REPRODUCTIONRESEARCH

Uterine blood flow and perfusion in mares with uterine cysts: effect of the size of the cystic area and age

J C Ferreira1, E L Gastal2 and O J Ginther1,2 1Eutheria Foundation, Cross Plains, Wisconsin 53528, USA and 2Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, 1656 Linden Drive, Madison, Wisconsin 53706, USA Correspondence should be addressed to E L Gastal; Email: [email protected]

Abstract

Transrectal color and power Doppler ultrasonography was used to study uterine blood flow and perfusion in mares with and without uterine cysts. Vascular perfusion of the and blood flow velocities, vascular perfusion, diameter,circumference, and area of a cross section of the mesometrial attachment were evaluated. To study the effect of internal cysts, two matched groups (cystic and control, nZ21 mares/group) were used. Uterine vascular perfusion in mares with cysts was less (P!0.0001) in the cystic than the noncystic region and less (P!0.0009) than that for controls. Mares with cysts had lower (P!0.04) pulsatility index (PI) and greater end diastolic velocity (EDV; P!0.03) and time-averaged maximum velocity (TAMV; P!0.05) of the mesometrial vessels than the controls. To study the effect of the size of internal uterine cystic area, paired mares were arranged in four groups (nZ8–11/group): small uterine cystic area (%275 mm2) versus controls and large uterine cystic area (O410 mm2) versus controls. A small uterine cystic area did not affect uterine hemodynamics. Mares with large uterine cystic area had lower PI (P!0.05) and greater peak systolic velocity (P%0.05), EDV (P!0.009), and TAMV (P!0.005). To study the effect of age, old versus young mares without cysts were compared (nZ11/group). Old mares had greater EDV (P!0.02) and TAMV (P!0.01) than young mares. Results demonstrated, for the first time in any species, reduced uterine vascular perfusion in mares with uterine cysts and a positive association between size of the cystic area and disturbed uterine hemodynamics. Reproduction (2008) 135 541–550

Introduction periglandular fibrosis (Kenney & Ganjam 1975, Ricketts 1975, Kenney 1978). Glandular cysts range from a few Uterine cysts have been described in diverse species of millimeters to 1 cm in diameter and are frequently found in animals, including horses, cattle, ewes, pigs, cats, dogs, pregnant mares. Lymphatic cysts start as small microscopic elephants, and humans (Dow 1959, 1962, Adams 1975, structures that enlarge from several millimeters to several Ricketts 1975, Al-Dahash & David 1977, Dallenbach- centimeters (Kenney & Ganjam 1975, Kenney 1978)and Hellweg 1987, Lipetz et al.1987, Agnew et al. 2004). In have reported mean diameters ranging from 2 to 48 mm mares, uterine cysts are fluid-filled structures commonly (Tannus & Thun 1995). Cysts are usually located at or near found in the three uterine layers or attached to the exterior the base of both horns (Bracher et al.1992, Eilts et al. 1995, surface (Ginther & Pierson 1984) of a normal or chronically Tannus & Thun 1995) in the ventral-most portion of the inflamed uterus (Kenney & Ganjam 1975). The cysts arise suspended uterus (Ginther & Pierson 1984). The reported from the (Kenney & Ganjam 1975, Ricketts incidence in horses ranges from 13 to 55% (Wilson 1985, 1975, Kenney 1978)ormyometrium(reviewedbyStanton Adams et al. 1987, Kaspar et al. 1987, Leidl et al. 1987, et al. 2004). Although the etiopathogenesis of uterine cysts Bracher et al. 1992, Tannus & Thun 1995). Uterine cysts is uncertain, it is likely that abnormal uterine blood flow appear to have clinical importance (Adams et al. 1987, can contribute to cyst formation as a result of inadequate Leidl et al.1987, Tannus & Thun 1995,reviewedby drainage due to poor venous return and angiosis of Stanton et al.2004, Yang & Cho 2007). arterioles (Schoon et al. 1999). Two additional origins for A higher frequency of uterine cysts in older mares uterine cysts have been suggested: (1) lymphatic cysts from (Adams et al. 1987, Leidl et al.1987, Bracher et al.1992) obstruction of lymphatic channels (Kenney & Ganjam and a progressive incidence of cysts as age advances 1975) or from a gravitational effect in the enlarged, (Tannus & Thun 1995) have been described. Increased age gravid, or post partum uterus, resulting in an impediment in mares has also been accompanied by increased severity to fluid drainage and ventral collection of lymph of endometrial fibrosis, increased uterine vascular dys- (reviewed by Stanton et al.2004)and(2)glandularcysts function, and reduced fertility (Doig et al.1981, Oikawa from endometrial glandular distension resulting from et al.1993, Nambo et al. 1995, Gru¨ninger et al. 1998,

q 2008 Society for Reproduction and Fertility DOI: 10.1530/REP-07-0447 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/30/2021 05:13:00AM via free access 542 J C Ferreira and others

