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Two-dimensional sonographic and Doppler changes in the uteri of bitches according to breed, estrus cycle phase, parity, and fertility
Accepted Manuscript Two-dimensional sonographic and Doppler changes in the uteri of bitches according to breed, estrus cycle phase, parity, and fertility Luana Azevedo de Freitas, Gustavo Lobato Mota, Herlon Victor Rodrigues Silva, Lúcia Daniel Machado da Silva PII:
S0093-691X(17)30128-0
DOI:
10.1016/j.theriogenology.2017.03.012
Reference:
THE 14035
To appear in:
Theriogenology
Received Date: 23 August 2016 Revised Date:
6 March 2017
Accepted Date: 14 March 2017
Please cite this article as: de Freitas LA, Mota GL, Silva HVR, da Silva LDM, Two-dimensional sonographic and Doppler changes in the uteri of bitches according to breed, estrus cycle phase, parity, and fertility, Theriogenology (2017), doi: 10.1016/j.theriogenology.2017.03.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
REVISED
ACCEPTED MANUSCRIPT 1
Two-dimensional sonographic and Doppler changes in the uteri of bitches
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according to breed, estrus cycle phase, parity, and fertility
3 Luana Azevedo de Freitas1*, Gustavo Lobato Mota1, Herlon Victor Rodrigues Silva1,
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Lúcia Daniel Machado da Silva1
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Fortaleza-CE, Brazil
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*[email protected]
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Laboratory of Carnivore Reproduction, Veterinary School, State University of Ceara,
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10 Abstract
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This study aimed to evaluate the two-dimensional and Doppler ultrasonographic
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features of bitches’ uteri according to breed, cycle phase, parity and fertility during
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the follicular and early diestrus phases. Thirty-nine pubertal bitches were divided into
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groups according to breed, parity, and fertility. Sonographic assessments started from
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the first day of vaginal bleeding and were performed weekly for a month. The
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diameter of the uterine body was measured longitudinally and the uterine
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characteristics observed sonographically were evaluated. The resistivity index (RI)
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and pulsatility index (PI) of the uterine artery were calculated and the spectral
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morphology analyzed subjectively. The uterine diameter increased from proestrus to
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the diestrus regardless of the breed or parity. Multiparous bitches of both breeds had
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higher uterine diameters than nulliparous bitches, except Fila Brasileiro bitches in
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estrus. In diestrus and proestrus, the uterine diameters were significantly the largest
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for multiparous bitches, followed by the primiparous bitches and smallest for
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nulliparous bitches. The uterine diameter was larger in Fila Brasileiro females than in
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ACCEPTED MANUSCRIPT French Bulldogs during estrus and diestrus. The RI did not differ during the different
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phases of the cycle for the same breed. However, the RI was higher in nulliparous and
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primiparous Fila Brasileiro females compared to French Bulldog females. Within Fila
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Brasileiro, multiparous bitches showed lower RI than nulliparous and primiparous
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bitches. Higher PI values were found during proestrus. All multiparous French
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Bulldog bitches had greater PI than nulliparous bitches, while multiparous Fila bitches
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had lower PI. Nulliparous and primiparous Fila bitches had larger PI larger than
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bitches with the same reproductive status of the breed French Bulldog. Infertile
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bitches had higher RI and PI than bitches considered fertile during the initial estrus
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and diestrus. Triphasic and types A, C, and D spectral morphologies were found in
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females who did not gestate; while pregnant females showed spectral morphologies of
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type C, B and D. Hence, it was concluded that the breed, the phase of estrus cycle and
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pregnancy history should be considered when studies the uterine artery flow during
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estrus and early diestrus using investigations such as Doppler assessment, which can
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be an important tool in diagnosing fertility in dogs.
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Key Words: Doppler; blood vessel; fertility; pregnancy; breed.
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1. Introduction
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Ultrasound examination of the uteri of bitches is an investigation routinely solicited
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for clinical reproductive evaluation [1,2]. Such exams permit the evaluation of size,
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echogenicity characteristics, contour of the uterine walls, and presence of luminal
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content. The size of the uterus varies depending on the animal's size, number of
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previous pregnancies, disease or wellness conditions, phase of the estrus cycle and
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pregnancy status [1,3]. However, few studies have considered and addressed these
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factors and applied them have considered to ultrasound images in dogs [4,5].
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ACCEPTED MANUSCRIPT Doppler ultrasound has found application as a non-invasive method in the assessment
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of uterine vasculature in different species [5-8]. Studies suggest that uterine perfusion
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regulates endometrial receptivity and influences successful implantation and
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maintenance of early pregnancy. Thus, good uterine blood supply preceding
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conception directly relates to fertility [9,10].
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Flow in the uterine artery is evaluated quantitatively by measuring the velocities,
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namely, peak systolic velocity (PSV), end diastolic velocity (EDV) and mean velocity
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(MV); this is followed by derivation of the indices, namely, resistivity (RI) and
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pulsatility index (PI). Qualitative evaluation is performed by assessing the spectral
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morphology. While velocity evaluates the blood flow behavior, these indices generate
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information on the impedance of the vessel and thus characterizes the vascular supply
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of a given organ [10]. Moreover, the study of flow by measuring the indices is
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accurate and provides replicability, especially when the study targets are vessels with
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small diameters or highly convoluted vessels, independent of the angle of sounding
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since they are based on the ratio between the speeds [11,12]. Some authors have
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demonstrated the relevance of spectral morphology evaluation to relate spectral
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descriptive aspects to infertility in women [13]. Although this is a subjective analysis
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for interpretation of flow, it permits evaluation when there is a diastolic change. In
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such cases, quantitative analysis does not provide precise flow information [9,12].
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Many studies have focused on Doppler evaluation of uterine arteries for a biological
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understanding of variations due to physiological or pathological events in women
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[9,10,12-17]. In dogs, studies have shown that there is a quantitative physiological
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variation in uterine flow during the estrus cycle [17,18], ovulation [18,20], deposit of
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semen [21], pregnancy [22] and post-partum [23], and in inflammatory processes
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[24]. Changes in the spectral morphology of the canine uterine artery were described
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ACCEPTED MANUSCRIPT during anestrus [19], and different wave patterns during the follicular and early luteal
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phase were found in dogs that did not become pregnant [20].
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Factors such as age influence changes in the flow of canine uterine artery and may
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have an impact on fertility [20] The phase of the estrus cycle can also influence the
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Doppler parameters [19]. However, factors influencing the size of the uterus and the
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uterine artery, such as the animal [25] and the pregnancy history [3, 25], factors that
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lead to reduced fertility such as a history of recurrent failures of pregnancies [15-17],
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uterine pathology [27], gestational changes [14, 26- 28], and ovarian pathology [29],
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have not been investigated to determine the influence of the canine uterine artery
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Doppler parameters on them. In this setting, this study aimed to evaluate the two-
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dimensional and Doppler ultrasonographic features of bitches’ uteri according to
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breed, cycle phase, parity and fertility during the follicular and early diestrus phases.
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2. Material and Methods
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2.1 Experimental Animals
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The experimental protocol (protocol number 5999508/2015) was approved by the
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Ethics Committee in Animal Experimentation of the State University of Ceará in
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accordance with the guidelines for the care and use of laboratory animals established
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by the Brazilian College of Animal Experimentation. Thirty-nine pubertal bitches
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were evaluated , consisting of the French Bulldog (n = 24) and Fila Brasileiro (n = 15)
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breeds. All included animals were healthy, with a body score of approximately 3.0,
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weighing between 10-75 kg, ages ranging from 8 months to 6 years, and belonging to
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private kennels. The animals were fed granulated canine commercial food and water
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ad libitum. Their reproductive history was known and/or had normal standard clinical
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ACCEPTED MANUSCRIPT examinations (physical inspection), laboratory (blood count) tests, and abdominal and
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pelvic ultrasound evaluations. All animals tested negative for brucellosis annually.
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The bitches were monitored using vaginal cytology and clinically; they were
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inseminated either artificially or, using known fertile males. The vaginal cytology was
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performed once a week by a single observer, beginning on the first day of bloody
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vaginal discharge until the identification of cytological diestrus. The smears were
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stained by Diff-Quick. The phases of the estrus cycle were defined based on the
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cytological characteristics [25]. Intravaginal inseminations were performed after
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obtaining the characteristic cytologic estrus on 3 alternate days using semen from
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animals with proven fertility. For those that were naturally mated, the male dog
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accompanied the bitch during the whole estrus.
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The animals were grouped according to categories: breed (French Bulldog or Fila
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Brasileiro), parity (nulliparous, primiparous or multiparous), and fertility history
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(fertile or infertile/sub-fertile). Bitches that did not conceive for a year or more after
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mating, had reproductive history of gestational failures, or had low prolificacy or
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reproductive alterations during the experiment were considered infertile or subfertile.
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A total of 21 French Bulldog bitches and 12 Fila Brasileiro bitches without clinical
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and historical anomalies of previous reproductive changes were allocated according to
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the order of parity into groups of 7 and 4 animals, respectively; for each breed, the
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uterine diameter and Doppler indices were evaluated. Six bitches (3 from each breed)
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were not included in this comparison as they showed sonographic changes during the
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stages of the evaluated cycle, endometritis and ovarian cyst, or a history of gestational
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failures.
