Morgan, 1981). The plasma cell and lymphocyte were the predominate inflammatory cells. Interpretation of Endometrial Cytology: Mares Endometrial cytology in the mare is used as a fast screening test prior to breeding and as a component of the BSE. Smears are evaluated primarily for the presence of inflammatory cells and for endometrial epithelial cell morphology. Stratified squamous epithelial cells, calcium carbonate crystals, and 21 unphagocytized bacteria represent cervicovaginal contamination. Reports in the current literature center around clinical applications of the swab smear. The swab is convenient, easy to handle, and less traumatic than a biopsy. The main disadvantage appears to be in smear quality and thus accurate interpretation. Swab smears contain. many disrupted and distorted epithelial cells which several authors have suggested are due to sampling, smearing, fixing, and staining techniques. The quality of the swab smear may be improved by pmemoistening the swab with sterile saline (Crickman and Pugh, 1986). The following are the reported components of the cytology smear . Epithelial Cells Couto and Hughes (1984) presented an excellent description: Nonciliated and ciliated cells are often observed in cervical/uterine smears. Among the most common nonciliated columnar cells are the mucus secreting cells. These cells may be elliptical, tall columnar, low columnar, or goblet-shaped. They may be found isolated or arranged in sheets. . . . All of the nonciliated columnar cells show affinity for the basic stains presenting a large purple to violet nucleus surrounded by light blue or clear, finely vacuolated cytoplasm. On the other hand, the columnar ciliated cells present an eosinophilic cytoplasm with a basally located darkly staining nucleus. 131 these cells, the ciliated border is slightly thickened and distant. They also describe the "honeycomb" arrangement as unique to endometrial epithelial cells. These appear as large 22 groups of cells with overlapping cytoplasms surrounded by mucus and single cells. Atypical or degenerate morphologic changes associated with infection, indwelling catheters, and/or uterine treatment have been reported by several authors (Couto and Hughes, 1984; 1aCour and Sprinkle, 1985; Freeman et al., 1986). Characteristic changes associated with acute and chronic inflammation have not been thoroughly depicted. Polymorphonuclear Leukocytes ¥ (PMN's) PMN's predominate in the smears of mares with bacteriological evidence of infection (Knudsen, 1964). Smears from normal mares contain few PMN's, although they will appear transiently after breeding or other uterine contamination (Digby, 1978). Peterson et al. (1969) described the cytologic reaction of the normal mare's endometrium to bacterial inoculation as a rapid (within 6 hourS), intense infiltration of phagocytotic PMN's that decreased within 72 hours. The PMN response persisted in barren mares, as did the number of recovered bacteria. Neutrophils, representative of’ an acute nonseptic inflammation, are mature and hypersegmented, while those involved in septic processes are pyknotic and karyolytic (Neely, 1983). The percentage of PMN's or ratio of PMN's to epithelial cells has been evaluated for significance 23 (Couto and Hughes, 1984; Asbury, 1984b), as has been the number of PMN's seen over a set number of fields (Brook, 1985). Asbury (1984b) stated that PMN's were significant when the ratio of epithelial cells to PMN's was below 10 to 1. 1aCour and Sprinkle (1985) reported a numerical category system (0 to 5) based primarily on the presence of PMN's. Their results indicated that as the category number increased, fertility decreased. Other Inflammatory Cells The occurrence of inflammatory cells other than PMN's in endometrial smears is uncommon. Lymphocytes and macrophages have been reported in chronic endometritis (Neely, 1983) although extensive descriptions or counts have not been done. Mares with chronic fibrotic endometritis may not have significant cytologic changes (Couto and Hughes, 1984). Multinucleated and vacuolated macrophages were consistently present along with PMN's in samples from foal heat mares (Brook, 1985; 1aCour and Sprinkle, 1985). Numerous lymphocytes were reported in smears from almost 10% of mares studied by Knudsen in 1964. He associated these findings with lymphatic stasis of the endometrium. 1aCour and Sprinkle (1985) reported that although scattered lymphocytes and plasma cells were seen in many smears, they did not appear to be significant. Mares with biopsies showing degenerative 24 glandular changes and stromal fibrosis in addition. to chronic and acute endometritis were classified as persistently endometritic by Watson et al. (1987). These mares had a higher percentage of PMN's and mononuclear cells (lymphocytes and monocytes) in uterine washings than normal mares. They suggested that the presence of increased numbers of mononuclear leukocytes in these mares was due to an increased stromal population. Eosinophils in cytologic smears were found to be associated with poor vulvar conformation in the mares examined by Slusher et al. (1984). Experimental introduction of air into the uteri of three mares resulted in an acute eosinophilic and neutrophilic response within 24 hours. Red Blood Cells Brook (1985) and Woolcock (1980) reported erythrocytes were numerous in cytologic smears from mares in foal heat and those with severe acute endometritis. A few red blood cells are frequently found in cervical/uterine smears especially in estrual mares, due to normal physiologic hyperemia of tissues and capillary fragility (Couto and Hughes, 1984). Erythrocytes may certainly be associated with trauma induced by cytologic sampling techniques. 25 Calcium Carbonate Crystals The presence of these crystals from urine in endometrial smears of mares is considered significant if there is abnormal vaginal slope, ‘urine in the vagina during estrus, or vaginal cervical hyperemia (Couto and Hughes, 1984). Freeman et al. (1986) described a cytologic pattern associated with urine pooling in mares. Endometrial smears contained calcium carbonate crystals, neutrophils, occasional vaginal epithelial cells, and very tall columnar epithelial cells with green cytoplasm termed "chlorocytes" (trichrome stain). These authors also mentioned the importance of differentiating calcium carbonate. crystals and talc crystals, which. frequently contaminate endometrial smears. Bacteria, Yeast, and Fungi Bacteria may be seen on cytologic smears and are significant in the presence of neutrophils. Gram staining of smears has been suggested as a reasonable procedure to hasten initiation of antibacterial therapy while awaiting culture results (Shin et al., 1979; Couto and Hughes, 1984; Crickman and Pugh, 1986). Fungal endometritis is a likely diagnosis when frequent hyphae or oval budding yeasts, and PMN's are present in endometrial smears. The oval thin walled yeast organisms are much larger than bacteria and stain gram positive (Carter, 1976). In a 26 limited study of five cases of mycotic endometritis in the mare, cytology was superior to biopsy in determining the presence of fungal elements (Freeman et al., 1986). Cytologic Detection of Cyclic Changes Seasonal changes in the histology of the mare's endometrium have been well described based on biopsy samples (Ricketts, 1975; Kenney, 1978; Gross and LeBlanc, 1984). Distinct epithelial, glandular, and stromal changes were associated with follicular and luteal status and corresponded, in a predictable manner, to estrus, diestrus, and anestrus in the normal mare. Seasonal anovulatory transition involves a variable period of time between the quiescence of anestrus and the metabolic activity of the ovulatory period and has not been well described histologically by any of these investigators. Efforts to determine the stage of cycle using cytologic smears have not been as successful as histologic techniques. The early work in cytology by Knudsen (1964) describes anestrous smears as inactive, with the nuclei of epithelial cells small and round. The diestrous smears were similar. Epithelial cells in smears from mares in estrus had larger eccentric nuclei with abundant cytoplasm. Strands of mucus originating from the epithelial cells were frequently seen in estrual smears. The presence of a few plasma cells was considered normal 27 near the time of ovulation, as was the appearance of a few lymphocytes towards the end of diestrus. Two reports (Britton, 1982; Couto and Hughes, 1984) described similar subtle changes. These investigators found that in diestrous samples, ciliated columnar epithelial cells