Schoon et al. 1999). Considering the reports of a high uterine artery (Bollwein et al.1998) and its branches, incidence of cysts and degenerative changes in the uterine the mesometrial attachment and vessels located in the artery walls in older mares, there may be a relationship endometrium and can be evaluated for among the presence of uterine cysts, decreased uterine the study of uterine hemodynamics (Silva et al. 2005, vascular perfusion, and pregnancy loss. However, no study Silva & Ginther 2006). Doppler ultrasonography is a has been reported on the direct effect of age on vascular potential technique for the study of the pathogenesis and perfusion of the uterine layers in mares. effects of treatment or preventive approaches for uterine Uterine cysts are readily imaged by ultrasound, and cysts. However, no report was found in any species on the transrectal ultrasonography has been an important advance- use of color Doppler ultrasonography to investigate the ment in the study of uterine cysts, providing detailed relationships between uterine blood flow and uterine cysts. research and clinical information (Ginther & Pierson 1984). The purposes of the present study using B-mode and Ultrasonically, internal cysts are characterized by an color Doppler ultrasonography (Fig. 1) in mares were to anechoic area (fluid) with an irregular echoic wall involving study the incidence of internal and external uterine cysts uterine tissue or a prominent bulge in the uterine lumen. and the relationship with blood flow. Specific goals were Individual, compartmentalized, or multilobulated sacs, to (1) examine in detail the prevalence of uterine cysts in pedunculated or not, can be imaged. External cysts usually young and old mares and determine the number, size, project outward from the outer surface of the endometrium and longitudinal and cross-sectional uterine locations (Ginther & Pierson 1984, Ginther 1995a). In mares, uterine of individual cysts (Part 1); (2) test the hypothesis that cysts also have been studied by necropsy, histology, uterine regions with cysts have low uterine vascular transrectal palpation, hysteroscopy, and fiber-optic tech- perfusion and determine the effects of cysts on spectral niques (Kenney & Ganjam 1975, Wilson 1985, Kaspar et al. blood flow velocity in the vessels of the mesometrial 1987, Leidl et al. 1987). attachment (Part 2); (3) test the hypothesis that a larger Color and power Doppler ultrasonography is a non- uterine cystic area is associated with changes in uterine invasive pulse wave technique that has been used recently vascular perfusion and uterine hemodynamics of the for transrectal study of blood flow in the reproductive tract than a smaller cystic area (Part 3); and of large domestic animals (Ginther 2007). In addition to the (4) test the hypothesis that older nulliparous mares

Figure 1 Diagram of an equine cystic uterus divided into nine segments and three portions with representative ultrasonograms. The uterus is divided into three portions: extremities (dark pink), central segments (intermediate pink), and cornual-body junction (light pink). The black dots within the uterine diagram symbolize internal uterine cysts. A cystic uterine region and an adjacent uterine region without cysts are represented by black and blue dashed oval circles respectively, in the left horn. (A) Cross- sectional images in B-mode, (B) power flow Doppler of a noncystic region, and (C) power flow Doppler of a cystic region are shown. (D) The mesometrial attachment of each horn was identified in B-mode and (E) delineated for measurements of the mesometrial diameter, circumference, and area (F) and vascular perfusion. (B and C) Uterine and (F) mesome- trial vascular perfusion were estimated accor- ding to the number and intensity of color spots in the vessels of the tissue. (G) The sample gate was placed in a vessel of the mesometrium to generate the Doppler frequency spectral graph that represents the changing velocities over time and depicts the arterial pulses generated during cardiac cycles; the width of the gate (1.5 mm) is exaggerated in the drawing.

