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Triplex doppler ultrasonography to describe the uterine arteries during diestrus and progesterone profile in pregnant and non-pregnant bitches of different sizes
Theriogenology 141 (2020) 153e160
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Triplex doppler ultrasonography to describe the uterine arteries during diestrus and progesterone profile in pregnant and non-pregnant bitches of different sizes J. Roos a, *, C. Aubanel a, Z. Niewiadomska a, L. Lannelongue b, C. Maenhoudt a, A. Fontbonne a a b
CERCA (Centre d’Etudes en Reproduction des Carnivores), Ecole Nationale V et erinaire d'Alfort, Maisons-Alfort Cedex, France Department of Public Health and Primary Care, University of Cambridge, Cambridge, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 May 2019 Received in revised form 29 August 2019 Accepted 30 August 2019 Available online 12 September 2019
Hemodynamics of uterine vascularization is modified throughout pregnancy to meet the increasing demand of the growing fetuses and triplex doppler ultrasonography is widely used in human medicine to study the uterine arteries and assess the fetal and placental conditions. The aim of our study was to confirm this observation in the bitch, to evaluate differences between bitches of different sizes and to study abnormal pregnancies. Forty-four bitches were monitored during the estrous period to determine ovulation and every 10 days from ovulation to 50 days post-ovulation: the resistivity (RI) and pulsatility (PI) indexes of the right uterine artery were measured as well as usual assessment of fetal development and follow up of the luteal function. Thirty-three out of forty-four bitches were pregnant, including 6 abnormal pregnancies (resorption of more than 10% of the embryos). We also divided them in four weight categories: 8 were small (<10 kg), 13 medium (10e25 kg), 13 large (>25e40 kg) and 10 were giant breeds (>40 kg). We observed that RI and PI decreased over time and were significantly lower for pregnant bitches compared to non-pregnant ones from 30 days post-ovulation. In contrast, RI and PI did not significantly vary with the size of the bitches and we could not determine a significant impact of abnormal pregnancies either. In conclusion, we found no significant difference related to the size of bitches in the RI and PI. The only significant difference between pregnant and non-pregnant bitches was observed from 30 days post-ovulation. © 2019 Elsevier Inc. All rights reserved.
Keywords: Doppler ultrasound Uterine artery Bitch Pregnancy Size
1. Introduction Throughout the pregnancy, the uterus and its vascularization change to respond to the growing demand of the fetuses [1e3]. These changes can be monitored by ultrasonography, using B-mode, colored and pulsed-wave Doppler. Tripplex doppler ultrasonography allows us to observe and characterize maternal and fetal blood flows by measuring several velocimetric factors: peak systolic velocity (PSV), end diastolic velocity (EDV) and mean velocity (MV). These parameters are then used to calculate their derivations called resistivity (RI) and pulsatility (PI) indexes: RI ¼ (PSV-EDV)/PSV and PI ¼ (PSV-EDV)/MV. A decrease in resistivity and pulsatility indexes reflects an increase in the organ perfusion [4].
* Corresponding author. E-mail address: [email protected] (J. Roos). https://doi.org/10.1016/j.theriogenology.2019.08.035 0093-691X/© 2019 Elsevier Inc. All rights reserved.
Doppler ultrasonography has been used in various species to describe umbilical, uterine and fetal vascularization. In women, characterization of RI and PI in uterine arteries has permitted to determine threshold values to anticipate negative outcome during pregnancy [5]. For example, a RI over 0.58 after the 24th week of pregnancy is a predictor of preeclampsia, intrauterine growth restriction and intrauterine death [6]. RI and PI in uterine arteries have also been monitored in ewes, goats, does, mares, cows and queens [7e13]. In those species, as in women, uterine arteries resistivity and pulsatility decrease during pregnancy and this has been confirmed in bitches for normal pregnancies by Nautrup in 1998 [14]. Similar observations have been made separately in Beagles, small, middle and large sizes bitches [15e18]. However, to the best of our knowledge, although the uterine vascularization has been well described in pregnant and nonpregnant bitches [19] as well as in bitches of different sizes [14e16], there is no analysis comparing different weight categories
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of bitches within the same study. Moreover, only a few studies on small populations have compared the evolution of RI and PI between pregnant and non-pregnant bitches [20,21] or in pregnant bitches with negative outcome (loss of some embryos or complete pregnancy arrest for example). To further complete previous reports [14e16,19e21], the first aim of our study was to compare the evolution of RI and PI over time in pregnant and non-pregnant bitches and to investigate if a difference could be observed between bitches of different weight categories. We also wanted to determine if a difference could be noticed in bitches that underwent an abnormal pregnancy. In a similar way, progesterone profiles of pregnant and nonpregnant bitches have already been described by previous authors. It has been shown that the progesterone blood level rises after ovulation and remains high for approximately two months during diestrus in pregnant but also in non-pregnant bitches. However, in non-pregnant bitches, the progesterone serum level starts to decrease earlier but more slowly than in pregnant bitches [22,23]. As bitches in our trial were monitored throughout their luteal phase, we also decided to analyze the evolution of the progesterone profiles of pregnant and non-pregnant bitches. 2. Material and methods This study was reviewed and approved by the ethical scientific comity of the Ecole Nationale Veterinaire d’Alfort (COMERC, trial registration number 2018-11-09).
