Farbdopplersonographische Untersuchungen an zystischen Ovarfollikeln beim Rind
INAUGURAL-DISSERTATION
zur Erlangung des Grades einer Doktorin der Veterinärmedizin
(Dr. med. vet.)
durch die Tierärztliche Hochschule Hannover
Vorgelegt von Alexandra Rauch aus Baguio / Philippinen
Hannover 2008
1. Gutachter: Univ. - Prof. Dr. Heinrich Bollwein 2. Gutachter: Univ. - Prof. Dr. Burkhard Meinecke
Tag der mündlichen Prüfung: 04. November 2008
Diese Arbeit wurde durch die Dr. Dr. h.c. Karl-Eibl-Stiftung gefördert.
Meinen Eltern
Meinen Eltern Meinen Eltern
Meinen Eltern
II. Publikation 12
Abstract 9
Introduction 10
Materials and Methods 12
Results 16
Discussion 19
Conclusion 22
Figures and tables 23
III. Zusammenfassung 30
IV. Erweiterte Zusammenfassung 32
V. Literatur 36
VI. Eigene Publikationen und Koautorenschaften im Rahmen dieser Arbeit 41
VII. Danksagung 42
Bl blood sampling
°C degree celsius
CA coloured area
cm² square centimetres
CL corpus luteum
COF cystic ovarian follicle COFs cystic ovarian follicles
DS Doppler sonography
EDTA ethylene diamine tetra-acetate
EIA Enzymgekoppelter Immunadsorptionstest
Ex examination
Fig. figure
GnRH gonadotropin-releasing hormone hCG human chorionic gonadotropin
Lut luteinized
MAD median absolute deviation
Max maximum
Med median
µg microgram
mg milligram
MHz megahertz
min minutes
Min minimum
ml millilitre
mm millimetre
n number
ng nanogram
NoLut non luteinized
PCOS polycystic ovary syndrome
persist persistent
PGF2α prostaglandin F2α
ROC receiver operating characteristic ROI region of interest
PPV positive predictive value rpm revolutions per minute SAS statistical analysis system
Se sensitivity
Sp specificity
TA total area
Temp temporary
TW thickness of wall
Vmax maximum velocity
x median
ZOF zystischer Ovarfollikel ZOFs zystische Ovarfollikel
17.08.2008. Abbildungen und Tabellen befinden sich am Ende des Manuskripts.
COLOUR DOPPLER SONOGRAPHY OF CYSTIC OVARIAN FOLLICLES IN COWS
Alexandra RAUCH1), Lars KRÜGER1), Akio MIYAMOTO²) and Heinrich BOLLWEIN1)
1) Clinic for Cattle, University of Veterinary Medicine Hannover, 30173 Hannover, Germany and
2) Graduate School of Animanl and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
Abstract
The goals of the present study were to investigate whether colour Doppler sonography can be used to differentiate temporary from persistent ovarian follicles and follicles with luteal tissue from follicles without luteal tissue and to assess the response of follicular cysts to administration of a gonadotropin releasing hormone (GnRH) analogue. Fifty-four cows having ovarian follicular structures with a diameter of >15 mm but no corpus luteum were included. These cows were examined via B- mode and colour Doppler sonography. The same examinations were repeated 10 to 12 days later, and the cows with follicular cysts (n=17) received a GnRH analogue.
Blood flow was measured before and 30 min after treatment. Ten to 12 days later, the response to treatment was assessed using B-mode sonography. While 31 of 54 follicles disappeared spontaneously (temporary follicles), 23 follicles persisted and were diagnosed as cystic ovarian follicles (COFs). There was no difference between temporary follicles and COFs in regard to total area, wall thickness or the perfused area. In the luteinized follicles (n=13), based on the plasma progesterone concentration, total area was twice as large, wall thickness was three times greater and the perfused area was 4.5 times larger than those of the non-luteinized follicles (n=41). The sensitivity of diagnosing luteinized follicles was 61.5% using B-mode sonography and 92.3% using colour Doppler sonography. Twelve cows responded to GnRH, and five cows did not. There was a trend (P=0.07) toward higher (59.3%) blood flow in the cyst wall 30 min after treatment in the responding cows compared with the non-responding cows. Our results showed that the perfused area more accurately reflects active luteal tissue than wall thickness. Thus, colour Doppler sonography is superior to B-mode sonography for differentiating follicular and luteal cysts and aids in the selection of treatment. However, exact prediction of COFs destined to regress or persist and the response of COFs to treatment with a GnRH analogue were not possible using colour Doppler sonography.
Key words: Blood flow, Cow, Cystic ovarian follicles, Doppler sonography, Gonadotropin releasing hormone (GnRH)
Introduction
Cystic ovarian follicles (COFs) occur with an incidence of 5.2 to 27.9% [1-7] and constitute one of the most important causes of reproductive failure in dairy cows.
Although cows with acute COFs may recover spontaneously [5, 8, 9], herd fertility is impaired considerably because the service rate is prolonged by 22 to 77 days [2, 3, 10].
A cystic follicle has been traditionally defined as an anovulatory follicle persisting for more than 10 days that has a diameter of >25 mm without luteal tissue [7, 11]. For the purpose of B-mode sonographic studies, a diameter of ≥20 mm [12, 13] or ≥17 mm [14, 15] and persistence for a minimum of seven days are used as criteria for COFs. Small cystic follicles with a diameter of 15 to 22 mm do not differ histologically from cystic follicles with a diameter of >25 mm [16]. Follicular cysts, which have a wall diameter of <3 mm and are accompanied by a plasma progesterone (P4) concentration of ≤1 ng/ml, are different from luteal cysts, which have a wall diameter of ≥3 mm and are accompanied by a serum P4 concentration of >1 ng/ml.
