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Intervillous Blood Flow in the Third Trimester Gravid Rhesus Monkey (Macaca mulatta): Use of Sonographic Contrast Agent and Harmonic Imaging
Placenta (2001), 22, 200–205 doi:10.1053 plac.2000.0605, available online at http://www.idealibrary.com on
Intervillous Blood Flow in the Third Trimester Gravid Rhesus Monkey (Macaca mulatta): Use of Sonographic Contrast Agent and Harmonic Imaging N. Ragavendraa,b and A. F. Tarantalc b
Department of Radiological Sciences, UCLA School of Medicine, Los Angeles, California and c California Regional Primate Research Center and Department of Pediatrics, University of California, Davis, California, USA Paper accepted 2 October 2000
A properly implanted and functioning placenta is essential for the normal outcome of pregnancy. As pregnancy advances, an increasing supply of maternal blood, which reaches the intervillous space of the placenta via the spiral arteries, is necessary for continued growth and development of the fetus. Presumably, deficient blood flow to the intervillous space can lead to placental ischaemia and an unfavourable outcome, such as pre-eclampsia. In this study, we used a primate model, where echocontrastenhanced harmonic imaging was utilized to demonstrate placental intervillous blood flow without visualization of fetal blood circulation within the chorionic villi. We propose that this technique, which requires further assessment of efficacy and safety prior to use in humans, is a potentially useful non-invasive clinical tool for assessing intervillous blood flow in the third trimester of pregnancy. 2001 Harcourt Publishers Ltd Placenta (2001), 22, 200–205
INTRODUCTION After implantation of a fertilized ovum in the uterus, trophoblast cells rapidly migrate into the decidua, invade the spiral arterioles (Brosens, Robertson and Dixon, 1967) and, eventually, form a placenta. The haemochorial human placenta (Figure 1) is a multilobular organ that consists of 40–60 cotyledons or placentones which are structural and functioning units of the placenta (Benirschke and Kaufmann, 1995). Each placentone (Figure 2) is composed of one villous tree-like structure with a corresponding centrifugally-perfused part of the intervillous space (Wilkin, 1954; Reynolds, 1966; Wigglesworth, 1968; Benirschke and Kaufmann, 1995). The central portion of the placentone provides a space (intervillous space) for the inflow of maternal arterial blood (Reynolds, 1966). After the blood is transported to the intervillous space by a single spiral artery (Freese, 1966; Wallenberg, 1973), the leading column of blood washes the surfaces of the villous tree before exiting via the endometrial veins (Ramsey, Corner and Donner, 1963; Schmid-Schonbein, 1988). As pregnancy advances, an increasing supply of maternal blood to the intervillous space is essential for continued growth of the fetus. However, in certain complications of pregnancies, such as pre-eclampsia (Dekker and Sibai, 1998; Roberts, 2000), a characteristic feature that is often seen in the placenta is a
To whom correspondence should be addressed at: Department of Radiological Sciences, 300 UCLA Medical Plaza, Suite 3102, Los Angeles, CA 90095-6969, USA. E-mail: [email protected] 0143–4004/01/020200+06 $35.00/0
reduced intervillous blood flow due to partial or complete failure of invasion of spiral arterioles by the cytotrophoblast (Brosens, Robertson and Dixon, 1972; Khong et al., 1986; Salafia et al., 1998). At the present time, there is need for an accurate, safe, non-invasive method to assess maternal blood flow to the intervillous space alone, thus excluding visualization of blood from the fetal circulation. The purpose of this study was to evaluate the utility of echocontrast enhanced grey scale harmonic imaging of the placenta in a primate model with similar developmental features when compared to humans (Tarantal and Gargosky, 1995). Here, we investigated intervillous blood flow alone while effectively eliminating the visualization of fetal blood circulating within the chorionic villi.
MATERIALS AND METHODS Animals All animal procedures conformed to the requirements of the Animal Welfare Act and protocols were approved prior to implementation by the Institutional Animal Use and Care Administrative Advisory Committee (AUCAAC) at the University of California at Davis. Three normally cycling, adult female rhesus monkeys (Macaca mulatta; n=3) weighing 8.6–10.4 kg, with a history of prior pregnancy were bred and identified as being pregnant using established methods 2001 Harcourt Publishers Ltd
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Figure 1. Diagrammatic representation of a portion of the primate (haemochorial) placenta.
(Tarantal and Hendrickx, 1988a). Pregnancy in the rhesus monkey is divided into trimesters by 55 days increments with gestational day (GD) 0–55 representing the first trimester, GD 56–110 representing the second trimester, and GD 111– 165 the third trimester (term GD 16510) (Tarantal and Gargosky, 1995). Rhesus monkeys were used because (a) the process of placentation in this species closely resembles that observed in the human (Ramsey and Harris, 1966; Myers, 1972) and (b) the gravid uterus of the monkey is readily accessible by transabdominal sonography (Tarantal and Hendrickx, 1988b).