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2.2 Technical examination
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metropolitan area of Fortaleza, Ceará, over 1 year. The equipment used was MyLab
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30 Vet Gold (Esaote, Genova, Italy) with a multi-frequency micro-convex probe of 5-
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10 MHz (LA523 reference-Esaote, Genova, Italy). All ultrasound evaluations were
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performed by the same operator. The evaluations were carried out, preferably in the
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morning, with the animals fasted for at least 8 hours. The bitches were placed in
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dorsal recumbency, without sedation. The abdominal and pelvic areas were shaved
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and a water soluble acoustic gel was applied.
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2.3 Two-dimensional ultrasound evaluation
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The bitches were evaluated once a week for 4 weeks. Assessments commenced on the
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first day of serosanguinous vaginal discharge (day 0), which marked the beginning of
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proestrus. The period of the estrus cycle was confirmed based on to the clinical signs,
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behavior, and vaginal cytology. The animals were grouped according to the phase of
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the cycle, breed and parity. The uterus was assessed for the presence or absence of
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intraluminal fluid. The longitudinal uterine diameter was measured (cm) at the body
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of the uterus. Ovaries were evaluated for the presence of cysts
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2.4 Doppler ultrasound evaluation
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After obtaining the two-dimensional images, Doppler was used to characterize the
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hemodynamics of the uterine artery on both sides of the uterine body.
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For color and spectral Doppler, we set and kept constant for all evaluations the
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insonation angle at 0°, color gain at 64% and the lowest wall filter to characterize the
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signal amplitude. For flow evaluation, we used a color map with the depth adequate
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for maximizing the frame rate, pulse repetition frequency of 9 to 18 Hz and sample
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ACCEPTED MANUSCRIPT size constant at 18 mm. The vessel was visualized longitudinally and the sample was
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measured in this plane.
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After identification of the arteries by color mapping, electronic markers (sample
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volume or gates) were placed in the central region of the vessel and the spectral
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Doppler was started. For analysis and to minimize data variability, 3 similar and
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consecutive waves were recorded. Blood flow was measured automatically by the
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ultrasound system using RI (PSV-EDV/PSV) [30] and PI (PSV-EDV/MV) [31] and
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by assessing the spectral morphology.
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The spectral morphology assessment was performed as follows [12]: Type A, a
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systolic peak followed by absent early diastolic wave, but with flow through the mid
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and late diastole until the next systole; Type B, systolic peak followed by continuous
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diastole to systole, absent late diastolic flow; Type C, the systolic peak followed by
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diastole, extending throughout the cardiac cycle to subsequent systole; Type D,
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systolic peak with absent early and late diastole, but with the mean diastolic flow;
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Type O, the systolic flow alone is identified, with characteristic high monophasic
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resistivity; Type N (3 phases), systolic peak followed by reverse-diastolic and positive
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flow.
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2.5 Statistical analysis
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The data addressing the sonographic parameters related to uterine sonogram
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characteristics were described qualitatively and quantitatively. The uterine diameter
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and the RI and PI were expressed as a mean with standard deviations. The comparison
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of uterine diameter, RI, and PI among the breeds, estrus cycle phase, reproductive
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status and fertility was performed using SPSS 19.0 software (SPSS Inc. Chicago, IL,
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USA). The data were subjected to the Shapiro-Wilk test for evaluation of normality
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and compared using bivariate analysis by Student T test and Kruskal-Wallis
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considering P values less than 0.05 as significant.
178 3. Results
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All bitches had normal estrus cycle and were mated. The average time of ultrasound
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assessment was 15 minutes, ranging from 10 to 20 minutes.
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The uterus was located dorsal to the urinary bladder and ventral to the colon. The
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uterine body was identified longitudinally as a tubular structure that could be
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visualized as a slight thickening with hypoechoic (edema) uterine walls during all
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stages. The layers of the uterus could be distinguished on the cross-section images due
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to reduced central echogenicity corresponding to the endometrium and a peripheral
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hyperechogenicity corresponding to the perimetrium (Figure 1). The uterine horns
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were identified as thin hypoechoic tubular structures in the lateral abdomen
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continuing along the line of the ovaries.
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A discrete amount of anechoic intraluminal content was visible during estrus in 18
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dogs of both breeds. However, only 1 French Bulldog bitch was diagnosed with
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endometritis; the presenting sonogram image was characterized by increased uterine
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wall contour irregularity, accompanied by a moderate amount of anechoic
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intraluminal fluid content. Two French Bulldog bitches presented with multiple
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ovarian cysts that visually manifested as multiple circular hypoechoic structures in the
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ovarian cortex measuring more than 3 mm in diameter.
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The uterine diameter increased from proestrus to diestrus regardless of the breed or
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parity. Multiparous bitches of both breeds had higher uterine diameters than
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nulliparous bitches, except for Fila Brasileiro bitches in estrus. In proestrus and
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ACCEPTED MANUSCRIPT diestrus, the uterine diameters of multiparous bitches were significantly greater than
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in primiparous females, which in turn was greater than in nulliparous females. In
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estrus, the uterine diameter of primiparous and multiparous Bulldogs did not differ
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and were significantly larger than in nulliparous females. For the Fila Brasileiro
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bitches in estrus, there was no difference in the uterine diameter based on parity. The
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uterine diameter was higher in Fila females than French Bulldog during estrus and
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diestrus, regardless of the parity (Table 1).
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The uterine arteries were identified in all bitches (Figure 1). No differences were
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found between the flow of the uterine artery on the right and left, based on grouped
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data. The RI of the uterine arteries did not differ between the phases of the cycle for
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either breed. The RI of the French Bulldog uterine artery did not differ in proestrus
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and estrus independent of the parity. However, the RI was significantly higher in
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diestrus in multiparous females compared to nulliparous and primiparous females. In
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Fila Brasileiro bitches, the RI during different phases of the cycle were similar in
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nulliparous and primiparous and significantly lower in multiparous females bitches.
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On comparing the breeds, it was observed that the RI in nulliparous and primiparous
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French Bulldog bitches were significantly lower than in the Fila Brasileiro bitches,
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while the RI of multiparous French Bulldog bitches did not differ from the RI of
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multiparous Fila Brasileiro bitches (Table 2).
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Larger PI values were notes during proestrus. All multiparous French Bulldog bitches
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registered PI greater than nulliparous bitches, while multiparous Fila Brasileiro
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bitches had lower IP. Nulliparous and primiparous Fila Brasileiro bitches registered
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greater PI than French Bulldog bitches with the same reproductive status (Table 3).
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The RI of fertile female dogs did not differ between the phases of the cycle. In
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infertile bitches, the RI in diestrus and estrus were significantly higher than in
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ACCEPTED MANUSCRIPT proestrus. The PI increased from proestrus to estrus and decreased from estrus to
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diestrus, both in fertile bitches as in those infertile. The RI and PI did not differ in
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proestrus between fertile and infertile bitches; however, they were significantly higher
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in infertile bitches during estrus and diestrus (Table 4). Females with endometritis (RI
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proestrus: 0.85; RI estrus: 0.89 and RI diestrus: 0.86 / PI proestrus: 2.63; PI estrus:
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2.80 and PI diestrus: 2.51) and ovarian cyst (RI proestrus: 0.88; RI estrus: 0.91 and RI
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diestrus: 0.86 / PI proestrus: 2.66 PI estrus: 2.88 and PI diestrus: 2.45) did not differ
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from fertile bitches.
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During this study, 26 dogs (66.66%) were diagnosed as pregnant, 17 being French
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Bulldogs and 9 Fila Brasileiro bitches. Most pregnant bitches showed type C spectral
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morphology during proestrus, estrus, and early diestrus. A Fila Brasileiro bitch that
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presented with fetal death at 42 days of gestation exhibited Type D spectral
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morphology. Spectral Type B were identified in a Fila Brasileiro bitch who had low
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prolificacy (2 pups) and multiple embryonic resorptions.
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Of Of the 7 Bulldog bitches that did not give birth, 1 had endometritis in the fourth
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week of evaluation, 2 had ovarian cysts, and 4 did not give birth because of a prior
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failure (history of gestational failures [2 nulliparous and 2 primiparous]). Of the 6 Fila
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Brasileiro that did not give birth, 3 had gestational failures without prior history
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(nulliparous), while the other 3 had a known history of gestational failures.
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The spectral morphology of the bitch who had endometritis was similar to those that
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gave birth (Type C). Bitches that had ovarian cysts had spectral morphologies
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between C and A types during the weekly assessments. The nulliparous bitches that
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did not give birth showed a type of flow in which only the systolic flow was
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identified, characterizing the spectrum as monophasic with high resistivity (type O or
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type D). Primiparous bitches that did not give birth had triphasic spectral
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morphologies that had a systolic peak, followed by reverse-diastolic and positive flow
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(type N). More details regarding the types of spectral morphologies may be found in
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Figure 2.
254 4. Discussion
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Factors that affect the uterine artery flow have already been described in young and
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mature dogs [20], amd after mating [21]; differences in the flow during different
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phases of the estrus cycle have been identified [19]. However, this is the first
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description of the uterine artery flow according to the breed, parity, and fertility in the
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period preceding conception, highlighting the importance of categorization of these
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factors in assessing the biological implications of the uterine artery Doppler study in
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canine reproduction.
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The uterine ultrasound description findings in this study agree with previous reports in
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the literature [1,4,32]. However, the presence of uterine intraluminal fluid during late
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estrus attributed to endometritis induced after mating [33] has not been related to an
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adverse effect on the fertility; this was confirmed in females who had reached term
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and showed no changes during pregnancy or postpartum.