While the positive predictive value (PPV) for follicular or luteal cysts diagnosed by transrectal palpation is 66.0%, it increases to 74.0% for follicular cysts and to 85.0%
for luteal cysts when B-mode ultrasonography is used [17]. An additional measurement of P4 values further increases the PPV for diagnosis of follicular and luteal cysts [18, 19].
Incorrect diagnosis, particularly with respect to differentiation of the two types of cysts, leads to inappropriate treatment [6]. Follicular cysts are treated with human chorionic gonadotropin (hCG), gonadotropin releasing hormone (GnRH) [20], while luteal cysts are treated with PGF2α or its analogue [20].
Cystic changes in the ovaries, called polycystic ovary syndrome (PCOS), also occur in women [21]. Diagnosis is based on the results of B-mode as well as colour Doppler sonography; during the menstrual cycle, the maximum blood flow velocity (Vmax) in the follicle and ovarian stroma is higher in women with PCOS than in women with normal ovaries [22]. Colour Doppler sonography has recently become important in bovine reproduction for investigation of the changes in follicular
circulation in the periovulatory period [23, 24]. The above studies were experimental in nature, but a recent review pointed out that the colour Doppler technique will likely become a cow-side tool for the practicing veterinarian in the near future [25].
The goals of the present study were to investigate the usefulness of colour Doppler sonography for differentiation of normal cyclic follicles from cystic ovarian follicles and of follicular cysts from luteal cysts and for early assessment of the efficacy of GnRH treatment.
Materials and Methods
Animals
This study was conducted from October 2005 to October 2006 in three dairy herds in Lower Saxony (Germany) and from November to December 2006 in one dairy herd in Brandenburg (Germany). The herd sizes were 80 (A), 90 (B), 105 (C) and 2559 (D) cows, and all cows calved year-round, with a mean annual milk production of 9,353 kg (A), 8,600 kg (B), 7,799 kg (C) and 8,948 kg (D) per cow, respectively. A total of 49 German Holstein and 5 German Black Pied cows were examined as described below. None of the examined animals had been treated for ovarian cysts before the first ultrasound examination.
Study design
All the cows from the four farms underwent a postpartum transrectal examination of the uterus and ovaries. Cows that calved a minimum of 28 days before the examination and had no clinical signs of endometritis, claw disease, metabolic disease or mastitis were selected for the study. Cows for which transrectal palpation indicated a follicular structure >15 mm and no corpus luteum (CL) were examined sonographically to confirm the findings. Cows for which these ovarian findings could be confirmed were included in the study, and the blood flow of the largest follicle was measured; this first examination was referred to as examination (Ex) 1 (Fig. 1). A second sonographic examination (Ex-2) of the ovaries was carried out 10 to 12 days later, by which time one of four situations had occurred: the largest follicle or a follicle other than the largest one present at Ex-1 had ovulated (classified as ovulation, with a CL and plasma P4 concentration >1.0 ng/ml); the largest follicle was luteinized (luteinization; P4 >1.0 ng/ml and wall diameter < 3 mm); all of the follicles >15 mm had regressed (regression); or the largest follicle had persisted and was the same size or larger compared with Ex-1 (persistence). Follicles that had ovulated, luteinized or regressed were referred to as temporary follicles (Temp), and the persistent follicles were referred to as Persist. The cows diagnosed with the latter type of follicles were considered to have COFs (follicular or luteal cysts) and were
examined further. The blood flow of the largest COF was quantified using colour Doppler sonography. Immediately afterwards, the cows were given 20 µg of a gonadotropin releasing hormone analogue (Buserelin; Receptal®, Intervet, Unterschleißheim, Germany) intramuscularly as an attempt to induce ovulation, and after 30 minutes (Ex-3), the blood flow was again quantified. The cows were examined 10 to 12 days later (Ex-4) for the presence of functional structures as at Ex-1 using transrectal palpation and sonography. Cows with a CL or luteinization of the largest COF were classified as responding to treatment (responding), and cows without a CL or no luteinization were categorized as non-responding.
Ultrasonography
Sonographic measurements were carried out using a portable LOGIQ Book XP ultrasound device (General Electrics Healthcare, Solingen, Germany) equipped with a 6 to 10 MHz linear probe (Model 1739-RS; General Electrics Yokogawa Medical Systems, Tokyo, Japan). All ultrasound examinations were conducted by the same investigator (A.R.). Each investigation included recording of images of both ovaries in different sections. The largest follicular structure was identified by B-mode sonography, and an ultrasonic cross-sectional image was frozen at its maximum area and saved for further off-line measurements. Colour blood flow mapping of the largest follicular structure in various transverse sections was carried out using Power Doppler mode. This mode expresses the number of red blood cells flowing through a blood vessel per unit of time. To minimize the variations in recording, the settings of the Power Doppler system were fixed and used for all examinations. The entire cross-sectional area of the follicular structure was visible within the Power Doppler sample box. After recording three video sequences with a duration of three seconds each, three single images without flash artifacts and with the maximum number of coloured areas were stored from these video sequences. Ultrasound images were exported in DICOM format into a laptop (Satellite M30X; Toshiba, Tokyo, Japan).
The total area (TA) and wall thickness (TW) of follicular structures with a diameter of at least 15 mm were measured on B-mode images using a computer-assisted image analysis program (PixelFlux; Chameleon-Software, Leipzig, Germany). Wall
thickness was taken as the mean of three measurements carried out at different wall locations. Based on the results of these measurements, follicles were defined as luteinized if the wall thickness was ≥3 mm and were categorized as non-luteinized if the wall thickness was <3 mm.