were more numerous than. nonciliated cells when compared to estrous samples. Recent publications concerning endometrial washes have delineated endometrial patterns which corresponded to winter anestrus (inactive pattern), estrus and diestrus (active pattern), and fall and spring transitional periods (Freeman et al., 1986; Roszel and Freeman, 1988). Small epithelial cells in cohesive flat groups with stripped nuclei near cytoplasmic fragments were predominant in the inactive pattern. The spring transitional pattern was described as a gradual change from cuboidal to columnar epithelial cells, with decreasing numbers of "apoptotic bodies" (cell fragments with degenerate nuclei) and stripped nuclei, with a few ciliated cytoplasmic tufts. Ciliated cytoplasmic tufts are assumed to be: cellular fragments of ciliated epithelial cells. Epithelial cells of the active pattern had oval basilar nuclei with multiply vacuolated foamy cytoplasms. These were found singly, cohesive, or in loose groups with a few ciliated cytoplasmic tufts present. During the fall transitional pattern, epithelial cell nuclei became hypochromatic, 28 pyknotic, and wider than the luminal tip of the cytoplasm. Ciliated cytoplasmic tufts were numerous and the smears gradually became like the inactive pattern. Interpretation of Endometrial Cultures: Women Infectious endometritis and endomyometritis are common problems in post-parturient women. The incidence ranges from 1-4% following vaginal delivery (Gibbs et al., 1980) to 85% in a group of low income women who had cesarean section following ruptured membranes and prolonged labor (DePalma et al., 1980). Duff et al., 1982, reported a 15% incidence of endomyometritis in middle-class patients who had undergone elective cesarean section. Postpartum uterine infection is thus a significant problem for the patient and obstetrician. Increasing morbidity and mortality rates in puerperal women have been suggested to be due to the recent rise in use of cesarean section (Pezzlo et al., 1979). Clinical signs used to determine systemic involvement and the need for evaluation and treatment, are fever and uterine tenderness more than 24 hours after delivery (Hoyme et al., 1986). Blood, urine, and guarded endometrial cultures are generally taken prior to institution of therapy. Infectious endometritis may affect the nonpuerperal patient as a result of (1) cervical biopsy or 29 surgery; (2) the transport of cervical flora into the endometrium by endometrial biopsy, hysterosalpingogram, instrumentation abortion or other such procedures; (3) ascending infections, such as in gonorrheal or chlamydial infection; and (4) blood-borne infection as in tuberculosis (Moyer, 1975). Development of chronic endometritis in women is rare due to cyclic menstrual bleeding and the presence of uterine bactericidal :mechanisms. Thus‘ acute: endometritis is transient and cannot be considered a significant cause of chronic infertility (Czernobilsky, 1978). For example, in a bacteriologic study of 75 uteri with intrauterine devices (IUD) at elective vaginal hysterectomy, 6 of 10 positive endometrial cultures were from patients in whom the IUD had been in place for less than 48 hours (Mishell et al., 1966). The other four positive endometrial cultures were from women in whom the IUD had been in place for 6 to 30 days duration. Endometrial cultures were sterile in all 15 women in whom the IUD had been in place longer than 30 days. The occurrence of a transient acute bacterial endometritis after placement of an IUD is well established. Chronic endometritis has been associated with use of the tailed IUD (Eschenbach, 1986), and also was found on outpatient biopsy in 9% of 899 patients undergoing screening for neoplasia (Greenwood and Morgan, 1981). The clinical complaint in 94% of these patients 30 was abnormal vaginal bleeding. Of the 69 women with chronic endometritis in this study who did not have subsequent hysterectomy, 72% became asymptomatic after dilatation and curettage or repeated endometrial biopsy. Nonspecific chronic endometritis in women has not been associated with subfertility (Czernobilsky, 1978). Interpretation of guarded endometrial cultures in women requires consideration of aerobic and anaerobic bacteria, chlamydia, and genital mycoplasmas. Many investigators stress the polymicrobial nature of infections in. postpartunl patients and particularly the importance of anaerobic bacteria. The most common aerobic isolates reported include Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Staphylococcus aureus, enterococci, Streptococcus pyogenes, Enterobacter cloacae, Proteus ndrabilis, Pseudomonas aeruginosa, Neisseria gonorrhea, and Gardnerella vaginalis (Gibbs and Huff, 1980). Anaerobic bacteria were isolated as frequently as aerobic bacteria in this study. The most common species isolated were from the Peptococci, Bacteroides, Clostridia, Peptostreptococci, Fusobacterium and Veillonella genera. Chlamydia trachomatis is most commonly associated with cervicitis yet may ascend to the endometrium and is a major cause of salpingitis in women (Watts and Eschenbach, 1988). Women with chlamydial endometritis often present 31 organism is responsible for a major component of female infertility manifested as a mild untreated postpartum infection and subsequent salpingitis. Chlamydia trachomatis is a well—established cause of urethritis in men, and cervicitis/endometritis in women with lower socioeconomic backgrounds and multiple sexual partners (Brunham et al., 1981). The role 'of the human genital mycoplasmas, Mycoplasma hominis and Ureaplasma urealyticum in genital infections is controversial and currently under intense investigation (Watts and Eschenbach, 1988). In a clinical, microbiologic study of women after vaginal delivery with late (7 to 42 days) postpartum endometritis, one group of investigators found that genital mycoplasmas were isolated from the endometrium in 8 of the 18 women and of these, 5 were in pure culture (Hoyme et al., 1986). Endometritis was diagnosed histologically in 14 of 30 women referred from a sexually transmitted disease clinic for evaluation of cervicitis (Paavonen et al., 1985). In their studies they found a highly significant correlation between the presences of M_. hominis (p < .05) and the level (p < .01) of serum antibodies to M; hominis in these patients with endometritis. 32 Interpretation of Endometrial Cultures: Mares Acute septic metritis occurs infrequently in postpartum mares. It is often associated with dystocia, retention of fetal membranes, and/or foaling in unsanitary conditions (Ricketts and Mackintosh, 1987) These mares may show depression, anorexia, fever, and laminitis associated with absorption of endotoxins from the uterus (Threlfall, 1984).' However, large numbers of potentially pathogenic organisms gain entrance to the uterus during foaling, breeding, veterinary examinations, and as a result of anatomical abnormalities (Asbury, 1984a). There is a transient acute endometritis, primarily neutrophilic and without systemic signs. The broodmare is prevailed upon to produce as many foals as possible during her lifetime. The response of a particular mare's endometrium to the multiple bacterial insults associated with breeding and foaling may be variable (Liu, 1988). Investigations in subfertile mares have resulted in the identification of mares which have poor uterine defense mechanisms and therefore are more susceptible to development of chronic endometritis (Ricketts, 1981; Liu, 1988). Liu (1988) has provided excellent evidence for local neutrophil dysfunction as the primary problem. Mares with normal transrectal palpations 33 and normal endometrial cultures, cytology, and biopsy are considered resistant. The incidence of endometrial infection in subfertile barren mares is high, and it remains a significant problem on breeding farms (Dimock and Edwards, 1928; Collins, 1964; Varadin, 1975). Transcervical endometrial cultures have been routinely used to evaluate normal mares prebreeding as well as mares with acute and chronic endometritis. Vaginal, cervical, and environmental contamination of the specimen must be considered during the sampling procedure and taken into account in the interpretation of the results. Scott et al. (1971) reported the results of vaginal cultures for aerobic bacteria from 100 slaughtered mares of unknown breeding history. Organisms isolated from the vaginal fornix included beta-hemolytic streptococci (S; zooepidemicus, S; eguisimilis), coliforms (E; ggliL Klebsiella pneumoniae), coagulase negative staphylococci, Staphylococcus aureus, alpha—hemolytic streptococci, and