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Downloaded from Bioscientifica.com at 09/30/2021 05:13:00AM via free access Effect of cysts on uterine blood flow 543 without cysts have lower uterine vascular perfusion than middle and uterine body middle (LM, RM and BM) and younger mares and determine the effects of age on cornual-body junction left, right horn posterior and spectral blood flow velocity in the vessels of the uterine body anterior (LP, RP and BA). The number of mesometrial attachment (Part 4). cysts did not differ between the internal and external cysts in the extremities and central segments, and internal cysts were more (P!0.005) predominant than external cysts in Results the cornual-body junction. There were more (P!0.0001) ! Part 1: prevalence and distribution of uterine cysts cysts (total) comprising a larger area (P 0.0001) in the central segments and cornual-body junction than in The prevalence of ultrasonographically detected uterine the extremities. Combinations of internal and external cysts in 76 mares in the three age groups is shown in cysts were detected in the majority (21 mares, 62%) of Table 1. Mean age of mares with uterine cysts was greater the 34 cystic mares. The frequency of mares with only than that in mares without detected cysts. The frequency internal cysts (23.5%) did not differ from that with only ! of mares with cysts increased (P 0.05) progressively external cysts (14.7%). The incidence of internal cysts in O with age, and mares 14 years had the highest frequency. the 21 mares increased (P!0.0001) from the extremities ! By contrast, the majority (P 0.008) of mares without to the cornual-body junction, whereas the external cysts cysts were 7–14 years. The size of the uterine cystic area ! O O were more (P 0.02) frequently observed in the central was greater (P 0.01) in mares 14 years than in mares segments. The mean age of the 21 mares with both 7–14 years. Although the size of the external cystic area G G 2 internal and external cysts (15.9 1.0 years) was greater (152 39 mm ) did not differ with the age, the size of the (P!0.007) than that for mares with only internal internal cystic area was greater (P!0.02) in mares O14 G G G 2 (10.3 1.2 years) and only external (12.3 2.9 years) years (523 165 mm ) than in mares 7–14 years cysts. The number of cysts was greater (P!0.0001) in the (121G47 mm2). The distribution of internal and external 21 mares (17.7G2.8 cysts) than that in mares with only uterine cysts is shown in Table 2. The mean number of internal cysts (8.0G1.2) and mares with only external cysts in mares with cysts ranged from 2 to 59 and the mean cysts (3.2G1.2). The uterine cystic area (mm2) of mares cystic area ranged from 28 to 3087 mm2. The number with only internal (65G11 mm2) and only external of internal cysts was greater (P!0.04) than that for (81G42 mm2) cysts did not differ, but the area was external cysts, and the mean diameter did not differ smaller (P%0.0002) than that in mares with both internal between internal (5.7G0.7 mm) and external cysts and external cysts (759G168 mm2). (5.5G0.3 mm). In regard to longitudinal locations, the frequency of both internal and external cysts was lower ! ! (P 0.004 and P 0.006 respectively) in the extremities Part 2: effect of cysts left, right horn anterior and uterine body posterior (LA, RA and BP) than in the central segments left, right horn The presence of internal cysts did not affect the vascular perfusion, diameter, circumference, and area of the cross G section of the mesometrial attachment (Table 3). The Table 1 Mean S.E.M. for frequency, number of uterine cysts, and age cystic group had a lower PI, lower RI (approached relationships in 76 mares. significance), greater end diastolic velocity (EDV), and Group greater time-averaged maximum velocity (TAMV) than End points Cystic* Noncystic Probability the controls. No difference between groups was found for peak systolic velocity (PSV) values. The percentage of Number of mares (%) 34 (44.7) 42 (55.3) NS Doppler signal in the cystic region was less than in the Mare age (years) 14.0G0.9 10.0G0.7 P!0.0002 Frequency of mares/age group (%) middle of the of controls (Table 3; Fig. 2). !7 years 2 (5.9)a 8 (19.0)a P!0.008 The percentage of uterine tissue with Doppler signals of 7–14 years 12 (35.3)b 23 (54.8)b P!0.009 c a vascular perfusion in the cystic group was also less O14 years 20 (58.8) 11 (26.2) P!0.03 ! No. of cysts/age group† (P 0.0001) in the cystic region than in the adjacent !7 years 1.5G0.5 – – region without cysts (Table 3; Fig. 3). No difference in 7–14 years 7.9G2.2a ––percentage of uterine vascular perfusion was detected O14 years 16.3G3.1b –– Uterine cystic area (mm2)/age group† between left and right horns in control mares (data not !7 years 42.4G14.1 – – shown), and the average was used in the analyses. The 7–14 years 222.9G81.9a ––region without cysts in the cystic group did not differ O G b 14 years 708.5 181.9 ––from the average for the two horns in the control group. a,b,cFrequencies or means within an end point are different (P!0.05). *Includes mares with internal and external cysts (nZ21), with only internal cysts (nZ8), and with only external cysts (nZ5). †The low Part 3: effect of size of cystic area ! number of mares with cysts 7 years did not allow statistic analyses % 2 between age categories. Comparison was done between mares with Mares with a small uterine cystic area ( 275 mm )had 2 2 cysts 7–14 and O14 years. an area of 180G24 mm (range 32–275 mm ). The www.reproduction-online.org Reproduction (2008) 135 541–550

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Table 2 Uterine distribution of cysts in mares*.