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One of the factors related to the increase in uterine diameter, due to endometrial
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cyclic activity during proestrus, estrus and diestrus is hormonal influence. The uterine
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tissue undergoes a variety of adaptive reactions in response to hormonal changes that
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occur at different stages of the estrus cycle [25], through organizing events and
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stratification of endometrial stroma; growth and differentiation of the myometrium;
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and coordinated development of the endometrial glands [34]. , apart from hormonal
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influence, the parity also determine the uterine size. A study of uterine morphometry
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in multiparous bitches demonstrated an increase in the endometrium and especially
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ACCEPTED MANUSCRIPT the myometrium sizes with increasing number of pregnancies [3]. The increased
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thickness of the uterine wall was subsequently confirmed by ultrasound [4]. As this
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study evaluated bitches of different breeds and sizes, other factors related to changes
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in the uterine size, such as the breed and size of the animal [25] were also identified.
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Uterine artery flow characteristics during the follicular phase have been reported in
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the past, exhibiting biphasic morphology with spectral peak flow during estrus and
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early luteal phase; this was identified in this study by evaluating the PI. Studies have
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suggested that uterine flow is a dependent cycle in women [9], mares [5], and cows
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[6]; it is believed that cyclic variations in the uterine artery flow are attributed to the
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concentration of progesterone and estrogen principally, and other flow regulating
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factors such as nitric oxide. However, in this study, even with an increase in the
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uterine size over the phases studied, no RI differences were identified in both breeds,
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unlike PI, which showed higher values during proestrus, the estrogen dominant phase.
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Based on these findings, recent works in women have emphasized greater reliability
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in the use of PI for the diagnosis of unexplained infertility [17].
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Previous canine studies were suggestive of a fluctuation in the uterine artery Doppler
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values during different stages of the estrus cycle [18-20]; this was not found in the
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current study due to the applied experimental design; hence, subtle differences that
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might have occurred daily were not identified weekly. Furthermore, another
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interesting aspect in the comparison of breeds and parity was that the RI and PI values
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of nulliparous and primiparous Fila Brasileiro bitches were higher than the French
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Bulldog bitches; however, there were no differences between multiparous bitches.
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This may be explained by assuming that parity and breed can influence the Doppler
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indices; however, after successive pregnancies, the influence of RI is lost.
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diestrus; they have maximum uterine flow during these periods [12]. At this time,
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important physiological events such as ovulation [18], sperm reception [21], and
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embryo implantation [13, 14] might occur; these require adequate blood perfusion for
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successful reproduction.
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Data shown in this work combined quantitative evaluation of obtaining the indices
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and qualitative evaluation of analyzing the spectral morphology. Thus, a high RI and
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change in spectral morphology of the diastolic flow might indicate poor perfusion in
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the vascular bed, which can in turn lead to failures in the design and maintenance of
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the developing conceptus (change in placental formation), as identified in Fila
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Brasileiro bitches with changes during pregnancy [9, 12, 26, 35]. Thus, our results
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demonstrate the role of uterine vascularization, and the importance of Doppler studies
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in pre-obstetric evaluation as a predictor of adversity before and during pregnancy
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[11-16].
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In a classification proposed by Dickey [35], there were 6 spectral morphologies in the
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uterine artery flow: A, B, C, D, O, and N, related to fertility and infertility. The
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triphasic flow was not reported with regard to the flow of uterine artery, but was
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described with regard to the ductus venous flow (transient vessel during fetal life)
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[11,12, 35]. It corresponds to the ventricular systole (1st wave), atrial contraction (2nd
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wave-reverse peak), and ventricular diastole (3rd wave), resulting in a pressure
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reduction, preceding the diastole, which leads to distension of the contracted vessel,
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which in turn leads to poor tissue perfusion [35]. In this study, bitches that had this
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wave morphology had a history of recurrent pregnancy failures and did not give birth.
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In this work, nulliparous bitches that did not give birth showed type O flow. This
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spectrum of the uterine artery flow patterns has already been described in pre-pubertal
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ACCEPTED MANUSCRIPT children and individuals [36], but it can also be identified in those with fertility
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problems who undergo in vitro fertilization programs [12]. In this study, its
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visualization was related to the early and reproductive immaturity (first heat) or the
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presence of anovulatory cycles in these dogs, mostly identified in the Fila Brasileiro,
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which is a giant breed, consequently showing late puberty [25].
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In many species, after the breeding, there is a transient inflammatory response to
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remove the excess sperm, bacteria, and other contaminants from the uterus. Some
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dogs have increased influx of neutrophils due to increased vascularization after
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mating, reducing fertility and leading to endometritis induced by mating. It has been
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reported in the literature that these females have a higher RI after breeding than
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healthy females [24]. In this study, no changes were noted in the RI of bitches that
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presented with endometritis; this most likely was due to the evaluation not being
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performed right after mating/insemination. Another factor related to infertility is the
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presence of ovarian cysts. In women, ovarian polycystic disease has been described as
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a cause of infertility, not only due to hormonal changes, but also due to increased
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resistance resulting from ovarian and uterine tissue vascularization [37]. In this work,
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only two-dimensional changes could be identified by inspection of intra-ovarian cysts
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and hence, no difference in the flow was identified.
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Thus, it is concluded that the flow of the normal uterine artery differs depending on
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the breed, phase of the cycle, reproductive status, and history. Moreover, Doppler
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studies during the follicular and early luteal phase can be an important tool in the
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diagnosis of infertility in dogs with unexplained causes of reproductive failure.
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4. References
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[1] Davidson AP, Baker TW. Reproductive ultrasound of dogs and tom. Top
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ACCEPTED MANUSCRIPT Companion Anim Med 2009;24:64-70.
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[2] Groppetti D, Vegetti F, Bronzo V, Pecile A. Ultrasonographic biometry in german
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shepherd dog for gestacional age estimation. Anim Reprod Sci 2015; 152:117-22.
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[3] Augsburger RH, Kurzi M. Histomorphologic and morphometric evaluation of the
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uterine horns in nulliparous and multiparous beagles. Am J Vet Res 2004; 65:552-58.
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[4] England GC, Yeager AE. Ultrasonographic appearance of the ovary and uterus of
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the bitch during oestrus, ovultation and early pregnancy. J Reprod Fertil Suppl
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1993;47:107-17.
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[5] Bollwein H, Weber F, Kolberg B, Stolla R. Uterine and ovarian blood flow during
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[6] Bollwein H, Meyer HHD, Maierl J, Weber F, Baumgartner U, Stolla R.
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cycle. Theriogenology 2000; 53:1541-52.
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[7] Honnens A, Niemann H, Herzog K, Paul V, Meyer HHD, Bollwein H. Doppler
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sonography of the uterine arteries during a superovulatory regime in cattle uterine
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blood flow in superovulated cattle. Theriogenology 2008;70:859-67.
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[8] Pereira BS, Freire LMP, Pinto JN, Domingues SFS, Silva LDM. Triplex Doppler
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evaluation of uterine arteries in cyclic and pregnant domestic cats. Anim Reprod Sci
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[9] Goswamy RK; Steptoe RP. Doppler ultrasound studies of the uterine artery in
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spontaneous ovarian cycles. Hum Reprod 1988;3:721–6.
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[10] El-Masny A, Abou-Salem N, El-Shenoufy. Doppler study of uterine
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hemodynamics in women with unexplained infertility. Eur J Obstet Gynecol Reprod
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Biol 2013;171:84-7.
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[11] Schärbele W. Ultrasonography in vascular diagnosis. 1st ed. Berlin: Springer;
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[12] Dickey RP, Hower JF, Matulich EM, Brown GT. Effect of standing on non-
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pregnant uterine blood flow. Ultrasound Obstet Gynecol 1994;4:480-7.
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[13] Goswamy RK, Williams G, Steptoe PC. Decreased uterine perfusion—a cause of
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infertility. Hum Reprod 1988;3:955-9.
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[14] Lang U, Scott BR, Braems G, Zygmunt M, Künzel W, Clark KE. Uterine blood
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flow—a determinant of fetal growth. Eur J Obstet Gynaecol Reprod Biol
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2003;110:55–61.
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[15] Kurjak A, Kupesic-Urek S, Schulman H, Zalud I. Transvaginal color flow
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Doppler in the assessment of ovarian and uterine blood flow in infertile women. Fertil
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Steril 1991;56:870-3.
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[16] Steer CV, Tan AL, Mason BA, Campbell S. Midluteal-phase vaginal color
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Doppler assessment of uterine artery impedance in a subfertile population. Fertil Steril
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1994;61:53-68.
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[17] Razik MA, Farag MAH, Sheta M. Uterine and ovarian arteries blood flow during
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the mid luteal phase in women with unexplained infertility. Middle East Fertil Soc J
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2015;20:209-12.
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[18] Barbosa CC, Souza MB, Scalercio SRRA, Silva TFP, Domingues SFS, Silva
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LDM. Ovarian and uterine periovulatory Doppler ultrasonography in bitches. Pesq
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Vet Bras 2013;33:1144-50.
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[19] Alvarez-Clau A, Liste R. Ultrasonographic characterization of the uterine artery
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in the nonestrus bitch. Ultrasound Med Biol 2005;31:1583-7.
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[20] Freeman SL, Russo M, England GCW. Uterine artery blood flow characteristics
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assessed during oestrus and the early luteal phase of pregnant and non-pregnant
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bitches. Vet J 2013;197:205-10.
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ACCEPTED MANUSCRIPT [21] England GCW, Russo M, Freeman SL. The bitch uterine response to semen
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deposition and its modification by male accessory gland secretions. Vet J
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2012;195:179-84.