Table 1 Breeds of the bitches included in the protocol. Breeds
Number of bitches
Chihuahua Parson Russel terrier Shetland shepherd Shipperke Dashund
2 1 3 1 1
Bull terrier French Bulldog Stafforshire bullterrier Beagle
1 1 2 9
American staffordshire terrier Dalmatian Australian shepherd Xoloitzcuintle Golden Retriever White swiss shepherd German shepherd Boxer Akita Inu Doberman Crossed breed (shepherd dog)
1 1 1 1 1 1 3 1 1 1 1
Great Dane Caucasian shepherd Leonberg Cane Corso English Mastiff Newfoundland dog Bernese mountain dog
2 1 2 1 2 1 1
2.1. Animals and determination of ovulation Animals were included from March 2018 to November 2018 and they had all been seen in consultation in the Alfort Veterinary School for ovulation timing. The protocol was offered to all owners provided they agree to commit to the five successive appointments needed for the trial. The process to detect ovulation always included repeated vaginal smears and blood collections for progesterone serum level measurement (clot activator containing dry vial) using an enzymelinked fluorescence assay (ELFA, MiniVidas® automated analyser, Biomerieux, France) [24]. Ovarian ultrasound exam was also performed. Ovulation was considered to occur when the progesterone serum level was around 10 ng/mL [25] and, provided the bitch could undergo the exam, when a clear change in the aspect of the ovaries was detected by ultrasonography [26]. Breeding was done either by natural mating or artificial insemination with fresh, cooled (for one bitch) or frozen (for one bitch) semen according to the owners' wishes. Whelping was either performed by natural way at home, by emergency or elective Csection (for breeds at risk of dystocia or individual bitches having suffered from a previous dystocia) [27]. During this clinical trial, the animals lived in their usual environment with their owners who received advice on pregnancy nutrition and prevention of parasites during pregnancy but these parameters could not be controlled. To diagnose an eventual hypoluteoidism, a blood sample was collected at the time of each pregnancy control to measure the progesterone serum level using the same assay as for ovulation timing. For statistical analysis, bitches were divided in four weight categories (see Tables 1 and 2): small (<10 kg), medium (10e25 kg), large (>25e40 kg) and giant breeds (>40 kg). 2.2. Tripplex Doppler ultrasonography Animals were examined every 10 days from the ovulation date to
the 50th day post-ovulation (corresponding to C1 at 10 days, C2 at 20 days, C3 at 30 days, C4 at 40 days and C5 at 50 days post-ovulation). The ultrasound device used was a LOGIC F8 (Scil, General Electric Healthcare, France) with a microconvexe probe, 6e10 mHz. Frequency (PRF) and wall filter (WF) were as followed: PRF: 6.4, WF: 11.We did not change these parameters or the gain between bitches but depth was changed according to the bitch's size. During each consultation, a global clinical examination of the dam was done and an ultrasound examination was performed to control the ovaries and the uterus (especially for presence of intraluminal liquid), to make the pregnancy diagnosis (at second control) and to monitor fetal vitality in pregnant bitches. The number of fetuses and the eventual images of embryonic resorptions were recorded (Fig. 1). Pregnancies were retrospectively classified as normal or abnormal (defined for this study by the loss of more than 10% of the embryos) [28]. This was coupled with Doppler measurements on the right uterine artery. Bitches were placed in dorsal recumbency in a cushion (ProfilVet, Coveto, France) and those that did not tolerate the position for 15 min (average duration of the exam) were excluded from the trial. Hairs were not clipped since some of the animals could compete in dog shows (especially if not pregnant) but alcohol was used to wet the hairs and the skin and to prepare the echographic window on the right caudal part of the animal close to the bladder. Only the right artery was studied. Acoustic gel was applied on the transducer and on the skin. Color Doppler was used to identify uterine vessels close to the most caudal part of the uterine body below the bladder and next to the main vessels (see Fig. 2). After identification, pulsed-waved Doppler was used to obtain the uterine artery waveforms (defined as the spectrum obtained with the pulsed wave Doppler mode characterizing the vascular flow) and resistivity and pulsatility indexes were automatically calculated by the machine. At least three measurements of RI and PI per examination were obtained and the mean value was calculated. Figs. 2 and 3 illustrate the anatomy and the usual image obtained with the color and pulsed-wave Doppler.
J. Roos et al. / Theriogenology 141 (2020) 153e160
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Table 2 Number of bitches per size groups and number of pregnant and non-pregnant bitches in each subgroups. Total number
Pregnant
Total Normal pregnancies Abnormal pregnancies
Non-pregnant
Total sample
Small group
Medium group
Large group
Giant group
44
8
13
13
10
33 27 6 11
6 5 1 2
11 11 0 2
9 6 3 4
7 5 2 3
Fig. 1. Illustration of an embryonic resorption in a bitch.
2.3. Statistical analysis A two-ways repeated measures ANOVA test was used to analyze if RI and PI evolve differently over time between different subgroups (pregnant versus non-pregnant, size subgroups or normal versus abnormal pregnancies). We carried out Tukey's HSD post hoc tests to compare subgroups at specific time points. Normality checks were also performed and the conditions were met.
whelping), one Doberman (100%, 7/7 of embryonic loss), one Akita Inu (75%, 3/4 of embryonic loss), one Great Dane (50%, 5/10 of embryonic loss) and one Leonberg (25%, 2/8 of embryonic loss). Regarding the clinical examination, the authors noted that the right uterine artery was more difficult to observe at day 10 postovulation for non-pregnant bitches compared to pregnant bitches and during the whole luteal phase for non-pregnant bitches. Moreover, examination on giant breeds like English Mastiff bitches appeared to be far more difficult than on small bitches since the artery is probably too deep to have a good Doppler signal.
3. Results 3.2. Evolution of the indexes in pregnant and non-pregnant bitches 3.1. General results A total of 44 bitches of 27 different breeds were included, presented in Table 1. Among the 44 bitches, 33 were pregnant, 11 were not. Eight out of forty-four bitches were classified as small (6 were pregnant), 13/44 as medium (11 were pregnant), 13/44 as large (9 were pregnant) and 10/44 as giant (7 were pregnant), see Table 2. Nine bitches were excluded from the study at different times. The reasons for their exclusion as well as the time when they were excluded are summarized in Table 3. Table 4 indicates the number of bitches at each control of the study. Six out of thirty-three pregnancies were classified as abnormal (more than 10% of the embryos undergoing resorption): one Chihuahua (1/3 of embryonic loss), one White Swiss Shepherd (43%, 3/7 of embryonic loss and 1 mummified fetus discovered at
The evolutions of RI and PI overtime were first compared between pregnant and non-pregnant bitches. Mean values are summarized in Table 5. Both indexes decreased during the course of pregnancy (Fig. 4.) and exhibited different behaviors between pregnant and non-pregnant bitches from the third control (C3, 30 days post ovulation p < 0.0001 for both RI and PI). In particular, since the values of RI were significantly different between pregnant and non-pregnant bitches from C3 onwards (p ¼ 0.0001 at C3, p ¼ 0.0054 at C4 and p ¼ 0.0080 at C5), we investigated some thresholds at C3 that could help differentiate pregnant bitches from non-pregnant ones. If we set the threshold to 0.72, the sensitivity of such classification rule would be 91% and its specificity 100%. With a threshold equal to 0.76, the sensitivity is up to 97% but the specificity is down to 82%.
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Cervix Uterine body
Bladder
Right uterine artery Fig. 2. Details of the anatomy of the bitch's genital tract (courtesy Pr Celine Robert).
Fig. 3. Example of the usual uterine artery color and pulsed-wave Doppler image (measurement at C2).
