Detection of pregnancy and monitoring patterns of uterine and fetal growth in the marmoset monkey (Callithrix jacchus) by real-time ultrasonography.код для вставкиСкачать
American Journal of Primatology 361-13 (1995) RESEARCH ARTICLES Detection of Pregnancy and Monitoring Patterns of Uterine and Fetal Growth in the Marmoset Monkey (Callithrixjacchus) by Real-Time Ultrasonography A.-K. OERKE, A. EINSPANIER, AND J.K. HODGES Department of Reproductive Biology, German Primate Centre, Gottingen, Germany The purpose of this study was to evaluate the potential of real-time ultrasonography for detecting and monitoring pregnancy in the common marmoset, Ultrasound was performed transabdominally on unsedated females using a 7.5 MHz linear or intraoperative sector probe. Pregnancies were timed using progesterone measurements in twice weekly blood samples to determine the day of ovulation. Eight pregnancies were examined three times a week until day 30, twice a week until day 80, and once a week until birth. Detection of pregnancy was possible on day 15, on average, by the appearance of a double endometrial echo indicating fluid accumulation in the uterus. The uterine lumen thus formed was first measurable on day 21 on average. Diameter of the pregnant uterus was significantly greater than that of the nonpregnant organ by day 38. Thereafter, the dimensions of uterus and uterine lumen showed a similar pattern of growth until day 75, when measurements became inaccurate due to increasing uterine pliability. Embryonic development was characterized by the initial expansion of the gestation sacs and the appearance of individual embryos by day 33. Detection of heart beat a t approximately day 54 allowed confirmation of number and viability of the embryos. With the visualization of the skulls by day 82, it was possible to determine fetal position and to monitor fetal growth by measurement of the biparietal diameter. All pregnancies were of normal length (140-145 days) resulting in viable offspring. It can be concluded that ultrasonography is suitable for detecting and monitoring pregnancy in the marmoset monkey and as a non-invasive method, has the potential for being routinely used in this and other species of Callitrichidae. o 1995 Wiley-Liss, Inc. Key words: pregnancy, ultrasonography, marmoset monkey INTRODUCTION The common marmoset (CuZZithrixjucchus) is a small New World monkey of the family Callitrichidae. Due to its adaptability to, and fertility in captivity, it is widely used as an experimental animal in biomedical research and, more recently, Received for publication February 28, 1994;revision accepted September 19,1994. Address reprint requests to Ann-Kathrin Oerke, Department of Reproductive Biology, German Primate Centre, Kellnerweg 4,37077 Gdttingen, Germany. 0 1995 Wiley-Liss, Inc. 2 / Oerke et al. in conservation biology as a model for endangered species. For both research and colony management purposes, practical and reliable methods for detecting and monitoring pregnancy are needed. Since female marmosets show neither menstruation nor estrus swellings and mating occurs throughout most of pregnancy [Hearn & Lunn, 19751, there are no reliable visible signs of reproductive status. Consequently, endocrine methods based on either plasma or urinary hormone analysis [Chambers & Hearn, 1979; Harlow et al., 1983; Eastman et al., 1984; Heger & Neubert, 19881have been used for this purpose. With respect to pregnancy detection, hormone determinations can be informative from day 16-20 onwards [Hodges et al., 1983; Hearn et al., 19881, but require serial blood sampling and are generally time consuming and expensive to perform. A much simpler diagnosis of pregnancy is possible by abdominal uterine palpation [Hearn & Lunn, 1975; Mitchell &Jones, 1975; Phillips & Grist, 19751 although the method can only be used with confidence four weeks after ovulation. Neither approach, however, allows accurate staging of pregnancy and their usefulness for assessing embryonic development, and fetal growth is limited. To date, the only opportunity to obtain this information has been from studies on material collected during surgical and/or terminal procedures [Phillips, 1976b; Chambers & Hearn, 1985; Smith et al., 1987; Merker et al., 1988; Moore et al., 19881. An alternative for detecting and monitoring pregnancy, as has been proven in human and domestic animals, is the use of real-time ultrasonography. In a non-invasive way this technique