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[22] Miranda SA, Domingues SFS. Conceptus ecobiometry and triplex Doppler
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ultrasonography of uterine and umbilical arteries for assessment of fetal viability in
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dogs. Theriogenology 2010;74:608–17.
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[23] Barbosa CC, Souza MB, Freitas LA, Silva TFP, Domingues SFS, Silva LDM.
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Assessment of uterine involution in bitches using B-mode and Doppler
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ultrasonography. Anim Reprod Sci 2013;139:121-6.
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[24] England GCW, Moxon R, Freeman SL. Delayed uterine fluid clearance and
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reduced uterine perfusion in bitches with endometrial hyperplasia and clinical
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management with post-mating antibiotic. Theriogenology 2012;78:1611-7.
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[25] Johnston SD, Root Kustritz MV, Olson PNS. Canine and feline theriogenology,
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1st ed. Saunders: Philadelphia, 2001.
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[26] Blanco PG, Rodríguez R, Rube A, Arias DO, Tórtora M, Díaz JD, Gobello C.
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Doppler ultrassonographic assessment of maternal and fetal blood flow in abnormal
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canine pregnancy. Anim Reprod Sci 2011;126:130-135.
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[27] Wahab HA, El-Din DA, Zain E, Abdelgancy M, Youssef MAFM. Uterine artery
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Doppler and subendometrial blood flow in patients with unexplained recurrent
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miscarriage. Middle East Fertil Soc J 2011;16:209-14.
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[28] Krishna U, Bhalerao S. Placental Insufficiency and Fetal Growth Restriction. J
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Obstet Gynaecol India 2011;61:505-11.
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[29] Ozkan S, Vural B, Caliskan E, Bodur H, Türköz E, Vural F. Color doppler
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sonographic analysis of uterine and ovarian blood flow in women with polycystic
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ovary syndrome. J Clin Ultrasound 2007;35:305-13.
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ACCEPTED MANUSCRIPT [30] Pourcelot L. Velocimetrie ultrasonore Doppler. 1st ed... Paris: Inserm, 1974.
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[31] Gosling RG, King DH. Arterial assessment by Doppler-shift ultra- sound. Proc R
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Soc Med 1974;67:447–9.
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[32] England GC, Yeager AE. Ultrasonographic appearance of the ovary and uterus of
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the bitch during oestrus, ovultation and early pregnancy. J Reprod Fertil Suppl. 1993;
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47: 107-17.
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[33] Freeman SL, Green MJ, England GCW. Prevalence and effect of uterine luminal
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free fluid on pregnancy and litter size in bitches. Theriogenology 2013;80:73-6.
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[34] Filant J, Spencer T. Uterine glands: biological roles in conceptus implantation,
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uterine receptivity and decasualization. Int J Dev Biol 2014;58:107-16.
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[35] Dickey RP. Doppler ultrasound investigation of uterine and ovarian blood flow
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in infertility and early pregnancy. Hum Reprod 1997;3:467- 503.
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[36] Ziereisen F, Heinrichs C, Dufour D, Saerens M, Avni EF. The role of Doppler
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evaluation of the uterine artery in girls around puberty. Pediatr Radiol 2001,31:712-9.
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[37] Bostanci MS, Sagsoz N, Noyan V, Yucel A, Goren K. Comprasion of Ovarian
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Stromal and Uterin Artery Blood Flow Measured by Color Doppler Ultrasonography
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in Polycystic Ovary Syndrome Patients and Patients With Ultrasonographic Evidence
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of Polycystic. J Clin Gynecol Obstet 2013;2:20-6.
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Figure legends
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Figure 1: (A). Two-dimensional ultrasound image showing a longitudinal section of
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the uterine body of a bitch in proestrus (B) Uterine layers identified in transverse
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section (C) Doppler with color mapping showing the uterine artery bordering the
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uterine body in a bitch in estrus.
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the uterine artery spectral morphologies of 39 bitches during proestrus, estrus, and
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diestrus. (A) Type A, a systolic peak followed by absent early diastolic wave but with
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flow during mid and late diastole until the next systole. (B) Type B, systolic peak
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followed by continuous diastole to systole, but absent late diastolic flow. (C) Type C,
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systolic peak is followed by diastolic, extending throughout the cardiac cycle to the
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subsequent systole. (D) Type D, systolic peak with no early and late diastolic flow,
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but with the mean diastolic flow. (E) Type O, systolic flow alone is identified, with
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characteristic monophasic high resistivity. (F) Type N (3 phases), systolic peak
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followed by reverse-diastolic and positive flow.
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Table 1: Mean ± standard deviation of canine uterine body diameter (cm) in longitudinal section according to parity and breed during different
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phases of the estrus cycle Uterine body diameter (cm) French Bulldog (n = 21)
Fila Brasileiro (n = 12)
Nulliparous
Primiparous
Multiparous
Nulliparous
Primiparous
Multiparous
Poestrus
0.81±0.07aA
0.94±0.04aB
1.03±0.04aC
0.80±0.06aA
0.98±0.03aB
1.06±0.09aC
Estrus
0.91±0.08bA*
1.10±0.12bB*
1.17±0.08bB*
1.33±0.10bA*
1.30± 0.19bA* 1.33±0.18bA*
Diestrus
1.12±0.07cA*
1.29±0.15cB*
1.53±0.25cC*
1.66±0.17cA*
1.84±0.11cB*
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Phases
1.99±0.13cC*
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Different superscript lower case letters in the same column indicate that there were differences among phases of the cycle in the same breed (P < 0.05).
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* It refers to the difference between breeds.
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Table 2: Mean ± standard deviation of resistivity index of the canine uterine artery according to parity and breed during different phases of the
Resistivity index French Bulldog (n = 21)
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estrus cycle
Fila Brasileiro (n = 12)
Nulliparous
Primiparous
Multiparous
Nulliparous
Primiparous
Poestrus
0.80±0.09aA*
0.85±0.07aA*
0.87±0.06aA
0.93±0.03aA*
0.96±0.02aA* 0.83±0.03aB
Estrus
0.88±0.06aA*
0.83±0.03aA*
0.90±0.04aA
0.98±0.07aA*
0.94±0.08aA* 0.83±0.06aB
Diestrus
0.74±0.14aA*
0.80±0.04aA*
0.86±0.09aB
0.97±0.07aA*
0.91±0.06aA* 0.81±0.09aB
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Multiparous
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Different superscript lower case letters in the same column indicate that there were differences among phases of the cycle in the same breed (P < 0.05).
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Table 3: Mean ± standard deviation of pulsatility index of the canine uterine artery according to parity and breed during different phases of the
Pulsatility index French Bulldog (n = 21)
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estrus cycle
Fila Brasileiro (n = 12)
Nulliparous
Primiparous
Multiparous
Nulliparous
Primiparous
Poestrus
2.51±0.21aA*
2.62±0.26aA*
2.86±0.24aB
3.46±0.28aA*
3.85±0.17aB* 2.56±0.26aC
Estrus
1.98±0.18bA*
2.39±0.15bB*
2.64±0.20bC
3.06±0.19ab*
3.50±0.26bB* 2.07±0.21bC
Diestrus
2.01±0.19bA*
2.29±0.23bB*
2.43±0.27bB
3.13±0.21aA*
2.93±0.22cB* 1.91±0.29bC
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Multiparous
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Different superscript lower case letters in the same column indicate that there were differences among phases of the cycle in the same breed (P < 0.05).
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Table 4: Mean ± standard deviation of resistivity and pulsatility index of the canine uterine artery of fertile (n = 28) and infertile (n = 11) bitches
Fertile
Infertile
Poestrus
0.88±0.12aA
0.89±0.14aA
Estrus
0.93±0.13aA
1.10±0.16bB
Diestrus
0.85±0.14aA
Fertile
Infertile
2.66±0.16aA
2.72±0.21aA
2.87±0.24bA
3.44±0.19bB
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Phases
Pulsatility index
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Resistivity index
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during different phases of the estrus cycle
1.07±0.18bB
2.51±0.22cA
3.19±0.31cB
Different superscript lower case letters refer to the comparison of the RI or PI in the same reproductive status among the phases of cycle (P <
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REVISED
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1 2
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We aimed to evaluate the two-dimensional and Doppler ultrasonographic features of bitches’ uteri
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Multiparous bitches had higher uterine diameters than nulliparous bitches.
5
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In diestrus and proestrus, uterine diameters were significantly the largest for
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multiparous bitches, followed by the primiparous bitches and smallest for nulliparous
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bitches. •
•
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•
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The breed, the phase of estrus cycle and pregnancy history must be taken into
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consideration when one intends to study the flow of the uterine artery.
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Spectra morphology of type A, C, D, and triphasic was found in females who did not gestate, while pregnant females showed spectra of type C, B and D.
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Infertile bitches had higher IR and IP than bitches considered fertile during the initial estrus and diestrus.
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French Bulldog females.