Echocontrast agent The sonographic contrast agent, Aerosomes (ImaRx Pharmaceutical Corporation, Tucson, AZ, USA), used in this study (Unger et al., 1992) consists of phospholipid-coated microbubbles that range in size from 1.03 m to 4.62 m. The concentration of bubbles in the undiluted agent is approximately 1.5109 microbubbles per millilitre. The suspension is available for research use in animals. The mean size of the microbubble is less than that of a red blood cell but is sufficiently large to remain within the vascular space (Wilson et al., 2000). Once deposited in the intravascular space, the echocontrast agent moves freely in the vascular tree, successfully crosses the pulmonary vascular bed and survives
repeated circulation through the body. The gas (microbubble) entrapped within the liposomes is perfluropropane, which is an inert gas often used in eye surgery (Ai and Gardner, 1993). These microbubbles are strong reflectors of the ultrasound beam thereby providing the necessary sonographic contrast effect (Unger et al., 1992).
Sonographic technique Harmonic imaging is a novel sonographic technique that utilizes harmonic echoes generated by microbubbles that resonate in an ultrasonic field (Burns, Powers and Fritzsch, 1992; Forsberg et al., 1993; Burns, 1994). A gas-filled microbubble that undergoes non-linear oscillations in an ultrasonic field emits echoes at twice (and other multiples of) the transmitted ultrasound frequency, whereas the blood within vessels, which is devoid of microbubbles, does not emit harmonic echoes. Thus, by sending an ultrasound beam at one frequency, e.g. 3 MHz, and receiving the harmonic echo at twice the original transmitted frequency, e.g. 6 MHz, echocontrast enhanced harmonic imaging can selectively track the intravascular movement of microbubbles. This technique enables detection of blood flow without requiring the use of Doppler methods (Ragavendra et al., 1997). Furthermore, superior spatial resolution afforded by grey scale imaging enables better and more
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appendicular skeleton, viscera, membranes, placenta, amniotic fluid), were incorporated, as previously described. All measurements were compared to normative growth curves for rhesus fetuses.
Echocontrast study
Figure 2. The inset from Figure 1 shows the components of a placentone, illustrating Wilkin’s (Wilkin, 1954) concept of an ‘implantation crown’. The branches of chorionic villi containing fetal vessels (fv), are bathed in a sea of maternal blood in the intervillous space (ivs). The maternal arterial blood entering the intervillous space via a single spiral artery (sa) (Freese, 1966; Wallenburg et al., 1973) percolates through the villous tree, much like flow of a liquid in a porous medium (Schmid-Schonbein, 1988), before exiting via the endometrial veins (ev). The force of arterial blood aids the forward flow of blood in the intervillous space (Reynolds, 1966; Freese, 1969). Modified and reproduced with permission from Reynolds (1966).
accurate delineation of echocontrast filled blood vessels than is currently possible with colour or power Doppler imaging.
Fetal assessments All pregnancies were sonographically assessed (Advanced Technology Laboratories, Ultramark 9 with HDI ) in order to confirm normal growth and development prior to the study (Tarantal and Hendrickx, 1988b). The dams were administered ketamine hydrochloride intramuscularly (10 mg/kg) for these and subsequent ultrasound examinations. Standard sonographic measurements of the fetal head (biparietal and occipitofrontal diameters, area and circumference), abdomen (area and circumference) and limbs (humerus and femur lengths), in addition to gross anatomical evaluations (axial and
On the day of the study, each animal was administered telazol intramuscularly and supplementary ketamine as required. An indwelling catheter was placed in a maternal peripheral (antecubital) vein for administration of the sonographic contrast agent. Monitoring of maternal electrocardiogram, systemic blood pressure and mixed venous oxygen saturation was initiated prior to the study. Fetal heart rate was assessed prior to and then at 5, 10 and 15 min post-injection. Each monkey was studied twice and received two bolus injections (30 min apart) of Aerosomes (0.05 ml/kg) via the indwelling catheter followed by transabdominal harmonic imaging of the placenta. All sonographic studies were performed using an ATL 5000 Ultrasound System (Advanced Technology Laboratories, Bothell, WA, USA) by employing a C5-2 curvilinear transducer and by using software (ATL Ultrasound) for harmonic imaging. The transmission frequency of the C5-2 ultrasound transducer was set at 3 MHz and the receiving frequency was set at 6 MHz. The time-gain compensation curve and gain settings were optimized for grey scale harmonic imaging. Low MI (mechanical index) in the range of 0.1–0.3 was selected for echocontrast enhancement of intervillous blood flow. During the study, the ultrasound transducer was held in a constant position in order to obtain optimal images of the placenta. Real-time harmonic images of the primary disc of the rhesus placenta were recorded on S-VHS videotape immediately prior to, during, and post-injection. The fetal abdomen, in particular the umbilical vein, was also monitored intermittently by harmonic imaging for signs of the echocontrast agent in the fetal circulation. Presence or absence of the echocontrast agent in the draining veins in the parametrium was not assessed. At the termination of the sonographic study, two fetuses were removed by hysterotomy, one fetus was monitored sonographically for approximately 1 month, and then removed by hysterotomy. A complete tissue harvest (thymus, spleen, liver, lymph nodes, pancreas, adrenals, kidneys, lung, heart, brain, small and large intestine) and necropsy (Tarantal et al., 1995) were performed in all fetuses. Fetal gross measures were assessed and all organs weighed and compared to a historical database for comparable aged control fetuses. Representative sections of all tissues were preserved in formalin, embedded and sectioned at 6 m, then stained with haematoxylin and eosin (H&E) for histological assessments. RESULTS Seven seconds after the maternal intravenous bolus injection of the echocontrast agent, harmonic imaging of the placenta
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Figure 3. (A) Baseline, grey scale transverse sonogram of gravid uterus of rhesus monkey. aw: anterior abdominal wall; p: placenta; af: amniotic fluid; f: fetus. The maximal thickness of the primary disc of the placenta shown here is 1.5 cm. (B–L) Sequential, harmonic images of the placenta obtained at 7, 9, 10, 11, 13, 15, 16, 21, 24, 31 and 47 sec, respectively, after maternal intravenous bolus injection of echocontrast agent (Aerosomes ). The echocontrast agent appears as white dots on the harmonic image. Note enhancement (beginning in frame B) of a myometrial artery (straight arrows), shown as an interrupted white line coursing parallel to the basal plate. The intervillous space (arrowhead) becomes visible at 9 sec, reaching maximal enhancement at 15 sec, after the injection. The intervillous space measured about 1 cm in diameter. Another intervillous space (curved arrow in frame E) is incompletely visualized (see text for discussion). Real time images of intervillous blood flow can be viewed on the internet.