The same software was also used to assess blood flow, which was expressed as the total area of coloured pixels in the wall (CA). For this purpose, the whole follicular structure and its blood flow area were chosen as the region of interest (ROI), and the coloured area within this ROI was calculated (Fig. 3). The means from three images were used for further evaluation of follicular / cystic blood flow.
Plasma progesterone assay
Blood samples were obtained from the coccygeal vein before scanning at Ex-1, Ex-2 and Ex-4. Blood was collected into evacuated EDTA tubes (BD Vacutainers; BD, Plymouth, UK) and transported in a styrofoam box with ice packs to the laboratory within 8 h. Plasma was separated at 4 C by centrifugation at 3,000 rpm for 15 min and stored at -20 C until analysis of the plasma level of P4. The P4 concentration was estimated by enzyme immunoassay as reported previously [26]. In brief, 20 µl plasma was measured directly using a monoclonal antibody developed in rats and the enzyme progesterone-3-(O-carboxymethyl oxime)-horseradish-peroxidase. The sensitivity of P4 measurements in terms of the lowest detectable concentration significantly different from zero was 0.6 ng/ml. The sensitivity of the test in terms of the 50% intercept was 1.9 ng/ml. All intra- and interassay coefficients of variation were less than 10%.
To distinguish between follicular structures with and without luteal tissue, a plasma P4
cut-off of 1.0 ng/ml was used. Concentrations of 1.0 ng/ml and lower (P4 low) defined follicles without luteal tissue (NoLut) and follicular cysts; concentrations greater than 1.0 ng/ml (P4 high) defined follicles with luteal tissue (Lut) and luteal cysts. Plasma P4
concentrations were also used to calculate sensitivity, specificity and the positive predictive value [27] of diagnoses based on sonographic findings.
Statistical analysis
Statistical analyses were carried out using StatView Version 5.0 and Statistical Analysis System 9.1 (SAS Institute, Cary, NC, USA). The Shapiro-Wilk test was used to test data for normal distribution. Cross sectional areas (TA), wall thickness (TW) and blood flow area (CA) were expressed as median ± median absolute deviation (MAD). Wilcoxon rank sum tests were conducted to determine differences in measurements between cows with temporary follicles and cows with persistent follicles in blood flow, cross-sectional area and wall thickness. Additionally, comparisons were made between cows with and without luteal tissue in the follicle wall. The cut-off value for blood flow to distinguish between follicles with and without luteal tissue was calculated by ROC analysis and Youden index. Blood flow areas before and after therapy with GnRH were compared using the Wilcoxon signed rank test. The absolute changes in blood flow were defined as the difference in CA before and after treatment. Additionally, percent changes in blood flow relative to the levels at Ex-2 were calculated. P ≤0.05 was considered significant.
Results
Clinical data comparison
At Ex-1, the mean age of the cows was 46.2 ± 17.9 months; 18 were in their 1st lactation, 14 were in their 2nd, 15 were in their 3rd and seven were in their 4th to 6th (x=4.4) lactation. The first examination (Ex-1) was carried out on 44 cows (81.5%) at a mean of 33 days (range 28 to 42 days) postpartum, seven cows (13.0%) at a mean of 57 days (range 52 to 63 days) postpartum and three cows (5.6%) at 80, 111 and 360 days postpartum.
Of the 54 follicles seen at Ex-1, 23 (42.6%) were still present at Ex-2, and 31 (57.4%) had regressed spontaneously (Fig. 2).
The maximum cross-sectional area of the largest follicle in 53 cows at Ex-1 ranged from 1.5 cm² to 16.1 cm², which corresponded to 13.8 mm to 45.3 mm in diameter (Table 1). The cross-sectional area of the largest follicle in 27 cows (50.9%) was less than 4.9 cm² (diameter <25 mm).
Based on the P4 concentration at Ex-1, 23 cows in the Temp group and 18 in the Persist group were retrospectively classified as NoLut (Fig. 2). Thus, at Ex-1, 41 of the 54 cows (75.9%) had no active luteal tissue. The Lut group consisted of 13 cows (24.1%); eight were from the Temp group, and five were from the Persist group (Fig.
2). At Ex-2, 18 (78.3%) of the persistent follicles were identified as NoLut, and five were identified as (21.7%) Lut based on the P4 value.
Eight cows had a follicular wall thickness of ≥3 mm and a concurrent P4
concentration of >1ng/ml. Of the other 46 cows with a wall thickness of <3 mm, 41 had a P4 concentration of ≤1ng/ml, and five had a P4 concentration of >1ng/ml (Table 2).
For technical reasons, measurement of blood flow was not possible in one cow at Ex-1. Thus, there are results for wall thickness, cross-sectional area and P4
concentrations for 54 cows, but only blood flow data for 53 animals at Ex-1.
Comparison of temporary and persistent follicles at Ex-1
At Ex-1, follicles that at Ex-2 were subsequently defined as Temp did not differ significantly from those subsequently defined as Persist with regard to cross-
sectional area (P=0.42), wall thickness (P=0.27) or blood flow (P=0.39), regardless of whether the wall was luteinized or not (Table 1). In the Lut group, the walls of Temp follicles were three times thicker (P=0.05) than the walls of persistent Lut follicles (Table 1). In the NoLut group, there was no significant difference in wall thickness between Temp and Persist follicles (P=0.68).
Comparison of follicles with and without luteal tissue at Ex-1
The cross-sectional area, wall thickness and blood flow of the Lut follicles were two times (P=0.0003), three times (P<0.0001) and 4.5 times (P<0.0001) those of the NoLut follicles, respectively (Table 1).
The cut-off value for the perfused area to differentiate Lut and NoLut follicles with the P4 level as the gold standard was 0.9 cm².
