Fetal endocrine responses to chronic placental embolization in the late-gestation ovine fetus

Fetal endocrine responsesto chronic placental fetus late-gestation in the ovine embolization Robert Gagnon, MD, * John Challis, PhD, DSc, ' Laura Johnston, ACT, 8 and Laurence Fraher, PhD"

London,Ontafio, Canada OBJECTIVE:The purpose of this study was to examine the effect of chronic fetal placental embolization on the fetal corficotropin, cortisol, and catecholaminesconcentrations and on myometdai contractility pattern. STUDY DESIGN: Fourteen fetal sheep were studied (seven embolized. seven controls) for 10 days between 0.84 and 0.91 of gestation. Daily Injections of nonradioactivemicrospheres were performed to decrease fetal arterial oxygen content by 30% to 35% of the preembolizationvalue. Umbilical artery Doppler flow velocity waveformswere measured daily. RESULTS:Chronic fetal placental embolization produced progressive fetal hypoxemia (p < 0.001) with increase In Indicative 25% Doppler flow placental a In of velocity waveforms umbilical artery changes increase fetal hypoxemia In there (p 0.01). to was a progressive response chronic < resistance vascular In baseline fetal plasma norepinephrine concentration (p < 0.001). There was a transient fourfold to fivefold Increase In baseline fetal plasma cortisol levels concomitant with a significant decrease In baseline immunoreactivecorticotropin between days 7 and 9 of embolization (both p<0.05), with a in frequency the in increase by day 10. There contracture 57% myometrial to a was values control return embolized group when compared with controls (p = 0.001). CONCLUSIONS: During repetitive chronic placental damage that led to fetal hypoxemia, the fetal endocrine environment changed with time In a direction that would prevent the onset of premature (Am OasTET GYNEcoL J delivery. hypothalamic-pituitary-adrenal the premature and axis of activation 1994;170:929-38.)

Key words: Chronic fetal hypoxia, placental insufficiency,fetal hormonal response Umbilical artery velocimetry has been used as an indirect measure of placental function and to identify fetusesat higher risk of intrauterine death and requir' The clinical situation in human ing closer surveiRance. pregnanciesassociatedwith intrauterine growth restriction (lUGR) is usually characterized by an elevated ' Models of vascular resistance. umbilical-placental have fetal chronic" placental embolization aCUte2or been developed in the sheep fetus, to understand the fetal cardiovascularadaptations in responseto chronic in fetal hypoxemia. damage that result placental ' and From the Departmentsof Obstetrics/Gratcology and Physiology, Medicine and Biochtmis"j' St. joseph's Health Centre, Lawson Researchinstitute, Afedical Research Council Group in Fetal and Neonatal Health and Development, University of Western Ontario. Supported by the Medical Research Council of Canada and the Physicians' Servicesincorporated Foundation. Presentedat the Fortieth Annual Meeting of the Societyfor Gyntcologic Investigation, Toronto, Ontano, Canada, March 31-April 3, 1993. Receivedfor publicationjune 4,1993; revisedSeptember29,1993, acceptedOctober 15,1993, Reprint requests:Robert Gagnon, AID, Department of Obstetricsand Gynaecoloo St.jostph's Health Centre, 268 Grosvenor St., London, Ontario, Canada N6A 4V2. Copynght 0 1994 by Mosby-Year Book, Inc. 611152275 0002-9378194 $3.00 +0

In the late-gestation ovine fetus endocrine responses to acute hypoxemia include increases in the fetal ' (AMI), argiplasma concentration of corticotropin nine vasopressin,*' glucocorticoids,' ` and catecholamines! The purpose of the current study was to examine the effect of progressivechronic fetal hypoxemia induced by fetal placental embolization on fetal AC1`H, cortisol, and catecholamine concentrations. These hormones play a key role in the redistribution of blood flow to vital organs during acute hypoxemia in the late-gestation ovine fetus."' Because of the reported increased risk of preterm tabor associatedwith IUGR" we also determined whether chronic fetal placental embolization would alter the pattern of myometrial contractility. Material and methods

Surgical procedures. Fourteen fetal lambs of mixed breed (seven in the embolized group, seven in the between 118 surgically prepared group) were control days). but All days 147 (term 121 one of gestation and (one set of unexpected twins in the control group) were singletons. Eweswere given 600 mg of thiopental sodium (Abbott, Montreal) intravenously; the eweswere 929