revealed the presence of linear columns of echocontrast laden maternal blood coursing parallel to the basal plate of the placenta, presumably representing a myometrial artery [Figure 3(B–I)]. Two seconds later (9 sec post-injection), a vertically oriented vessel (spiral arteriole) and a cavity-like structure (intervillous space) became evident [Figure 3(C)]. Complete filling of the intervillous space occurred by 15 sec [Figure 3(G)] after intravenous injection of the echocontrast agent.
The dimensions of the intervillous space were 1.1 cm 1.0 cm1.0 cm. The filling of the intervillous space with maternal blood laden with contrast agent occurred continuously and as a uniform stream. While the entry of echocontrastladen maternal blood into the intervillous cavity was clearly seen, its disappearance from the intervillous space was poorly visualized. On any given image plane, only a few intervillous spaces were found to be filled with the echocontrast agent.
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At 10 min following the bolus intravenous injection, the echocontrast agent was undetectable in the placenta. Harmonic imaging of the fetal abdomen failed to reveal any sign of the echocontrast agent in the umbilical vein. This suggests that there was no transplacental passage of Aerosomes into the fetal circulation. Assessments of fetal heart rates upto 15 min post-injection indicated no significant changes. The digitized version of a representative portion of the videotape can be viewed on the internet. All fetal/placental measurements and specimens collected at necropsy were within normal limits, and no evidence of adverse effects were apparent grossly or histologically.
DISCUSSION During the 1950s and 1960s, cine-angiography was used to assess placental circulation in both animals and humans (Borell, Fernstrom and Westman, 1958; Ramsey, Corner and Donner, 1963; Eskes, Stotte and Seelen, 1965). However, this technique was eventually deemed unsuitable for clinical use in humans because of fetal exposure to ionizing radiation, and thus was abandoned. Doppler methods, such as uterine arterial Doppler velocimetry, have been used successfully for noninvasive assessment of normal and abnormal uteroplacental circulation (Fleischer, Schulman and Farmakides, 1986; Coleman, McCowan and North, 2000). This technique does not permit direct visualization of intervillous blood flow, but rather provides an overall and indirect measurement of vascular impedance of the uteroplacental circulation. Further, its ability to accurately predict pre-eclampsia has not been universally accepted (Chien et al., 2000). While colour Doppler and power Doppler sonography of the placenta, with or without the use of a sonographic contrast agent, have been successfully employed also for visualizing intervillous blood flow (Pretorius et al., 1998; Schmiedl et al., 1998; Simpson et al., 1998; Orden, Gudmundsson and Kirkinen, 1999), its clinical utility for assessing intervillous blood flow alone is limited due to: (a) interference from the Doppler signals emanating from the fetal blood circulating within adjacent chorionic vessels, and/or (b) ‘blooming’ artifacts (Forsberg et al., 1999) that manifest during echocontrast enhanced colour and/or power Doppler imaging.
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In our study, grey scale harmonic imaging of the placenta, after maternal administration of echocontrast agent, afforded clear delineation of only the intervillous blood flow, and thus is a potentially useful method for assessing uteroplacental ischaemia. The entry into the intervillous space of maternal blood, laden with echocontrast agent, occurred as a continuous stream and not as the ‘jets’ or ‘spurts’ of blood, as previously reported in the cine-angiographic studies of Borell, Fernstrom and Westman (1958) and Ramsey, Corner and Donner (1963). The continuous stream-type of movement seen in our study is in agreement with the observations of Myers (1972), Moll (1981), and Schmid-Schonbein (1988). While the flow of blood into the intervillous space was clearly visualized, the outflow of the echocontrast agent from the intervillous space to maternal veins was poorly (and sometimes not) visualized. This is probably due to dilution of the echocontrat agent and/or loss of the agent (bubble rupture) caused by the ultrasound beam (Bouakaz et al., 1999). On any one imaging plane, blood flow in only a few intervillous spaces was observed, even though many more were expected to be filled with blood, since 16–24 placentones are known to exist in the placenta of the rhesus monkey (Ramsey and Donner, 1980a). We speculate that lack of visualization of greater numbers of placentones may be due to sporadic and intermittent closure of some of the intervillous spaces caused by physiological uterine contractions (Borell et al., 1965; Ramsey and Donner, 1980b; Bower et al., 1991). In summary, grey scale harmonic imaging of the third trimester placenta, performed in a primate model after maternal intravenous administration of a bubble-based sonographic contrast agent, afforded excellent delineation of intervillous blood flow. Since no echocontrast agent was present in the chorionic vessels, fetal placental circulation was not visualized by this technique. Further studies to determine the safety of this technique, particularly the issue of ultrasound-induced bioeffects in the presence of microbubbles (Miller and Bao, 1998; Miller and Geis, 1998; AIUM, 2000), are essential prior to performing such studies in humans. Clinically, echocontrast enhanced harmonic imaging of the placenta may improve the prediction of pre-eclampsia by facilitating the detection of uteroplacental ischaemia at an earlier stage of pregnancy than is possible with current methods.