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IR was higher in nulliparous and primiparous females of Fila Brasileiro compared to
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S0093-691X(17)30128-0
DOI:
10.1016/j.theriogenology.2017.03.012
Reference:
THE 14035
To appear in:
Theriogenology
Received Date: 23 August 2016 Revised Date:
6 March 2017
Accepted Date: 14 March 2017
Please cite this article as: de Freitas LA, Mota GL, Silva HVR, da Silva LDM, Two-dimensional sonographic and Doppler changes in the uteri of bitches according to breed, estrus cycle phase, parity, and fertility, Theriogenology (2017), doi: 10.1016/j.theriogenology.2017.03.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
REVISED
ACCEPTED MANUSCRIPT 1
Two-dimensional sonographic and Doppler changes in the uteri of bitches
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according to breed, estrus cycle phase, parity, and fertility
3 Luana Azevedo de Freitas1*, Gustavo Lobato Mota1, Herlon Victor Rodrigues Silva1,
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Lúcia Daniel Machado da Silva1
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Fortaleza-CE, Brazil
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*[email protected]
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Laboratory of Carnivore Reproduction, Veterinary School, State University of Ceara,
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10 Abstract
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This study aimed to evaluate the two-dimensional and Doppler ultrasonographic
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features of bitches’ uteri according to breed, cycle phase, parity and fertility during
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the follicular and early diestrus phases. Thirty-nine pubertal bitches were divided into
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groups according to breed, parity, and fertility. Sonographic assessments started from
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the first day of vaginal bleeding and were performed weekly for a month. The
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diameter of the uterine body was measured longitudinally and the uterine
18
characteristics observed sonographically were evaluated. The resistivity index (RI)
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and pulsatility index (PI) of the uterine artery were calculated and the spectral
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morphology analyzed subjectively. The uterine diameter increased from proestrus to
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the diestrus regardless of the breed or parity. Multiparous bitches of both breeds had
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higher uterine diameters than nulliparous bitches, except Fila Brasileiro bitches in
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estrus. In diestrus and proestrus, the uterine diameters were significantly the largest
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for multiparous bitches, followed by the primiparous bitches and smallest for
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nulliparous bitches. The uterine diameter was larger in Fila Brasileiro females than in
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ACCEPTED MANUSCRIPT French Bulldogs during estrus and diestrus. The RI did not differ during the different
27
phases of the cycle for the same breed. However, the RI was higher in nulliparous and
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primiparous Fila Brasileiro females compared to French Bulldog females. Within Fila
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Brasileiro, multiparous bitches showed lower RI than nulliparous and primiparous
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bitches. Higher PI values were found during proestrus. All multiparous French
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Bulldog bitches had greater PI than nulliparous bitches, while multiparous Fila bitches
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had lower PI. Nulliparous and primiparous Fila bitches had larger PI larger than
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bitches with the same reproductive status of the breed French Bulldog. Infertile
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bitches had higher RI and PI than bitches considered fertile during the initial estrus
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and diestrus. Triphasic and types A, C, and D spectral morphologies were found in
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females who did not gestate; while pregnant females showed spectral morphologies of
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type C, B and D. Hence, it was concluded that the breed, the phase of estrus cycle and
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pregnancy history should be considered when studies the uterine artery flow during
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estrus and early diestrus using investigations such as Doppler assessment, which can
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be an important tool in diagnosing fertility in dogs.
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Key Words: Doppler; blood vessel; fertility; pregnancy; breed.
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1. Introduction
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Ultrasound examination of the uteri of bitches is an investigation routinely solicited
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for clinical reproductive evaluation [1,2]. Such exams permit the evaluation of size,
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echogenicity characteristics, contour of the uterine walls, and presence of luminal
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content. The size of the uterus varies depending on the animal's size, number of
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previous pregnancies, disease or wellness conditions, phase of the estrus cycle and
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pregnancy status [1,3]. However, few studies have considered and addressed these
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factors and applied them have considered to ultrasound images in dogs [4,5].
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ACCEPTED MANUSCRIPT Doppler ultrasound has found application as a non-invasive method in the assessment
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of uterine vasculature in different species [5-8]. Studies suggest that uterine perfusion
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regulates endometrial receptivity and influences successful implantation and
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maintenance of early pregnancy. Thus, good uterine blood supply preceding
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conception directly relates to fertility [9,10].
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Flow in the uterine artery is evaluated quantitatively by measuring the velocities,
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namely, peak systolic velocity (PSV), end diastolic velocity (EDV) and mean velocity
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(MV); this is followed by derivation of the indices, namely, resistivity (RI) and
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pulsatility index (PI). Qualitative evaluation is performed by assessing the spectral
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morphology. While velocity evaluates the blood flow behavior, these indices generate
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information on the impedance of the vessel and thus characterizes the vascular supply
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of a given organ [10]. Moreover, the study of flow by measuring the indices is
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accurate and provides replicability, especially when the study targets are vessels with
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small diameters or highly convoluted vessels, independent of the angle of sounding
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since they are based on the ratio between the speeds [11,12]. Some authors have
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demonstrated the relevance of spectral morphology evaluation to relate spectral
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descriptive aspects to infertility in women [13]. Although this is a subjective analysis
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for interpretation of flow, it permits evaluation when there is a diastolic change. In
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such cases, quantitative analysis does not provide precise flow information [9,12].
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Many studies have focused on Doppler evaluation of uterine arteries for a biological
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understanding of variations due to physiological or pathological events in women
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[9,10,12-17]. In dogs, studies have shown that there is a quantitative physiological
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variation in uterine flow during the estrus cycle [17,18], ovulation [18,20], deposit of
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semen [21], pregnancy [22] and post-partum [23], and in inflammatory processes
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[24]. Changes in the spectral morphology of the canine uterine artery were described
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phase were found in dogs that did not become pregnant [20].
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Factors such as age influence changes in the flow of canine uterine artery and may
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have an impact on fertility [20] The phase of the estrus cycle can also influence the
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Doppler parameters [19]. However, factors influencing the size of the uterus and the
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uterine artery, such as the animal [25] and the pregnancy history [3, 25], factors that
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lead to reduced fertility such as a history of recurrent failures of pregnancies [15-17],
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uterine pathology [27], gestational changes [14, 26- 28], and ovarian pathology [29],
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have not been investigated to determine the influence of the canine uterine artery
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Doppler parameters on them. In this setting, this study aimed to evaluate the two-
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dimensional and Doppler ultrasonographic features of bitches’ uteri according to
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breed, cycle phase, parity and fertility during the follicular and early diestrus phases.
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2. Material and Methods
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2.1 Experimental Animals
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The experimental protocol (protocol number 5999508/2015) was approved by the
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Ethics Committee in Animal Experimentation of the State University of Ceará in
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accordance with the guidelines for the care and use of laboratory animals established
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by the Brazilian College of Animal Experimentation. Thirty-nine pubertal bitches
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were evaluated , consisting of the French Bulldog (n = 24) and Fila Brasileiro (n = 15)
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breeds. All included animals were healthy, with a body score of approximately 3.0,
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weighing between 10-75 kg, ages ranging from 8 months to 6 years, and belonging to
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private kennels. The animals were fed granulated canine commercial food and water
100
ad libitum. Their reproductive history was known and/or had normal standard clinical
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pelvic ultrasound evaluations. All animals tested negative for brucellosis annually.
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The bitches were monitored using vaginal cytology and clinically; they were
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inseminated either artificially or, using known fertile males. The vaginal cytology was
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performed once a week by a single observer, beginning on the first day of bloody
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vaginal discharge until the identification of cytological diestrus. The smears were
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stained by Diff-Quick. The phases of the estrus cycle were defined based on the
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cytological characteristics [25]. Intravaginal inseminations were performed after
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obtaining the characteristic cytologic estrus on 3 alternate days using semen from
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animals with proven fertility. For those that were naturally mated, the male dog
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accompanied the bitch during the whole estrus.
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The animals were grouped according to categories: breed (French Bulldog or Fila
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Brasileiro), parity (nulliparous, primiparous or multiparous), and fertility history
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(fertile or infertile/sub-fertile). Bitches that did not conceive for a year or more after
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mating, had reproductive history of gestational failures, or had low prolificacy or
116
reproductive alterations during the experiment were considered infertile or subfertile.
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A total of 21 French Bulldog bitches and 12 Fila Brasileiro bitches without clinical
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and historical anomalies of previous reproductive changes were allocated according to
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the order of parity into groups of 7 and 4 animals, respectively; for each breed, the
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uterine diameter and Doppler indices were evaluated. Six bitches (3 from each breed)
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were not included in this comparison as they showed sonographic changes during the
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stages of the evaluated cycle, endometritis and ovarian cyst, or a history of gestational
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failures.
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2.2 Technical examination
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metropolitan area of Fortaleza, Ceará, over 1 year. The equipment used was MyLab
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30 Vet Gold (Esaote, Genova, Italy) with a multi-frequency micro-convex probe of 5-
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10 MHz (LA523 reference-Esaote, Genova, Italy). All ultrasound evaluations were
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performed by the same operator. The evaluations were carried out, preferably in the
131
morning, with the animals fasted for at least 8 hours. The bitches were placed in
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dorsal recumbency, without sedation. The abdominal and pelvic areas were shaved
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and a water soluble acoustic gel was applied.
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2.3 Two-dimensional ultrasound evaluation
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The bitches were evaluated once a week for 4 weeks. Assessments commenced on the
137
first day of serosanguinous vaginal discharge (day 0), which marked the beginning of
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proestrus. The period of the estrus cycle was confirmed based on to the clinical signs,
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behavior, and vaginal cytology. The animals were grouped according to the phase of
140
the cycle, breed and parity. The uterus was assessed for the presence or absence of
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intraluminal fluid. The longitudinal uterine diameter was measured (cm) at the body
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of the uterus. Ovaries were evaluated for the presence of cysts
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2.4 Doppler ultrasound evaluation
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After obtaining the two-dimensional images, Doppler was used to characterize the
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hemodynamics of the uterine artery on both sides of the uterine body.