930 Gagnon et al.

intubated and maintained on a closed-circuit anesthesia system with 0.5% to 1.5% halothane (Halocarboti, North Augusta, S.C. ) and a 50: 50 (vol/vol) mixture of oxygen and nitrous oxide, with a flow rate between 2 hindlimb 3 The L/min. uterus was exposed, and a and was exteriorized. Polyvinyl catheters (V4, Bolab, Lake Havasu City, Ariz., 3F, 0.33 mm outside diameter) were inserted through the fetal femoral artery into the descending abdominal aorta, approximately 2 cm below the renal arteries and I to 2 cm above the common umbilical artery, and into the inferior vena cava through the femoral vein. Ilie correct position of the fetal catheters was confirmed at postmortem examination. A polyvinyl catheter (VI 1, Bolab) was sutured to the exterior of the fetal hindlimb to record amniotic pressure. Teflon-coated stainless steel wire electrodes (Cooner, Chatsworth, Calif) were sewn into the myometrium for continuous recording of uterine electromyographic activity in 13 animals (seven in the embolized group and six in the control group), as previously described. " All catheters were exteriorized through the flank of the ewe, and the abdomen was closed in layers. Polyvinyl catheters (VI 1, Bolab) were also placed in the femoral artery and vein of the ewe. At surgery and for 3 days thereafter the ewe received intramuscular injections of 4 ml of Pen-di-Strep (200,000 IU of sodium penicillin G and 250 mg dihydrostreptomycin/mI, Rogar, London, Ontario). One milliliter of'Crystapen (1,000,000 lU penicillin G, Ayerst, Montreal) was injected daily for 3 days into the fetal femoral vein and into the amniotic sac. After surgery the sheep were housed in individual metabolic cages with hay and water available ad libitum. Ewes were maintained on a 12-hour light-dark cycle and were allowed at least 4 days to recover from surgery before the experiments began. This study was approved by the Animal Care Committees of St. Joseph's Health Centre and of the University of Western Ontario in accordance with the guidelines of the Canadian Council on Animal Care. Experimental protocol. On the fourth post recovery day (range 122 to 125 days) animals were assigned randomly to either an embolized (n = 7) or a control group (n = 7). After a 2-hour recording period (8 to 10 -%m), nonradiolabeled carbonized latex 15 ýtm microspheres suspended in dextran and diluted with sterile saline solution (0.1 ml of microspheres in 0.9 ml of' saline solution = 1.83 million microspheres per milliliter) were sonicated and injected into the experimental fetuses over a 2-hour period (I 0. ýmto 12 noon) through the descending aorta. Microscopic inspection of the microspheres in each vial indicated that there were no rnicroaggregates of' spheres. Microspheres were inje(ted daily for 10 davs, and the number of micro. spheres iniected was adjusted to decrease the fetal

Mardi 1994 Ain I OWet (; vne(ol

arterial oxygen content (Cao2, millimoles per liter) by 30% to 35% of the preembolization value. Control fetuses were injected over the same period with the vehicle diluted in sterile saline solution. Paired maternal (7.0 ml) and fetal (3.0 ml) femoral arterial blood of samples were taken daily at 9 AM for measurement glucose, lactate, oxygen content, Poll P('021 pH, hematACTH, ocrit, immunoreactive cortisol, epinephrine, norepinephrine, and dopamine. During the 2-hour embolization

0.5 mi of fetal blood was taken approximately every 15 minutes (total 4.0 ml) to measure arterial gases and oxygen content until fetal Ca02 was period

3Wc to 35% below control values. Fetal arterial oxygen content was measured in the absence of uterine electromyographic activity. The animals were then allowed to recover until the next day. The ewe was killed on day 10 at 4 Pm, and the uterus and its contents were dissected, and weighed. The fetal brain, removed, heart, thymus, lungs, thyroid, liver, adrenals, and kidneys were immediately weighed to the nearest 0.00 1 gm with an electronic weight scale (model PC440, Nlettlar, Zurich) and calculated in grams per kilogram of body weight.