ACKNOWLEDGEMENTS We wish to thank Gustaaf Dekker, MD for manuscript review and comments; Sue Kahler, Michele Permenter and Rick Stetson for technical assistance; Lynne Olson, Chris Gray, Gina Zangla and Dave Nelson for illustrations; Barbara Schick for editorial assistance, and Mary Grooms for manuscript preparation. Supported in part by the Rigler Foundation.
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Ragavendra et al.: Intervillous Blood Flow in Rhesus Monkey Placenta Bouakaz A, Frinking PJ, DeJong N & Bom N (1999) Noninvasive measurement of the hydrostatic pressure in a fluid filled cavity based on the disappearance of micrometer sized free gas bubbles. Ultrasound Med Biol, 25, 1407–1415. Bower S, Campbell S, Vyas S & McGirr C (1991) Braxton-Hicks contractions can alter uteroplacental perfusion. Ultrasound Obstet Gynecol, 1, 46–49. Brosens I, Robertson WB & Dixon HG (1967) The physiological response of the vessels of the placental bed to normal pregnancy. J Pathol Bact, 93, 569–579. Brosens IA, Robertson WB & Dixon JD (1972) The role of the spiral arteries in the pathogenesis of preeclampsia. Obstet Gynecol Annu, 1, 177–191. Burns PNT, Powers J & Fritzsch F (1992) Harmonic imaging: new imaging and Doppler method for contrast enhanced ultrasound. Radiology, 185, 142 (abstr). Burns PNT (1994) Ultrasound contrast agents in radiological diagnosis. Radiol Med, 87, 71–82. Chien PF, Arnott N, Gordon A, Owen P & Khan KS (2000) How useful is uterine artery Doppler flow velocimetry in the prediction of preeclampsia, intrauterine growth retardation and perinatal death? Br J Obstet Gynecol, 107, 196–208. Coleman MA, McCowan LM & North RA (2000) Mid-trimester uterine artery Doppler screening as a predictor of adverse pregnancy outcome in high risk women. Ultrasound Obstet Gynecol, 15, 7–12. Dekker GA & Sibai BM (1998) Etiology and pathogenesis of preeclampsia: current concepts. Amer J Obstet Gynecol, 179, 1359–1375. Eskes T, Stolte L & Seelen J (1965) Radioangiographic studies of the maternal circulation in the human placenta. Arch Gynaek, 200, 735–743. Fleischer A, Schulman H & Farmakides G (1986) Uterine artery Doppler velocimetry in pregnant women with hypertension. Am J Obstet Gynecol, 154, 806–813. Forsberg F, Newhouse VL, Liu JB, Merton DA, Dickerson KS & Halpern EJ (1993) In vivo application of contrast enhanced harmonic imaging. Radiology, 189, 285 (abstr). Forsberg F, Liu JB, Burns PN, Merton DA & Goldberg BB (1999) Artifacts in ultrasonic contrast agent studies. J Ultrasound Med, 13, 357–365. Freese UE (1966) The fetal-maternal circulation of the placenta. I. Histomorphologic, plastoid injection, and x-ray cinematographic studies on human placentas. Am J Obstet Gynecol, 94, 361–366. Freese UE (1969) The fetal-maternal placental circulation. A redefinition of the utero-placental vascular relationship and the intervillous space in the human and the rhesus monkey. In The Foeto-Placental Unit (Eds) Pecile A & Finzi C, p. 18. Amsterdam: Excerpta Medica Foundation. Khong TY, De WF, Robertson WB & Brosens IA (1986) Inadequate vascular response to placentation in pregnancies complicated by preeclampsia and small for gestational age infants. Br J Obstet Gynecol, 93, 1049–1059. Miller DL & Bao S (1998) The relationship of scattered subharmonic, 3.3 MHz fundamental and second harmonic signals to damage to monolayer cells by ultrasonically activated Albunex . J Acoust Soc Am, 103, 1183– 1190. Miller DL & Geis RA (1998) Enhancement of ultrasonically induced hemolysis by perfluorocarbon-based compared to air-based echo contrast agents. Ultrasound Med Biol, 24, 285–292. Moll W (1981) Physiologie der maternen plazentaren Durchblutung. In Die Plazenta des Menschen (Eds) Becker V, Schiebler TH & Kubli F, p. 172. Stuttgart: Thieme. Myers RE (1972) The gross pathology of the rhesus monkey placenta. J Reprod Med, 9, 171–198. Orden MR, Gudmundsson S & Kirkinen P (1999) Intravascular ultrasound contrast agent: an aid in imaging intervillous blood flow? Placenta, 20, 235–240.