Cross-sectional location†

Longitudinal location‡ Internal (no. cysts/mare) External (no. cysts/mare) Total (no. cysts/mare) Cystic area (mm2) Overall 7.9G1.5x 4.6G0.9y 12.5G2.1 497.6G118.2 Extremities 0.9G0.2a 0.7G0.3a 1.6G0.3a 60.7G21.9a Central segments 3.1G0.8b 2.3G0.5b 5.4G0.9b 205.3G54.2b Cornual-body junction 3.9G0.7b,x 1.6G0.4b,y 5.5G0.9b 231.6G56.5b a,b,cFrequencies or means with different letters within a column are different (P!0.02). x,yMeans within a row (internal versus external) with different letters are different (P!0.005 to P!0.04). *Includes mares with internal and external cysts (nZ21), with only internal cysts (nZ8), and with only external cysts (nZ5). Total number of mares is 34. †Internal and external cysts were determined in cross-sectional examinations. Internal cysts were located within the endometrium and/or myometrium. External cysts were located outside and apparently attached to the perimetrium. ‡Uterus was divided into three uterine portions and each portion consisted of three uterine segments. individual cysts were fewer (P!0.004) in number and Discussion less (P!0.007) in diameter in the small cystic area than in The use of color or power Doppler ultrasonography has the large cystic area (Table 4). No significant differences become one of the best available and reliable techniques between mares with a small cystic area and the paired for the diagnoses and studies of uterine hemodynamics controls were found at the mesometrial attachment for in vivo (Ginther 2007). The present study demonstrates for spectral end points, vascular perfusion, cross-sectional the first time in any species the relationships between diameter, circumference, or area, except for a lower RI uterine cysts and uterine hemodynamics, using ultrasono- (approached significance) in the group with a small cystic graphic Doppler technology. The findings can be helpful area. Uterine vascular perfusion percentage in the cystic for understanding the origin and effects of cysts. New region was less (P!0.04) than that in the adjacent region information was obtained with regard to the reduced without cysts and also less (P!0.02) than that in the control mares (Table 4; Fig. 3). The vascular perfusion uterine perfusion in cystic areas and the absence of a percentage in uterine regions without cysts did not differ from the percentage in controls. Table 3 MeanGS.E.M. extent of mesometrial vascular perfusion and Mares with a large uterine cystic area (O410 mm2) blood flow velocities and uterine vascular perfusion in mares with hadanareaof1110G314 mm2 (range 411–2854 mm2). internal uterine cysts versus control mares without detected cysts. The large uterine cystic area did not affect vascular Group perfusion, diameter, circumference, and area (mm2)of End points Internal cysts* Control* Probability the mesometrial attachment compared with the paired controls (Table 4). The PI was lower (P!0.05) and the RI Number of mares 21 21 – Mesometrial attachment † was not different when compared with the control group. Cross-sectional view The PSV (P%0.05), EDV (P!0.009), and TAMV (P!0.005) Vascular perfusion 2.9G0.2 3.0G0.2 NS were greater at the mesometrial attachment in the cystic (scores 1–4) G G group than in the paired controls (Table 4; Fig. 4). Diameter (mm) 17.1 0.6 17.1 0.6 NS Circumference (mm) 66.2G1.8 65.7G1.7 NS Differences in vascular perfusion between regions and Area (mm2) 240.2G15.3 218.6G12.9 NS groups were similar to those for the small cystic area. Spectral end points PI (index) 1.60G0.08 1.80G0.08 P!0.04 RI (index) 0.71G0.02 0.75G0.02 P!0.09 G G Part 4: effect of age in mares without cysts PSV (cm/s) 13.6 0.7 12.7 0.6 NS EDV (cm/s) 3.7G0.3 3.1G0.2 P!0.03 TAMV (cm/s) 6.3G0.4 5.4G0.3 P!0.05 In mares without detected cysts, the mean ages in the ‡ G G Uterine vascular perfusion (%) young and old groups were 6.7 0.7 and 16.6 0.5 Cystic region 7.6G1.2a 14.1G1.6§ P!0.0009 years respectively. A gated artery of the mesometrial Adjacent to cystic 12.7G1.6b NS attachment had a greater EDV (P!0.02) and TAMV region ! G G (P 0.01) in old mares (3.40 0.3 and 5.81 0.4 cm/s NS, not significant. PI, pulsatility index; RI, resistance index; PSV, peak respectively) than in young mares (2.65G0.1 and systolic velocity; EDV, end diastolic velocity; TAMV, time-averaged 4.68G0.2 cm/s respectively). A positive correlation maximum velocity. a,bUterine vascular perfusion means with different letters within a column are different (P%0.0001). (rZ0.44; P!0.04) between age and TAMV was found * ! The presence or absence of external cysts was not considered in the and EDV tended (P 0.07) to be positively correlated cystic group. Controls had neither internal nor external cysts and were (rZ0.38) with age. No other significant differences paired with mares with cysts of similar age. †Vessels of the mesometrial ‡ between young and old mares were detected for any of attachment averaged for the two uterine horns. Percentage of endometrium and myometrium with color Doppler signals of blood the other end points measured at the mesometrial flow. §Average for left and right horns of mares without cysts was used attachment and uterus (data not shown). in the analyses.

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Figure 3 Selected power flow Doppler ultrasonograms of uterine horn cross sections of (A, C and E) cystic regions and (B, D and F) noncystic adjacent regions in three mares. Vascular perfusion was lower in cystic regions than in adjacent regions without cysts in mares with small or large cystic areas.

Figure 2 (A, C, E and G) Selected B-mode and (B, D, F and H) power flow Doppler mode ultrasonograms of cross sections of the uterine To our knowledge, this is the first ultrasonographic study horns of (A and B) one mare without cysts and (C–H) three mares with to consider internal and external cysts separately. The cysts. B-mode ultrasonography was used to determine the presence and majority of the mares with cysts had both internal and location of uterine cysts. (C–F) Internal and (G and H) external uterine external cysts. The size of cysts was not affected by their cysts are shown. Uterine cysts were identified as (C and D) single or location (internal or external). However, external cysts (E–H) multiple and compartmentalized anechoic fluid-filled structures were present in lower number/mare and were smaller in with irregular borders. Power Doppler ultrasonography was used to estimate uterine vascular perfusion. The uterus of the mare (B) without area than internal cysts. The extremities of the uterus were cysts had greater uterine vascular perfusion than uterus of mares (D, F least affected by internal and external cysts. The cornual- and H) with cysts. body junction was more affected by internal than external cysts. Considering only mares with both internal and detected effect of age on uterine vascular perfusion in external cysts, a progressive increase in the frequency of mares without cysts. internal cysts from the extremities to the cornual-body The frequency of mares with cysts in this study was junction of the uterus was found and agrees with previous greater than that reported previously (Adams et al. 1987, reports (Bracher et al.1992, Eilts et al.1995, Tannus & Kaspar et al. 1987, Leidl et al. 1987, Eilts et al. 1995, Thun 1995) that suggested a higher prevalence of Tannus & Thun 1995). A progressive increase in the endometrial cysts in the caudal portion of the uterine frequency of mares with cysts with the advancing of age horns. During the embryo mobility phase (11–15 days after (Tannus & Thun 1995) and a lower mean age of mares ovulation; Ginther 1983a, Leith & Ginther 1984), the without cysts (Adams et al. 1987, Leidl et al. 1987, embryo has been observed to move through cystic areas Bracher et al. 1992, Eilts et al. 1995) agrees with our (Ginther & Pierson 1984). However,it is unknown whether findings. Although the overall number of cysts and the extensive cysts could interfere with embryo mobility and size of the cystic area increased with age, our findings thereby with the requisite exposure of the entire uterus to indicated that old age was associated with increase in the the mobile embryo in the prevention of luteolysis internal cystic area, whereas the size of the external cystic (McDowell et al.1988). Fixation of the embryo occurs area did not differ between mares of intermediary versus almost always in the caudal portion of one of the uterine advanced age. horns (Ginther 1983b), but it is unknown whether cysts in www.reproduction-online.org Reproduction (2008) 135 541–550