Femoral arterial blood pressure, inferior vena cava blood pressure, amniotic fluid pressure, and uterine electromyographic activity were recorded continuously between 8 Amand 4 P. m daily with pressure transducers (Statham model P-23 ID, Gould, Oxnard, Calif. ) and a chart recorder (model 7, Grass Instrument, Quincy, Mass.). Fetal heart rate (FHR) was derived from the femoral arterial blood pressure record. Mean arterial blood pressure referenced to amniotic fluid pressure was calculated as diastolic plus 0.4 of systolic minus diastolic pressure. Electrical signals were processed with a common mode rejection preamplifier (model 7P51 1], Grass). FHR was measured from the pressure pulse with a carchotachometer (model 744B, Grass). Uterine electromyographic activity was displayed directly on the chart recorder after passage through a passive handpass filter, 0.3 to 30 liz, on the preamplifier. Umbilical artery flow velocity waveform recording. A real-time Duplex scanner (Ultramark 8, Advanced Technology, Bothell, Wash.) with a 3.5 Nlilz sector scanner was used to measure umbilical artery flow velocity waveforms. After baseline maternal and fetal blood sampling at 9 with the ewe standing, contact -%M jelly was spread over the lower abdomen and tile transducer applied oil the surface of the maternal abdomen to visuali7e the umbilical cord. The Doppler beam was moved toward the umbilical arterv within 5 cm from the fetal abdominal insertion of the umbilical cord. Ten waveforms were recorded and stored on standard 0.5 inch VIIS tape for off-line analysis. The resistance index (S - INS where S is pcak-sys(olic flow velocity and D is end-cliastolic flow veiocitv)" was used

Gagnon et al. 931

Volume 170, Number 3 Am I Obstet Gynecol

as an indirect measure of placental vascular resistance daily. In for 10 the recorded waveforms and averaged one animal of the control group twin gestation precluded reliable Doppler flow velocity recordings. Analytic measurements. Maternal and fetal arterial blood samples were drawn into heparinized syringes and placed on ice. Fetal arterial P02,Pco2,and pH were measured with a blood gas analyzer (ABU, Radiometer, Copenhagen) with measurementscorrected to a fetal temperature of 39.5* C. Arterial oxygen saturation duplicate in hemoglobin (0.3 ml) were measured and device hernoximeter Oxy, (Radiometer). OSN12 an with gen content wascalculatedwith a capacity of 1.34 ml of blood hemoglobin. Wiole glucose of gram oxygen per in lactate triplicate with were made measurements and membrane-bound glucose oxidase and D-lactatedehydrogenase, respectively (model 23A, Yellow Springs instrument, Yellow Springs, Ohio). Ilematocrit was measured after centrifugation (Autocrit Ultra 3, Clay Adams, Becton-Dickinson,Parsippany,NJ. ). one milliliter of fetal and maternal blood wascentrifuged in plastic tubes at 15OOgfor 12 minutes at V C. The plasma was stored at -20* C. Immunoreactive AMI and cortisol were measured with radioimmunoassayspreviously described and validated for fetal intraassay " The combined plasma. sheep maternal and 7% interassay were and variation coefficients of and 13%, respectively. Fetal (1.0 ml) and maternal (5.0 ml) blood samples for the determination of catecholamineconcentrations were transferred to plastic tubes containing glutathione--ethylenediaminetetraaceticacid and centrifuged at 15OOg for 12 minutes at V C. The plasma C and stored until analyzed. at removed was -70" Norepinephrine, epinephrine, and dopamine were detection by measured electrochemical subsequently fractionliquid high-pressure chromatography after ation. " Briefly, plasma samples(0.5 ml fetal and 1.0 in] maternal plasma) were spiked with 500 pg of the internal standard 3,4-dihydrobenzylamineand thereafter treated with 100 mg of aluminum oxide at pH 8.7 to absorb all catecholamines.After centrifugation and removal of supernatant the pellet waswashedthree times Tris-ethylenediaminctetraacetic 4 acid at mmol/L with p1l 8.7, and then the catecholamineswere extracted from the aluminum oxide with 150 ILI of glacial acetic Fifty 3.0. microliters of this extract wasthen at pH acid liquid high-pressure chroto reverse-phase subjected matography on a Bondapak column (30 x 0.39 cm, Waters, Mississauga,Ont. ) cluted with 50 mmol/L sodium acetate, 20 mmoVL citric acid, 0.135 mmol/L sodium ethylenediaminctetraacetic acid, 1.0 mmoVL di-n-butyalmine in watcr/methanol (95: 5 vol/vol) at pH 4.3 (containing 3.75 mmol/L sodium-I -octane-sulpho1.0 flow ion a rate of reagent) at an pairing nate as