diptheroids. Pathogenic aerobic bacteria reported to be significant in equine endometritis include beta-hemolytic streptococci, E; coli, Klebsiella spp., Pseudomonas spp., Proteus spp., coagulase positive Staphylococcus spp., and Cognebacterium spp. (Threlfall, 1984). Bacillus cereus has also been reported (Matros and Ansari, 1986); however, 34 the recovery of Bacillus spp. has generally been interpreted as nonpathogenic contamination (Shin et al., 1979; Ball, 1988). Other organisms named as nonpathogenic contaminants include nonhemolytic and alpha-hemolytic streptococci, Streptococcus faecalis, (Enterococcus faecalis), coliforms, Staphylococcus albus, Corynebacterium spp., Anthrocoides spp., and Neisseria spp. (Ricketts, 1981). An unclassified species of Corynebacterium was reported as the causative agent in an endometritic mare; however, the authors relate that these organisms are generally regarded as contaminants (Blue and Hannwacker, 1984). Yeast and fungal endometritis occur most often in mares which have either received prolonged antibiotic therapy or in which the uterus is in a chronic, debilitated state (Neely, 1983). Significant yeast and fungal organisms are Candida spp. (C; albicans, Q; parapsilosis), Aspergillus spp., M252; spp., and Allescheria boydii (Blue, 1983). Culture for mycoplasmas and ureaplasmas requires special media and a longer incubation time. Few investigators have attempted to isolate these organisms from the equine genital tract. The only paper concerning genital ureaplasmas in horses resulted in 6 isolations of Ureaplasma spp. from clitoral sinus swabs of 27 subfertile 35 mares in North America (Ball at al., 1988). Three of the six mares also had these organisms isolated from a partially guarded uterine flush. Twenty-three of 24 normal mares had negative clitoral swab and flush cultures for ureaplasma. Ball et al. (1988) also cultured specificallly for mycoplasmas. Mycoplasma spp. were identified in the uterine flush of one subfertile mare and the clitoral swab of another. A group of investigators from Australia isolated Mycoplasma spp. from an aborted fetus, from vaginal swabs in 12 of 19 mares, and from penile swabs in 2 of 4 stallions (Moorthy et al., 1976, 1977). Nine of the mares and two of the stallions had ulcerative genital lesions, which were very similar to those seen with coital exanthema (Herpes type III) in North America. Mycoplasma spp. were isolated from four mares and one stallion with lesions and were .not isolated in five: mares and one stallion with lesions. The authors referenced two European studies which found a low incidence of mycoplasma isolation from mares and stallions. A study of 337 mares on six Ontario breeding farms found M; subdolum in the clitoral fossa of 72 mares (21.4%), and M; equigenitalium in the clitoral fossa of 49 mares (14.5%) (Bermudez et al., 1987). Mycoplasmas were most evident on a pony farm (74.4% incidence) and on a Standardbred farm (41.9%). Eleven maiden mares were free of mycoplasmas. Infertility 36 in the mares was not associated with the occurrence of these organisms in the clitoral fossa. Some gram-negative anaerobic bacteria are commensals on the mucous membranes of the genital tract and may represent 90% of the bacteria in the intestine (Carter, 1976). Despite the reported significance of anaerobic bacteria in human endometritis, traditional endometrial culturing techniques in mares have only considered the aerobic bacteria. Two investigators have recently reported results regarding anaerobic culturing of genital specimens. Over a 10 year period, only 3 of 1000 endometrial swabs resulted in anaerobic bacterial growth (Threlfall, 1987). All three of these mares conceived without treatment. Anaerobic bacteria were isolated from 96% of 113 penile swabs from stallions in a study reported by Ricketts and Mackintosh (1987). They suggested that these bacteria are regularly introduced into the mares' uterus at breeding and foaling, and may act as opportunists in the postpartum period. Bacteroides fragilis was the most frequent isolate from clitoral swabs of which 15 of 15 were positive. Anaerobes were isolated from 42% of 335 endometrial swabs. Ten of 18 samples with cytologic evidence of acute endometritis (Z 0.5% PMN's), and without aerobic growth, yielded pure growth of an anaerobe (7 B; fragilis, 2 Peptococcus spp. and 1 Cl. 37 perfringens). However, endometrial swabs in this study were taken using a vaginal speculum which may have decreased the accuracy of the cultures. Endometrial swabs were positive for anaerobes in 21 of 41 maiden mares in this study and none of these had cytological evidence of inflammation. Bacteroides fragilis accounted for 81% of these isolations. Positive cultures for anaerobic bacteria in 50% of the maidens, all without endometritis, indicates that their sampling technique was very likely contaminated by cervicovaginal flora. Statement of Purpose Examination of the literature highlights many considerations pertinent to endometrial evaluation of the mare. The rapid progression of cytologic sampling techniques in women from simple aspiration to more sophisticated methods was accompanied by an increase in accuracy. All recent techniques sample a: larger endometrial surface and are guarded to prevent contamination by cervical cells. Unguarded uterine lavages for culture were shown to be contaminated (Duff et al., 1983) and normal saline solutions were found to be bactericidal (Rein and Mandell, 1973). Thus guarded techniques and fluids other than saline are preferred for uterine lavage. Swab techniques are notably absent. 38 It is apparent in the equine literature that direct endometrial sampling using guarded techniques is necessary (Scott et al., 1971; Newcombe, 1978; Blanchard et al., 1981). Partially guarded uterine flush techniques have been used extensively for research (Zavy et al., 1978) and also for bacteriologic and cytologic sampling (Ball at al., 1988). The latter obtained more pathogenic organisms (higher CFU's) from flush solutions than from swabs in subfertile mares, yet culture of the recovered fluid also resulted in isolation. of' many contaminants found in the caudal reproductive tract. .All reports concerning endometrial cultures in mares are the result of sampling of the uterine body. Because of its proximity to the vagina and cervix, the quality of samples from the uterine body may be compromised even with good sampling technique. The simple endometrial wash technique described by Freeman and Roszel (1988) covered a larger surface area than swabs and provided the first detailed accounts of cytologic change relative to stage of cycle in the mare. The impact of this work is diminished, however, due to the lack of transrectal palpation, teasing, and hormonal analysis data, and the apparent reliance upon endometrial biopsy results and time of year to separate mares into cycling, transitional, and anestrous phases. 39 The main objective of this study was to develop a uterine horn lavage (UHL) technique for cytologic and bacteriologic evaluation of the mare's endometrium. Such a technique has not been previously reported. It is a necessity that the technique is guarded to minimize cervicovaginal contamination. The hypothesis is that by sampling the large surface area of a uterine horn, a less contaminated sample for culture and a more accurate cytologic sample than current swab techniques can be obtained. MATERIALS AND METHODS Mare Selection and Management Eight nonpregnant light-horse—breed mares were selected from a university teaching herd based on general health, reproductive history, endometrial biopsy category, and guarded endometrial swab culture results. All mares had category I or II endometria as determined by biopsy, and no ‘uterine jpathogens were isolated on guarded endometrial swab culture. Three mares had category I endometria, four were category IIA's, and one was a IIB. Mares with poor reproductive histories, multiple foalings, or poor vulvar conformation were excluded. All mares were maintained on pasture supplemented with hay. Vaccination and deworming practices were routine. Timing of Sampling For investigation of the right uterine horn) lavage (UHL) technique, mares were sampled once in estrus, diestrus, and anestrus. Sampling consisted of transrectal palpation, endometrial swab culture, endometrial swab cytology, right UHL, and endometrial biopsy, respectively. Teasing and mare selection began in July. Every other day teasing, through direct 40 41 stallion contact (chute), began no less than 30 days prior to estrous sampling and continued for at least one estrus after the diestrous sampling. Anestrous samplings were performed in January and February in four mares, while four continued to cycle. For the estrous sampling, mares were in the third or fourth day of estrus with transrectal palpations indicating the presence of a follicle greater than 35 mm, decreasing uterine tone, and a relaxing cervix. Subsequent diestrous sampling took place on the mare's ninth or tenth day of negative tease, with palpation indicating the absence of any follicles greater than 35 mm, increased uterine tone and a closed cervix. Anestrous samples were collected when cycling ceased. This was based on teasing records and multiple transrectal palpations revealing small inactive ovaries and the uterus and cervix decreased in tone. Venous blood was collected prior to each sampling, allowed to clot at room temperature, and certrifuged (1300 G x 5 min) prior to immediate serum separation. Samples were frozen at —17°C for later progesterone and urea nitrogen analysis. Diestrous progesterone (P values greater ) 4 than 4 ng/ml, estrous values less than 1.5 ng/ml and anestrous values less than 1.5 ng/ml were established as parameters considered to be supportive of the reproductive status. 