Table 3 Bitches excluded throughout the trial, time of their exclusion and justification. Bitch
Time of exclusion
Reason for the exclusion
Beagle Beagle English Mastiff Bernese mountain dog Bull terrier Shetland Akita Inu Golden retriever Beagle
After After After After After After After After After
Did not tolerate the manipulation Did not tolerate the manipulation Non-pregnant and the owner refused to come for the next controls Non-pregnant and the owner refused to come for the next controls Pyometra diagnosed at the third controle Non-pregnant and the owner refused to come for the next controls Singleton pregnancy confirmed at the third control / decision of medical abortion Singleton pregnancy confirmed at the third control / decision of medical abortion Not available for the fifth control
C1 C1 C2 C2 C2 C3 C3 C3 C4
J. Roos et al. / Theriogenology 141 (2020) 153e160
and their evolution over time as illustrated by Fig. 6 (p ¼ 0.27 for RI and p ¼ 0.40 for PI). In order to be sure that there was no problem of including in the data the nine bitches that did not fully follow the protocol, we recalculated the results after excluding them and found that they would still be similar: the weight group doesn't affect significantly the evolution of PI and RI (p ¼ 0.06 for RI and p ¼ 0.31 for PI) and neither does abnormal pregnancies (p ¼ 0.49 for RI and p ¼ 0.60 for PI).
Table 4 Number of pregnant and non-pregnant bitches at each time point. Number of bitches
C1
C2
C3
C4
C5
Pregnant Non-pregnant
33 11
31 11
30 9
28 8
27 8
157
3.3. Evolution of the indexes according to the bitches' size and normal vs abnormal pregnancy Comparison of the RI and PI over time between the different weight groups (see Table 2) did not reveal any significant difference (p ¼ 0.11 for RI and p ¼ 0.48 for PI). Fig. 5 shows the results of the individual bitches and average per size group. When comparing normal and abnormal pregnancies (6/33 bitches), no significant difference was observed between RI and PI
3.4. Evaluation of the progesterone serum level evolution in pregnant and non-pregnant bitches Fig. 7 shows the evolution of the progesterone serum levels for pregnant and non-pregnant bitches. Even though the levels
Table 5 Average (±standard deviation) of the uterine artery resistivity and pulsatility (RI and PI) at different controls (C1, C2, C3, C4, and C5 being respectively the controls at 10, 20, 30, 40 and 50 days post-ovulation) in pregnant and non-pregnant bitches. For C3, C4 and C5 values from pregnant and non-pregnant bitches were statistically different. C1
Pregnant Non-pregnant
C2
C3
C4
C5
RI
PI
RI
PI
RI
PI
RI
PI
RI
PI
0.77 ± 0.15 0.82 ± 0.11
2.63 ± 1.72 3.18 ± 1.93
0.74 ± 0.09 0.77 ± 0.16
2.20 ± 1.01 3.13 ± 2.15
0.61 ± 0.09 0.88 ± 0.11
1.22 ± 0.49 5.16 ± 3.59
0.57 ± 0.12 0.78 ± 0.15
1.08 ± 0.51 2.77 ± 1.76
0.52 ± 0.12 0.93 ± 0.19
0.89 ± 0.4 4.43 ± 2.91
Fig. 4. Box plot illustrating the evolution of the resistivity (RI) and pulsatility (PI) indexes over time in pregnant and non-pregnant bitches.
Fig. 5. Evolution of the resistivity index over time per weight categories for unproblematic pregnancies. Narrow lines represent the individual results, bold lines the evolutions of the averages.
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Fig. 6. Evolution of the resistivity index over time in normal and abnormal pregnancies. Narrow lines represent the individual results, bold lines the evolutions of the averages.
Fig. 7. Evolution of progesterone serum levels (P4) over time in pregnant and non-pregnant bitches. Narrow lines represent the individual results, bold lines the evolutions of the averages.
decreased in both groups, it was significantly higher for pregnant bitches (p ¼ 0.0002), especially after the third control. 4. Discussion This study confirms the observations of previous authors: the uterine blood flow increases during pregnancy in bitches as in other species [15,17,20]. Table 6 illustrates the comparison between uterine artery resistivity measured in our trial and in two other
studies. If our results are slightly different from those found by Batista et al. (2018), they are in or very close to their defined reference ranges for small size bitches and they are similar to those of Feliciano et al. (2014). In our study, only the right artery was studied given that several previous studies already showed that no significant difference was observed between the right and left arteries [2,15,21]. In those studies, it was assumed that the distribution of implantation of embryos was symmetric and we did not observe a great difference
Table 6 Comparison of reference ranges in RI of different studies in the literature for pregnant bitches. 4, 5, 6 and 7 correspond respectively to the 4th, 5th, 6th and 7th weeks of pregnancy. Reference
Number of bitches
Particularities of the animals
4
5
6
7
Feliciano et al. (2014) Mean ± standard deviation Batista et al. (2018) Mean [5th-95thquartiles] Our study Mean ± standard deviation
26
Brachycephalic breeds, all sizes (5e25 kg)
/
0.6 ± 0.1
0.5 ± 0.1
0.5 ± 0.1
52
Small sizes (1.5e6.5 kg)
0.72 [0.64e0.79]
/
0.62 [0.52e0.66]
33
All breeds and sizes (2e65 kg)
0.61 ± 0.09
/
0.66 [0.57 - 0.75] 0.57 ± 0.12
0.52 ± 0.12
J. Roos et al. / Theriogenology 141 (2020) 153e160
between the two horns in our population. However, this was not calculated here so we only assumed this symmetry to measure only the right artery. Very few studies have compared, in the same trial, the evolution of the uterine artery RI and PI between pregnant and non-pregnant bitches. In their studies, Freeman et al. (2013) included 20 Labrador bitches and measured uterine vascularization during estrus and the very early luteal phase. They noticed a significant difference in RI and PI between pregnant (n ¼ 14) and non-pregnant (n ¼ 6) bitches [21]. Similarly, we observed a significant difference from the 30th day post-ovulation onward. Moreover, a threshold value could be determined between the two groups at this control. Thus, we could imagine that measurement of RI and PI could be a new criterion in the pregnancy diagnosis especially in giant bitches in which, even at 30 days post-ovulation, diagnosis by ultrasonography of a singleton pregnancy may be challenging. Although the population size in this study is not large enough to provide a definitive value for the classification threshold, the analyses that we conducted are promising and command further investigations. It also has to be taken into account that, as specified in subjective results, the realization of Doppler ultrasound of the uterine artery in giant breeds appears to be more difficult in