ml/min. The column cluent was monitored with a model N1460electrochemical detector (Waters) in the oxidative mode at a potential difference of 0.6 V. The data N1740 moda model with peaks were quantitated ule (Waters) and compared with those of authentic reference standards of norepinephrine, epinephrine, dopamine, and 3,4-dihydrobenzylamine.The intraassay and interassay coefficients of variation were 8% and IM, respectively. Data analysis. The mean fetal arterial blood pressure corrected for intraamniotic pressureand FIIR wasanalyzed every 5 minutes over the 2-hour period between8 and 10 Amdaily and averaged. Uterine contractures were visually identified from the chart recording and consisted of rhythmic bursts of electromyographic activity, The incidence, number, and mean duration of uterine electromyographicactivity bursts wascalculated for each of the four 2-hour epochs recorded daily between 8 Amand 4 Pm. In the control group there was a significant negative correlation between the instantaneous FIIR value and the umbilical artery resistanceindex (RI) (r = -0.41, p<0.001, slope -0.00129 RI/beats/min). Therefore the umbilical artery resistanceindex was corrected in both embolized and control groups for an instantaneous FHR value of 160 beats/min (RI160).Changesin blood blood Rl,, and arterial gases artery umbilical O, pressure, MR, fetal endocrine measurements, and uterine electromyographic activity were analyzedwith a two-way correlation matrix analysis of variance with Soft5V, BMDP Statistical (BMDP measures repeated ware, Los Angeles)comparing the effect of time, group (embolized vs control), and interaction between group and time. Logarithmic transformation of the ACT11, data was performed to and catecholaminc cortisol, data if the necessary.In one control animal normalize catecholaminesamplescould not be processedbecause of technical difficulty. If a significant effect of group or time was found (p < 0.05), within-animal comparisons were conducted with multiple comparison Tukey's I test," and between-group comparisons were made with unpaired t tests. All results are presented as means ± SEM for the number of fetusesstudied. Results Fetal morphometry and umbilical artery flow velocity wayeforms. Table I summarizesfetal morpbometric measurements at delivery. 'Mere was no significant difference in total fetal body weight betweenthe embolized and control groups. However, in the embolized fetuses the combined adrenal weight was significantly higher than in the control group (P < 0.05). Fig. I illustrates an example of the increase in umbilical artery Rll,, observed during repetitive fetal placental embolization. Two-way analysis of variance

932

Gagnon et al

L, T, 1,

P r)

I

)1), t,. l ('Nileml

DAY 1 RI=0.38

DAY 8 RI=0.68

Fig. 1. Example ofincrease in umbilical arterý resistance index (RI) between days I and 8 of chronic letal placental embolization.

Table 1. Morphometric

data at postmortem examination (mean ± SEM

Gestationalage (days) Fetal body weight (kg) Fetal organ weight (gm/kg body weight) Brain Heart Thymus Lungs Fhvroid Liver Adrenals Kidneys Cotyledonaryweight (gm)

Controlgroup (n = 7)

Embohzed group (n = 7)

134.5 0.3 -!: 4.48 0.36 -t:

134.0 t 0.5 4.37 0.24 --t

11.37 1.07 -t 7.38 t 0.43 1.10 ýt 0.25 29.82 :t1.60 0.22 ± 0.03 28.94 ± 1.90 0.10 0.01 --t 6.46 -t 0.71 386 ± 16

11.57 7.80 1.18 -t 27.90 -t 0.21 -t 33.94 ± 0.19 -t 5.94 -t 349 ±

0.47 0.42 0.12 1.23 0.02 3.15 0.03* 0.33 31

< 0.05 compared with controls.