205 Pretorius DH, Nelson TR, Baergen RN, Pai E & Cantrell C (1998) Imaging of placental vasculature using three-dimensional ultrasound and color Doppler: a preliminary study. Ultrasound Obstet Gynecol, 12, 45–49. Ragavendra N, Chen H, Powers JE, Nilawat C, Robert JM, Carangi C & Laifer-Narin SL (1997) Harmonic imaging of porcine intraovarian arteries using sonographic contrast medium: initial findings. Ultrasound Obstet Gynecol, 9, 266–270. Ramsey EM, Corner GW & Donner MW (1963) Serial and cineradiographic visualization of maternal circulation in the primate (hemochorial) placenta. Am J Obstet Gynecol, 86, 213. Ramsey EM & Harris JWS (1966) Comparison of uteroplacental vasculature and circulation in the rhesus monkey and man. Carnegie Contrib Embryol, 38, 59–70. Ramsey EM & Donner MW (1980a) Structure of the chorionic villi. In Placental Vasculature and Circulation (Eds) Ramsey EM & Donner MW, p. 31. Philadelphia: W.B. Saunders Company. Ramsey EM & Donner MW (1980b) Angiography. In Placental Vasculature and Circulation (Eds) Ramsey EM & Donner MW, p. 56. Philadelpha: W.B. Saunders Company. [See also videotape available from American College of Obstetricians and Gynecologists, Washington, DC.] Reynolds SRM (1966) Formation of fetal cotyledons in the hemochorial placenta. A theoretical consideration of the functional implications of such an arrangement. Am J Obstet Gynecol, 94, 425–439. Roberts JM (2000) Preeclampsia: what we know and what we do not know. Sem Perinatol, 24, 24–28. Salafia CM, Pezzullo JC, Ghindini A, Poez-Zeno JA & Whittington SS (1998) Clinical correlations of patterns of placental pathology in preterm preeclampsia. Placenta, 19, 67–72. Schmid-Schonbein H (1988) Conceptual proposition for a specific microcirculatory problem: maternal blood flow in hemochorial multivillous placentae as percolation of a porous medium. Trophoblast Res, 3, 17–38. Schmiedl UP, Komarnski K, Winter TC, Luna JA, Cyr DR, Ruppenthal G & Schlief R (1998) Assessment of fetal and placental blood flow in primates using contrast enhanced ultrasonography. J Ultrasound Med, 17, 75–80. Simpson NAB, Nimrod C, DeVermette R, Leblanc C & Fourier J (1998) Sonographic evaluation of intervillous flow in early pregnancy: use of echo-enhancement agents. Ultrasound Obstet Gynecol, 11, 204–208. Tarantal AF (1995) Effects of viral virulence on intrauterine growth in SIV-infected fetal rhesus macaques (Macaca mulatta). J AIDS Human Retrovirol, 10, 129–138. Tarantal AF & Gargosky SE (1995) Characterization of the insulin-like growth factor axis in the fetal macaque (Macaca mulatta and Macaca fascicularis): A cross-sectional study. Growth Regulation, 5, 190–198. Tarantal AF & Hendrickx AG (1988a) Use of ultrasound for early pregnancy detection in the rhesus and cynomolgus macaque (Macacamulatta and Macaca fascicularis). J Med Primatol, 17, 105–112. Tarantal AF & Hendrickx AG (1988b) Prenatal growth in the cynomolgus and rhesus macaque (Macaca fascicularis and Macaca mulatta): A comparison by ultrasonography. Am J Primatol, 15, 309–323. Unger EC, Lund PJ, Shen DH, Fritz T, Yellowhair D & New TE (1992) Nitrogen filled liposomes as a vascular ultrasound contrast agent: preliminary evaluation. Radiology, 185, 453–456. Wallenberg HCS, Hutchinson DL, Schuler HM, Stolte LAM & Janssens J (1973) The pathogenesis of placental infarction II: An experimental study in the rhesus monkey. Am J Obstet Gynecol, 116, 841–846. Wigglesworth JS (1968) Vascular organisation of the human placenta. Nature, 216, 1120–1121. Wilkin P (1954) Contribution a l’etude de la circulation placentaire d’origine foetale. Gynecologie et Obstetrique, 53, 239–263. Wilson SR, Burns PN, Muradali D, Wilson JA & Xiaoming L (2000) Harmonic hepatic US with microbubble contrast agent. Radiology, 215, 153–161.