practice than in other breeds. One of the potential limitations of this study is the relatively low number of animals in each weight categories and in the group of abnormal pregnancies. This trial relies on the owners' wish to participate to the study and we did not manage to include more animals in our study period. For those two parameters (size of bitches and abnormal pregnancies) it would be very interesting to continue the trial including more animals to increase the statistical power of the analysis. Perhaps for this reason, we did not observe a significant difference in uterine perfusion between pregnant bitches of different weights despite Batista et al. (2018) suggesting the opposite after comparing their data to those found in the literature. Similarly, our results could not confirm the link between RI/PI and negative outcome in pregnancies, as observed in human medicine [6,29]. Furthermore, we did not find the same data as previous publications trying to predict resorptions of embryos [1,20,30]. Additionally, we did not take into account several criteria like age, parity of bitches and litter size. For example, according to some authors, the age of the dam seemed to have a negative effect on uterine perfusion (RI superior in old bitches during pregnancy) [21]. Moreover, if RI and PI did not seem to be influenced by litter size, there was an effect on PSV and EDV in small bitches [15]. In queens, it appears that uterine perfusion is higher in the horn when there are more fetuses [31]. No such observation has been done in bitches so far. In pharmacologically induced abortion in bitches and queens, Blanco et al. (2009 and 2016) described an increase in RI after medical treatment. Only a few studies could be done on natural negative outcome during pregnancy or neonatal mortality (in opposition to pharmacologically induced). Blanco et al. (2011) observed that in abnormal pregnancies (defined in their study as gestational arrest between 52 and 60 from estimated luteinizing hormone peak, or pregnancies that went to term but with perinatal death greater than 60% within the first 48 h after parturition), RI decreased throughout the luteal phase but to a smaller extent than in normal pregnancies and even increased from 40 days of pregnancy onward [2]. Another criterion that could be taken into account is the description of the waveforms, but this was not done in our trial. Waveforms of uterine arteries have been well described in bitches and queens [18,21,31]. The presence of a diastolic notch (characterized on the waveforms by a protodiastolic incisure) has been associated with negative outcome in canine and human pregnancies [6,18]. In queens, Pereira et al. (2012) suggested that
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the presence of an end diastolic notch was normal before the end of organogenesis and should disappear during the second half of pregnancy. Its persistency could be then a predictor of a negative outcome [31,32]. Our results regarding the progesterone serum levels (higher levels for pregnant bitches) are in accordance with what has been previously described with higher levels for pregnant bitches [22]. Nevertheless, in a study on 73 bitches (of different size and age) the author could not find a significant difference in progesterone blood levels when comparing pregnant and non-pregnant bitches regardless to the stage of diestrus [33]. It is important to emphasize that some owners of non-pregnant bitches stopped participating to the trial after the third control when the non-pregnanct status was confirmed. Indeed, as we relied on the owners' willingness to take part in the study, it was difficult to perform more examinations on non-pregnant bitches. 5. Conclusion To our knowledge, this is the first study comparing the evolution of Doppler uterine arteries characteristics in pregnant and nonpregnant bitches and in bitches of different sizes. Values of uterine arteries resistivity and pulsatility are similar in bitches of different sizes. There is a need to confirm these results in a larger number of animals, especially in bitches developing an abnormal pregnancy. Indeed, significant difference in RI and PI may allow the clinicians to anticipate a negative outcome in pregnancies. Conflicts of interest The author declares no conflict of interest. Acknowledgements The author would first like to thank Dr Dan Cahen who gives his great help in teaching the ultrasonographic techniques to found and measure uterine arteries in women and for his wise advice. The authors would like to thank their colleagues who participated actively to this study. References [1] Blanco P, Arias D, Rube A, Barrena J, Corrada Y, Gobello C. An experimental model to study resistance index and systolic/diastolic ratio of uterine arteries in adverse canine pregnancy outcome. Reprod Domest Anim 2009;44:164e6. https://doi.org/10.1111/j.1439-0531.2009.01369.x. rtora M, Díaz JD, et al. Doppler [2] Blanco PG, Rodríguez R, Rube A, Arias DO, To ultrasonographic assessment of maternal and fetal blood flow in abnormal canine pregnancy. Anim Reprod Sci 2011;126:130e5. https://doi.org/10.1016/ j.anireprosci.2011.04.016. [3] Browne VA, Julian CG, Toledo-Jaldin L, Cioffi-Ragan D, Vargas E, Moore LG. Uterine artery blood flow, fetal hypoxia and fetal growth. Philos Trans R Soc B Biol Sci 2015;370. https://doi.org/10.1098/rstb.2014.0068. 20140068e20140068. [4] Guerin du Masgenet B, Robert Y, Coquel P, Bourgeot P. Echographie en pratricale. Elsevier; 2014. https://doi.org/10.1016/C2013-0-18949-0. tique obste [5] Velauthar L, Plana MN, Kalidindi M, Zamora J, Thilaganathan B, Illanes SE, et al. First-trimester uterine artery Doppler and adverse pregnancy outcome: a meta-analysis involving 55 974 women: UtA Doppler prediction of adverse pregnancy outcome. Ultrasound Obstet Gynecol 2014;43:500e7. https:// doi.org/10.1002/uog.13275. [6] Cnossen JS, Morris RK, ter Riet G, Mol BWJ, van der Post JAM, Coomarasamy A, et al. Use of uterine artery Doppler ultrasonography to predict pre-eclampsia and intrauterine growth restriction: a systematic review and bivariable metaanalysis. CMAJ Can Med Assoc J J Assoc Medicale Can 2008;178:701e11. https://doi.org/10.1503/cmaj.070430. [7] Beltrame RT, Covre C, Littig LB, Martins A de B, Quirino CR, Junior AB, et al. Transrectal Doppler sonography of uterine blood flow in ewes during pregnancy. Theriogenology 2017;91:55e61. https://doi.org/10.1016/j.theriogenology.2016. 12.026. [8] Bollwein H, Baumgartner U, Stolla R. Transrectal Doppler sonography of uterine blood flow in cows during pregnancy. Theriogenology 2002;57: 2053e61.