indicated a significant effect of time (p < 0.0001) and embolization (p = 0.008) on the umbilical artery Rf,,,. The mean umbilical artery RI,,, increased progressively from 0.50 ± 0.02 on day I preembolization to 0.66 :t- 0.02 on day 8 (p < 0.05); it remained significantiv above controls until day 10 (Fig. 2). However, it was not until day 8 ofembolization that the umbilical artery RI,, ) remained consistently higher than that of controls. The mean number of microspheres injected during the entire 10-day period to decreasefetal Cao., by 30% to 35% was 51.4 ±- 12.0 million (range 13.1 to 98.6 million). The number of microspheres in jected , decreased significantly from 11.6 ±: 2.5 million on day I to 0.6 --t 0.3 million on day 10 (p = 0.001, Fig. 3). Fetal blood gases,glucose, FHR, and arterial blood pressure. Fetal placental embolization causedprogressi%efetal hypoxcinia (analysis of variance effect of group and time for Cao, and Po.,,all p<0.00 1, Fig. 4). The mean fetal Cao.,decreasedfrom 3.3 ± 0.2 mmol/l. before embolization to 2.2 ± 0.2 mmol/L on clav 10 (P < 0.05). There was a partial recovery in the fetal Cao2of the "basal" sample on each day after emboliza-

tion until day 8, after which the fall in fetal Cao, after embolization remained stable. Fetal Cao, remained unchanged in the control group (Table 11). Changes in fetal arterial Po2 during embolization paralleled the changes observed in arterial oxygen content (Fig. 4). Fetal blood glucose tended to decrease from 1.0 :1- 0.1 mmol/L on day I to 0.8 ± 0.1 mmol/L on day 10 (p = 0.089) in the embolizcd group and did not change in controls (Table 11).Fetal hemoglobin and hematocrit the experiments remained unchanged throughout (control values 9.2 ± 0.5 gm/dl and 31.3% ± 4.2%, respectively). analysis of variance indicated a significant effect of time but not of treatment on fetal arterial pH (p < 0.001) and base excess (p < 0.01). The mean fetal arterial pH decreased from 7.35 ± 0.01 on day I to Two-way

7.31 ± 0.01 on day 10 (p < 0.05, Fig. 4 and 'Fable 11). The fall in fetal pH was due entirely to a small and progressive decrease in fetal arterial base excess from 0.98 ± 0.56 mmol/I. on clav I to - 1.05 ± 0.47 mmoUL on day 10 (p < 0.05) without any significant change in fetal Pc02 (Table 11).

Fig. 5 demonstrates the changes in FHR and fetal

Volume 170, NUMbeT Am] Obstet Gynecol

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0.50

0.45 11111111111 123456789

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Experimental

Day

Fig. 2. Umbilical artery resistance index (corrected at FHR value of 160 beats/min = Rl,, O) (Mean :t SEM) before (day 1) and during (horizontal bar) fetal placental embolization. Asterisk, Significantly greater than preembolization (day 1) and control values (p < 0.05).

arterial blood pressure in embolized and control groups. The overall mean baseline FHR decreased from 172 t3 beats/min on day I before embolization to 154 t4 beats/min (11% fall) on day 10 (analysis of Fig. 5). Conversely, the overall variance, P<0.001, mean fetal arterial blood pressure increased progressivelv from 43.6 ± 1.0 mm Hg on day I to 52.6 ± 1.5 mm Hg (20% increase) on day 10 (analysis of variance, Fig. 5), consistent with the maturational p<0.001, changes in FtIR and fetal arterial blood pressure recently described in the late-gestation ovine fetus. " Fetal and maternal catecholamine measurements. Before embolization the mean fetal plasma norepinephrine was 410 t 105 pg/ml, which was not significantiv different from 381 ± 81 pg/ml in the control group (Fig. 6). During repetitive fetal placental embolization the mean fetal plasma norepinephrine concentration increased progressively above the control group to a maximum of 1040 ± 248 pg/ml on day 9 and then remained significantly elevated above controls at 897 :t 273 pg/ml on day 10 (analysis of vatiance, effect of time p<0.000 1 and effect of group p=0.0 17, Fig. 6). In the control group the mean fetal plasma norepinephrine remained unaltered. There was no significant change in the mean fetal plasma epinephrine (control values 50 ± 15 pg/ml) or dopamine (control values 228 ± 131 pg/ml) concentrations in either the embolized or control groups. Mean maternal plasma norepinephrine concentration (1217 ± 273 pg/ml) was higher than that in fetal plasma on day I and did not change significantly

20 CL ul

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Fig. 3. Mean number of microspheres (: L SEM) injected daily into fetal placental circulation.