enables direct visualization of the reproductive organs, providing immediate and reproducible results. Among non-human primates, ultrasound has been mainly applied to macaques [Peterson et al., 1972; Sabbagha et al., 1975; Nyland et al., 1984; Cho et al., 1987; Korte et al., 1988; Tarantal & Hendrickx, 1988a, 1988b, 1988~; Shimizu, 1988; Conrad et al., 19891. Its potential for use with the much smaller New World monkeys, Callitrichidae, has not been accurately assessed. An initial attempt at using ultrasound for examination of pregnancy in the marmoset is mentioned in Du Boulay and Wilson [19881, but details of the study are not given. The aims of the present investigation were, therefore, (i) to evaluate the potential of ultrasonography for detecting and monitoring pregnancy in the marmoset monkey, and (ii) to provide additional information on the characteristics of embryonic development and fetal growth in this species by use of a non-invasive method. METHODS Animals A total of eight pregnancies was examined in five marmoset monkeys. Females lived with males either as pairs or in family groups. Four females were multiparous having had normal pregnancies resulting in viable offspring. One female was nulliparous prior to the onset of the study. Animals were maintained in the primate facilities at the German Primate Centre under conditions described by Heistermann et al. [19931. Timing of Pregnancy Ovarian cycles were monitored by measurement of progesterone in blood samples taken twice a week. Plasma was assayed by a direct, non-extraction enzymeimmunoassay [Hodges et al., 19881modified by Heistermann et al. [19931.The day of ovulation (day 0) was defined as the day before progesterone levels increased above a value of 10 ng/ml as originally reported by Harlow et al. . The day after ovulation (day 1) was taken to be the first day of pregnancy. Due to the Ultrasonography of Pregnancy in the Marmoset / 3 6 Fig. 1. Diagrammatic representation of a marmoset uterus in cross section as visualized by ultrasound. 1 = abdominal wall (arrow indicates direction of scan); 2 = uterus; 3 = endometrium; 4 = uterine lumen; 5 & 6 indicate measurements of ventro-dorsal and transverse diameter, respectively. frequency of blood sampling however, an error of * 1day in estimating the time of ovulation and therefore gestation age is to be expected. Ultrasonography Females were examined by ultrasound three times a week until day 30, twice a week between day 30 and 80, and once a week from day 80 until birth. Ultrasonography was performed transabdominally on unsedated and unshaved animals. In order to minimize movements during the scanning procedure, the females were restrained on a padded frame to which they had been conditioned during a training period prior to the study. Animals tolerated the device well and appeared relatively relaxed. Fruit or pap was offered as a reward during each examination. Ultrasonography was carried out with a Picker CS 9000 real-time scanner fitted with two probes operating a t a frequency of 7.5 MHz. An intraoperative sector probe was used to provide detailed images of early pregnancy, whereas a linear probe was preferred for surveys of late gestational stages. Examinations took 5-10 minutes. After warmed transmission gel had been applied thickly to the abdomen of the animal the probe was placed perpendicular to the Iong axis of the female in the deep pelvic region and moved slowly in a cranial direction. Uterine appearance was documented and images were printed on photopaper and recorded on videotape. All measurements were made from cross-sections of the uterus (see Fig. 1).Diameter (ventro-dorsal and transverse) and circumference of the uterus were taken with internal calipers, the values of which were used by the system to automatically calculate volume (method of ellipse) and area. Embryonic development was monitored and fetal growth recorded by regular measurements of the transverse diameter of the skulls (biparietal diameter = BPD). Neonates were weighed and measured for comparison with data for offspring not exposed to ultrasound during intrauterine development. Data Analysis Statistical comparisons between uterine measurements in pregnant and nonpregnant females were made using an unpaired t-test. A P value of <0.05 was taken to indicate a significant difference. 