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Table 4 MeanGS.E.M. extent of mesometrial vascular perfusion and blood-flow velocities and uterine vascular perfusion in mares with a small (%275 mm2) or large (O410 mm2) internal uterine cystic area compared to controls.

Small cystic area* Large cystic area*

End points Cystic Control Cystic Control Number of mares 11 11 8 8 Individual cysts Number 4.5G0.9a – 18.3G3.8b – Diameter (mm) 5.6G0.5a – 7.9G0.6b – Mesometrial attachment † Cross-sectional results Vascular perfusion (score 1 to 4) 3.1G0.3 3.2G0.2 2.8G0.3 2.8G0.3 Diameter (mm) 18.3G0.6 18.1G0.8 16.1G0.8 16.1G0.8 Circumference (mm) 70.1G2.1 68.5G2.7 63.9G2.7 62.7G1.9 Area (mm2) 270.5G16.9 244.7G21.3 220.8G25.7 191.4G8.4 Spectral results PI (index) 1.57G0.11 1.76G0.11 1.49G0.08x 1.81G0.15y RI (index) 0.70G0.02x 0.75G0.02y 0.71G0.02 0.75G0.03 PSV (cm/s) 12.7G1.0 13.6G0.8 14.8G1.3x 11.9G1.1y EDV (cm/s) 3.6G0.3 3.4G0.3 4.2G0.5x 2.8G0.2y TAMV (cm/s) 5.8G0.5 5.9G0.5 7.2G0.6x 5.0G0.4y Uterine vascular perfusion (%)‡ Cystic region 8.9G2.1c,x 15.6G2.4y,§ 6.9G1.3c,x 12.3G2.0y,§ Adjacent to cystic region 12.3G2.4d,y 12.8G1.9d,y

PI, pulsatility index; RI, resistance index; PSV, peak systolic velocity; EDV, end diastolic velocity; TAMV, time-averaged maximum velocity. a,bMeans with different letters between the two cystic groups are different (P!0.007). c,dUterine vascular perfusion means with different letters within a column are different (P!0.04). x,yMeans within a pair of columns (cystic versus control) with different letters are different (P!0.05), except that the difference approaches significance (P!0.07) for the comparison of the RI index between the small cystic area and its control. *The presence or absence of external cysts was not considered in the cystic group. Controls had neither internal nor external cysts and were paired with mares of similar age with internal cysts. †Vessels of the mesometrium attachment averaged for the two uterine horns. ‡Percentage of endometrium and myometrium with color Doppler signals of blood flow. §Average for left and right horns of mares without cysts were used in the analyses. the fixation area would interfere with the development of 2007). The greater sensitivity allows the evaluation of the fixed embryonic vesicle. vessels with small diameter or slow blood flow that do not The capability of color and power mode Doppler appear on a conventional color flow image because of ultrasonography as an effective method for detecting the incompatible velocity ranges and Doppler angles differences in uterine vascular supply and perfusion (Ginther 2007). The earlier study using color Doppler between mares with and without cysts was demonstrated imaging and scoring from 1 to 4 found constant low for the first time in any species. To our knowledge, this is endometrial vascular perfusion from 1 to 16 days after the first study on uterine vascular perfusion based on ovulation in nonpregnant mares; however, in pregnant power mode Doppler ultrasonography in farm animals; mares, an increased uterine vascularity was observed previous studies used the color flow mode (Silva et al. after 11 days (Silva et al. 2005). Therefore, to evaluate 2005, Silva & Ginther 2006). Power Doppler increases potential weak vascular perfusion with minimum artifacts the sensitivity for displaying blood flow within the soft in cystic regions, the percentage of color signals was tissue three- to fivefold, compared with conventional evaluated in the power mode for the study of uterine color display for Doppler ultrasonography (Ginther vascular perfusion. In addition, color Doppler signals

Figure 4 Spectral color Doppler graphs from a gate (indicated by an arrow) placed in a color signal from a vessel of the mesometrial attachment of a mare (A) without and (B) with cysts. Spectral results are given in the boxes. Mares with uterine cystic area O410 mm2 (B) had a lower PI and greater velocities for PSV, EDV, and TAMV at the mesometrial attachment when compared with their paired controls (A). There was no difference for spectral end points between mares with uterine cystic area %275 mm2 and their controls.