(1437 ± 467 pg/ml on day 10 of embolization). The mean maternal plasma epinephrine and dopamine concentrations remained unaltered throughout the study (control values 145 t 28 pg/ml and IIIt 36 pg/ml, respectively). Fetal and maternal ACTH and cortiscil. Two-way analysis of'variance demonstrated a significant effect of time (p < 0.05) and a significant interaction between group and time (p < 0.05) on both fetal immunoreactive ACTH and cortisol values (Fig. 7). The mean baseline fetal plasma immunoreactive ACTH concentration was 34 ±8 pg/ml on day I in the embolized group; it remained unchanged until day 6. On day 7 it decreased significantly below preembolization values, to 3.1 ± 0.5 pg/ml. Concentrations remained below controls until day 9 (P < 0.05), although at day 10 they

934

Gagnon et al.

March 1994 Am j Obstet Gynecol

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Fig. 4. Fetal femoral arterial oxygen content, P0.2,and 1)[I (mean ± SEM) before and at end of each of daily fetal placentalembolizations(arrows).'I'liere waspartial recoveryin fetal oxygenationuntil day 8, after which both fetal arterial oxygen content and tension remained stable.

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were no longer significantly different (Fig. 7). In contrast, the mean fetal plasma cortisol concentration rose from 3.0 ± 0.5 ng/ml on day I to 17.6 ± 8.2 ng/ml on day 7 and 22.9 ± 12.0 ng/ml on day 9 (both p<0.05). On day 10 the mean fetal plasma cortisol concentration was not significantly different between the two groups (Fig. 7). In the control group mean fetal plasma immunoreactive ACTH remained unaltered (control value 21 ±6 pg/ml), and mean fetal plasma cortisol rcmained unchanged from 3.6 :t1.3 ng/mi on day I until day 10, at which time mean fetal plasma cortisol increased significantly to 8.6 ± 1.1 ng/ml (P < 0.05). This is consistent with maturation of the fetal hypothalarnic-pituitary-adrenal axis occurring after 130 days' gestation in the ovine fetus. " There was no significant effect of time or embolization on maternal plasma immunoreactive ACTH and cortisol concentrations, which remained unaltered throughout the study (control values 40 -_ 12 pg/ml and 12 ±5 ng/ml, respectivelv). Uterine electromyographic activity. 'I"he mean inci-

Gagnon et al.

Voltunt- 170, Nuniber 3 Am j Obstet Gynecol

935

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Fig. 6. Baseline fetal norepinephrine concentrations (mean ýL SEM) before (day 1) and during fetal placental embolization (horizontal bar). Asterisk, Significantly greater than preembolization (day 1) and control values (p < 0.05).

Table 11. Maternal and fetal arterial blood gases, oxygen concentration, glucose, and lactate before (clav 1) and after 10 days (day 10) of fetal placental embolization (mean ± SEM) I

I Control (11- 7) pH P(: 02 (min Hg) P02 (min

Hg)

Oxygen content (mmol/L) Glucose (mmoUL) Lactate (mmol/L) Embolized (n = 7) pH No., (min Hg) P02

(MM

t1g)

Oxygen content (mmoVL) Glucose (mmol/L) Lactate (mmol/L)

Matemal

DaY II

7.46 33.6 108.1 6.1 2.6 0.8

= 0.01 ± 1.3 4.1 --t :t0.2 :t0.1 ± 0.2

7.47 :t0.02 34.5 ± 2.4 113.7 :t5.7

6.0 t 0.3 2.6 :t0.2 1.0 ± 0.3

Fekd

An 10

1

Day I

I

Day 10

7.44 33.8 115.5 5.5 2.6 0.8

0.02 -t :t1.5 ± 1.7 ± 0.5 --t 0.1 :t0.1

7.35 50.8 23.0 3.4 1.0 1.5

0.01 1.6 0.8 0.1 0.1 0.1

7.31 t 0.010 1.9 50.5 1.8 22.5 0.2 3.0 1.0 :t0.2 0.2 1.9

7.42 36.8 111.7 5.4 2.6 1.2

0.01 1.3 :t8.0 ± 0.3 ± 0.1 :t0.6

7.36 49.0 22.6 3.3 1.0 1.9

0.01 :t1.6 ± 1.2 ± 0.2 ± 0.1 ± 0.4

7.32 0.01* 50.5 0.8 18.0 t 1.0* 2.2 0.2 0.8 0.1 1.8 0.3

< 0.05 compared with day 1.