42 Guarded UHL Catheter: Materials and Assemny The components of this apparatus include the outer tube and cap, two stylets, and the silicone balloon catheter. The outer tubing is composed of cellulose acetate buterate (CAB) tubing2 66 cm long. Edges on both ends were rounded with a small jewelry grinder and burrs removed with a rat-tail file. Piped steam was used to heat uniformly a 7.5 cm section of the tubing centered 15 cm from one end. The tubing was then gently bent to approximately a 55 degree angle, without compromising the lumen and permitted to cool. A cap was formed for the distal end of the tubing by cutting a plastic instrument guard3 down to a 2 cm total length and pushing it over the end of the tubing several times to round its shape and form a tight, yet releasable, seal. A 12 mm long cut was made 1 cm and parallel to the edge of this cap through which a 7.5 cm length of split Silastic® tubing4 was pulled. Both ends of this split tubing were pulled taut and attached to a scored area of the CAB tubing with 8 to 10 tight wraps of 23 ga stainless steel suture wire. The split tubings' tension.‘was adjusted such. that it would retract the detached cap behind the end of the CAB tubing. A rigid stylet to pop off this cap was made from brazing wires, polyethylene tubinge, and a plastic tab. 43 A 70 cm length of the wire was covered with the polyethylene tubing. A flame applied to one end caused a 3 mm ball of polyethylene to form. The round 12 mm diameter plastic tab was drilled in the center and fitted to the opposite end of this rigid stylet. To give the custom made 10 Fr. balloon catheter7 stiffness, a .95 mm coiled wire stylet was threaded the entire length. Maximum inflation of the balloon was 100 cc creating a 47 mm outside diameter (Figure 1). With air completely evacuated from the balloon and the stylet in place, the silicon catheter was passed into and through the CAB tubing to the capped end. The rigid stylet was then passed beside this catheter until the balled end contacted the CAB tubing cap. A tapered 9 cm wide sheath. made from a clear plastic disposable sleeve covered the entire catheter and was folded over the capped end. To accommodate the heat sensitive CAB tubing, a commercial ethylene oxide gas sterilizer8 was used on its cold cycle (37°C for 5 hours). The packaged catheter was then aerated for 48 hours prior to use. Sampling Procedures For each sampling, mares were placed in stocks, venous blood was taken, and 20 mg acepromazine and 200 mg xylazine were administered intravenously. Transrectal palpation results were recorded. Care was taken to 44 .uflm mo 00 ooH nuflz UmumHmcfl cooHHmn can cuocm>©m uuvwnumo nufl3 ”zoaum .msuwus oucfl couscouucw ma uu>on< .msumumoom mom>m~ anon ucfiuuu: cucumaurr.~ musmwm 46 remove fecal material to facilitate transrectal manipulation during placement of the UHL catheter. The mare was then prepared for vaginal procedures: gauze tail wrap, tail tie, and subsequent washing of the vulva and perineunn Mild soap, roll cotton, and alternate rinses with warm water were used to cleanse the perineum from the midline of the vulva outward to approximately 10 cm on each side. Washing continued until squeezed out cotton remained grossly clean after wiping the length of the vulva. Sterile disposable sleeves and sterile obstetrical lubricant9 were: ‘used for all vaginal procedures. The first three of the following four procedures were completed within 15 minutes. 1. Endometrial swab culture: With the tip of a guarded uterine swab1 placed under the thumb: of the sleeved hand and sterile lubricant applied to the back of the hand, the instrument was guided into the anterior vagina. The cervix was located and penetrated with the index finger to the internal os. The culture instrument was passed along the finger and 2.5 cm into the uterine body. The inner sheath was exposed and the swab- introduced until slight resistance was met. The swab was gently rotated several times and contact maintained with the endometrial surface for 30 seconds. With the swab and sheath withdrawn, the instrument was removed and the O swab introduced into an aerobic Culturette.1 Swabs were 47 labeled with accession number and date and refrigerated immediately. 2. Endometrial swab cytology: The swab culture procedure was repeated with fresh equipment with the exception that the swab was exposed outside the mare and 11 and premoistened with several drops of sterile saline, the 30 second endometrial contact was omitted. The swab was rotated gently several times against the endometrial surface and withdrawn. The swab was immediately rolled lightly three times across two glass slides labeled with the accession number and date. Slides were fixed while wet with an aerosol spray fixative.12 3. Right uterine horn lavage: Mares were rinsed and checked for cleanliness prior to UHL. With the left arm and hand in a sterile sleeve, the guarded catheter was removed from its sterile package. The cap attached to the outer tubing was loosened. The narrow plastic sheath remained folded loosely over the end. Sterile lubricant was applied to the back of the hand to assist placement into the anterior vagina. The curve in the outer tubing was orientated vertically with the tip pointing cranioventrally. The cervix was located digitally. The outer plastic sheath remained in the vagina while the capped apparatus was advanced (Figure 2). 48 cap a retractor plastic sheath Inflation valve outer tubing ”0} rigid stylet ’L/I ”WW 6 balloon catheter flexible stylet Figure 2. Position of the guarded UHL apparatus in the uterine body 49 The left arm was then removed, relubricated and inserted rectally to a position above the bifurcation of the uterine horns. The fingers and palm of the left hand were curled under the base of the right horn to assist placement. By rotating the outer tube counter-clockwise using the right hand (outside of the mare) and gently advancing it, the still-capped tubing was placed at the base of the right horn. With the left hand relaxed and the fingers of the right hand stabilizing the outer tubing, the rigid stylet was depressed with the right thumb. This stylet pushed the cap off the CAB tubing within the right horn. The rigid stylet was withdrawn. A laboratory assistant then advanced the balloon catheter until a marked decrease in resistance was felt as the cuff extended beyond the CAB tubing. In this position, the cuff was inflated with 100 cc of air (Figure 3). The cuff could then be identified transrectally in the right horn. The right hand was used to stabilize the end of the silicone catheter protruding from the mare's vulvar lips to facilitate removal of the flexible inner stylet. A syringe13 14 containing 50 cc of phosphate buffered saline solution was attached to the catheter. The entire 50 cc was introduced into the right horn lumen and gently aspirated by syringe until resistance was detected. The left hand remained relaxed in the rectum and was not used to assist in fluid recovery. The 50 Figure 3. Final placement and inflation of the cuff in the right uterine horn 51 syringe was detached, the cuff allowed to deflate, and the apparatus removed. Lavage fluid samples with volumes less than 20 ml were split evenly. With larger volume samples, 13 - 15 mls were designated for culture and 10 mls for cytology. Fluid samples for culture were immediately transferred to a capped, sterile, plastic centrifuge tube15 and refrigerated. The fluid for cytologic analysis was concentrated 5 times using a centrifuge.16 As the centrifuge was allowed to reach top speed (1300 G), it was turned off and allowed to stop on its own. This required 45-60 seconds. The top 80% of the fluid (4 - 8 ml) was aspirated by a 10 cc syringe and 3 1/2 Fr. tomcat catheter.” The remaining 1 - 2 ml concentrated sample was refrigerated for transport to the laboratory. A 2 ml unconcentrated aliquot was retained for nucleated cell count. In mares with greater than 90% recovery, 5 ml of unconcentrated lavage fluid was centrifuged at 1300 G for 10 minutes. The supernatant was removed and frozen for urea nitrogen analysis. 