4 / Oerke et al. Fig. 2. Ultrasonogram of the uterus in a nonpregnant (a) and a n early pregnant (b)marmoset monkey. Note the central linear echo in the nonpregnant organ in contrast to the presence of a double endometrial echo during early pregnancy (arrows). Uterine dimensions in both (a) and (b) are 6 x 8 mm as measured in ventro-dorsal and transverse section. RESULTS Detection of Pregnancy In ultrasound images, the uterus of nonpregnant marmosets appeared as a dark, oval structure giving a sparse echo. The nonpregnant organ was characterized by the presence of a single central bright line reflecting the close apposition of the endometrial surface (Fig. 2a). The mean diameter of the nonpregnant uterus during the luteal phase of the cycle was 5.7 2 1.0 mm and 8.2 & 0.8 mm as measured in ventro-dorsal and transverse sections, respectively (n = 20 from 6 animals). The first detectable change in the ultrasound image of the uterus during pregnancy was the appearance of a double endometrial echo as the endometrial surface began to be separated by the fluid-containing, and therefore, weakly echogenic uterine lumen. This can be seen in Figure 2b as a double bright line separated by a dark region in the centre. The figure provides an example of the appearance of the uterus in a pregnant female 20 days after ovulation. The time at which a double endometrial echo was first visible in the eight pregnancies studied ranged between 12 and 18 days after ovulation, with a mean 2 SD of 15.2 2 2.5 days. Changes in Uterine Measurements The diameter of the uterus was recorded during the first 90 days of pregnancy. Changes in the transverse dimension are shown in Figure 3a and 3b for mean and individual data, respectively. Results for measurements in ventro-dorsal section (mean values) are illustrated in Figure 5. No obvious increase in uterine diameter by either measurement was discernable during the first month of pregnancy after which the uterus displayed a continuous growth pattern. Mean values for uterine diameter during gestation were significantly elevated over those in nonpregnant females on day 38 in both dimensions. In general, the ventro-dorsal dimension (Fig. 5) was smaller and showed a less pronounced increase when compared to the transverse diameter (Fig. 3a). As the endometrial surface began to separate during early pregnancy, a small fluid-filled lumen within the uterus became visible (Fig. 2b). On average, the uterine lumen was first measurable on day 21 (mean ? SD; 20.7 ? 2.3 days) with Ultrasonography of Pregnancy in the Marmoset I 5 45 50 40 1 Mean f S.D. (n = 8 ) - n 2 35- v 30 - 3 25- s 2 .,-I k s 20 - 15 - NP lo- 1 I 5 01 a I I I I I I I I I I I 0 10 20 30 40 50 60 70 80 90 100 50 45 o - Singleton A 40 - V 0 A T . Twins Triplets h g 35- k 4 30- A 5 V *. s 8 &% Z.X&BX n$$*Gg"" l o5 - T V 8". m # b A yAm VAV A 15 - 01 0 *llTxA im 20 - .rl 5 o h .P 25- .A 2 vn . A mm v v 0 V I I I I I I I I I I I 0 10 20 30 40 50 60 70 80 90 100 Days of pregnancy Fig. 3. Changes in transverse uterine diameter during the first 90 days of pregnancy in the marmoset monkey. Mean values ( * SD) of eight pregnancies are shown (a).Data have been plotted at 5-day intervals. Mean value (t SD) for the diameter of the nonpregnant uterus (NP, ). is given for comparison. Corresponding data for individual pregnancies are shown (b) 0 singleton, A V 0 twins, A V triplets. 6 / Oerke et al. 40 L P) r 17 2 5 I- -- 4 ; 20 d 0 a I I I I I I I 1 I I 10 20 30 40 50 60 70 80 90 100 a 40 o 35 E E v k Singleton A V 0 a A V . Twins Triplets v . 30 25 ;20 A. 0 0 . AA d .a . ma 15 0 E fie 10 T mmv ... A c V17vA VA *>aV V mM A ro om I CPA A AV A 0) 5 A 0 v a. 0 a ma P) 4 El A I 0 0 V 0 0 . V OA w a m ~ o v o am m a P v 5 - 8 0 n worn D n-worn I 0 b - 0 10 O Il U 20 - 0 0 I 1 I I I I I 30 40 60 60 70 80 90 I 100 Days of pregnancy Fig. 4. Changes in transverse diameter of the uterine lumen during the first 90 days of pregnancy in the marmoset monkey. Mean values ( L SD) of eight pregnancies are shown (a).Data have been plotted at 5-day intervals. Corresponding data for individual pregnancies are shown (b) 0 singleton, A V 0 twins, A 7 m triplets. Ultrasonography of Pregnancy in the Marmoset / 7 25 - uterus T o uterine lumen 20 h 1 1 5 - v h 4 al 10 - N P a 1 5 - 0 I 0 10 20 30 40 I I I I I I 50 60 70 80 90 100 Days of pregnancy Fig. 5. Pattern of growth of the uterus (0 external diameter) and uterine lumen (0 internal diameter) as indicated by measurements in the ventro-dorsal section during the first 90 days of pregnancy in the marmoset monkey. Values are mean i SD (n = 