dence of uterine electromyogTaphic activity and the mean duration of uterine co'ntractures per 2; hours for the 2 hours before the onset of embolization (8 to 10 AM) and for the three subsequent 2-hour epochs day I 4 (10. %. are shown on Fig. 8. The mean on to Pm) m daily incidence and duration of uterine contractures per 2 hours was measured until day 10. Two-way analysis of vanance demonstrated a significant effect of time (p = 0.003) and embolization (P = 0.016) on the incidence of uterine electromyographic activity, which inof' creased from 10.5% ± 3.5% to a maximum 19.0% ± 2.2% between 2 and 4 hours after (he onset of embolization on day I (Fig. 8). The incidence of uterine

electromyographic activity remained above that of the it 10, time was (lay at which group until control 17.317t± 1.3% per 2 hours in the embolized group compared with 11.6% ± 1.9% in controls (p < 0.05, Fig. 8). The mean duration of uterine contractIII 1-esper 2 hours remained unchanged throughout study (control value 5.2 ± 0.5 minutes, Fig. 8). The increase in the incidence of uterine electromyographic activity was due entirely to an increase in the mean number of uterine contractures. These reached a maximum of' 5.3 ± 0.6 per 2 hours by 4 to 6 hours after the onset of embolization on day I and averaged 4.4 ± 0.3 per 2 hours in the embolized group compared with 2.8 ± 0.3

Gagnon et al.

936

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%larch 1994 Obstet Gynecol

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Experimental Day Fig. 7. Baseline fetal ACTH and fetal cortisol concentrations (mean t SEM) before (day 1) and during fetal placental embolization (horizontal bar). Asterisk, Significantly different from preembolization (day 1) and control values (p < 0.05).

in the control group during the following 9 days (analysis of variance, effect of group, p=0.001).

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EXPERIMENTAL DAY Fig. S. Incidence, number, and duration of utefine contractures per 2 hours (mean t SEM) before (8 to 10 AM,day 1) and during (hortzant& bar) fetal placental embolization. In embolized group incidence and number ofuterine contractures was approximately 50% higher than in control group. Asterisk, Significantly different from preembolization (day 1) and control values (p < 0.05).

lization in the ovine fetus at 0.84 to 0.91 of gestation produced progressive fetal hypoxemia with progressive changes in the umbilical artery Doppler How velocity waveforms indicative of elevated placental vascular resistance. Interestingly, fetal oxygenation consistently improved before the subsequent embolization up to day 8, after which fetal arterial oxygen content remained 30% to 35% below control values. It is now well accepted that abnormal umbilical artery flow velocity waveforms suggestive of elevated placental vascular resistance are associated with obliteration of the small arterioles of the tertiary stem villi. " The cause of this is still unknown. We used fetal placental embolization to obliterate the fetal microcirculation of the placenta, as previously described by Trudinger et al. ' However we achieved daily a predetermined degree of fetal hypoxemia rather than injecting a predetermined number of microspheres. The number of microspheres necessary to achieve our goal was on average four times higher than that used previously. " Although a reduction in fetal arterial oxygen content was always obtained, the fetuses partially recovered before the next embolization (Fig. 3). It is possible that the initial fetal hypoxernia resulted from a mismatch between the umbilical-placental blood flow and the uteroplacental blood flow, followed by a redistribution of blood flow within the