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pregnant bitches. Vet J Lond Engl 1997 2013;197:205e10. https://doi.org/ 10.1016/j.tvjl.2013.02.015. Concannon PW. Reproductive cycles of the domestic bitch. Anim Reprod Sci 2011;124:200e10. https://doi.org/10.1016/j.anireprosci.2010.08.028. Onclin K, Verstegen JP. Secretion patterns of plasma prolactin and progesterone in pregnant compared with nonpregnant dioestrous beagle bitches. J Reprod Fertil Suppl 1997;51:203e8. England G, Concannon PW. Determination of the optimal breeding time in the bitch: basic considerations. In: Recent Advances in Small Animal Reproduction. International Veterinary Information Service; 2002 . www.ivis.org. A1231.0602. Brugger N, Otzdorff C, Walter B, Hoffmann B, Braun J. Quantitative determination of progesterone (P4) in canine blood serum using an enzyme-linked fluorescence assay. Reprod Domest Anim Zuchthyg 2011;46:870e3. https:// doi.org/10.1111/j.1439-0531.2011.01757.x. Fontbonne A. In vivo ovulation, oocyte maturation and fertilisation in the bitch. AgroParisTech; 2008. Roos J, Maenhoudt C, Zilberstein L, Mir F, Borges P, Furthner E, et al. Neonatal puppy survival after planned caesarean section in the bitch using aglepristone as a primer: a retrospective study on 74 cases. Reprod Domest Anim Zuchthyg 2018;53(Suppl 3):85e95. https://doi.org/10.1111/rda.13353. England GCW, Russo M. Ultrasonographic characteristics of early pregnancy failure in bitches. Theriogenology 2006;66:1694e8. https://doi.org/10.1016/ j.theriogenology.2006.01.028. Aardema MW, Wolf BTHMD, Saro MCS, Oosterhof H, Fidler V, Aarnoudse JG. Quantification of the diastolic notch in Doppler ultrasound screening of uterine arteries. Ultrasound Obstet Gynecol 2000;16:630e4. https://doi.org/ 10.1046/j.1469-0705.2000.00289.x. Blanco PG, Vercellini R, Rube A, Rodríguez R, Arias DO, Gobello C. Evaluation of feline uterine and umbilical arteries blood flow in a pharmacologically induced abnormal gestation model. Theriogenology 2016;86:2323e7. https:// doi.org/10.1016/j.theriogenology.2016.07.028. Pereira BS, Freire LMP, Pinto JN, Domingues SFS, Silva LDM. Triplex Doppler evaluation of uterine arteries in cyclic and pregnant domestic cats. Anim Reprod Sci 2012;130:99e104. https://doi.org/10.1016/j.anireprosci.2011. 12.019. Brito AB, Miranda SA, Ruas MR, Santos RR, Domingues SFS. Assessment of feline fetal viability by conceptus echobiometry and triplex Doppler ultrasonography of uterine and umbilical arteries. Anim Reprod Sci 2010;122: 276e81. https://doi.org/10.1016/j.anireprosci.2010.09.003. Marinelli L, Rota A, Carnier P, Da Dalt L, Gabai G. Factors affecting progesterone production in corpora lutea from pregnant and diestrous bitches. Anim Reprod Sci 2009;114:289e300. https://doi.org/10.1016/j.anireprosci.2008. 10.001.
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Triplex doppler ultrasonography to describe the uterine arteries during diestrus and progesterone profile in pregnant and non-pregnant bitches of different sizes J. Roos a, *, C. Aubanel a, Z. Niewiadomska a, L. Lannelongue b, C. Maenhoudt a, A. Fontbonne a a b
CERCA (Centre d’Etudes en Reproduction des Carnivores), Ecole Nationale V et erinaire d'Alfort, Maisons-Alfort Cedex, France Department of Public Health and Primary Care, University of Cambridge, Cambridge, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 May 2019 Received in revised form 29 August 2019 Accepted 30 August 2019 Available online 12 September 2019
Hemodynamics of uterine vascularization is modified throughout pregnancy to meet the increasing demand of the growing fetuses and triplex doppler ultrasonography is widely used in human medicine to study the uterine arteries and assess the fetal and placental conditions. The aim of our study was to confirm this observation in the bitch, to evaluate differences between bitches of different sizes and to study abnormal pregnancies. Forty-four bitches were monitored during the estrous period to determine ovulation and every 10 days from ovulation to 50 days post-ovulation: the resistivity (RI) and pulsatility (PI) indexes of the right uterine artery were measured as well as usual assessment of fetal development and follow up of the luteal function. Thirty-three out of forty-four bitches were pregnant, including 6 abnormal pregnancies (resorption of more than 10% of the embryos). We also divided them in four weight categories: 8 were small (<10 kg), 13 medium (10e25 kg), 13 large (>25e40 kg) and 10 were giant breeds (>40 kg). We observed that RI and PI decreased over time and were significantly lower for pregnant bitches compared to non-pregnant ones from 30 days post-ovulation. In contrast, RI and PI did not significantly vary with the size of the bitches and we could not determine a significant impact of abnormal pregnancies either. In conclusion, we found no significant difference related to the size of bitches in the RI and PI. The only significant difference between pregnant and non-pregnant bitches was observed from 30 days post-ovulation. © 2019 Elsevier Inc. All rights reserved.
Keywords: Doppler ultrasound Uterine artery Bitch Pregnancy Size
1. Introduction Throughout the pregnancy, the uterus and its vascularization change to respond to the growing demand of the fetuses [1e3]. These changes can be monitored by ultrasonography, using B-mode, colored and pulsed-wave Doppler. Tripplex doppler ultrasonography allows us to observe and characterize maternal and fetal blood flows by measuring several velocimetric factors: peak systolic velocity (PSV), end diastolic velocity (EDV) and mean velocity (MV). These parameters are then used to calculate their derivations called resistivity (RI) and pulsatility (PI) indexes: RI ¼ (PSV-EDV)/PSV and PI ¼ (PSV-EDV)/MV. A decrease in resistivity and pulsatility indexes reflects an increase in the organ perfusion [4].
* Corresponding author. E-mail address: [email protected] (J. Roos). https://doi.org/10.1016/j.theriogenology.2019.08.035 0093-691X/© 2019 Elsevier Inc. All rights reserved.
Doppler ultrasonography has been used in various species to describe umbilical, uterine and fetal vascularization. In women, characterization of RI and PI in uterine arteries has permitted to determine threshold values to anticipate negative outcome during pregnancy [5]. For example, a RI over 0.58 after the 24th week of pregnancy is a predictor of preeclampsia, intrauterine growth restriction and intrauterine death [6]. RI and PI in uterine arteries have also been monitored in ewes, goats, does, mares, cows and queens [7e13]. In those species, as in women, uterine arteries resistivity and pulsatility decrease during pregnancy and this has been confirmed in bitches for normal pregnancies by Nautrup in 1998 [14]. Similar observations have been made separately in Beagles, small, middle and large sizes bitches [15e18]. However, to the best of our knowledge, although the uterine vascularization has been well described in pregnant and nonpregnant bitches [19] as well as in bitches of different sizes [14e16], there is no analysis comparing different weight categories
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of bitches within the same study. Moreover, only a few studies on small populations have compared the evolution of RI and PI between pregnant and non-pregnant bitches [20,21] or in pregnant bitches with negative outcome (loss of some embryos or complete pregnancy arrest for example). To further complete previous reports [14e16,19e21], the first aim of our study was to compare the evolution of RI and PI over time in pregnant and non-pregnant bitches and to investigate if a difference could be observed between bitches of different weight categories. We also wanted to determine if a difference could be noticed in bitches that underwent an abnormal pregnancy. In a similar way, progesterone profiles of pregnant and nonpregnant bitches have already been described by previous authors. It has been shown that the progesterone blood level rises after ovulation and remains high for approximately two months during diestrus in pregnant but also in non-pregnant bitches. However, in non-pregnant bitches, the progesterone serum level starts to decrease earlier but more slowly than in pregnant bitches [22,23]. As bitches in our trial were monitored throughout their luteal phase, we also decided to analyze the evolution of the progesterone profiles of pregnant and non-pregnant bitches. 2. Material and methods This study was reviewed and approved by the ethical scientific comity of the Ecole Nationale Veterinaire d’Alfort (COMERC, trial registration number 2018-11-09).