8).Mean (? SD) ventro-dorsal diameter of the nonpregnant uterus (NP, B) is given for comparison. Note relatively constant relationship between external and internal measurements, indicating lack of change in thickness of uterine wall. a range between day 17 and 24. Its dimension when first measurable was 1 mm in ventro-dorsal and 2 mm in transverse section. Only slight expansion of the uterine lumen occured until day 30 of gestation after which a progressive increase in diameter was seen. Changes in the diameter of the uterine lumen during the first 90 days of pregnancy are shown in Figures 4a (transverse section) and 5 (ventrodorsal section). Comparison of the growth pattern of the uterus and uterine lumen (Fig. 5) indicates that the uterine wall maintained its thickness throughout the period studied. In all measurements taken, an increase in variability was noted with advancing pregnancy (see Figs. 3, 4, and 51, probably due to the increasing pliability of the uterus beyond day 70. Although data for individual animals indicate a tendency for all dimensions recorded to be larger in triplet than in twin or singleton pregnancies (see Figs. 3b and 4b), the data pool was too small to permit statistical analysis. Measurements after day 90 were not possible due to the size of the image of the uterus exceeding that of the ultrasound monitor. Embryonic and Fetal Development Within the first month of pregnancy, the increase in size of the uterine lumen largely reflects the growth of the fluid-filled gestation sacs. The presence of multiple gestation sacs was notable by the appearance of fine echos representing the interface of adjoining structures. Individual embryos first became visible on day 33 (mean ? SD; 33.2 t 3.1 days, range 28-38) and were seen as small, bright structures within the dark uterine cavity. Confirmation of the number and viability of the embryos was possible by detection of embryonic heart beat from on average day 54 of pregnancy (mean 2 SD; 54.2 t 2.0 days, range 52-57). 8 I Oerke et al. Fig. 6. Ultrasound image of twin fetal heads (facing left) on day 112 of pregnancy. Skulls appear of different size due to differing positions of fetuses within the uterus. Accurate measurement of body structures was difficult due to the usual presence of multiple embryos or fetuses. Individual fetal skulls could, however, be identified and measured from day 82 of pregnancy (mean r SD; 81.9 f 2.8 days, range 76-84). The image of two fetal heads on day 112 of pregnancy is given in Figure 6. The pattern of growth of the fetal skulls as determined by measurements of transverse diameter (biparietal diameter = BPD) is shown in Figure 7. The initial rise in BPD appears to occur at a faster rate than that seen over the last month of gestation. No obvious differences between BPD measurements for singleton, twin, or triplet fetuses were apparent (Fig. 7b, mean values for twins and triplets shown). Measurements of BPD by ultrasound examination during the week before parturition (mean f SD; 17 2 0.9 mm) compared well with those obtained by direct measurements of neonates (mean SD; 19 2 0.6 mm). * DISCUSSION The results of this study demonstrate that real-time ultrasonography is a practical and reliable method for the early detection and monitoring of pregnancy in the marmoset monkey. Frequent and regular scannings of pregnant females appeared to have no influence on the progression and outcome of gestation. All pregnancies ended in normal births producing viable offspring (1 x singleton, 3 x twins, 4 x triplets) after gestation lengths ranging from 140 to 145 days (mean 142 days). In the non-fertile cycle of the marmoset monkey, the uterus was characterized by a single central bright echo as has also been described for the nonpregnant organ in women [Callen et al., 19791 and rhesus and cynomolgus macaques [Morgan et al., 1987; Tarantal & Hendrickx, 1988a; Foster et al., 19921. The first indication of pregnancy in the marmoset was the appearance of a double endometrial echo in the uterus on day 15 on average. Similar uterine changes forming a thick double-line during early pregnancy in the marmoset were reported in brief by Du Boulay and Wilson  although information on the exact time at which pregnancy diagnosis was possible was not given. The double echo reflects the separation of the endometrial surfaces presumably caused, a t least initially, by an accumulation of maternally derived secretions. Since implantation