Using B-mode sonography, the sensitivity of diagnosing Lut follicles was 61.5%, and the specificity was 100%. Using colour Doppler sonography, the sensitivity was 92.3%, and the specificity was 100% (Table 2).
Follicular blood flow at Ex-2 and Ex-3 in cows that responded and those that did not respond to treatment
The mean perfused area at Ex-2 and Ex-3 was of 0.4 cm² ± 0.2 MAD. There was no significant difference at Ex-2 (P=0.46) and Ex-3 (P=0.14) between the responding and non-responding cows.
The absolute change in the perfused area between Ex-2 and Ex-3 was not associated with the outcome of treatment (Table 3). There was no difference in blood flow before and after GnRH administration in the responding (P=0.31) and non- responding (P=0.69) cows.
There was a trend toward a difference in the percent change in the perfused area in the responding cows between Ex-2 and Ex-3 (P=0.07); the relative blood flow of eight responding cows increased by a mean of 59.3%, whereas it decreased by a mean of 19.7% in four of the responding cows (Fig. 4). In contrast, the percent change in the perfused area of the non-responding cows did not differ between Ex-2 and Ex-3 (P=0.50); the relative blood flow increased by a mean of 15.9% in two cows and
decreased by a mean of 16.8% in three cows (Fig. 4).
Discussion
In our study, 57% of follicles >15 mm had disappeared by 10 days after the initial examination, which is similar to rates in other studies. Spontaneous regression of COFs occurred in 61% [8], 48% [9] and 38.6% [5] of the cows in previous studies during the puerperal period. Based on the plasma P4 concentrations at the first examination (Ex-1), 78% of the cows had follicles without luteal tissue, and the remaining 22% had follicles with luteal tissue. Other similar studies have reported relative frequencies of follicular cysts of 58% [28] and 73% [29]. However, the criteria for diagnosis of ovarian cysts were different in these studies; Carroll et al. [28]
defined cysts to be > 2.5 cm in diameter with persistence of at least 8 days, while the diagnosis of Bartolome et al. [29] was based on a size of at least approximately 18 mm in diameter.
Assessment of the size of non-luteinized and luteinized follicles by B-mode sonography could not be used to predict whether the follicle would persist or regress spontaneously. The same prediction was also not possible based on the blood flow by colour Doppler sonography. Kawashima et al. [24] studied the development of dominant follicles in the first 20 days postpartum and were unable to predict their outcome (ovulation or persistence) based on their size and local blood flow. If ovulation did not occur in the first 20 days postpartum, the cow was considered anovulatory. The dominant follicle had the same mean diameter, with local blood flow independent of whether ovulation occurred or not. Wall thickness could be used to predict the likelihood of spontaneous regression in cows that had luteal tissue in the follicle wall. The wall was markedly thicker in follicles that regressed than in those that persisted. A possible explanation for this is that some of these structures were incorrectly diagnosed and may have been corpora lutea with a central cavity rather than luteal cysts. Corpora lutea with a central cavity usually have an oval cross- section, irregular indentations and a central cavity of up to 20 mm in diameter [30].
Corpora lutea with a central cavity are normal cyclical structures and therefore regress after 10 to 14 days. They are not always easily differentiated from luteal cysts.
The sensitivity of diagnosing active luteal tissue in a follicle wall using B-mode sonography was 61.5% in the present study, sensitivities of 31.5 [29], 86.7 and 91.5% [31, 32] have been reported in other studies. Possible reasons for the marked variation are different cut-off values for P4 and different examination intervals. By contrast, the specificity of B-mode sonography was 100%, which means that all follicles determined to be luteinized via sonography also had high P4 concentrations.
In relation to the relatively low sensitivity, it should be noted that some follicular walls
<3 mm were associated with a P4 concentration >1.0 ng/ml. Therefore, the cut-off for wall thickness of 3 mm may have been chosen incorrectly and may have to be lowered. Another possible explanation is that the CL went undetected in one of the ovaries. The sensitivity of diagnosing luteal tissue using colour Doppler sonography was 92.3% in the present study, which was considerably higher than that of B-mode sonography. This indicated that many cows with a follicular wall thickness of <3 mm and a P4 >1.0 ng/ml had a greater follicular blood flow than cows with follicles without luteal tissue. This confirmed the findings of Miyamoto et al. [33] and points to increased blood flow in luteal cysts compared with follicular cysts. In luteal cysts, the theca interna becomes luteinized, and the luteal cells appear histologically similar to those of an intact CL [34-36]. A well-developed vascular network surrounds the large luteal cells of a cyst [34], and this probably supports luteinization of the thecal cells [35]. Active angiogenesis associated with luteinization has also been observed in colour Doppler sonographic studies of the early CL that have shown a progressive increase in perfused area and blood flow velocity [23]. The perfused area of a 5-day- old CL is several-fold higher than that of an ovulatory follicle [23].
Administration of a synthetic GnRH analogue is the treatment of choice for follicular cysts [7] and has a success rate of approximately 80% [7]. An increase in plasma luteinizing hormone (LH) concentration always occurs and can be detected as early as 30 min after administration of GnRH or an analogue to cows with COFs [37-39].
This indicates that the mechanism of LH release from the pituitary is not altered in cows with COFs. However, an increase in the LH concentration 2 h after GnRH does not necessarily amount to a successful treatment. In one study, although all the cows
had an increase in LH concentration, three of the 25 cows did not form a CL nor did the wall of the cyst luteinize, and three others had delayed CL development [40].