Table 1 Breeds of the bitches included in the protocol. Breeds
Number of bitches
Chihuahua Parson Russel terrier Shetland shepherd Shipperke Dashund
2 1 3 1 1
Bull terrier French Bulldog Stafforshire bullterrier Beagle
1 1 2 9
American staffordshire terrier Dalmatian Australian shepherd Xoloitzcuintle Golden Retriever White swiss shepherd German shepherd Boxer Akita Inu Doberman Crossed breed (shepherd dog)
1 1 1 1 1 1 3 1 1 1 1
Great Dane Caucasian shepherd Leonberg Cane Corso English Mastiff Newfoundland dog Bernese mountain dog
2 1 2 1 2 1 1
2.1. Animals and determination of ovulation Animals were included from March 2018 to November 2018 and they had all been seen in consultation in the Alfort Veterinary School for ovulation timing. The protocol was offered to all owners provided they agree to commit to the five successive appointments needed for the trial. The process to detect ovulation always included repeated vaginal smears and blood collections for progesterone serum level measurement (clot activator containing dry vial) using an enzymelinked fluorescence assay (ELFA, MiniVidas® automated analyser, Biomerieux, France) [24]. Ovarian ultrasound exam was also performed. Ovulation was considered to occur when the progesterone serum level was around 10 ng/mL [25] and, provided the bitch could undergo the exam, when a clear change in the aspect of the ovaries was detected by ultrasonography [26]. Breeding was done either by natural mating or artificial insemination with fresh, cooled (for one bitch) or frozen (for one bitch) semen according to the owners' wishes. Whelping was either performed by natural way at home, by emergency or elective Csection (for breeds at risk of dystocia or individual bitches having suffered from a previous dystocia) [27]. During this clinical trial, the animals lived in their usual environment with their owners who received advice on pregnancy nutrition and prevention of parasites during pregnancy but these parameters could not be controlled. To diagnose an eventual hypoluteoidism, a blood sample was collected at the time of each pregnancy control to measure the progesterone serum level using the same assay as for ovulation timing. For statistical analysis, bitches were divided in four weight categories (see Tables 1 and 2): small (<10 kg), medium (10e25 kg), large (>25e40 kg) and giant breeds (>40 kg). 2.2. Tripplex Doppler ultrasonography Animals were examined every 10 days from the ovulation date to
the 50th day post-ovulation (corresponding to C1 at 10 days, C2 at 20 days, C3 at 30 days, C4 at 40 days and C5 at 50 days post-ovulation). The ultrasound device used was a LOGIC F8 (Scil, General Electric Healthcare, France) with a microconvexe probe, 6e10 mHz. Frequency (PRF) and wall filter (WF) were as followed: PRF: 6.4, WF: 11.We did not change these parameters or the gain between bitches but depth was changed according to the bitch's size. During each consultation, a global clinical examination of the dam was done and an ultrasound examination was performed to control the ovaries and the uterus (especially for presence of intraluminal liquid), to make the pregnancy diagnosis (at second control) and to monitor fetal vitality in pregnant bitches. The number of fetuses and the eventual images of embryonic resorptions were recorded (Fig. 1). Pregnancies were retrospectively classified as normal or abnormal (defined for this study by the loss of more than 10% of the embryos) [28]. This was coupled with Doppler measurements on the right uterine artery. Bitches were placed in dorsal recumbency in a cushion (ProfilVet, Coveto, France) and those that did not tolerate the position for 15 min (average duration of the exam) were excluded from the trial. Hairs were not clipped since some of the animals could compete in dog shows (especially if not pregnant) but alcohol was used to wet the hairs and the skin and to prepare the echographic window on the right caudal part of the animal close to the bladder. Only the right artery was studied. Acoustic gel was applied on the transducer and on the skin. Color Doppler was used to identify uterine vessels close to the most caudal part of the uterine body below the bladder and next to the main vessels (see Fig. 2). After identification, pulsed-waved Doppler was used to obtain the uterine artery waveforms (defined as the spectrum obtained with the pulsed wave Doppler mode characterizing the vascular flow) and resistivity and pulsatility indexes were automatically calculated by the machine. At least three measurements of RI and PI per examination were obtained and the mean value was calculated. Figs. 2 and 3 illustrate the anatomy and the usual image obtained with the color and pulsed-wave Doppler.
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Table 2 Number of bitches per size groups and number of pregnant and non-pregnant bitches in each subgroups. Total number
Pregnant
Total Normal pregnancies Abnormal pregnancies
Non-pregnant
Total sample
Small group
Medium group
Large group
Giant group
44
8
13
13
10
33 27 6 11
6 5 1 2
11 11 0 2
9 6 3 4
7 5 2 3
Fig. 1. Illustration of an embryonic resorption in a bitch.
2.3. Statistical analysis A two-ways repeated measures ANOVA test was used to analyze if RI and PI evolve differently over time between different subgroups (pregnant versus non-pregnant, size subgroups or normal versus abnormal pregnancies). We carried out Tukey's HSD post hoc tests to compare subgroups at specific time points. Normality checks were also performed and the conditions were met.
whelping), one Doberman (100%, 7/7 of embryonic loss), one Akita Inu (75%, 3/4 of embryonic loss), one Great Dane (50%, 5/10 of embryonic loss) and one Leonberg (25%, 2/8 of embryonic loss). Regarding the clinical examination, the authors noted that the right uterine artery was more difficult to observe at day 10 postovulation for non-pregnant bitches compared to pregnant bitches and during the whole luteal phase for non-pregnant bitches. Moreover, examination on giant breeds like English Mastiff bitches appeared to be far more difficult than on small bitches since the artery is probably too deep to have a good Doppler signal.
3. Results 3.2. Evolution of the indexes in pregnant and non-pregnant bitches 3.1. General results A total of 44 bitches of 27 different breeds were included, presented in Table 1. Among the 44 bitches, 33 were pregnant, 11 were not. Eight out of forty-four bitches were classified as small (6 were pregnant), 13/44 as medium (11 were pregnant), 13/44 as large (9 were pregnant) and 10/44 as giant (7 were pregnant), see Table 2. Nine bitches were excluded from the study at different times. The reasons for their exclusion as well as the time when they were excluded are summarized in Table 3. Table 4 indicates the number of bitches at each control of the study. Six out of thirty-three pregnancies were classified as abnormal (more than 10% of the embryos undergoing resorption): one Chihuahua (1/3 of embryonic loss), one White Swiss Shepherd (43%, 3/7 of embryonic loss and 1 mummified fetus discovered at
The evolutions of RI and PI overtime were first compared between pregnant and non-pregnant bitches. Mean values are summarized in Table 5. Both indexes decreased during the course of pregnancy (Fig. 4.) and exhibited different behaviors between pregnant and non-pregnant bitches from the third control (C3, 30 days post ovulation p < 0.0001 for both RI and PI). In particular, since the values of RI were significantly different between pregnant and non-pregnant bitches from C3 onwards (p ¼ 0.0001 at C3, p ¼ 0.0054 at C4 and p ¼ 0.0080 at C5), we investigated some thresholds at C3 that could help differentiate pregnant bitches from non-pregnant ones. If we set the threshold to 0.72, the sensitivity of such classification rule would be 91% and its specificity 100%. With a threshold equal to 0.76, the sensitivity is up to 97% but the specificity is down to 82%.