in the marmoset does not commence before day 11after 20 - 18 - 14 - 12 - 10 - 16 Ei -E Ultrasonography of Pregnancy in the Marmoset / 9 n k 8 - 6 4 2 0 ' 70 I I I I I I I I 80 90 100 110 120 130 140 150 18 16 14 12 Ei Ei 10 20 8 0 ; . ; * 0 0 0 . n k D 8 - 0 0 8 . . * 0 0 5 Neonates 0 0 Singleton n = l Twins n=3 Triplets n=4 0 0 0 O 0 6 - 1 0 0 4 2 0 ' b I I I I I I I 1 80 90 100 110 120 130 140 150 70 Days of pregnancy Fig. 7. Changes in biparietal diameter (BPD) of fetal marmoset skulls between day 80 of pregnancy and birth. Mean values (+ SD) for eight pregnancies are demonstrated (a). Data for singleton (0) and mean values for are shown (b).The last point in each graph is the BPD of neonates. twins (0)and triplets (m) 10 / Oerke et al. ovulation [Moore et al., 19851, and an early post-attachment blastocyst has a total diameter of less than 1mm [Summers et al., 19931, ultrasonographic visualization of the conceptus a t this early stage is unlikely. In other primates an accumulation of fluid in the early pregnant uterus leads to comparable ultrasonograms. In rhesus and cynomolgus macaques the initial indication of pregnancy is an irregularity of the central linear echo [Tarantal & Hendrickx, 1988al followed by the appearance of intrauterine fluid [Tarantal, 19921. The early pregnant uterus of Macaca fascicularis shows changes in endometrial echogenicity, swelling of the central line [Conrad et al., 19891,and a lumen detectable as a dark gap [Korte et al., 19881. In other studies in macaque monkeys, however, the presence of a gestation sac at around day 20 is reported as being the first sign of pregnancy visible by ultrasound [Nyland et al., 1984; Cho et al., 1988; Shimizu, 19881. Based on the present results, prenatal development in the marmoset monkey as followed by ultrasonography can be conveniently divided into three phases: expansion of the gestation sacs, embryonic development, and fetal growth. The period represented by the expansion of the gestation sacs is associated with further accumulation of intrauterine fluid and can be seen by ultrasound as an increased separation of the endometrial surfaces. Although it is possible to identify gestation sacs from day 21 onwards, their number cannot be accurately determined a t this stage. Repeated scannings during this first phase of pregnancy indicate a gradual increase in the size of the uterine lumen whilst the external dimensions of the organ remain relatively constant. The time a t which an increase in mean uterine diameter can be determined (i.e., between day 33 and 38) approximates to that at which pregnancy diagnosis is first possible by abdominal palpation [Hearn & Lunn, 1975; Mitchell & Jones, 1975; Phillips & Grist, 19751. The embryos themselves become visible by ultrasonography between day 28 and 38 of pregnancy. This can be taken to represent the start of the second gestational phase which appears to include all stages of embryonic development described by Phillips [1976b] and Merker et al. . Although the embryos can be clearly seen within the uterus, details of embryonic structures are not possible to monitor by ultrasound at this stage. A notable event, however, is the onset of the heart beat on day 54, on average, which is important in allowing confirmation of the number of individual embryos. Since, according to histological sections, the heart in the marmoset is not fully developed until day 60170 [Phillips, 1976b; Merker et al., 19881it would thus appear that the onset of the heart beat precedes the completion of anatomical development of the organ. From a diagnostic point of view, detection of heart beat is of importance in providing the first reliable indication of embryonic viability and also being useful as a routine check throughout the course of gestation. Although embryonic development cannot be monitored in sufficient detail to enable staging of pregnancy, changes in uterine size provide useful information in this regard. As illustrated in Figure 5, the diameters of the uterus and uterine lumen display similar, regular growth curves between days 35 and 90. Since, however, both measurements show an increase in variability after about day 75, the most reliable time for using ultrasonographical measurements of the uterus for staging pregnancy would be within the period 35-75 days gestation. Earlier studies using transabdominal palpation described similar patterns of uterine growth to those obtained here [Hearn & Lunn, 1975; Mitchell 