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Downloaded from Bioscientifica.com at 09/30/2021 05:13:00AM via free access Effect of cysts on uterine blood flow 547 within the endometrium are commonly inadequate for contraction in response to a severe increased arterial the production of spectral waveforms (Silva et al. 2005). resistance (reviewed by Frohlich 1983). The increased Therefore, blood flow indices and velocities collected blood velocities detected in the mesometrial attachment from the arteries of the mesometrial attachment have and the decreased blood flow perfusion in uterine cystic been used as an indirect method for the evaluation of regions in the present study indicates that vascular uterine blood flow and perfusion (Silva et al. 2005, defects may play a role in the development of cysts. This Ginther 2007). is supported by histological studies that suggested that The lower vascular perfusion in cystic regions when angiosis of arterioles in the endometrium and lymphan- compared with adjacent noncystic regions and with the giectasia are possible precursors of lymphatic cysts middle segments of both horns in mares without cysts (reviewed by Schoon et al. 1999). Therefore, it is likely supported the hypothesis that cysts are spatially associ- that the cysts were a response rather than a cause of the ated with reduced uterine vascularity. The association vascular dysfunction. In addition to the vascular between cysts and reduced uterine vascular perfusion dysfunction (Schoon et al. 1999), the presence of was local; the reduced perfusion did not extend to other lymphatic (Kenney & Ganjam 1975, reviewed by regions of the uterus. The reduced uterine vascular Stanton et al. 2004) and glandular (Kenney & Ganjam perfusion for both small (%275 mm2)andlarge 1975, Ricketts 1975, Kenney 1978) distention may also (O410 mm2) cystic areas when compared with the contribute to continued development of uterine cysts. controls of similar age, and reproductive status did not The hypothesis that old mares without cysts have support the hypothesis that local reduced vascular lower uterine vascular perfusion than noncystic young perfusion is more pronounced when the cystic area is mares was not supported. The mares did not have a large. Speculatively, reduced uterine vascular perfusion history of pregnancy or parturition during the past 10 in the vicinity of cysts as well as interference with years, unlike mares of similar age on breeding farms, and embryo dynamics and implantation could contribute to this may account for the lack of difference in vascularity. infertility and loss or impairment of growth of the con- It has been suggested that age may influence the ceptus. In this regard, endometrial receptivity, conceptus incidence of uterine angiopathies in mares (Oikawa implantation, and pregnancy development are depen- et al. 1993, Nambo et al. 1995, Schoon et al. 1999). dent on an adequate uterine circulation in humans Although mild uterine angiopathies may occur indepen- (Sterzik et al. 1989, Chien et al. 2004). In addition, dent of age, the severity of uterine angiosis has been impairment fetal growth due to restricted blood supply associated with the number of pregnancies and parturi- has been described in sheep (Harding et al. 1985). tions in addition to the age in mares (Gru¨ninger et al. Mares with small uterine cystic area versus paired 1998). A higher uterine resistance in multiparous mares controls of similar age and reproductive status were not than uniparous mares has been reported (Bollwein et al. different in the mesometrial blood flow velocities (PSV, 1998); however, the relationship between uterine EDV, and TAMV) and PI or in the vascular perfusion, vascular perfusion and age was not studied. Although diameter, circumference, and area of a cross section of the uterus has the ability for restoration post partum, the mesometrial attachment. However, mares with a elastosis of the arterial wall is a frequent degenerative large uterine cystic area had increased mesometrial PSV, change of uterine vessels in multiparous aged mares EDV, and TAMV and decreased PI when compared with (Gru¨ninger et al. 1998). Aging induces a series of the paired controls. These findings support the structural, architectural, and compositional modifi- hypothesis that a large uterine cystic area has a greater cations in the vasculature even in healthy individuals effect on uterine hemodynamics than a small area. The (rats: Guyton et al. 1983; humans: reviewed by Lakatta increased mesometrial blood flow velocities (PSV, EDV, 2002), and these modifications are associated with a and TAMV) may have been a response to the increased decrease in vascular elasticity (Tao et al. 2004)andan resistance to the uterine blood flow (lower perfusion) in increase in thickness of the arterial wall (Vaitkevicius mares with large uterine cystic area. Oxidative stress is a et al. 1993). Studies in humans have demonstrated that critical factor in processes involving ischemia/reper- both arterial stiffness and speed of the blood flow fusion (Ferrari et al. 1990) and has been correlated with increase progressively with the advancing of age in endothelial dysfunction and increase in vascular tone of healthy individuals (Avolio et al. 1985, Vaitkevicius et al. peripheral vessels (Yang et al. 1998, Zhou et al. 2005). 1993, Mitchell et al. 2004). Therefore, an age-related Furthermore, greater blood flow velocities (EDV and increased stiffness or decreased distensibility of the PSV) have been found in arterial segments with arterial walls may have contributed to the greater narrowed lumen (Zwiebel & Perrelito 2005). The mesometrial blood flow velocities (EDV and TAMV) in elevated mesometrial PSV might also reflect a compen- older mares in the present study. satory increase in cardiac systole from the increased In conclusion, power mode Doppler ultrasonography uterine vascular resistance (decreased perfusion) as well was effective for the evaluation of the uterine vascular as resistance at the mesometrial attachment. The perfusion in mares with and without cysts. Uterine cystic myocardium has the ability to increase its force of regions had lower uterine vascular perfusion than www.reproduction-online.org Reproduction (2008) 135 541–550