LH has been shown to increase ovarian blood flow in rats [41], rabbits [42] and sheep [43]. Transrectal Doppler sonography has been used to show an increase in blood flow to the follicular wall after administration of a GnRH analogue in cows; the blood flow to the ovulatory follicle increased simultaneously with an acute increase in the plasma LH concentration [23]. These observations suggest an association between administration of GnRH and ovarian blood flow. Most likely, administration of GnRH results in an LH peak, which in turn leads to an increase in ovarian blood flow.
However, this proposed sequence of events was not substantiated in all the cows with COFs in the present study. Some cows had almost no change in blood flow to the cystic follicular wall 30 minutes after GnRH administration. Some cows had an increase and others a decrease in the perfused area. Failure of LH to induce an increase in blood flow may have resulted from structural or functional differences between normal dominant follicles and COFs.
Comparison of follicular blood flow before and after treatment allowed us to predict, to a certain extent, response to treatment. In contrast to the non-responding cows, the responding cows tended to have an increase in the perfused wall area. The responding cows having no clear increase in blood flow probably did not respond to the GnRH analogue. Luteinization or ovulation that occurred without an increase in blood flow may not have been attributable to hormonal treatment but may have occurred spontaneously.
Conclusion
In conclusion, the perfused area more accurately reflects active luteal tissue than wall thickness. Thus, colour Doppler sonography is superior to B-mode sonography for differentiating follicular and luteal cysts and aids in the selection of treatment.
However, exact prediction of COFs destined to regress or persist and the response of COFs to treatment with a GnRH analogue were not possible using colour Doppler sonography.
Figures and tables
Fig. 1 Schematic illustration of the examination and treatment at different time points (Ex-1,
Ex-2, Ex-3, Ex-4) for the identification of follicles that were present for less (temporary) than or at least ten days (persistent).
Bl = Blood sampling; B = B-mode sonography; DS = Doppler sonography;
a = time interval of 10 to 12 days; b = time interval of 30 minutes.
GnRH was given only to cows with follicles that persisted from Ex-1 to Ex-2.
Bl B DS GnRH
E3 E4
E2 E1
DS Bl
B DS
Bl B
a b a
Ex-1 Ex-2 Ex-3 Ex-4
Fig. 2 Schematic illustration of the assignment of animals (only cows with follicles ≥ 15 mm in
diameter) to groups at different time points (Ex-1, Ex-2, Ex-4).
Temp = follicles present less than ten days; (temporary); Persist = follicles persisting for at least ten days = COFs (persistent); NoLut = follicles without luteal tissue (P4 ≤1ng/ml); Lut = follicles with luteal tissue (P4 >1ng/ml).
Responding = cows with a CL or luteinization at Ex-4; Non-responding = cows without a CL or no luteinization at Ex-4.
* Measurement of blood flow not possible in one animal at Ex-1.
Ex-1
Ex-2
Ex-1
Ex-2
Ex-4
Fig. 3 Colour Doppler sonographic image of a luteinized cyst with blood flow (coloured
pixels) in the cyst wall.
The white line delineates the region of interest (ROI), used for measurements of blood flow (coloured pixels).
Fig. 4 Relative changes in blood flow (coloured area [CA]) in the follicle wall before (Ex-2)
and 30 minutes after treatment (Ex-3) with GnRH, shown as dotted line and bars, respectively.
Black bars represent the cows with a positive ovarian response ten days after treatment (n=12). White bars show the cows with no response (n=5).
Table 1 Total area of follicle (TA), thickness of wall (TW) and blood flow (coloured area [CA]) on the
day of first examination (Ex-1). Follicles were categorised into temporary (Temp) follicles, persistent (Persist) follicles and, on the basis of the P4 concentrations, follicles with luteal tissue (Lut) or without luteal tissue (NoLut). Data are expressed as median (Med), median absolute deviation (MAD), minimum (Min) and maximum (Max).
Lut/
NoLut Med MAD Min Max Med MAD Min Max Med MAD Min Max
Temp Persist Temp + Persist
NoLut n 23 23 23 23 17 17 17 17 40 40 40 40
TA (cm²) 3.2 0.9 1.7 11.2 4.1 2.3 1.5 16.1 3.6* 1.5 1.5 16.1 TW (mm) 1.0 0.2 0.5 2.6 0.9 0.1 0.7 1.5 1.0* 0.3 0.5 2.6 CA (cm²) 0.4 0.1 0.2 0.8 0.4 0.2 0.1 0.9 0.4* 0.1 0.1 0.9
Lut n 8 8 8 8 5 5 5 5 13 13 13 13
TA (cm²) 8.5 1.8 5.2 11.5 10.2 3.4 3.2 14.4 8.6# 1.9 3.2 14.4 TW (mm) 5.2a 1.4 1.1 7.0 1.4 b 0.4 1.0 4.1 3.2# 2.1 1.0 7.0 CA (cm²) 1.8 0.1 1.4 2.5 1.6 0.5 0.7 2.6 1.8# 0.3 0.7 2.6
NoLut+Lut n 31 31 31 31 22 22 22 22 53 53 53 53
TA (cm²) 3.9 1.9 1.7 11.5 6.1 3.3 1.5 16.1 4.7 2.3 1.5 16.1 TW (mm) 1.2 0.4 0.5 7.0 1.0 0.2 0.7 4.1 1.1 0.3 0.5 7.0 CA (cm²) 0.5 0.2 0.2 2.5 0.44 0.3 0.1 2.6 0.5 0.3 0.1 2.6
Within rows and columns, values with different superscripts differ (P≤0.05 and P≤0.01, respectively).