8z Jones, 1975; Phillips & Grist, 19751. Direct comparison of uterine dimensions obtained by ultrasound and transabdominal palpation with respect to the stage of pregnancy is however difficult due to the use of varying methods for monitoring time of ovulationlconception. Ultrasonography of Pregnancy in the Marmoset / 11 Nevertheless, the data as shown here tend to confirm the findings of Hearn and Lunn [19751 and Mitchell and Jones [19751, whereas the values given by Phillips and Grist [19751possibly overestimate the actual stage of gestation. The dificulty of obtaining reliable uterine measurements between day 70 and 90 as experienced in the present study using ultrasound, is also reported for transabdominal palpation [Mitchell & Jones, 1975; Phillips & Grist, 19751. Whilst the increasing softness and pliability of the uterus is clearly an important factor, the results presented here indicate that the number of embryos is also likely to be an additional source of inter-individual variation. Since, however, the data pool in the present investigation was limited, the extent of this variation and relevance in practical terms could not be assessed. Little difference was found between the information obtained from measurements of ventro-dorsal and transverse dimensions. Combined measurements of the uterus and uterine lumen in both planes would, however, be likely to result in a more reliable assessment of stage of pregnancy, than by determination of any single parameter. The calculation of area or volume did not, in our experience, provide useful additional information, and manual tracking of circumference contains a high degree of error and is not recommended. Coincident with the end of the period of embryonic development, which according to Phillips [1976b] and Merker et al. (19881 occurs at around day 80, the fetal skulls become visible by ultrasound. This represents the beginning of the third ultrasonographically determined phase of prenatal development. The time at which heads can be detected appears to precede the onset of ossification, since in an earlier radiological study by Phillips [1976al, skeletal formation was first noted at around day 110. Mitchell and Jones  and Phillips and Grist  were able to transabdominally palpate heads between day 90 and 120, the same period during which differentiation of singleton from twin and triplet pregnancies using this method was reported by Hearn and Lunn [19751. Although other body parts also become easier to distinguish by ultrasonography after day 90, the presence of multiple fetuses usually causes difficulties in monitoring individual structures. The skulls, however, provide reliable information about fetal number and position in the uterus. Movements of the fetal heads provide an additional means of confirming fetal viability. Based on the measurements of the biparietal diameter (BPD); fetal age and delivery are predictable, although a decrease in accuracy must be accepted due to a less pronounced growth of the skulls from day 120 onwards. This tendency is also indicated by the ultrasound data published by Du Boulay and Wilson [19881, whereas Chambers and Hearn [ 19851, using material collected at hysterectomy, found a continuous increase in the width of the fetal heads until birth. For corresponding pregnancy stages the head size as measured by abdominal palpation [Phillips & Grist, 19751in general, appears to be smaller. Dimensions of the heads in neonates in this study, however, were comparable to fetal BPD measurements in the week before birth. CONCLUSIONS 1. Ultrasonography is a practical and reliable non-invasive method for detecting and monitoring pregnancy in Callithrix jacchus and can be safely applied in longitudinal studies. 2. Detection of pregnancy is possible as early as 12 days after ovulation (day 15 on average) i.e., from the time of implantation onwards. 3. Pregnancy can be staged most accurately between days 35 and 75 by measurements of external and internal uterine diameters. 12 / Oerke et al. 4. Monitoring of fetal growth is reliable by measurements of the biparietal diameter from around day 80. ACKNOWLEDGMENTS Special thanks to Dorothea Blank and Cornelia Casper for looking after the animals and helping during all of the examinations. Dr. Michael Heistermann is gratefully acknowledged for advice with the progesterone assay. REFERENCES Callen, P.W.; De Martini, W.J.; Filly, R.A. 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