Downloaded from Bioscientifica.com at 09/30/2021 05:13:00AM via free access 548 J C Ferreira and others noncystic regions. A large uterine cystic area (mm2) was attachment were being scanned in a slow continuous motion associated with lower PI and higher blood flow velocities several times (Ginther 2007). The uterus was divided into nine in the mesometrial attachment. In mares without cysts, imaginary segments of similar length (Ginther 1995a)as aging increased the pulse wave velocities (EDV and follows: LA, LM, LP, RA, RM, RP, BA, BM, and BP (Fig. 1). TAMV) in the mesometrial attachment but did not alter The nine segments were utilized to identify three distinctive uterine blood flow resistance and vascular perfusion of uterine portions as follows: extremities (LA, RA, and BP), the uterine layers. The results indicated that uterine central segments (LM, RM, and BM), and cornual-body vascular disturbances should be considered as an junction (LP, RP, and BA). Scanning started at the caudal additional potential cause of uterine cysts. portion of the uterus (close to the ), and the transducer was directed toward each horn as described (Ginther 1995a). Longitudinal and cross-sectional planes of the uterine body, Materials and Methods horns, and mesometrial attachment were acquired. To obtain a cross section of the mesometrial attachment and its accom- Animals panying vessels, the transducer was positioned dorsocaudally within 1 cm of the surface of each horn, directed cranially, and Animals were handled in accordance with the United States rotated on its vertical axis (Silva et al. 2005, Silva & Ginther Department of Agriculture Guide for Care and Use of 2006). Mares were slightly sedated during Doppler scans with Agricultural Animals in Research. The study was done during detomidine hydrochloride (1 mg per mare, i.v., Dormosedan; November in the Northern Hemisphere (latitude 43 8N). The Pfizer Animal Health, Philadelphia, PA, USA) to optimize mares (nZ76) were mixed breeds of large ponies and apparent ultrasound color images and minimize the presence of artifacts pony–horse crosses weighing 270–410 kg (mean 300G6 kg) and were 3–25 years of age (mean 11.8G0.6 years). The score due to animal movement. for body condition for all mares was high throughout the For the spectral Doppler mode, the velocity range limit was experiment (scoreR7; Henneke et al. 1983). Mares were kept set at 19.9 cm/s and the Doppler filter at 100 Hz (Silva et al. under natural light in an open shelter and outdoor paddock and 2005). However, the setting for the range of flow velocity were maintained on alfalfa/grass hay with access to water and detection was adjusted, when indicated, to obtain the optimal trace-mineralized salt. Mares had docile temperament and no spectral graph (Ginther 2007). The sample cursor or gate was macroscopic abnormalities of the reproductive tract as set at a width of 1.5 mm. The angle of insonation was determined by ultrasound examination (Ginther 1995a), except unknown. The gate was placed within the image of a lumen that some had uterine cysts. Only one examination per mare of a vessel in the mesometrial attachment of each uterine horn, was used. Reproductive status was based on previous records using the most prominent color signal and pulse repetition obtained every day or alternate day by ultrasound scanning of frequency as a guide. A Doppler spectrum with three cardiac the and uterus (Ginther 1995b). The age of mares was cycles was generated, and the optimal cycle was used for estimated from dental characteristics (American Association of spectral measurements (Fig. 1). This procedure was done two Equine Practitioners Manual 2002). more times, and the mean of the three measurements for each spectral end point was used in the statistical analyses.

Ultrasonography Uterine vascular perfusion Mares were scanned using a duplex B-mode (gray scale) and pulsed-wave color Doppler ultrasound instrument (Aloka SSD- Vascular perfusion of the uterus (all layers combined) was 3500; Aloka America, Wallingford, CT, USA) equipped with a estimated subjectively by one operator from the cross sections finger-mounted 7.5 MHz convex-array transducer (UST-995- of a horn using the power flow function. The following 7.5). The principles, techniques, and interpretation of B-mode ultrasound settings were used: velocity range limit, 6.22 cm/s; examination of the mare reproductive tract have been reviewed flow filter, 3; and frame rate, 6 Hz. Only the color signals that (Ginther 1995b). The brightness and contrast controls of the appeared to be within the limits of the uterus were considered. monitor and the gain controls of the scanner were standardized The uterine vascular perfusion was estimated subjectively using to constant settings (Gastal et al. 1998). the percentage of uterine tissue with color signals of blood flow The color and power flow modes were utilized to display during real-time cross-sectioning of the uterus in a continuous signals for blood flow in the vessels of the endometrium, span of 1 min. Multiple cross sections were viewed because of myometrium, perimetrium, and mesometrial attachment animal and uterine movements and the variation resulting from (Fig. 1). In the power Doppler mode, the degree of uterine angles of insonation. In mares without cysts (controls), regions vascular perfusion was based on the estimated proportion of of the middle segments of left and right horns were used. In tissue with color spots, as described previously and validated mares with cysts, uterine vascular perfusion was estimated at a using the color Doppler mode in mares (Silva et al. 2005). The cyst location with the most prominent internal cysts versus the color gain setting was kept constant. Real-time B-mode and adjacent uterine region without internal uterine cysts. color and power flow mode images of continuous scans were captured with an online digital videotaping system and stored Evaluation of the mesometrial attachment for selection of images for illustrative purposes. The B-mode and color and power Doppler uterine end points Each entire mesometrial attachment was scanned in a slow were evaluated while the entire uterus and mesometrial continuous motion for 1 min. The dimensions of a cross section