4. Endometrial biopsy: The vulva and perineum were cleansed again following UHL. An endometrial biopsy was taken from the junction of the right horn and uterine body. The vaginal technique was used with a biopsy forcep18 designed for use in the mare. Endometrial tissue obtained was maintained in Bouin's solution for 12 - 24 hours, prior to trimming and transfer to 10% 52 buffered neutral formalin” Biopsies were categorized without knowledge of mare or stage of cycle, using a previously described system (Kenney and D019, 1986), with the aid of an experienced pathologist. Laboratory Procedures Endometrial Swab Cytology All slides were stained within twelve hours of sampling with an automatic stainer19 utilizing modified Wright's stain. Slides were scanned under low (100x) and high dry (400x) powers for cellular areas and rbc/tuft scores. These scores were calculated by averaging RBC's or ciliated cytoplasmic tufts per high power field (hpf) over three cellular fields. tuft/rbc score 0-1 1 per hpf 2-4 2 5-7 3 > 7 4 Nucleated cell differentials and morphologic determinations were made under oil immersion (1000x). Average differentials were calculated from two or more 100 cell differentials including intact endometrial epithelial cells, stripped epithelial cells (nuclei only), PMN's, lymphocytes, monocytes, and eosinophils. Clumps of epithelial cells large enough that individual cells could not be counted were excluded. 53 UHL Fluid Cytology The 1.-:2 ml sample of concentrated lavage fluid was submitted for cytocentrifugation within 90 minutes of collection. Two slides were made using 0.3 ml aliquots in a cytospin machinezo (95 G for 10 min.) Air dried slides were stained with the same automatic stainer utilizing a modified Wright's stain. Tuft and rbc scores, differentials, and morphologic determinations were performed as described for swab cytology. UHL Fluid Nucleated Cell Count A 2 ml aliquot of unconcentrated lavage fluid was submitted for nucleated cell count. A clean cover glass was positioned on the hemocytometer. After discarding the first few drops, both sides of the hemocytometer chamber were filled with the undiluted lavage fluid. The filled hemocytometer was allowed to sit in a moistened covered petri dish for 5 minutes. Nine large squares (1 mm) were counted on each side. Results were reported as the number of nucleated cells per microliter (cubic millimeter). total # cells Cell count/“1 ' total # squares Microbiology-Swab Each swab was used to inoculate blood agar enriched with 1% yeast extract and 1% horse serum 54 (EBA)21, phenylethyl alcohol agar (PEA)22, and MacConkey agar (MAC)23 plates within two hours of sampling. The 24 enriched swab was then placed in a thioglycollate broth with 1% hemin and 1% vitamin K. EBA and PEA plates were incubated in a 5% CO incubator at 37 C. The MAC plate 2 and thioglycollate broth were incubated in an aerobic incubator at 37 C. Plates and broth were examined at 24 and 48 hours. After 48 hours incubation PEA and MAC plates without growth were discarded. Plates with growth were analyzed for different colony types and colony forming units were estimated as <5, <10, <50, (100, >100, or >1,000 for each colony type. 'The EBA. plate and thioglycollate broth were incubated for 72 hours before being discarded as no growths. Different colony types were subcultured and Gram stained. Identification was based on biochemical testing using conventional and microtubezs'26 methods. Cultures without growth on agar plates or in the broth at 72 hours were considered no growths. The broth was subcultured onto plate media if turbidity was present and the original agar plates were negative. Antibiotic susceptibility testing was not performed. Microbiology-UHL Six to fifteen mls of UHL fluid were placed in a sterile capped centrifuge tube, immediately refrigerated 55 and transported to the laboratory on ice. Plating was completed within two hours of sampling. The sample was centrifuged” at 7° C for 15 minutes (3000 G). Supernatant was decanted, leaving .5 to 1.9 ml of fluid in the centrifuge tube. This remaining fluid and any cellular material were mixed by gentle aspiration in and out of a sterile glass pipette. This fluid was divided between the primary quadrants of EBA, PEA, and MAC plates. A flamed loop was used to spread the fluid throughout the quadrant. Streaking, incubation, colony counts, and identification were as for swab microbiology. Progesterone (P4) Assay Serum P4 concentrations were determined by radioimmunoassay, using a commercially available kit.28 In the laboratory used, serum was extracted using petroleum ether (to remove species' differences in protein binding) and equine standards were prepared. The techniques for ether extraction have been recently described (Refsal et al., 1987). Blood Urea Nitrogen Analysis Urea nitrogen concentrations of sera and UHL fluid BUN concentrations were determined using a commercially available BUN reagent kit and chemistry analyzer.29 56 Statistical Analysis Data are expressed as mean 1 SEM. To evaluate differences in recovery of fluid, mean tuft scores, and mean rbc scores between stages of the cycle, data were analyzed using an analysis of variance-~one way classification. If the F-test was significant at p < .05 then the means were compared by computing several least significant differences (Steel and Torrie, 1980). Results of bacterial cultures of guarded endometrial swabs and UHL fluid were expressed as growth or no growth, and differences evaluated using the Chi square analysis. The paired Student's t-test was used to analyze cytologic differentials. All differences were evaluated at the 5 percent level of significance. 57 FOOTNOTES 1McCullough Swab® ; McCullough Cartwright, Barrington, IL. 2Cellulose acetate buterate tubing; 3/8" I.D. x 1/2" O.D. x 1/16" wall, Almac Plastics, Grand Rapids, MI. 3Plastic instrument; guard; Oxboro-Medical Int., catalog #601-365. 4Silastic medical grade tubing; 1/8" I.D. x 1/4" OD., Dow Corning Corp., Midland, MI. 5Brazing wire; Anaconda 997; Michigan Welding Supply Inc., Lansing, MI. 6PE—260; Clay Adams, Parsippany, NJ. 7Silicone catheter 10 Fr., length 1 meter, with Aire-Cuf®; Bivona, Inc., Gary IN. esteri-Vacc> gas sterilizer 400C; 3M Medical Products Division, st. Paul, MN. 9Ster-L-JelC‘D; McCullough Cartwright, Barrington, IL. 10Culturettec"); with modified Stuart's bacterial transport media .5 m1, American Scientific Products, McGraw Park, IL. llNaCl inj. USP 0.9%; Abbott Laboratories, N. Chicago, IL. 12Spray-CyteO water soluble aerosol fixative; Clay Adams, Parsippany, NJ. 13B-D 60 cc syringe; Becton Dickinson and Co., Rutherford, NJ. l4D-—PBS (1x) 100 ml; Gibco Labs, Grand Island, NY. 58 15Centrifuge tube, plastic — 15 ml; Dow Corning Corp., Midland, MI. l6Triac centrifuge; Clay Adams, Parsippany, NJ. l7Tomcat catheter, open end 3 1/2 Fr.; Monoject, Div. of Sherwood Medical, St. Louis, MO. 18Pilling Uterine Biopsy Forcep; Pilling Surgical Instruments, Fort Washington, PA. 19Hema-tek 1000; Miles Lab Inc., Elkhart, IN. 20Cytocentrifuge; Shandon Southern Instrument Corp., Sewickey, PA. 21EBA per liter; composed of Brain Heart Infusion Agar 52 gm (Becton Dickinson, Cockeysville, MD), Yeast Extract 110 gm (Becton Dickinson, Cockeysville, MD), Ultrapure water 1,000 ml, horse serum 10 ml (heat inactivated at 56 C for 30min), defibrinated sheep blood 50 ml. 22Phenylethyl Alcohol Agar (Becton Dickinson, Cockeysville, MD), with 5% defibrinated sheep blood added. 23MacConkey Agar, Difco Laboratories, Detroit, MI. 24Thioglycollate broth per liter; composed of Thioglycollate: Medium 'without indicator 30 gm (Becton Dickinson, Cockeysville, MD), 1% Hemin solution .5 ml (.1 gm of hemin in 10 ml ultrapure water with .5 ml of 1 N NaOHO), Vitamin K solution .1 ml (1 gm of Vitamin K in 99 ml of absolute ethanol), Ultrapure water 1000 ml. 25API 20E®; Analytab Products, Plainview, NY. 26Rapid STREP®; Analytab products, Plainview, NY. 27Sorvall®RT 6000 B refrigerated centrifuge and A-500 fixed angle rotor; Du Pont Co., Wilmington, Delaware. 