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For color and spectral Doppler, we set and kept constant for all evaluations the
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insonation angle at 0°, color gain at 64% and the lowest wall filter to characterize the
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signal amplitude. For flow evaluation, we used a color map with the depth adequate
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for maximizing the frame rate, pulse repetition frequency of 9 to 18 Hz and sample
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ACCEPTED MANUSCRIPT size constant at 18 mm. The vessel was visualized longitudinally and the sample was
152
measured in this plane.
153
After identification of the arteries by color mapping, electronic markers (sample
154
volume or gates) were placed in the central region of the vessel and the spectral
155
Doppler was started. For analysis and to minimize data variability, 3 similar and
156
consecutive waves were recorded. Blood flow was measured automatically by the
157
ultrasound system using RI (PSV-EDV/PSV) [30] and PI (PSV-EDV/MV) [31] and
158
by assessing the spectral morphology.
159
The spectral morphology assessment was performed as follows [12]: Type A, a
160
systolic peak followed by absent early diastolic wave, but with flow through the mid
161
and late diastole until the next systole; Type B, systolic peak followed by continuous
162
diastole to systole, absent late diastolic flow; Type C, the systolic peak followed by
163
diastole, extending throughout the cardiac cycle to subsequent systole; Type D,
164
systolic peak with absent early and late diastole, but with the mean diastolic flow;
165
Type O, the systolic flow alone is identified, with characteristic high monophasic
166
resistivity; Type N (3 phases), systolic peak followed by reverse-diastolic and positive
167
flow.
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2.5 Statistical analysis
170
The data addressing the sonographic parameters related to uterine sonogram
171
characteristics were described qualitatively and quantitatively. The uterine diameter
172
and the RI and PI were expressed as a mean with standard deviations. The comparison
173
of uterine diameter, RI, and PI among the breeds, estrus cycle phase, reproductive
174
status and fertility was performed using SPSS 19.0 software (SPSS Inc. Chicago, IL,
175
USA). The data were subjected to the Shapiro-Wilk test for evaluation of normality
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and compared using bivariate analysis by Student T test and Kruskal-Wallis
177
considering P values less than 0.05 as significant.
178 3. Results
180
All bitches had normal estrus cycle and were mated. The average time of ultrasound
181
assessment was 15 minutes, ranging from 10 to 20 minutes.
182
The uterus was located dorsal to the urinary bladder and ventral to the colon. The
183
uterine body was identified longitudinally as a tubular structure that could be
184
visualized as a slight thickening with hypoechoic (edema) uterine walls during all
185
stages. The layers of the uterus could be distinguished on the cross-section images due
186
to reduced central echogenicity corresponding to the endometrium and a peripheral
187
hyperechogenicity corresponding to the perimetrium (Figure 1). The uterine horns
188
were identified as thin hypoechoic tubular structures in the lateral abdomen
189
continuing along the line of the ovaries.
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A discrete amount of anechoic intraluminal content was visible during estrus in 18
192
dogs of both breeds. However, only 1 French Bulldog bitch was diagnosed with
193
endometritis; the presenting sonogram image was characterized by increased uterine
194
wall contour irregularity, accompanied by a moderate amount of anechoic
195
intraluminal fluid content. Two French Bulldog bitches presented with multiple
196
ovarian cysts that visually manifested as multiple circular hypoechoic structures in the
197
ovarian cortex measuring more than 3 mm in diameter.
198
The uterine diameter increased from proestrus to diestrus regardless of the breed or
199
parity. Multiparous bitches of both breeds had higher uterine diameters than
200
nulliparous bitches, except for Fila Brasileiro bitches in estrus. In proestrus and
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202
in primiparous females, which in turn was greater than in nulliparous females. In
203
estrus, the uterine diameter of primiparous and multiparous Bulldogs did not differ
204
and were significantly larger than in nulliparous females. For the Fila Brasileiro
205
bitches in estrus, there was no difference in the uterine diameter based on parity. The
206
uterine diameter was higher in Fila females than French Bulldog during estrus and
207
diestrus, regardless of the parity (Table 1).
208
The uterine arteries were identified in all bitches (Figure 1). No differences were
209
found between the flow of the uterine artery on the right and left, based on grouped
210
data. The RI of the uterine arteries did not differ between the phases of the cycle for
211
either breed. The RI of the French Bulldog uterine artery did not differ in proestrus
212
and estrus independent of the parity. However, the RI was significantly higher in
213
diestrus in multiparous females compared to nulliparous and primiparous females. In
214
Fila Brasileiro bitches, the RI during different phases of the cycle were similar in
215
nulliparous and primiparous and significantly lower in multiparous females bitches.
216
On comparing the breeds, it was observed that the RI in nulliparous and primiparous
217
French Bulldog bitches were significantly lower than in the Fila Brasileiro bitches,
218
while the RI of multiparous French Bulldog bitches did not differ from the RI of
219
multiparous Fila Brasileiro bitches (Table 2).
220
Larger PI values were notes during proestrus. All multiparous French Bulldog bitches
221
registered PI greater than nulliparous bitches, while multiparous Fila Brasileiro
222
bitches had lower IP. Nulliparous and primiparous Fila Brasileiro bitches registered
223
greater PI than French Bulldog bitches with the same reproductive status (Table 3).
224
The RI of fertile female dogs did not differ between the phases of the cycle. In
225
infertile bitches, the RI in diestrus and estrus were significantly higher than in
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ACCEPTED MANUSCRIPT proestrus. The PI increased from proestrus to estrus and decreased from estrus to
227
diestrus, both in fertile bitches as in those infertile. The RI and PI did not differ in
228
proestrus between fertile and infertile bitches; however, they were significantly higher
229
in infertile bitches during estrus and diestrus (Table 4). Females with endometritis (RI
230
proestrus: 0.85; RI estrus: 0.89 and RI diestrus: 0.86 / PI proestrus: 2.63; PI estrus:
231
2.80 and PI diestrus: 2.51) and ovarian cyst (RI proestrus: 0.88; RI estrus: 0.91 and RI
232
diestrus: 0.86 / PI proestrus: 2.66 PI estrus: 2.88 and PI diestrus: 2.45) did not differ
233
from fertile bitches.
234
During this study, 26 dogs (66.66%) were diagnosed as pregnant, 17 being French
235
Bulldogs and 9 Fila Brasileiro bitches. Most pregnant bitches showed type C spectral
236
morphology during proestrus, estrus, and early diestrus. A Fila Brasileiro bitch that
237
presented with fetal death at 42 days of gestation exhibited Type D spectral
238
morphology. Spectral Type B were identified in a Fila Brasileiro bitch who had low
239
prolificacy (2 pups) and multiple embryonic resorptions.
240
Of Of the 7 Bulldog bitches that did not give birth, 1 had endometritis in the fourth
241
week of evaluation, 2 had ovarian cysts, and 4 did not give birth because of a prior
242
failure (history of gestational failures [2 nulliparous and 2 primiparous]). Of the 6 Fila
243
Brasileiro that did not give birth, 3 had gestational failures without prior history
244
(nulliparous), while the other 3 had a known history of gestational failures.
245
The spectral morphology of the bitch who had endometritis was similar to those that
246
gave birth (Type C). Bitches that had ovarian cysts had spectral morphologies
247
between C and A types during the weekly assessments. The nulliparous bitches that
248
did not give birth showed a type of flow in which only the systolic flow was
249
identified, characterizing the spectrum as monophasic with high resistivity (type O or
250
type D). Primiparous bitches that did not give birth had triphasic spectral
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morphologies that had a systolic peak, followed by reverse-diastolic and positive flow
252
(type N). More details regarding the types of spectral morphologies may be found in
253
Figure 2.
254 4. Discussion
256
Factors that affect the uterine artery flow have already been described in young and
257
mature dogs [20], amd after mating [21]; differences in the flow during different
258
phases of the estrus cycle have been identified [19]. However, this is the first
259
description of the uterine artery flow according to the breed, parity, and fertility in the
260
period preceding conception, highlighting the importance of categorization of these
261
factors in assessing the biological implications of the uterine artery Doppler study in
262
canine reproduction.
263
The uterine ultrasound description findings in this study agree with previous reports in
264
the literature [1,4,32]. However, the presence of uterine intraluminal fluid during late
265
estrus attributed to endometritis induced after mating [33] has not been related to an
266
adverse effect on the fertility; this was confirmed in females who had reached term
267
and showed no changes during pregnancy or postpartum.
268
One of the factors related to the increase in uterine diameter, due to endometrial
269
cyclic activity during proestrus, estrus and diestrus is hormonal influence. The uterine
270
tissue undergoes a variety of adaptive reactions in response to hormonal changes that
271
occur at different stages of the estrus cycle [25], through organizing events and
272
stratification of endometrial stroma; growth and differentiation of the myometrium;
273
and coordinated development of the endometrial glands [34]. , apart from hormonal
274
influence, the parity also determine the uterine size. A study of uterine morphometry
275
in multiparous bitches demonstrated an increase in the endometrium and especially
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ACCEPTED MANUSCRIPT the myometrium sizes with increasing number of pregnancies [3]. The increased
277
thickness of the uterine wall was subsequently confirmed by ultrasound [4]. As this
278
study evaluated bitches of different breeds and sizes, other factors related to changes
279
in the uterine size, such as the breed and size of the animal [25] were also identified.