Intervillous Blood Flow in the Third Trimester Gravid Rhesus Monkey (Macaca mulatta): Use of Sonographic Contrast Agent and Harmonic Imaging N. Ragavendraa,b and A. F. Tarantalc b
Department of Radiological Sciences, UCLA School of Medicine, Los Angeles, California and c California Regional Primate Research Center and Department of Pediatrics, University of California, Davis, California, USA Paper accepted 2 October 2000
A properly implanted and functioning placenta is essential for the normal outcome of pregnancy. As pregnancy advances, an increasing supply of maternal blood, which reaches the intervillous space of the placenta via the spiral arteries, is necessary for continued growth and development of the fetus. Presumably, deficient blood flow to the intervillous space can lead to placental ischaemia and an unfavourable outcome, such as pre-eclampsia. In this study, we used a primate model, where echocontrastenhanced harmonic imaging was utilized to demonstrate placental intervillous blood flow without visualization of fetal blood circulation within the chorionic villi. We propose that this technique, which requires further assessment of efficacy and safety prior to use in humans, is a potentially useful non-invasive clinical tool for assessing intervillous blood flow in the third trimester of pregnancy. 2001 Harcourt Publishers Ltd Placenta (2001), 22, 200–205
INTRODUCTION After implantation of a fertilized ovum in the uterus, trophoblast cells rapidly migrate into the decidua, invade the spiral arterioles (Brosens, Robertson and Dixon, 1967) and, eventually, form a placenta. The haemochorial human placenta (Figure 1) is a multilobular organ that consists of 40–60 cotyledons or placentones which are structural and functioning units of the placenta (Benirschke and Kaufmann, 1995). Each placentone (Figure 2) is composed of one villous tree-like structure with a corresponding centrifugally-perfused part of the intervillous space (Wilkin, 1954; Reynolds, 1966; Wigglesworth, 1968; Benirschke and Kaufmann, 1995). The central portion of the placentone provides a space (intervillous space) for the inflow of maternal arterial blood (Reynolds, 1966). After the blood is transported to the intervillous space by a single spiral artery (Freese, 1966; Wallenberg, 1973), the leading column of blood washes the surfaces of the villous tree before exiting via the endometrial veins (Ramsey, Corner and Donner, 1963; Schmid-Schonbein, 1988). As pregnancy advances, an increasing supply of maternal blood to the intervillous space is essential for continued growth of the fetus. However, in certain complications of pregnancies, such as pre-eclampsia (Dekker and Sibai, 1998; Roberts, 2000), a characteristic feature that is often seen in the placenta is a
To whom correspondence should be addressed at: Department of Radiological Sciences, 300 UCLA Medical Plaza, Suite 3102, Los Angeles, CA 90095-6969, USA. E-mail: [email protected] 0143–4004/01/020200+06 $35.00/0
reduced intervillous blood flow due to partial or complete failure of invasion of spiral arterioles by the cytotrophoblast (Brosens, Robertson and Dixon, 1972; Khong et al., 1986; Salafia et al., 1998). At the present time, there is need for an accurate, safe, non-invasive method to assess maternal blood flow to the intervillous space alone, thus excluding visualization of blood from the fetal circulation. The purpose of this study was to evaluate the utility of echocontrast enhanced grey scale harmonic imaging of the placenta in a primate model with similar developmental features when compared to humans (Tarantal and Gargosky, 1995). Here, we investigated intervillous blood flow alone while effectively eliminating the visualization of fetal blood circulating within the chorionic villi.
MATERIALS AND METHODS Animals All animal procedures conformed to the requirements of the Animal Welfare Act and protocols were approved prior to implementation by the Institutional Animal Use and Care Administrative Advisory Committee (AUCAAC) at the University of California at Davis. Three normally cycling, adult female rhesus monkeys (Macaca mulatta; n=3) weighing 8.6–10.4 kg, with a history of prior pregnancy were bred and identified as being pregnant using established methods 2001 Harcourt Publishers Ltd
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Figure 1. Diagrammatic representation of a portion of the primate (haemochorial) placenta.
(Tarantal and Hendrickx, 1988a). Pregnancy in the rhesus monkey is divided into trimesters by 55 days increments with gestational day (GD) 0–55 representing the first trimester, GD 56–110 representing the second trimester, and GD 111– 165 the third trimester (term GD 16510) (Tarantal and Gargosky, 1995). Rhesus monkeys were used because (a) the process of placentation in this species closely resembles that observed in the human (Ramsey and Harris, 1966; Myers, 1972) and (b) the gravid uterus of the monkey is readily accessible by transabdominal sonography (Tarantal and Hendrickx, 1988b).
Echocontrast agent The sonographic contrast agent, Aerosomes (ImaRx Pharmaceutical Corporation, Tucson, AZ, USA), used in this study (Unger et al., 1992) consists of phospholipid-coated microbubbles that range in size from 1.03 m to 4.62 m. The concentration of bubbles in the undiluted agent is approximately 1.5109 microbubbles per millilitre. The suspension is available for research use in animals. The mean size of the microbubble is less than that of a red blood cell but is sufficiently large to remain within the vascular space (Wilson et al., 2000). Once deposited in the intravascular space, the echocontrast agent moves freely in the vascular tree, successfully crosses the pulmonary vascular bed and survives
repeated circulation through the body. The gas (microbubble) entrapped within the liposomes is perfluropropane, which is an inert gas often used in eye surgery (Ai and Gardner, 1993). These microbubbles are strong reflectors of the ultrasound beam thereby providing the necessary sonographic contrast effect (Unger et al., 1992).
Sonographic technique Harmonic imaging is a novel sonographic technique that utilizes harmonic echoes generated by microbubbles that resonate in an ultrasonic field (Burns, Powers and Fritzsch, 1992; Forsberg et al., 1993; Burns, 1994). A gas-filled microbubble that undergoes non-linear oscillations in an ultrasonic field emits echoes at twice (and other multiples of) the transmitted ultrasound frequency, whereas the blood within vessels, which is devoid of microbubbles, does not emit harmonic echoes. Thus, by sending an ultrasound beam at one frequency, e.g. 3 MHz, and receiving the harmonic echo at twice the original transmitted frequency, e.g. 6 MHz, echocontrast enhanced harmonic imaging can selectively track the intravascular movement of microbubbles. This technique enables detection of blood flow without requiring the use of Doppler methods (Ragavendra et al., 1997). Furthermore, superior spatial resolution afforded by grey scale imaging enables better and more
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appendicular skeleton, viscera, membranes, placenta, amniotic fluid), were incorporated, as previously described. All measurements were compared to normative growth curves for rhesus fetuses.