Table 2 Reliability of diagnosis of follicles with (Lut) and without (NoLut) luteal tissue using B-
mode (B) and Doppler (DS) sonography compared to plasma progesterone concentration (cut-off = 1 ng/ml), expressed as positive predictive value (PPV), negative predictive value (NPV), sensitivity (Se) and specificity (Sp).
n a b c d PPV NPV Se Sp
NoLut B 54 41 5 0 8 89.1 100 100 61.5
Lut B 54 8 0 5 41 100 89.1 61.5 100
NoLut DS 53 40 1 0 12 97.6 100 100 92.3
Lut DS 53 12 0 1 40 100 97.6 92.3 100
The gold standard for B-mode sonography was wall thickness with a cut-off-point of 3 mm.
The gold standard for Doppler sonography was a coloured area with a cut-off-point of 0.9 cm².
a = true positive; b = false positive, c = false negative; d = true negative.
PPV, [a/(a+b)]*100; NPV, [d/(c+d)]*100; Se, [a/(a+c)]*100; Sp, [d/(d+b)]*100.
Table 3 Blood flow area (CA) in the walls of follicles without luteal tissue (NoLut) before and
30 minutes after treatment with GnRH. Cows with a CL or luteinization of the largest COF were classified as responding to treatment (responding) and cows without a CL or luteinization as non-responding.
n Med MAD Min Max
Responding and non responding
Before GnRH 17 0.4 0.2 0.1 1.3
30 min after GnRH 17 0.4 0.2 0.1 1.5
Difference / / / 0.2
Responding
before GnRH 12 0.5 0.3 0.1 1.3
30 min after GnRH 12 0.6 0.2 0.3 1.5
difference 0.1 0.1 0.2 0.2
Non responding
Before GnRH 5 0.4 0.1 0.2 0.8
30 min after GnRH 5 0.3 0.1 0.1 0.9
Difference 0.1 / 0.1 0.1
Data were expressed as median (Med), median absolute deviation (MAD), minimum (Min) and maximum (Max).
III. Zusammenfassung
Alexandra Rauch (2008)
Farbdopplersonographische Untersuchungen an zystischen Ovarfollikeln beim Rind
Hintergrund: Das Ziel dieser Studie war es herauszufinden, ob sich die Farbdopplersonographie eignet, temporär vorkommende von persistierenden Ovarfollikeln zu unterscheiden, zwischen Follikeln mit Lutealgewebe und ohne Lutealgewebe zu differenzieren und bei Follikelzysten frühzeitig Aussagen über den mittels eines GnRH Analogons zu erwartenden Therapieerfolg machen zu können.
Material und Methode: Es wurden 54 Tiere, welche follikuläre Strukturen auf den Ovarien mit einem Durchmesser von mehr als 15 mm und kein Corpus Luteum besaßen, in die Studie aufgenommen. Diese Kühe wurden mittels B-Bild- und Dopplersonographie untersucht. Zehn bis zwölf Tage später wurde die Untersuchung wiederholt und Tiere mit Follikelzysten (n=17) mit einem GnRH Analogon behandelt.
Der follikuläre Blutfluss wurde vor und eine halbe Stunde nach der Therapie gemessen. Nach weiteren zehn bis zwölf Tagen wurde der Erfolg der Therapie mittels B-Bild-Sonographie überprüft. Als Therapieerfolg wurde das Vorhandensein von Lutealgewebe gewertet. Zu jedem Untersuchungszeitpunkt wurden Blutproben entnommen, um den Progesterongehalt (P4) im Plasma zu bestimmen.
Ergebnisse: Insgesamt bildeten sich zwischen erster und zweiter Untersuchung 31 von 54 Follikeln spontan zurück (temporäre Follikel). Bei den 23 persistierenden Follikeln wurde die Diagnose zystischer Ovarfollikel (ZOF) gestellt. Es gab keine Unterschiede in der Querschnittsfläche, in der Wanddicke und in der durchbluteten Fläche zwischen temporären Follikeln und ZOFs. In 13 von 54 Fällen wurde bei der Erstuntersuchung anhand des Progesteronspiegels ein luteinisierter Follikel diagnostiziert. Bei den luteinisierten Follikeln war die Querschnittsfläche doppelt so groß, die Wand dreimal so dick und die durchblutete Fläche um das 4,5-fache größer als bei den nicht luteinisierten Follikeln (n=41). Die Sensitivität für die Diagnose
„luteinisierter Follikel“ lag bei der B-Bild-Sonographie bei 61,5% und bei der Dopplersonographie bei 92,3%. Bei zwölf der 17 mit GnRH behandelten Tiere war
die Therapie erfolgreich. Diese Tiere zeigten eine halbe Stunde nach der GnRH- Injektion tendenziell (P=0,07) einen höheren Blutfluss in der Zystenwand als vor der Therapie.
Schlussfolgerung: Die Ergebnisse zeigen, dass aktives Lutealgewebe über die durchblutete Fläche besser bestimmt werden kann als über die Wanddicke. Somit eignet sich die Dopplersonographie besser als die B-Bild Sonographie, Follikel- von Luteinzysten zu unterscheiden und hilft bei der Auswahl der geeigneten Therapie.
Jedoch sind mit Hilfe der Dopplersonographie weder exakte Aussagen darüber zu treffen, ob Follikel persistieren oder sich spontan zurückbilden werden, noch ob die Therapie mit einem GnRH Analogon erfolgreich sein wird.
Schlüsselwörter: Doppler, Blutfluss, Zystische Ovarfollikel, Rind, GnRH
IV. Erweiterte Zusammenfassung
Zystische Ovarfollikel (ZOFs) sind mit einer Inzidenzrate von 5,2 bis 27,9% [1-7]
einer der wichtigsten Gründe für eine herabgesetzte Fruchtbarkeit in Milchvieh- herden. Obwohl früh auftretende ZOFs sich oft wieder spontan zurückbilden [5, 8, 9], führen sie zu einer Verlängerung der Güstzeit um 22 bis 77 Tage [2, 3, 10] und haben damit doch einen erheblichen Einfluss auf die Herdenfruchtbarkeit.