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Cervix Uterine body
Bladder
Right uterine artery Fig. 2. Details of the anatomy of the bitch's genital tract (courtesy Pr Celine Robert).
Fig. 3. Example of the usual uterine artery color and pulsed-wave Doppler image (measurement at C2).
Table 3 Bitches excluded throughout the trial, time of their exclusion and justification. Bitch
Time of exclusion
Reason for the exclusion
Beagle Beagle English Mastiff Bernese mountain dog Bull terrier Shetland Akita Inu Golden retriever Beagle
After After After After After After After After After
Did not tolerate the manipulation Did not tolerate the manipulation Non-pregnant and the owner refused to come for the next controls Non-pregnant and the owner refused to come for the next controls Pyometra diagnosed at the third controle Non-pregnant and the owner refused to come for the next controls Singleton pregnancy confirmed at the third control / decision of medical abortion Singleton pregnancy confirmed at the third control / decision of medical abortion Not available for the fifth control
C1 C1 C2 C2 C2 C3 C3 C3 C4
J. Roos et al. / Theriogenology 141 (2020) 153e160
and their evolution over time as illustrated by Fig. 6 (p ¼ 0.27 for RI and p ¼ 0.40 for PI). In order to be sure that there was no problem of including in the data the nine bitches that did not fully follow the protocol, we recalculated the results after excluding them and found that they would still be similar: the weight group doesn't affect significantly the evolution of PI and RI (p ¼ 0.06 for RI and p ¼ 0.31 for PI) and neither does abnormal pregnancies (p ¼ 0.49 for RI and p ¼ 0.60 for PI).
Table 4 Number of pregnant and non-pregnant bitches at each time point. Number of bitches
C1
C2
C3
C4
C5
Pregnant Non-pregnant
33 11
31 11
30 9
28 8
27 8
157
3.3. Evolution of the indexes according to the bitches' size and normal vs abnormal pregnancy Comparison of the RI and PI over time between the different weight groups (see Table 2) did not reveal any significant difference (p ¼ 0.11 for RI and p ¼ 0.48 for PI). Fig. 5 shows the results of the individual bitches and average per size group. When comparing normal and abnormal pregnancies (6/33 bitches), no significant difference was observed between RI and PI
3.4. Evaluation of the progesterone serum level evolution in pregnant and non-pregnant bitches Fig. 7 shows the evolution of the progesterone serum levels for pregnant and non-pregnant bitches. Even though the levels
Table 5 Average (±standard deviation) of the uterine artery resistivity and pulsatility (RI and PI) at different controls (C1, C2, C3, C4, and C5 being respectively the controls at 10, 20, 30, 40 and 50 days post-ovulation) in pregnant and non-pregnant bitches. For C3, C4 and C5 values from pregnant and non-pregnant bitches were statistically different. C1
Pregnant Non-pregnant
C2
C3
C4
C5
RI
PI
RI
PI
RI
PI
RI
PI
RI
PI
0.77 ± 0.15 0.82 ± 0.11
2.63 ± 1.72 3.18 ± 1.93
0.74 ± 0.09 0.77 ± 0.16
2.20 ± 1.01 3.13 ± 2.15
0.61 ± 0.09 0.88 ± 0.11
1.22 ± 0.49 5.16 ± 3.59
0.57 ± 0.12 0.78 ± 0.15
1.08 ± 0.51 2.77 ± 1.76
0.52 ± 0.12 0.93 ± 0.19
0.89 ± 0.4 4.43 ± 2.91
Fig. 4. Box plot illustrating the evolution of the resistivity (RI) and pulsatility (PI) indexes over time in pregnant and non-pregnant bitches.
Fig. 5. Evolution of the resistivity index over time per weight categories for unproblematic pregnancies. Narrow lines represent the individual results, bold lines the evolutions of the averages.
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Fig. 6. Evolution of the resistivity index over time in normal and abnormal pregnancies. Narrow lines represent the individual results, bold lines the evolutions of the averages.
Fig. 7. Evolution of progesterone serum levels (P4) over time in pregnant and non-pregnant bitches. Narrow lines represent the individual results, bold lines the evolutions of the averages.