Echocontrast study
Figure 2. The inset from Figure 1 shows the components of a placentone, illustrating Wilkin’s (Wilkin, 1954) concept of an ‘implantation crown’. The branches of chorionic villi containing fetal vessels (fv), are bathed in a sea of maternal blood in the intervillous space (ivs). The maternal arterial blood entering the intervillous space via a single spiral artery (sa) (Freese, 1966; Wallenburg et al., 1973) percolates through the villous tree, much like flow of a liquid in a porous medium (Schmid-Schonbein, 1988), before exiting via the endometrial veins (ev). The force of arterial blood aids the forward flow of blood in the intervillous space (Reynolds, 1966; Freese, 1969). Modified and reproduced with permission from Reynolds (1966).
accurate delineation of echocontrast filled blood vessels than is currently possible with colour or power Doppler imaging.
Fetal assessments All pregnancies were sonographically assessed (Advanced Technology Laboratories, Ultramark 9 with HDI ) in order to confirm normal growth and development prior to the study (Tarantal and Hendrickx, 1988b). The dams were administered ketamine hydrochloride intramuscularly (10 mg/kg) for these and subsequent ultrasound examinations. Standard sonographic measurements of the fetal head (biparietal and occipitofrontal diameters, area and circumference), abdomen (area and circumference) and limbs (humerus and femur lengths), in addition to gross anatomical evaluations (axial and
On the day of the study, each animal was administered telazol intramuscularly and supplementary ketamine as required. An indwelling catheter was placed in a maternal peripheral (antecubital) vein for administration of the sonographic contrast agent. Monitoring of maternal electrocardiogram, systemic blood pressure and mixed venous oxygen saturation was initiated prior to the study. Fetal heart rate was assessed prior to and then at 5, 10 and 15 min post-injection. Each monkey was studied twice and received two bolus injections (30 min apart) of Aerosomes (0.05 ml/kg) via the indwelling catheter followed by transabdominal harmonic imaging of the placenta. All sonographic studies were performed using an ATL 5000 Ultrasound System (Advanced Technology Laboratories, Bothell, WA, USA) by employing a C5-2 curvilinear transducer and by using software (ATL Ultrasound) for harmonic imaging. The transmission frequency of the C5-2 ultrasound transducer was set at 3 MHz and the receiving frequency was set at 6 MHz. The time-gain compensation curve and gain settings were optimized for grey scale harmonic imaging. Low MI (mechanical index) in the range of 0.1–0.3 was selected for echocontrast enhancement of intervillous blood flow. During the study, the ultrasound transducer was held in a constant position in order to obtain optimal images of the placenta. Real-time harmonic images of the primary disc of the rhesus placenta were recorded on S-VHS videotape immediately prior to, during, and post-injection. The fetal abdomen, in particular the umbilical vein, was also monitored intermittently by harmonic imaging for signs of the echocontrast agent in the fetal circulation. Presence or absence of the echocontrast agent in the draining veins in the parametrium was not assessed. At the termination of the sonographic study, two fetuses were removed by hysterotomy, one fetus was monitored sonographically for approximately 1 month, and then removed by hysterotomy. A complete tissue harvest (thymus, spleen, liver, lymph nodes, pancreas, adrenals, kidneys, lung, heart, brain, small and large intestine) and necropsy (Tarantal et al., 1995) were performed in all fetuses. Fetal gross measures were assessed and all organs weighed and compared to a historical database for comparable aged control fetuses. Representative sections of all tissues were preserved in formalin, embedded and sectioned at 6 m, then stained with haematoxylin and eosin (H&E) for histological assessments. RESULTS Seven seconds after the maternal intravenous bolus injection of the echocontrast agent, harmonic imaging of the placenta
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Figure 3. (A) Baseline, grey scale transverse sonogram of gravid uterus of rhesus monkey. aw: anterior abdominal wall; p: placenta; af: amniotic fluid; f: fetus. The maximal thickness of the primary disc of the placenta shown here is 1.5 cm. (B–L) Sequential, harmonic images of the placenta obtained at 7, 9, 10, 11, 13, 15, 16, 21, 24, 31 and 47 sec, respectively, after maternal intravenous bolus injection of echocontrast agent (Aerosomes ). The echocontrast agent appears as white dots on the harmonic image. Note enhancement (beginning in frame B) of a myometrial artery (straight arrows), shown as an interrupted white line coursing parallel to the basal plate. The intervillous space (arrowhead) becomes visible at 9 sec, reaching maximal enhancement at 15 sec, after the injection. The intervillous space measured about 1 cm in diameter. Another intervillous space (curved arrow in frame E) is incompletely visualized (see text for discussion). Real time images of intervillous blood flow can be viewed on the internet.
revealed the presence of linear columns of echocontrast laden maternal blood coursing parallel to the basal plate of the placenta, presumably representing a myometrial artery [Figure 3(B–I)]. Two seconds later (9 sec post-injection), a vertically oriented vessel (spiral arteriole) and a cavity-like structure (intervillous space) became evident [Figure 3(C)]. Complete filling of the intervillous space occurred by 15 sec [Figure 3(G)] after intravenous injection of the echocontrast agent.