28Coat--A-Count®; Diagnostic Products Corp., Los Angeles, CA. 296emini® BUN Reagent Kit, ENI Gemini Chemistry Analyzer; Electro-nucleonics‘a, Fairfield, NJ. RESULTS Teasing and transrectal palpation findings indicated that all mares had regular overt estrual behavior, palpable follicular growth, and corresponding changes in uterine and cervical tone as they entered the study in July. All eight mares were sampled in estrus (mean P4 = 0.70 ng/ml) and diestrus (mean P4 - 10.18 ng/ml). Three of the 8 mares returned to estrus within 4 days of the diestrous sampling (mean days in diestrus = 13.3), while the other 5 maintained a mean diestrous length of 17.4 days. Only 4 mares went into winter anestrous (mean P4 = 0.51 ng/ml), while 4 continued to cycle. Anestrous mares were sampled according to protocol in January and February. The anestrous mares were not sampled during the anovulatory transitional phase. Endometrial biopsies taken during mare selection, and as the last procedure in each sampling, revealed glandular and epithelial changes consistent with the mares' stage of cycle. Acute endometritis was not diagnosed, nor were leukocytic infiltrations beyond mild to moderate mononuclear types observed. The predominant pathologic changes were a mild diffuse mononuclear 59 60 infiltration of the stratum compactum, and infrequent scattered fibrotic foci within the stratum spongiosum. Mare #503 had the lowest biopsy category with an average of 3 fibrotic foci per 5.5 mm field accompanied by overall low gland density. This mare began and ended the study as a category IIB. Two mares shifted from an endometrial biopsy category of IIA at preliminary sampling to a category I at the final or anestrual biopsy. Four mares decreased one-half biopsy category (I to IIA, IIA to IIB) during this 6-month period. Biopsy category changes are listed in Table 1. All mares remained in category I or II. Table 1.--Endometrial Biopsy Category Shifts Mare No. Preliminary Estrus Diestrus (figegiggi) 125 IIA IIA IIA 11A 220 I IIA IIA IIA+ 308 I I IIA IIA 412 IIA IIA IIA IIB+ 503 118 118 IIA IIB+ 621 IIA I IIA I 728 IIA IIA IIA 1+ 822 I IIA I IIA +During winter anestrus 61 Fluid recovery during right uterine horn lavage (UHL) was highly variable ranging from 24 - 98% (mean = 57 i 23). Differences between mean percent fluid recovery in estrous (71 i 22), diestrous (56 i 24), and anestrous (80 i 29) groups were not statistically significant. Figure 4 presents fluid recovery results from all groups combined. Five of 7 lavages with less than 50% recovery were from mares with an average right horn diameter of 45 mm or larger. Eight of 9 lavages with greater than 80% recovery came from mares with 40 mm or less average right uterine horn diameters prior to sampling. Recovered UHL fluid was clear with no appreciable cellularity prior to concentration. Nucleated cell counts of UHL fluid averaged 5.3 i 5.5 cells/pl, with a range of 0 to 48 cells/til. Seventeen of 20 nucleated cell counts were less than 10 cells/111. Statistically significant differences in nucleated cell counts were not found for estrous, diestrous, or anestrous groups. Aerobic cultures of centrifuged UHL fluid resulted in significantly more (p < .05) no growths than the guarded endometrial swab technique (75% vs 35%). Results are presented in Figure 5. Colony forming units (CFU) for the 22 total isolates (Figure 6) were less than 5 CFU in 15, less than 50 CFU in 5, and only from broth in 2. The isolates from endometrial swab culture, in 62 10- frequency (number of lavages) % recovery (20 samples) Figure 4. Percent recovery of UHL fluid 63 m swab UHL 0000 990 €€€€§§B§3¥3¥€ cdddddddxxwie c333¥03%0.9 0 av n3RRfififififiéfléfififléfiéfiéfiéfififi‘Qfi ee .fifibfibfihfihfibbh% 0 JV y999$£f$£f$f99L9999988839... eoeeeeeeeeeeee eeeeeeeeeeeee. Addllfiwififikkbfi OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 0000000000000 OOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII OOOOOOOOOOOOOOOOO 111111 d 11111 (ca-aeooeeeeeea-eateteaaeaaaa 1 111111111 I‘d-aaaeoaaaaaoaaaeoa a. ‘lldluaaoooaeaaaaaaa aaaaa aaeaeaeonaaoo-oeo aaoaaaaaeeat...neaaaaeaaoaaaaaoaaeoaaaaaaaaoaoaeaaeaaaeaa aaa oaoaaa eaaaeoaaa aaaaaaaaaaa aaaaaeaaaeaaeeeaeaaaaeeaeeeaaeeaeeoaaaaaaaaa a a aaaaa aaoae-aaaaeaaaaaaeeaaa.aaeaa.aaaaeeeeaeaeaaaaaaaeeeaaaaaaaaoaa aaeeoea .- aaoa aaaaaaaa ea aaaaaaa aaaaaaaaaaaaoaaeeaaaoaaeaaeaaaeeaaee a ...... ........ ........ ........... ...... ..... I CC... ............... ...... ..... .Av .09 000 cfxv ee .8 $23.30 .o eon—.55 Caucuses. eee ee economic. e e e e e 993333390e p _ — — L WOW.} 9 W 000000 000000000000 OOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOO ooooooooooooooooooooo a3&a&&&&&&¢ ......O.......... oeooeeo. oe§3333§333§83 a??333333?3 09090099000 9900. ’.’ ... two Isolates one Isolate no growth (40 samples) 1 cultures lates from endometria Number of 130 Figure 5. 64 '9 .4 .a to: O .4 . .0. lb (\ O: oeeeeo reesewewewe. 9 ° e. x... ‘0 e‘ dddddi G‘ &V '60... ‘ «seas.» as... so ..oevxxé ‘ N N 1‘ ‘ N o Weueueueue“ . V 90 ".‘.‘.‘.‘.4 \\\° veeeeeeeeee. Q 9 555555 A? Av ) we 8 V.‘ ‘ ‘.‘.‘..‘l.i‘m‘:.‘.‘ E.‘ ‘.1.¢.0 .1. w h ufififflfiflfihfihfibfifihfih nP r 90.0.0.0.OOODOOOOOOOOOOQOOO.0‘0‘01. Q& m x... .8 . l‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ E C G C 0 I a c a o G s 88.eeeeeexx.".x.x.".".x.x o .9} D 909.9990DOD.99.0....OOOOOOOOOOO‘O‘O‘O‘O‘OO.A § 4 O l\ xxxo seems“.”sews."«assesses \. e ............9......werewvfissssas. 00 O o. c .9 AW C ............................................................O.. ... 0. § 0 .§§§§%eeeeeeI???93¥9&3323¥333990§?3§ fi? &\ 9000 9900090 _ . uxawwNwwwmnuwfiruwwauwwwwwwhnuarwuww .nr b F p F b p n P b p 0o Iv Aw Qu at al .0 .56 . 6:23.02 ..o 39:55 Econ—co: Isolation frequency from endometrial culture Figure 6. 65 decreasing order of occurrence were Corynebacterium spp. (5), alpha hemolytic streptococci (3), coliforms (3), coagulase negative staphylococci (2), and one each of beta hemolytic streptococcus, E; coli, Bacillus sp., and Lactobacillus sp. The isolates from UHL fluid cultures were Corynebacterium spp. (2), coagulase negative staphylococcus (2), and an organism biochemically similar to Gardnerella vaginalis (1). The Corynebacterium species isolated were .nonhemolytic, nonmotile and catalase positive. The Gardnerella vaginalis isolate was Gram negative, pleomorphic, nonhemolytic, catalase negative and oxidase negative. There was no apparent correlation between number of CFU's nor kind of bacterial isolate between the UHL and swab results. Cytocentrifugation. of' UHL fluid resulted in a significantly higher (p < .05) mean percentage of intact epithelial cells than swab cytology (81.4 i 54 vs 51.3 + 15.2). Figure 7 represents data from each of the three groups. The mean number of PMN/100 cells was similar for UHL and swab cytology (2.25 i 1.4 vs 0.5 + 0.7). This difference was not significant at the 5% level. The mean tuft score (number of ciliated cytoplasmic tufts per hpf) was significantly higher (p < .05) for estrous UHL cytology slides (2.5) than for diestrous UHL (1.1), estrous swab (1.1), and diestrous swab (1.0) cytology slides (Figure 8). 66 wswab cytology mean 90 ‘ UHL cytospin mean 80‘- 70' 60 30- % intact epithelial cells 20‘- 10— Estrus Diestrus Anestrus n=8 n=8 n=4 Figure 7. Percent intact epithelial cells from endometrial cytology 67 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO .................... m swab smear UHL— cytospin .0.000000000000000000000I ........... 0 ............... O 0 0 0 0 0 0 OOOOOOOOOOOOOOOOOOOOOO — 2 oeoom on. .m. h .m w r w hualnflqlql .8 O r 0.05) e m mm fl _ m a... wH 8 “U o . . em 01 s 0. . 0.00 00 00000.00..0.00”.”.”0”.”.”.”0”0 000 ........ .0000. 000.000.000.00... 0 0.00.00.00.00.. 0.000000000000000 ..0 ...... .0 IIIIII 000 0 0 0 0.0000000... ......... 00.000. ““.“‘. Ifififi33fi33. veeeeeeee. 333333333 553333335 0 Diestrus Anestrus 0.000.000.000000000 0000000000000.000000 00... ........... 0.0. 000 ........ 0 ..... .0. 000.000.0000...000000000000.0.000.000... 0.... ..... ... lllll 00 0.0.0.0...000. 0 ........ 0 0. 0 0 I I ‘ N ‘ ‘ ‘ ‘ ‘ ‘ ‘ ddddddiii diidddid: .......... ...... ............. OOOOOOOOOOOOO . U ‘ . . ‘ ‘ U ‘ ‘ A 933?3333?3 3333333?33 0000000000000000000. Estrus Soon :3 n=4 n=8 Tuft and red blood cell scores from endometrial cytology n=8 Figure 8. 