Als ZOFs werden gewöhnlich anovulatorische Follikel mit einem Durchmesser von mehr als 25 mm, ohne Vorhandensein eines Corpus luteums und mit einer Persistenz von mehr als zehn Tagen [7, 11] bezeichnet. In mittels B-Bild Sonographie durchgeführten Studien wurden bereits Follikel ab einem Durchmesser von 20 mm [12, 13], bzw. sogar ab 17 mm [14, 15] als ZOFs definiert, wobei diese mindestens sieben Tage persistieren mussten. Man unterscheidet üblicherweise zwischen Follikelzysten und Luteinzysten. Erstere besitzen eine Wanddicke von weniger als 3 mm und einen Progesteronspiegel (P4) von höchstens 1 ng/ml; letztere weisen eine Wanddicke von 3 mm und größer und einen P4 Wert von mehr als 1 ng/ml auf [16].
Die Genauigkeit ZOFs zu unterscheiden hängt von der verwendeten Methode ab.
Mittels rektaler Palpation werden für Follikel- bzw. Luteinzysten jeweils positive prädiktive Werte (PPV) von 66% erreicht [17]. Mit Hilfe der B-Bild-Sonographie erzielt man für Follikelzysten einen PPV von 74%, für Luteinzysten sogar von 85% [17].
Fehldiagnosen bezüglich der beiden Zystenformen führen zu ungeeigneten Therapieansätzen [6]. Während Follikelzysten mit Hormonpräparaten wie hCG oder GnRH behandelt werden, stellen PGF2α und seine Analoga die Mittel der Wahl zur Behandlung von Luteinzysten dar [20].
Das Ziel dieser Studie war es herauszufinden, ob sich Farbdopplersonographie eignet um normal zyklische Follikel von zystischen Ovarfollikeln und Follikelzysten von Luteinzysten zu differenzieren und um frühzeitig Aussagen über die Effektivität einer GnRH-Behandlung zu machen.
Die sonographischen Untersuchungen erfolgten an insgesamt 54 Tieren mit einem tragbaren LOGIQ Book XP (General Electrics Healthcare, Solingen, Germany) und einem 6 bis 10 MHz Linearschallkopf (General Electrics Yokogawa, Medical Systems, Ltd., Model 1739-RS, Japan) in vier Milchviehherden in Nord- und Ostdeutschland. Um Kühe mit zystischen Ovarfollikeln zu erfassen wurden Tiere der vier Herden ausgewählt, bei denen die Geburt mindestens 28 Tage zurücklag und bei denen mindestens ein Follikel mit einem Durchmesser von mehr als 15 mm und kein Corpus luteum auf den Ovarien darstellbar war. Bei Kühen mit einem derartigen Befund wurde bei dieser Erstuntersuchung (Ex-1) die Durchblutung in der Wand des größten Follikels im Power Doppler Modus aufgenommen (Fig. 1). Dieser Modus gibt die Anzahl der Erythrozyten wieder, die pro Zeiteinheit durch ein Gefäß fließen.
Später wurde mit einem Bildbearbeitungsprogramm die farbige Fläche als Maß für die Follikeldurchblutung bestimmt (Fig. 3). Zusätzlich wurden die Wanddicke und die Querschnittsfläche des größten Follikels anhand von B-Bildern gemessen. Zehn bis zwölf Tage später (Ex-2) wurden die Ovarien erneut sonographisch untersucht (Fig. 1). Follikel, die ovulierten, luteinisierten oder sich zurückbildeten wurden als temporäre Follikel (temp) bezeichnet. Blieb die Größe des größten Follikels gleich oder nahm sie zu, wurde dieser als persistierender Follikel (persist) definiert und die Blutflussmessung wiederholt. Anschließend wurden dem Tier 20 µg des GnRH – Analogons Buserelin (Receptal®, Intervet, Unterschleißheim) intramuskulär verabreicht und die Blutflussmessung eine halbe Stunde später wiederholt. Zehn bis zwölf Tage nach der Applikation von Buserelin wurde der Therapieerfolg mittels B- Bild-Sonographie überprüft (Fig. 1). War dabei ein Corpus luteum oder ein luteinisierter Follikel nachweisbar, wurde dies als Therapieerfolg (responding) angesehen. Alle anderen Befunde wurden als Therapiemisserfolg (non-responding) gewertet.
An jedem Untersuchungstag wurde eine Blutprobe entnommen und der Progesterongehalt (P4) im Blutplasma nach der von Prakash [26] beschriebenen Methode mittels EIA bestimmt. P4-Konzentrationen von mehr als 1,0 ng/ml definierten Follikel und Zysten mit Lutealgewebe; P4-Konzentrationen von höchstens 1,0 ng/ml definierten Follikel und Zysten ohne Lutealgewebe.
Von den 54 Follikeln, welche bei Ex-1 festgestellt wurden, waren zum Zeitpunkt Ex-2 31 (57,4%) nicht mehr nachweisbar (Fig. 2). In anderen Studien bildeten sich 61%
[8], 48% [9] und 38,6% [5] der ZOFs spontan zurück. Bei 23 (42,6%) der Follikel war eine Persistenz von mehr als zehn Tagen zu beobachten und damit konnte bei diesen die Diagnose zystischer Ovarfollikel (ZOF) gestellt werden.
Die Querschnittsfläche des größten Follikels zum Zeitpunkt Ex-1 variierte zwischen 1,5 cm² und 16,1 cm², was einem Durchmesser von 13,8 mm und 45,3 mm entspricht (Table 1).