The dimensions of the intervillous space were 1.1 cm 1.0 cm1.0 cm. The filling of the intervillous space with maternal blood laden with contrast agent occurred continuously and as a uniform stream. While the entry of echocontrastladen maternal blood into the intervillous cavity was clearly seen, its disappearance from the intervillous space was poorly visualized. On any given image plane, only a few intervillous spaces were found to be filled with the echocontrast agent.
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At 10 min following the bolus intravenous injection, the echocontrast agent was undetectable in the placenta. Harmonic imaging of the fetal abdomen failed to reveal any sign of the echocontrast agent in the umbilical vein. This suggests that there was no transplacental passage of Aerosomes into the fetal circulation. Assessments of fetal heart rates upto 15 min post-injection indicated no significant changes. The digitized version of a representative portion of the videotape can be viewed on the internet. All fetal/placental measurements and specimens collected at necropsy were within normal limits, and no evidence of adverse effects were apparent grossly or histologically.
DISCUSSION During the 1950s and 1960s, cine-angiography was used to assess placental circulation in both animals and humans (Borell, Fernstrom and Westman, 1958; Ramsey, Corner and Donner, 1963; Eskes, Stotte and Seelen, 1965). However, this technique was eventually deemed unsuitable for clinical use in humans because of fetal exposure to ionizing radiation, and thus was abandoned. Doppler methods, such as uterine arterial Doppler velocimetry, have been used successfully for noninvasive assessment of normal and abnormal uteroplacental circulation (Fleischer, Schulman and Farmakides, 1986; Coleman, McCowan and North, 2000). This technique does not permit direct visualization of intervillous blood flow, but rather provides an overall and indirect measurement of vascular impedance of the uteroplacental circulation. Further, its ability to accurately predict pre-eclampsia has not been universally accepted (Chien et al., 2000). While colour Doppler and power Doppler sonography of the placenta, with or without the use of a sonographic contrast agent, have been successfully employed also for visualizing intervillous blood flow (Pretorius et al., 1998; Schmiedl et al., 1998; Simpson et al., 1998; Orden, Gudmundsson and Kirkinen, 1999), its clinical utility for assessing intervillous blood flow alone is limited due to: (a) interference from the Doppler signals emanating from the fetal blood circulating within adjacent chorionic vessels, and/or (b) ‘blooming’ artifacts (Forsberg et al., 1999) that manifest during echocontrast enhanced colour and/or power Doppler imaging.
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In our study, grey scale harmonic imaging of the placenta, after maternal administration of echocontrast agent, afforded clear delineation of only the intervillous blood flow, and thus is a potentially useful method for assessing uteroplacental ischaemia. The entry into the intervillous space of maternal blood, laden with echocontrast agent, occurred as a continuous stream and not as the ‘jets’ or ‘spurts’ of blood, as previously reported in the cine-angiographic studies of Borell, Fernstrom and Westman (1958) and Ramsey, Corner and Donner (1963). The continuous stream-type of movement seen in our study is in agreement with the observations of Myers (1972), Moll (1981), and Schmid-Schonbein (1988). While the flow of blood into the intervillous space was clearly visualized, the outflow of the echocontrast agent from the intervillous space to maternal veins was poorly (and sometimes not) visualized. This is probably due to dilution of the echocontrat agent and/or loss of the agent (bubble rupture) caused by the ultrasound beam (Bouakaz et al., 1999). On any one imaging plane, blood flow in only a few intervillous spaces was observed, even though many more were expected to be filled with blood, since 16–24 placentones are known to exist in the placenta of the rhesus monkey (Ramsey and Donner, 1980a). We speculate that lack of visualization of greater numbers of placentones may be due to sporadic and intermittent closure of some of the intervillous spaces caused by physiological uterine contractions (Borell et al., 1965; Ramsey and Donner, 1980b; Bower et al., 1991). In summary, grey scale harmonic imaging of the third trimester placenta, performed in a primate model after maternal intravenous administration of a bubble-based sonographic contrast agent, afforded excellent delineation of intervillous blood flow. Since no echocontrast agent was present in the chorionic vessels, fetal placental circulation was not visualized by this technique. Further studies to determine the safety of this technique, particularly the issue of ultrasound-induced bioeffects in the presence of microbubbles (Miller and Bao, 1998; Miller and Geis, 1998; AIUM, 2000), are essential prior to performing such studies in humans. Clinically, echocontrast enhanced harmonic imaging of the placenta may improve the prediction of pre-eclampsia by facilitating the detection of uteroplacental ischaemia at an earlier stage of pregnancy than is possible with current methods.
ACKNOWLEDGEMENTS We wish to thank Gustaaf Dekker, MD for manuscript review and comments; Sue Kahler, Michele Permenter and Rick Stetson for technical assistance; Lynne Olson, Chris Gray, Gina Zangla and Dave Nelson for illustrations; Barbara Schick for editorial assistance, and Mary Grooms for manuscript preparation. Supported in part by the Rigler Foundation.
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