68 Mucus strands were commonly seen in estrous swab smears, yet were absent in UHL cytology slides. Epithelial cells in anestrous cytology slides (UHL and swab) were smaller in overall size with a larger nucleus to cytoplasm ratio than epithelial cells from estrous or diestrous groups. Total cells present on cytology slides were less for UHL than with swab smears, and UHL slides from one sampling contained too few cells to count differentials (< 200). Determinations of urea nitrogen content in lavage fluid from 5 samplings with greater than 90% fluid recovery resulted in no detectable levels. Serum urea nitrogen values at sampling were within the normal range (mean = 22). DISCUSSION The overall goal of this study was to develop a safe, efficient uterine horn lavage technique which would accurately depict the cytologic and bacteriologic status of the mare's endometrium. Vaginal placement of the apparatus into the uterine body followed by transrectal support of the right horn during continued advancement, was necessary for consistent positioning of the balloon. The orientation of the bend in the capped end of the CAB tubing was also critical to the success of the technique. The instrument was easily introduced into the uterine body with the bend in the vertical plane. Rotating the tubing counterclockwise to orientate the bend in a horizontal plane facilitated entrance into the right horn. Safety A goal of any reproductive examination, employing techniques which invade the tubular genital tract, is to minimize insult to the endometrium. Inflammatory cells indicating endometritis were not detected in the diestrous samples collected 10 1x: 12 days after estrous UHL and other techniques (swab, biopsy). It is likely that UHL alone will cause a transient inflammatory 69 70 response as does intrauterine saline infusion (Bennett et al., 1980). The occurrence of estrus in 4 of the mares shortly after diestrous sampling was anticipated due to the multiple cervicovaginal procedures at each sampling. Early return to estrus is an established effect of saline infusion and endometrial biopsy in the mare (Hurtgen and Ganjam, 1979). However, uterine flushing alone (with no parallel procedures), and done on a range of days post- ovulation, was insufficient stimulation to shorten the length of diestrus in mares (Zavy et al., 1978; Strzemienski and Kenney, 1984)., As UHL requires minimal cervicovaginal stimulation, and transrectal. ‘uterine manipulation only to facilitate catheter placement, it is likely that UHL will not shorten diestrus if used as a single technique. Although biopsy categories have been. well correlated with foaling rates, the slight changes in biopsy category during this study were considered to be due to seasonal changes in the endometrium rather than a compromise of fertility. Two groups of investigators have demonstrated that anestrual endometrial changes, (decreased stromal edema, increased nonfibrotic gland nesting) depress the biopsy category using current criteria (Shideler et al., 1982; Gross and LeBlanc, 1984). 71 The safety of the UHL technique described here is further supported by the establishment of first cycle pregnancies in two of the mares after natural service in March. Both mares had completed the present study in February. Yolk sac growth and embryo development were normal ultrasonographically until elective abortion using prostaglandins at day 30 of gestation. Other mares were not bred. An additional pregnancy resulted in a maiden mare which entered the study in August and was sampled in estrus according to protocol. Eight days following UHL she was removed from the study, receiving an embryo from a category III donor. She remained pregnant and foaled without complications. Recovery Poor fluid recovery ((50%) during UHL wes associated with right horn diameters greater than or equal to 45 mm, while efficient UHL (>80%) was associated with right horn diameters of 40 mm or less. The cuff of the UHL catheter in this study inflates to a 47 run diameter when filled with 100 cc of air. In the mares with larger ‘uteri, and subsequent poor recovery, the inflated cuff could be felt in the right uterine horn, yet during UHL, fluid could be observed dribbling out of the CAB tubing. As the open, cranial tip of the CAB tubing rests in the space just caudal to the cuff placed 72 in the right horn, escaping lavage fluid was directed into it indicating the horn was not completely blocked. It is interesting that embryo recovery using uterine horn lavage in the cow is accomplished with similar techniques using only 10 - 18 cc of air in the cuff, resulting in a cuff diameter of 24 - 30 mm. Care must be taken in the cow to prevent overinflation of the cuff and splitting of the endometrium (Mapletoft, 1986). The mare UHL recovery findings indicate the need for larger diameter cuffs and the use of transrectal palpation to estimate minimum cuff inflation, to monitor catheter placement, and possibly prevent endometrial damage. Cell Counts Normal nucleated cell counts for equine uterine lavage fluid have not been previously defined. The results of this study infer that normal cell counts for UHL fluid are <10 nucleated cells/U1. Only 3 lavages in this study had cell counts >10 cells/ ul and 2 of these were from lavages with. poor recovery indicating some doubt that the cells were exclusively from the uterine horn. Although the estrous group had the highest mean cell count (9.9 cells/ pl), the differences among groups were not statistically significant. 73 Cytology In human obstetrics, cytocentrifugation has been suggested to concentrate fluids with low cellularity (Gravlee, 1975). It has also been used extensively in bronchoalveolar lavage of human (Hunninghake et al., 1979) and equine (Derksen et al., 1985) patients. Cytocentrifugation did not provide adequate concentration of cells from UHL samples until an initial 5—fold concentration of the retrieved fluid was performed. Although the UHL cytology slides were still less cellular than swab smears, initial concentration followed by cytocentrifugation provided adequate cytologic specimens (>200 cells) in all but one of the lavages. The significantly higher percentage of intact versus stripped epithelial cells in UHL slides indicates that cells available are of good quality. The value of a detailed assessment for subtle morphologic abnormalities and for differential counts, even when cell counts are within the normal range, was pointed out by Christopher et al. (1988). Using cytocentrifugation, they found abnormalities in cell type or morphology in 24.5% of canine cerebrospinal fluid samples with cell counts in the normal range (<8 cells/ul). Extensive characterization of epithelial cell morphology was not attempted in this study. The subtle, seasonal cellular changes have been thoroughly described 74 for normal mares by two experienced cytologists (Roszel and Freeman, 1988). However, the occurrence of significantly higher ciliated cytoplasmic tuft scores in estrous UHL cytology slides in this study conflicts with some of their findings. They did not include separate patterns for estrus and diestrus, but their active pattern, associated with cycling mares, did not identify an increase in the number of ciliated cytoplasmic tufts. Numerous ciliated cytoplasmic tufts were identified with their fall transitional pattern and to a lesser extent their spring transitional pattern. in: consideration of this, one must be aware that the estrous samplings in our study were performed in September and early October. However, all 8 mares were actively cycling and ovulating at this time as evidenced by progesterone analysis, teasing, and transrectal palpation results. Endometrial biopsies did not reveal changes associated with transition (cubodial or low columnar epithelium, deep gland atrophy, nonfibrotic nests) in these mares. Diestrous UHL samples taken in this study during this same time period had low numbers of ciliated cytoplasmic tufts. The significance of increased numbers of ciliated cytoplasmic tufts in estrous UHL samples is not clear. Perhaps the findings of several investigators that intact ciliated epithelial cells are more numerous in diestrual smears (Britton, 1982; Couto and Hughes, 1984) indicates 75 that the ciliated epithelial cells of the mare's estrous endometrium are more fragile resulting in large numbers of ciliated cytoplasmic tufts (fragments). The cytologic findings in this study need to be evaluated in cycling normal mares in the height of the breeding season to confirm these findings. The higher mean percentage of PMN's in UHL versus swab cytology slides was not associated with endometritis in biopsies or by positive cultures in any of the mares. Increased PMN's in UHL samples thus may be a result of (1) the increased endometrial surface: area sampled in UHL, (2) a