decreased in both groups, it was significantly higher for pregnant bitches (p ¼ 0.0002), especially after the third control. 4. Discussion This study confirms the observations of previous authors: the uterine blood flow increases during pregnancy in bitches as in other species [15,17,20]. Table 6 illustrates the comparison between uterine artery resistivity measured in our trial and in two other
studies. If our results are slightly different from those found by Batista et al. (2018), they are in or very close to their defined reference ranges for small size bitches and they are similar to those of Feliciano et al. (2014). In our study, only the right artery was studied given that several previous studies already showed that no significant difference was observed between the right and left arteries [2,15,21]. In those studies, it was assumed that the distribution of implantation of embryos was symmetric and we did not observe a great difference
Table 6 Comparison of reference ranges in RI of different studies in the literature for pregnant bitches. 4, 5, 6 and 7 correspond respectively to the 4th, 5th, 6th and 7th weeks of pregnancy. Reference
Number of bitches
Particularities of the animals
4
5
6
7
Feliciano et al. (2014) Mean ± standard deviation Batista et al. (2018) Mean [5th-95thquartiles] Our study Mean ± standard deviation
26
Brachycephalic breeds, all sizes (5e25 kg)
/
0.6 ± 0.1
0.5 ± 0.1
0.5 ± 0.1
52
Small sizes (1.5e6.5 kg)
0.72 [0.64e0.79]
/
0.62 [0.52e0.66]
33
All breeds and sizes (2e65 kg)
0.61 ± 0.09
/
0.66 [0.57 - 0.75] 0.57 ± 0.12
0.52 ± 0.12
J. Roos et al. / Theriogenology 141 (2020) 153e160
between the two horns in our population. However, this was not calculated here so we only assumed this symmetry to measure only the right artery. Very few studies have compared, in the same trial, the evolution of the uterine artery RI and PI between pregnant and non-pregnant bitches. In their studies, Freeman et al. (2013) included 20 Labrador bitches and measured uterine vascularization during estrus and the very early luteal phase. They noticed a significant difference in RI and PI between pregnant (n ¼ 14) and non-pregnant (n ¼ 6) bitches [21]. Similarly, we observed a significant difference from the 30th day post-ovulation onward. Moreover, a threshold value could be determined between the two groups at this control. Thus, we could imagine that measurement of RI and PI could be a new criterion in the pregnancy diagnosis especially in giant bitches in which, even at 30 days post-ovulation, diagnosis by ultrasonography of a singleton pregnancy may be challenging. Although the population size in this study is not large enough to provide a definitive value for the classification threshold, the analyses that we conducted are promising and command further investigations. It also has to be taken into account that, as specified in subjective results, the realization of Doppler ultrasound of the uterine artery in giant breeds appears to be more difficult in practice than in other breeds. One of the potential limitations of this study is the relatively low number of animals in each weight categories and in the group of abnormal pregnancies. This trial relies on the owners' wish to participate to the study and we did not manage to include more animals in our study period. For those two parameters (size of bitches and abnormal pregnancies) it would be very interesting to continue the trial including more animals to increase the statistical power of the analysis. Perhaps for this reason, we did not observe a significant difference in uterine perfusion between pregnant bitches of different weights despite Batista et al. (2018) suggesting the opposite after comparing their data to those found in the literature. Similarly, our results could not confirm the link between RI/PI and negative outcome in pregnancies, as observed in human medicine [6,29]. Furthermore, we did not find the same data as previous publications trying to predict resorptions of embryos [1,20,30]. Additionally, we did not take into account several criteria like age, parity of bitches and litter size. For example, according to some authors, the age of the dam seemed to have a negative effect on uterine perfusion (RI superior in old bitches during pregnancy) [21]. Moreover, if RI and PI did not seem to be influenced by litter size, there was an effect on PSV and EDV in small bitches [15]. In queens, it appears that uterine perfusion is higher in the horn when there are more fetuses [31]. No such observation has been done in bitches so far. In pharmacologically induced abortion in bitches and queens, Blanco et al. (2009 and 2016) described an increase in RI after medical treatment. Only a few studies could be done on natural negative outcome during pregnancy or neonatal mortality (in opposition to pharmacologically induced). Blanco et al. (2011) observed that in abnormal pregnancies (defined in their study as gestational arrest between 52 and 60 from estimated luteinizing hormone peak, or pregnancies that went to term but with perinatal death greater than 60% within the first 48 h after parturition), RI decreased throughout the luteal phase but to a smaller extent than in normal pregnancies and even increased from 40 days of pregnancy onward [2]. Another criterion that could be taken into account is the description of the waveforms, but this was not done in our trial. Waveforms of uterine arteries have been well described in bitches and queens [18,21,31]. The presence of a diastolic notch (characterized on the waveforms by a protodiastolic incisure) has been associated with negative outcome in canine and human pregnancies [6,18]. In queens, Pereira et al. (2012) suggested that
159
the presence of an end diastolic notch was normal before the end of organogenesis and should disappear during the second half of pregnancy. Its persistency could be then a predictor of a negative outcome [31,32]. Our results regarding the progesterone serum levels (higher levels for pregnant bitches) are in accordance with what has been previously described with higher levels for pregnant bitches [22]. Nevertheless, in a study on 73 bitches (of different size and age) the author could not find a significant difference in progesterone blood levels when comparing pregnant and non-pregnant bitches regardless to the stage of diestrus [33]. It is important to emphasize that some owners of non-pregnant bitches stopped participating to the trial after the third control when the non-pregnanct status was confirmed. Indeed, as we relied on the owners' willingness to take part in the study, it was difficult to perform more examinations on non-pregnant bitches. 5. Conclusion To our knowledge, this is the first study comparing the evolution of Doppler uterine arteries characteristics in pregnant and nonpregnant bitches and in bitches of different sizes. Values of uterine arteries resistivity and pulsatility are similar in bitches of different sizes. There is a need to confirm these results in a larger number of animals, especially in bitches developing an abnormal pregnancy. Indeed, significant difference in RI and PI may allow the clinicians to anticipate a negative outcome in pregnancies. Conflicts of interest The author declares no conflict of interest. Acknowledgements The author would first like to thank Dr Dan Cahen who gives his great help in teaching the ultrasonographic techniques to found and measure uterine arteries in women and for his wise advice. The authors would like to thank their colleagues who participated actively to this study. References [1] Blanco P, Arias D, Rube A, Barrena J, Corrada Y, Gobello C. An experimental model to study resistance index and systolic/diastolic ratio of uterine arteries in adverse canine pregnancy outcome. Reprod Domest Anim 2009;44:164e6. https://doi.org/10.1111/j.1439-0531.2009.01369.x. rtora M, Díaz JD, et al. Doppler [2] Blanco PG, Rodríguez R, Rube A, Arias DO, To ultrasonographic assessment of maternal and fetal blood flow in abnormal canine pregnancy. Anim Reprod Sci 2011;126:130e5. https://doi.org/10.1016/ j.anireprosci.2011.04.016. [3] Browne VA, Julian CG, Toledo-Jaldin L, Cioffi-Ragan D, Vargas E, Moore LG. Uterine artery blood flow, fetal hypoxia and fetal growth. Philos Trans R Soc B Biol Sci 2015;370. https://doi.org/10.1098/rstb.2014.0068. 20140068e20140068. [4] Guerin du Masgenet B, Robert Y, Coquel P, Bourgeot P. Echographie en pratricale. Elsevier; 2014. https://doi.org/10.1016/C2013-0-18949-0. tique obste [5] Velauthar L, Plana MN, Kalidindi M, Zamora J, Thilaganathan B, Illanes SE, et al. First-trimester uterine artery Doppler and adverse pregnancy outcome: a meta-analysis involving 55 974 women: UtA Doppler prediction of adverse pregnancy outcome. Ultrasound Obstet Gynecol 2014;43:500e7. https:// doi.org/10.1002/uog.13275. [6] Cnossen JS, Morris RK, ter Riet G, Mol BWJ, van der Post JAM, Coomarasamy A, et al. Use of uterine artery Doppler ultrasonography to predict pre-eclampsia and intrauterine growth restriction: a systematic review and bivariable metaanalysis. CMAJ Can Med Assoc J J Assoc Medicale Can 2008;178:701e11. https://doi.org/10.1503/cmaj.070430. [7] Beltrame RT, Covre C, Littig LB, Martins A de B, Quirino CR, Junior AB, et al. Transrectal Doppler sonography of uterine blood flow in ewes during pregnancy. Theriogenology 2017;91:55e61. https://doi.org/10.1016/j.theriogenology.2016. 12.026. [8] Bollwein H, Baumgartner U, Stolla R. Transrectal Doppler sonography of uterine blood flow in cows during pregnancy. Theriogenology 2002;57: 2053e61.
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