Bei 13 der 54 Follikel zu Ex-1 handelte es sich um luteinisierte Follikel (Fig 2). Diese Häufigkeit liegt im Bereich anderer Untersuchungen [28, 29].
Weder die Dicke der Wand, die Querschnittsfläche noch die durchblutete Fläche des Follikels gaben eindeutige Hinweise darauf, ob sich ein Follikel spontan zurückbildet oder weiterhin bestehen bleibt. Dieses Ergebnis ist vergleichbar mit denjenigen von Kawashima et al. [24], welche zwischen ovulierenden und persistierenden Follikeln keine Unterschiede in der Größe und Durchblutung feststellen konnten.
Eine zuverlässige Differenzierung war mittels Farbdopplersonographie zwischen luteinisierten Follikeln und Follikeln ohne Lutealgewebe möglich. Der Blutfluss war in luteinisierten Follikeln deutlich höher als in Follikeln ohne Lutealgewebe. Die Sensitivität, luteinisierte Follikel zu diagnostizieren lag mittels Farbdopplersonographie bei 92.3%, mittels B-Bild-Sonographie nur bei 61.5%
(Table 2).
Die Injektion mit GnRH führte bei zwölf Tieren zum Erfolg, bei fünf Tieren blieb dieser aus. Die Behandlung von ZOFs mit GnRH oder seinen Analoga führen immer zu einem LH Anstieg im Plasma, der bereits eine halbe Stunde post injectionem nachweisbar ist [23-25]. Bei ovulatorischen Follikeln konnte eine halbe Stunde nach Einwirkung eines GnRH Analogons neben dem LH-Anstieg im Plasma ein gesteigerter follikulärer Blutfluss dopplersonographisch aufgezeigt werden [23]. Die Vermutung liegt deshalb nahe, dass GnRH Injektionen einen LH Peak auslösen und dieser zu einer Steigerung des Blutflusses am Ovar führt. Dass diese Hypothese auch bei ZOFs gilt, konnte von uns nicht in jedem Fall bestätigt werden: Anhand des
Blutflusses konnte nur tendenziell (P=0.07) der Therapieerfolg hervorgesagt werden.
Acht der zwölf erfolgreich behandelten Tiere zeigten eine halbe Stunde nach der Behandlung tatsächlich einen Anstieg des prozentualen Blutflusses. Bei den restlichen vier Tieren war der follikuläre Blutfluss dagegen abgefallen (Fig. 4).
Zusammenfassend zeigt diese Untersuchung, dass die Follikeldurchblutung keinen Hinweis liefert, ob ein Follikel wirklich persistieren oder sich spontan zurückbilden wird. Die Dopplersonographie eignet sich besser als die B-Bild Sonographie, Follikel- von Luteinzysten zu unterscheiden und stellt damit ein hilfreiches Mittel dar um die richtige Therapie auszuwählen. Die Veränderung der durchbluteten Fläche in der Zystenwand lässt aber keine exakte Aussage über den Erfolg einer Therapie mit einem GnRH Analogon zu.
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VI. Eigene Publikationen und Koautorenschaften im Rahmen dieser Arbeit
1. Rauch A, Krüger L, Miyamoto A, Bollwein H. Colour Doppler sonography of cystic ovarian follicles in cows. Journal of Reproduction and Development (JRD) 2008; 54; 6.
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Budapest, Hungary. Abstract 378.
VII. Danksagung
Herrn Prof. Dr. Bollwein danke ich für die Überlassung des interessanten Themas und die Anleitung zum wissenschaftlichen Arbeiten. Seine allzeit gewährte Unterstützung und seine guten Ideen haben mich motiviert und an die Arbeit glauben lassen.
Besonders bedanken möchte ich mich bei Herrn Dr. Lars Krüger für seinen Beistand in schwierigen Situationen, sein sonniges Gemüt und seinen Scharfsinn bei der Durchführung des praktischen Teils und bei der Durchsicht der Arbeit.
Herzlichst bedanke ich mich bei den Betriebsleitern für die Bereitstellung von Datenmaterial, Versuchstieren und den Mitabeitern der Betriebe für ihre Hilfsbereitschaft. Hervorheben möchte ich an dieser Stelle Herrn S. Ludwig aus Dedelow und Herrn H.-G. Sander aus Mariensee, die mir das Arbeiten im Betrieb ungemein erleichtert haben.
Allen Assistenten der Klinik für Rinder, die für mich ein offenes Ohr hatten, möchte ich ein besonderes Dankeschön aussprechen. Im Besonderen Dr. Nicola Beindorff für die Betreuung im Labor und Dr. Kathrin Herzog, Jörn Dettmer und Dr. Georgios Tsousis in Computerfragen.
Meinen Mitdoktoranden danke ich für die unvergessliche Zeit; besonders der Rikli Doks Kochtruppe für die wunderbaren Mittagspausen, Anne für die gute Zusammenarbeit, Anika für die aufmunternden Autofahrten, Claudia für die Kaffeepausen in Mariensee, Katrin für ihre liebenswerte Unterstützung in Dedelow und Yvonne für ihre kompetente Hilfe im Labor.
Meinen Mitbewohnern Olly und Kai gilt besonderer Dank für ihre unendliche Geduld bei der Rettung aus Statistik- und Computerkrisen.
Kai danke ich, dass er mir Kraft gegeben und den Rücken in der Endphase freigehalten hat.
Ein großes Dankeschön geht an meine Freunde Alexandra, Christina, Sybille und Peter für die Hilfe beim Fertigstellen und Einreichen des Manuskriptes.
Bei meiner Oma und meinen Eltern möchte ich mich bedanken, dass sie immer für mich da waren und sie mir über die ganzen Jahre blind vertraut und an mich geglaubt haben.