slight inflammatory response to swab procedures prior to UHL and/or (3) peripheral blood contamination. It may be that UHL will provide a more sensitive cytologic method, and thus have a higher number of PMN's in normal mares than swab cytology techniques. Microbiology The significantly higher percentage of no growths in the culturing of UHL fluid versus swabs appears to indicate that the UHL samples were less contaminated by aerobic bacteria from the caudal tubular genital tract. As all organisms isolated in this study have been previously associated with contamination of endometrial cultures in mares (except for _C_;_. vaginalis), the UHL cultures appear to provide a more representative picture 76 of the microbiological status of the mares' uterine lumen. The ability of the UHL technique to evaluate the status of abnormal mares (infectious endometritis) has not been investigated. Results from the use of tampons (Stratton et al., 1979) and flushing (Ball et al., 1988) to culture the mares' uterus indicated that these techniques were more sensitive than endometrial swabs for particular organisms. Ball's findings from uterine flushing of 27 subfertile mares identified Corynebacterium spp. as the most frequent isolate. Both cytologic and histologic evidence of chronic inflammation were present in 8 of the 12 mares with this isolate. Corynebacterium spp. were also the most common bacterial isolate in this UHL study. The majority of isolations were less than 5 CFU's and from endometrial swabs . The isolations of Corynebacterium spp . were not associated with inflammatory cells in the corresponding cytologic or histologic samples of mares in this study. Gardnerella vaginalis was an unusual isolate in that it has not been reported from vaginal or endometrial cultures in mares. The role of this bacterium in nonspecific vaginitis (NSV) in women is 'unclear. .A clinical report has suggested an association between the isolation of g; vaginalis and NSV in women and also that women with this isolate and NSV respond well to 77 metronidazole therapy (Pheifer et al. 1978), while another report suggests that no relationship exists between. E; vaginalis and clinical signs of vaginitis (McCormack et al., 1977). No studies inferred that this organism was a uterine pathogen. The UHL cytology slides corresponding to our isolation of g; vaginalis indicated possible vaginal contamination of the sample (a few squamous and parabasal epithelial cells) . The isolate may represent part of the vaginal flora in this particular mare. Urea Nitrogen Analysis The urea nitrogen in UHL fluid and serum was measured in selected lavages to determine if this component of serum would reflect the degree of dilution of intrauterine fluid present. at lfifl3. Albumin (Weinberger et al., 1978) and potassium (Mandel et al., 1976) levels have been used as reference standards for reporting immunoglobulin levels in bronchoalveolar lavage fluids. Proteins of low to intermediate molecular weight ((150,000 daltons) have been shown to be present in human lung lavage effluents in proportion to their concentrations in serum (Bell et al., 1979). Urea has been shown to be a normal constituent of uterine fluid in rats (McRae and Kennedy, 1982) and cows (Ferguson et al., 1986) and may be reflective of serum concentrations. Of 78 the five UHL samples submitted none had detectable urea nitrogen. Determination of the degree of dilution by UHL was not accomplished. Summary A guarded uterine horn lavage technique was developed in this study. The apparatus was able to block effectively the right horn in mares with smaller diameter uterine horns (<45 mm) and resulted in good recovery of fluid. A new catheter has been designed, the balloon of which can be to inflated to a 75 mm diameter to facilitate more efficient sampling in infertile. mares with larger uteri. Such a catheter will be necessary for future use of the technique in abnormal mares with larger uteri. Microbiologic results suggested that the technique was less contaminated than the guarded endometrial swab. Further studies are necessary to ensure that all bacteria are being retrieved from the fluid and to assess any effects the lavage fluid may have on bacterial recovery. The abnormal mare with bacterial or fungal endometritis will be the subject of the next studies with UHL. Uterine horn lavage provided adequate cytologic samples compared to swab methods. The fluid cytology slides contained fewer distorted and disrupted epithelial 79 cells, and were easier to interpret, as all cells from cytocentrifugation were concentrated in a central area on the slide. The results raise several questions relative to the clinical use of this technique. Such as: 1. Will the technique be more sensitive than swabbing of infected mares? 2. Can the apparatus be packaged in an economic manner and used properly by other veterinary practitioners? 3. Can methods be developed to simplify laboratory procedures for fluid samples? LI ST 01“ REFERENCES LIST OF REFERENCES Allen WE. Aspects of genital infection and swabbing techniques in the mare. Vet Rec 1979;104:228—231. Anderson B. Diagnosis of endometrial cancer. Clin Obstet Gynecol 1986;13:739-751. Ansari MM, Matros LE. Bacillus cereus: The pathogenic origin of infertility in mares? Vet Med 1986;July:657-658. Asbury AC. Pathogenesis and diagnosis of uterine infection in mares. Proceedings of the Western States Veterinary Conference, Feb 1984az9-14. Asbury AC. Endometritis diagnosis in the mare. Eg Vet Data l984b;5:166. Asbury AC, Halliwell REW, Foster GW, et al. Immunoglobulins in uterine secretions of mares with differing resistance to endometritis. Theriogenology 1980;14:299-308. Ayre, JE. A simple office test for uterine cancer diagnosis. Can Med Ass J 1944;51:17—22. Ball BA, Shin SJ, Patten VH, et al. Use of a low-volume uterine flush for microbiologic and cytologic examination of the mare's endometrium. Theriogenology 1988;29:1269-1283. Barbaro C. A simpler technique of uterine lavage for the study of endometrial cytology. Aust. N.Z.J. Obstet Gynaec. 1969;9:37-41. Bell DY, Spock A, Hook GCR. Plasma proteins of the bronchoalveolar lining from human lung. FASEB 1979;38:522. :Bennett DG, Poland HJ, Kaneps AJ, et a1. Reaction of the equine endometrium to intrauterine infusion, in Proceedings. 26th Ann Meeting, Am Assoc Equine Pract 1980;134-139. 80 81 Bermudez V, Miller R, Johnson W, et al. The prevalence of Mycoplasma spp. and their relationship to reproduCtive performance in equine herds in southern Ontario. J Reprod Fert Suppl 1987;35:671—673. Blanchard TL, Garcia MC, Hurtgen JP, Kenney RM. Comparison of two techniques for obtaining endometrial bacteriologic cultures in the mare. Theriogenology 1981;16:85-93. Blue MG. Mycotic invasion of the mare's uterus. Vet Rec 1983;113:131-132. Blue MG, Hannwacker MA. Endometritis in the mare caused by a coryneform organism--a case report and experimental studies. Cornell Vet 1984;74:331-343. Bouchardy C, Vassilakos P, Riotton G. Endometrial cytohistology by the pistol-aspiration technique: clinical applicability. Obstet Gynecol 1987;70:389- 393. Brandt GW, Manning JP. Improved uterine biopsy techniques for diagnosing infertility in the mare. VM/SAC 1969;64:977-893. Britton BA. Endometrial change in the annual reproductive cycle of the mare. J Reprod Fert Suppl 1982;32:175-180. Brook D. The diagnosis of equine bacterial endometritis. Compend Contin Ed Pract Vet 1984;6:300—306. Brook D. Cytological and bacteriological examination of the mare's endometrium. Eq Vet Sci 1985;5:16-22. Brunham RC, Irwin B, Stamm WE, Holmes KK. Epidemiological and clinical correlates of C; trachomatis and N. gonorrhoeae infection among women attending a STD EIinic. Clin Res 1981;29:47A. Carter GR. Essentials of Veterinary Bacteriology and Mycology. East Lansing, MI: Michigan State University Press, 1976;253-256, 139-140. Cary WH. A method of obtaining endometrial smears for study of their cellular content. Am J Obstet Gynecol 1943;46:422-424. 82 Casey MJ, Madden TJ. Endometrial screening of asymptomatic women by irrigation technique in the private gynecology office. Am Geriatrics Society 1977;15:118-124. Christopher MM, Perman V, Hardy RM. Reassessment of cytologic values in canine cerebrospinal fluid by use of cytocentrifugation. J Am Vet Med Assoc 1988;192:1726-1729. Collins SM. A study of the incidence of cervical and uterine infection in Thoroughbred mares in Ireland. 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Journal of Animal Science 1978;47:672-676. nICHran STATE UNIV. LIBRARIES lllll"Illllllllllllllllllllllllllllllllllllllllllllllllllll 31293005791532