280
Uterine artery flow characteristics during the follicular phase have been reported in
281
the past, exhibiting biphasic morphology with spectral peak flow during estrus and
282
early luteal phase; this was identified in this study by evaluating the PI. Studies have
283
suggested that uterine flow is a dependent cycle in women [9], mares [5], and cows
284
[6]; it is believed that cyclic variations in the uterine artery flow are attributed to the
285
concentration of progesterone and estrogen principally, and other flow regulating
286
factors such as nitric oxide. However, in this study, even with an increase in the
287
uterine size over the phases studied, no RI differences were identified in both breeds,
288
unlike PI, which showed higher values during proestrus, the estrogen dominant phase.
289
Based on these findings, recent works in women have emphasized greater reliability
290
in the use of PI for the diagnosis of unexplained infertility [17].
291
Previous canine studies were suggestive of a fluctuation in the uterine artery Doppler
292
values during different stages of the estrus cycle [18-20]; this was not found in the
293
current study due to the applied experimental design; hence, subtle differences that
294
might have occurred daily were not identified weekly. Furthermore, another
295
interesting aspect in the comparison of breeds and parity was that the RI and PI values
296
of nulliparous and primiparous Fila Brasileiro bitches were higher than the French
297
Bulldog bitches; however, there were no differences between multiparous bitches.
298
This may be explained by assuming that parity and breed can influence the Doppler
299
indices; however, after successive pregnancies, the influence of RI is lost.
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301
diestrus; they have maximum uterine flow during these periods [12]. At this time,
302
important physiological events such as ovulation [18], sperm reception [21], and
303
embryo implantation [13, 14] might occur; these require adequate blood perfusion for
304
successful reproduction.
305
Data shown in this work combined quantitative evaluation of obtaining the indices
306
and qualitative evaluation of analyzing the spectral morphology. Thus, a high RI and
307
change in spectral morphology of the diastolic flow might indicate poor perfusion in
308
the vascular bed, which can in turn lead to failures in the design and maintenance of
309
the developing conceptus (change in placental formation), as identified in Fila
310
Brasileiro bitches with changes during pregnancy [9, 12, 26, 35]. Thus, our results
311
demonstrate the role of uterine vascularization, and the importance of Doppler studies
312
in pre-obstetric evaluation as a predictor of adversity before and during pregnancy
313
[11-16].
314
In a classification proposed by Dickey [35], there were 6 spectral morphologies in the
315
uterine artery flow: A, B, C, D, O, and N, related to fertility and infertility. The
316
triphasic flow was not reported with regard to the flow of uterine artery, but was
317
described with regard to the ductus venous flow (transient vessel during fetal life)
318
[11,12, 35]. It corresponds to the ventricular systole (1st wave), atrial contraction (2nd
319
wave-reverse peak), and ventricular diastole (3rd wave), resulting in a pressure
320
reduction, preceding the diastole, which leads to distension of the contracted vessel,
321
which in turn leads to poor tissue perfusion [35]. In this study, bitches that had this
322
wave morphology had a history of recurrent pregnancy failures and did not give birth.
323
In this work, nulliparous bitches that did not give birth showed type O flow. This
324
spectrum of the uterine artery flow patterns has already been described in pre-pubertal
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ACCEPTED MANUSCRIPT children and individuals [36], but it can also be identified in those with fertility
326
problems who undergo in vitro fertilization programs [12]. In this study, its
327
visualization was related to the early and reproductive immaturity (first heat) or the
328
presence of anovulatory cycles in these dogs, mostly identified in the Fila Brasileiro,
329
which is a giant breed, consequently showing late puberty [25].
330
In many species, after the breeding, there is a transient inflammatory response to
331
remove the excess sperm, bacteria, and other contaminants from the uterus. Some
332
dogs have increased influx of neutrophils due to increased vascularization after
333
mating, reducing fertility and leading to endometritis induced by mating. It has been
334
reported in the literature that these females have a higher RI after breeding than
335
healthy females [24]. In this study, no changes were noted in the RI of bitches that
336
presented with endometritis; this most likely was due to the evaluation not being
337
performed right after mating/insemination. Another factor related to infertility is the
338
presence of ovarian cysts. In women, ovarian polycystic disease has been described as
339
a cause of infertility, not only due to hormonal changes, but also due to increased
340
resistance resulting from ovarian and uterine tissue vascularization [37]. In this work,
341
only two-dimensional changes could be identified by inspection of intra-ovarian cysts
342
and hence, no difference in the flow was identified.
343
Thus, it is concluded that the flow of the normal uterine artery differs depending on
344
the breed, phase of the cycle, reproductive status, and history. Moreover, Doppler
345
studies during the follicular and early luteal phase can be an important tool in the
346
diagnosis of infertility in dogs with unexplained causes of reproductive failure.
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347 348
4. References
349
[1] Davidson AP, Baker TW. Reproductive ultrasound of dogs and tom. Top
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[2] Groppetti D, Vegetti F, Bronzo V, Pecile A. Ultrasonographic biometry in german
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shepherd dog for gestacional age estimation. Anim Reprod Sci 2015; 152:117-22.
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[3] Augsburger RH, Kurzi M. Histomorphologic and morphometric evaluation of the
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uterine horns in nulliparous and multiparous beagles. Am J Vet Res 2004; 65:552-58.
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[4] England GC, Yeager AE. Ultrasonographic appearance of the ovary and uterus of
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the bitch during oestrus, ovultation and early pregnancy. J Reprod Fertil Suppl
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1993;47:107-17.
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[5] Bollwein H, Weber F, Kolberg B, Stolla R. Uterine and ovarian blood flow during
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the estrous cycle in mares. Theriogenology 2002;65:2129-38.
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[6] Bollwein H, Meyer HHD, Maierl J, Weber F, Baumgartner U, Stolla R.
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Transrectal Doppler sonography of uterine blood flow in cows during the estrous
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cycle. Theriogenology 2000; 53:1541-52.
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[7] Honnens A, Niemann H, Herzog K, Paul V, Meyer HHD, Bollwein H. Doppler
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sonography of the uterine arteries during a superovulatory regime in cattle uterine
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blood flow in superovulated cattle. Theriogenology 2008;70:859-67.
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[8] Pereira BS, Freire LMP, Pinto JN, Domingues SFS, Silva LDM. Triplex Doppler
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evaluation of uterine arteries in cyclic and pregnant domestic cats. Anim Reprod Sci
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2012;130:99-104.
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[9] Goswamy RK; Steptoe RP. Doppler ultrasound studies of the uterine artery in
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spontaneous ovarian cycles. Hum Reprod 1988;3:721–6.
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[10] El-Masny A, Abou-Salem N, El-Shenoufy. Doppler study of uterine
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hemodynamics in women with unexplained infertility. Eur J Obstet Gynecol Reprod
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[11] Schärbele W. Ultrasonography in vascular diagnosis. 1st ed. Berlin: Springer;
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[12] Dickey RP, Hower JF, Matulich EM, Brown GT. Effect of standing on non-
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pregnant uterine blood flow. Ultrasound Obstet Gynecol 1994;4:480-7.
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[13] Goswamy RK, Williams G, Steptoe PC. Decreased uterine perfusion—a cause of
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infertility. Hum Reprod 1988;3:955-9.
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[14] Lang U, Scott BR, Braems G, Zygmunt M, Künzel W, Clark KE. Uterine blood
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flow—a determinant of fetal growth. Eur J Obstet Gynaecol Reprod Biol
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2003;110:55–61.
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[15] Kurjak A, Kupesic-Urek S, Schulman H, Zalud I. Transvaginal color flow
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Doppler in the assessment of ovarian and uterine blood flow in infertile women. Fertil
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Steril 1991;56:870-3.
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[16] Steer CV, Tan AL, Mason BA, Campbell S. Midluteal-phase vaginal color
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Doppler assessment of uterine artery impedance in a subfertile population. Fertil Steril
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1994;61:53-68.
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[17] Razik MA, Farag MAH, Sheta M. Uterine and ovarian arteries blood flow during
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the mid luteal phase in women with unexplained infertility. Middle East Fertil Soc J
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2015;20:209-12.
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[18] Barbosa CC, Souza MB, Scalercio SRRA, Silva TFP, Domingues SFS, Silva
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LDM. Ovarian and uterine periovulatory Doppler ultrasonography in bitches. Pesq
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Vet Bras 2013;33:1144-50.
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[19] Alvarez-Clau A, Liste R. Ultrasonographic characterization of the uterine artery
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in the nonestrus bitch. Ultrasound Med Biol 2005;31:1583-7.
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[20] Freeman SL, Russo M, England GCW. Uterine artery blood flow characteristics
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assessed during oestrus and the early luteal phase of pregnant and non-pregnant
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bitches. Vet J 2013;197:205-10.
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ACCEPTED MANUSCRIPT [21] England GCW, Russo M, Freeman SL. The bitch uterine response to semen
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deposition and its modification by male accessory gland secretions. Vet J
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2012;195:179-84.
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[22] Miranda SA, Domingues SFS. Conceptus ecobiometry and triplex Doppler
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ultrasonography of uterine and umbilical arteries for assessment of fetal viability in
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dogs. Theriogenology 2010;74:608–17.
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[23] Barbosa CC, Souza MB, Freitas LA, Silva TFP, Domingues SFS, Silva LDM.
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Assessment of uterine involution in bitches using B-mode and Doppler
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ultrasonography. Anim Reprod Sci 2013;139:121-6.
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[24] England GCW, Moxon R, Freeman SL. Delayed uterine fluid clearance and
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reduced uterine perfusion in bitches with endometrial hyperplasia and clinical
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management with post-mating antibiotic. Theriogenology 2012;78:1611-7.