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Downloaded from Bioscientifica.com at 09/30/2021 05:13:00AM via free access Effect of cysts on uterine blood flow 549 of the mesometrial attachment of each side were obtained. The controls (nZ11) and large uterine cystic area (O410 mm2, selected cross section of the mesometrium was oblong and nZ8) paired with controls (nZ8). The diameter (mm) of each contained sections of vessels in various planes. No attempt was cyst was obtained from an average of height and width. To made to determine if a cross section represented a single vessel determine the area (mm2) of a cyst, the cyst was considered or multiple vessels. Measurements of the mesometrium were circular in shape, and the formula for area of a circle (pr 2) from performed from one still image in B-mode, using the split- mean diameter was utilized. The sum of the area for all internal screen function with the color mode image on one side to cysts was used to represent the total or combined internal facilitate location and delineation of the cross section (Ginther uterine cystic area. The same approach was used to determine 2007). The diameter (mm) was obtained from an average of the external uterine cystic area per animal. height and width. The area (mm2) and circumference (mm) of each cross section of the mesometrium was determined, using the scanner’s tracing function. The vascular perfusion of the Part 4: effect of age in mares without cysts attachment in the cross section was estimated subjectively by From the pool of 76 mares, 22 mares without cysts (uterine cysts one operator during scanning in the color flow Doppler mode, not detected) were used to study the effect of age on uterine and using a pulse repetition frequency of 8 Hz and constant color mesometrial vascular perfusion in the absence of cysts. The mares gain settings and scoring from 1 to 4 (minimal to maximal, were assigned into two age groups: young (3–10 years, 6.7G0.7 including fractional scores). years; nZ11) and old (15–21 years, 16.5G0.5 years; nZ11). Spectral assessment of the blood flow velocity in an artery at Mares of the intermediate age were not used in order to increase the mesometrial attachment was performed as described (Silva the age difference between the two age groups. None of the mares et al. 2005, Silva & Ginther 2006). The end points for the gated had foaled during the last 10 years, and 10 out of 11 young mares intramesometrial color signal were resistance index (RI), (!10 years) had never been bred. The remaining young mare had pulsatility index (PI), PSV, EDV, and TAMV. The meaning and an unknown reproductive history. formulas for these spectral end points are well established (Zwiebel & Pellerito 2005, Ginther 2007). Statistical analyses Part 1: prevalence and distribution of uterine cysts Paired and unpaired Student’s t-tests were used to locate differences within and between two groups respectively. The The prevalence of uterine cysts was determined in the pool of area (mm2) of internal and external cysts in different uterine 76 mares. The mares were assigned to one of the three age portions and the three age classes were analyzed by one-way ! O groups as follows: 7, 7–14, and 14 years (15–25 years). ANOVA and Duncan’s multiple range tests. Chi-square tests of Uterine cysts were classified, based on their cross-sectional independence were used to examine the differences in locations, as internal when located within the endometrium frequency data. Pearson’s correlation analyses were performed and/or myometrium, and external when located outside and between age and Doppler end points in part 4. A probability of apparently attached to the perimetrium. For longitudinal P%0.05 indicated that a difference was significant, and location of cysts, internal and external uterine cysts were probabilities between PO0.05 and %0.1 indicated that a assigned to each of the nine segments and three portions of the difference approached significance. Data are presented as uterus within each mare. meanGS.E.M., unless otherwise indicated.

Part 2: effect of cysts Acknowledgements From the pool of 76 mares, 21 pairs were used to study the This research (Project P6-ELG-06) was supported by the association between uterine cysts and uterine and mesometrial Eutheria Foundation Inc., Cross Plains, WI, USA. The authors vascular perfusion and mesometrial blood flow velocities (PSV, thank M O Gastal for assistance with statistical analyses and EDV, and TAMV). The age and number of days post-ovulation figures, R R Araujo for assistance with figures, and D Cooper for were balanced within pairs of control mares (uterine cysts not technical assistance with the management of the horse herd. detected) and mares with cysts. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. Part 3: effect of size of cystic area Out of the 21 pairs from part 2, 19 were used to evaluate the References association between the size of the cystic area (mm2) and the uterine and mesometrial vascular perfusion and mesometrial Adams NR 1975 A pathological and bacteriological abattoir survey of the reproductive tracts of merino ewes in Western Australia. Australian blood flow velocities (PSV, EDV, and TAMV). The mares were Veterinary Journal 51 351–354. assigned to a group in which the cystic area of the cystic Adams GP, Kastelic JP, Bergfelt DR & Ginther OJ 1987 Effect of uterine member of the pair was %275 mm2 (11 pairs) versus inflammation and ultrasonically-detected uterine pathology on fertility in O410 mm2 (8 pairs). Two pairs in which the cystic member the mare. Journal of Reproduction and Fertility 35 445–454. 2 Agnew DW, Munson L & Ramsay EC 2004 Cystic endometrial hyperplasia had an intermediary cystic area (312 and 360 mm ) were not in elephants. Veterinary Pathology 41 179–183. considered. Therefore, mares were arranged in four groups: American Association of Equine Practitioners Manual 2002 Official Guide small uterine cystic area (%275 mm2, nZ11) paired with for Determining the Age of the Horse, pp 26. Lexington, KY: AAEP. www.reproduction-online.org Reproduction (2008) 135 541–550

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