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[25] Johnston SD, Root Kustritz MV, Olson PNS. Canine and feline theriogenology,
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1st ed. Saunders: Philadelphia, 2001.
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[26] Blanco PG, Rodríguez R, Rube A, Arias DO, Tórtora M, Díaz JD, Gobello C.
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Doppler ultrassonographic assessment of maternal and fetal blood flow in abnormal
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canine pregnancy. Anim Reprod Sci 2011;126:130-135.
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[27] Wahab HA, El-Din DA, Zain E, Abdelgancy M, Youssef MAFM. Uterine artery
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Doppler and subendometrial blood flow in patients with unexplained recurrent
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miscarriage. Middle East Fertil Soc J 2011;16:209-14.
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[28] Krishna U, Bhalerao S. Placental Insufficiency and Fetal Growth Restriction. J
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Obstet Gynaecol India 2011;61:505-11.
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[29] Ozkan S, Vural B, Caliskan E, Bodur H, Türköz E, Vural F. Color doppler
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sonographic analysis of uterine and ovarian blood flow in women with polycystic
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ovary syndrome. J Clin Ultrasound 2007;35:305-13.
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ACCEPTED MANUSCRIPT [30] Pourcelot L. Velocimetrie ultrasonore Doppler. 1st ed... Paris: Inserm, 1974.
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[31] Gosling RG, King DH. Arterial assessment by Doppler-shift ultra- sound. Proc R
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Soc Med 1974;67:447–9.
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[32] England GC, Yeager AE. Ultrasonographic appearance of the ovary and uterus of
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the bitch during oestrus, ovultation and early pregnancy. J Reprod Fertil Suppl. 1993;
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47: 107-17.
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[33] Freeman SL, Green MJ, England GCW. Prevalence and effect of uterine luminal
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free fluid on pregnancy and litter size in bitches. Theriogenology 2013;80:73-6.
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[34] Filant J, Spencer T. Uterine glands: biological roles in conceptus implantation,
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uterine receptivity and decasualization. Int J Dev Biol 2014;58:107-16.
435
[35] Dickey RP. Doppler ultrasound investigation of uterine and ovarian blood flow
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in infertility and early pregnancy. Hum Reprod 1997;3:467- 503.
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[36] Ziereisen F, Heinrichs C, Dufour D, Saerens M, Avni EF. The role of Doppler
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evaluation of the uterine artery in girls around puberty. Pediatr Radiol 2001,31:712-9.
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[37] Bostanci MS, Sagsoz N, Noyan V, Yucel A, Goren K. Comprasion of Ovarian
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Stromal and Uterin Artery Blood Flow Measured by Color Doppler Ultrasonography
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in Polycystic Ovary Syndrome Patients and Patients With Ultrasonographic Evidence
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of Polycystic. J Clin Gynecol Obstet 2013;2:20-6.
444 445
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Figure legends
446
Figure 1: (A). Two-dimensional ultrasound image showing a longitudinal section of
447
the uterine body of a bitch in proestrus (B) Uterine layers identified in transverse
448
section (C) Doppler with color mapping showing the uterine artery bordering the
449
uterine body in a bitch in estrus.
18
ACCEPTED MANUSCRIPT 450 Figure 2: Graph (x-axis, flow rate in meters and the y-axis, time in seconds) showing
452
the uterine artery spectral morphologies of 39 bitches during proestrus, estrus, and
453
diestrus. (A) Type A, a systolic peak followed by absent early diastolic wave but with
454
flow during mid and late diastole until the next systole. (B) Type B, systolic peak
455
followed by continuous diastole to systole, but absent late diastolic flow. (C) Type C,
456
systolic peak is followed by diastolic, extending throughout the cardiac cycle to the
457
subsequent systole. (D) Type D, systolic peak with no early and late diastolic flow,
458
but with the mean diastolic flow. (E) Type O, systolic flow alone is identified, with
459
characteristic monophasic high resistivity. (F) Type N (3 phases), systolic peak
460
followed by reverse-diastolic and positive flow.
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REVISED
Table 1: Mean ± standard deviation of canine uterine body diameter (cm) in longitudinal section according to parity and breed during different
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phases of the estrus cycle Uterine body diameter (cm) French Bulldog (n = 21)
Fila Brasileiro (n = 12)
Nulliparous
Primiparous
Multiparous
Nulliparous
Primiparous
Multiparous
Poestrus
0.81±0.07aA
0.94±0.04aB
1.03±0.04aC
0.80±0.06aA
0.98±0.03aB
1.06±0.09aC
Estrus
0.91±0.08bA*
1.10±0.12bB*
1.17±0.08bB*
1.33±0.10bA*
1.30± 0.19bA* 1.33±0.18bA*
Diestrus
1.12±0.07cA*
1.29±0.15cB*
1.53±0.25cC*
1.66±0.17cA*
1.84±0.11cB*
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Phases
1.99±0.13cC*
TE D
Different superscript lower case letters in the same column indicate that there were differences among phases of the cycle in the same breed (P < 0.05).
AC C
* It refers to the difference between breeds.
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Different superscript capital letters in the same line indicate that there were differences among reproductive status in the same breed (P < 0.05).
ACCEPTED MANUSCRIPT
Table 2: Mean ± standard deviation of resistivity index of the canine uterine artery according to parity and breed during different phases of the
Resistivity index French Bulldog (n = 21)
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estrus cycle
Fila Brasileiro (n = 12)
Nulliparous
Primiparous
Multiparous
Nulliparous
Primiparous
Poestrus
0.80±0.09aA*
0.85±0.07aA*
0.87±0.06aA
0.93±0.03aA*
0.96±0.02aA* 0.83±0.03aB
Estrus
0.88±0.06aA*
0.83±0.03aA*
0.90±0.04aA
0.98±0.07aA*
0.94±0.08aA* 0.83±0.06aB
Diestrus
0.74±0.14aA*
0.80±0.04aA*
0.86±0.09aB
0.97±0.07aA*
0.91±0.06aA* 0.81±0.09aB
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Phases
Multiparous
TE D
Different superscript lower case letters in the same column indicate that there were differences among phases of the cycle in the same breed (P < 0.05).
AC C
* It refers to the difference between breeds.
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Different superscript capital letters in the same line indicate that there were differences among reproductive status in the same breed (P < 0.05).
ACCEPTED MANUSCRIPT
Table 3: Mean ± standard deviation of pulsatility index of the canine uterine artery according to parity and breed during different phases of the
Pulsatility index French Bulldog (n = 21)
RI PT
estrus cycle
Fila Brasileiro (n = 12)
Nulliparous
Primiparous
Multiparous
Nulliparous
Primiparous
Poestrus
2.51±0.21aA*
2.62±0.26aA*
2.86±0.24aB
3.46±0.28aA*
3.85±0.17aB* 2.56±0.26aC
Estrus
1.98±0.18bA*
2.39±0.15bB*
2.64±0.20bC
3.06±0.19ab*
3.50±0.26bB* 2.07±0.21bC
Diestrus
2.01±0.19bA*
2.29±0.23bB*
2.43±0.27bB
3.13±0.21aA*
2.93±0.22cB* 1.91±0.29bC
M AN U
SC
Phases
Multiparous
TE D
Different superscript lower case letters in the same column indicate that there were differences among phases of the cycle in the same breed (P < 0.05).
AC C
* It refers to the difference between breeds.
EP
Different superscript capital letters in the same line indicate that there were differences among reproductive status in the same breed (P < 0.05).
ACCEPTED MANUSCRIPT
Table 4: Mean ± standard deviation of resistivity and pulsatility index of the canine uterine artery of fertile (n = 28) and infertile (n = 11) bitches
Fertile
Infertile
Poestrus
0.88±0.12aA
0.89±0.14aA
Estrus
0.93±0.13aA
1.10±0.16bB
Diestrus
0.85±0.14aA
Fertile
Infertile
2.66±0.16aA
2.72±0.21aA
2.87±0.24bA
3.44±0.19bB
M AN U
Phases
Pulsatility index
SC
Resistivity index
RI PT
during different phases of the estrus cycle
1.07±0.18bB
2.51±0.22cA
3.19±0.31cB
Different superscript lower case letters refer to the comparison of the RI or PI in the same reproductive status among the phases of cycle (P <
TE D
0.05).
AC C
EP
Different superscript capital letters refer to the comparison between fertile and infertile reproductive status in the same phase (P < 0.05).
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
REVISED
ACCEPTED MANUSCRIPT HIGHLIGHTS
1 2
•
We aimed to evaluate the two-dimensional and Doppler ultrasonographic features of bitches’ uteri
3 •
Multiparous bitches had higher uterine diameters than nulliparous bitches.
5
•
In diestrus and proestrus, uterine diameters were significantly the largest for
RI PT
4
multiparous bitches, followed by the primiparous bitches and smallest for nulliparous
7
bitches. •
•
12
•
•
The breed, the phase of estrus cycle and pregnancy history must be taken into
EP
consideration when one intends to study the flow of the uterine artery.
AC C
15
Spectra morphology of type A, C, D, and triphasic was found in females who did not gestate, while pregnant females showed spectra of type C, B and D.
13
14
Infertile bitches had higher IR and IP than bitches considered fertile during the initial estrus and diestrus.
11
M AN U
French Bulldog females.
9
10
IR was higher in nulliparous and primiparous females of Fila Brasileiro compared to
TE D
8
SC
6
1