Volume 170, Number 3 Am j Obstet Gynecol

placental vascularbed during the recovery period. This situation would be analogous to the initial hypoxemia observed in adult humans during acute pulmonary embolism resulting from mismatch betweenlung ventilation and perfusion." Becauseumbilical blood flow was not continuously measured in this study, it is not possible to be certain whether umbilical blood flow and umbilical-placental vascular resistancewere acutely altered. A second explanation for the recovery in fetal oxygenation would be a reduction in fetal oxygen consumption by decreasingfetal breathing activity and fetal movements.However, the fetal lamb can adapt behaviorally to prolonged hypoxernia induced by restricting uteroplacental blood flow with a rapid return of fetal behavioral activity to control levels within 16 hours of hypoxemia," and fetal oxygen consumption is maintained." The pattern observed in the umbilical artery resistance index showed a progressive yet intermittent increasein the RI160,with a return to control values on days 5 and 7, followed by a steady increaseon days 8 through 10. This suggestedthat the control of placental vascular resistanceis a dynamic rather than a passive process and may change temporarily during chronic placental damage. Although speculative,it is possible that there is a "placental reserve" and that umbilical placental vascular resistance is increased marginally during embolization until a critical level of reduction in umbilical blood flow occurs that is detectable with Doppler velocimetry. It remains to be established whether corticotropin-releasing hormone, which increasesacutely during experimental restriction of uterine blood flow" at the time that umbilical blood flow increases," plays a role in the regulation of the placental vascular resistanceand umbilical blood flow. Clinically corticotropin-releasing hormone concentrations are elevated in amniotic fluid in pregnancies complicated with IUGR and placental insufficiency." Although we did not aim to produce IUGR, we expected that a reduction in fetal arterial oxygen tension of 5 mm Hg would lead to a significant reduction in fetal weight." " However, the onset of embolization was 124 days in the current study,which is later than in previous experiments.'- ' Therefore our study period was relatively late during gestation, when the fetal growth rate starts to decrease."' It is also possible that IUGF, a processthat might take months to develop in human pregnancy," would occur in the ovine fetus only when embolization has been started at an earlier gestational age and maintained for > 10 days. In addition, our data indicated that a sustainedelevation in placental vascularresistanceoccurred only after 8 daysof fetal placental embolization. We observed almost a doubling in fetal adrenal weight in the embolized fetuses.Ile mechanismof this

Gagnon et al. 937

remains unclear but suggeststhat hypertrophy or byperplasia occursunder chronic stressconditions. In vivo pulsatile ACM administration to the fetal lamb for 100 hours resulted in a twofold increase in fetal adrenal weight." However, in the current study fetal immunoreactiveAMI decreasedon day 7 coincidentally with a rise in fetal cortisol. One possibleexplanation would be that chronic placental damage might increasethe production or decreasedegradation of prostaglandin E2 (PGE2)by the placenta, fetal membranes,or endometrium. PGE2has a known stimulatory effect on myometrial contractility and raises plasma cortisol in fetal sheep at a time when the adrenals are still relatively unresponsiveto ACTH. " Although speculativebecause it wasnot measuredin this study, PGE2may play a role in the presenceof placental insufficiency and elevated placental vascular resistanceand could initiate premature activation of the fetal adrenals with subsequent negative feedback on ACTE release with subsequent return of fetal cortisol to control levels, as observedon day 10 (Fig. 7). This adaptive mechanism in the presence of chronic placental insufficiency would prevent the onset of parturition in the sheep fetus, which is known to be initiated by the maturation of the hypothalamic-pituitary-adrenal axis. If this negative feedback mechanism would fail, it is possible to speculate that the increase in myometrial activity combined with elevated fetal cortisol would eventually lead to premature parturition. The relationship betweennutrients and oxygen availability, decreasesin fetal plasma glucose,and enhanced prostaglandin PGE2 release and onset of labor has recently been suggested as a protective mechanism against a less-than-optimal intrauterine environment." The increase in the number of uterine contractures in the current study occurred between4 and 6 hours after the onset of embolization and persisted throughout the study period. An increasein PGE2has been reported at 4 to 6 hours of hypoxemia during sustainedrestriction of uteroplacental blood flow,` 2' and the release of prostaglandin F metabolites in amniotic fluid has been described after 30 minutes of hypoxia" and would therefore support the concept of increased PGE2 releaseor decreaseddegradation during chronic, repetitive placental embolization. Norepinephrine, which is predominantly an a-agonist, results in the stimulation of uterine contractions in the pregnant and the nonpregnant uterus." The progressiveincrease in fetal norepinephrine with chronic fetal embolization without any significant change in epinephrine may have led to an increasein myometrial contractility. The increase in norepinephrine observed during embolization was modest and progressive.'Mis contrastswith the fetal catecholamineresponseto acute hypoxia induced by restricting uteroplacental blood flow, which consistsof an increaseof tenfold to twenty-

938

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fold above baseline! There was no relationship between baseline FHR, which decreased in both control and embolized groups, and norepinephrine levels, which increased only in embolized fetuses. The source of the increased amount of circulating norepinephrine in this study was most probably the fetal adrenal medulla, although some could have originated from sympathetic neurons because of an increased sympathetic drive in the fetus. In summary, during repetitive fetal placental embolization resulting in fetal hypoxemia the umbilical artery resistance index progressively increased, suggesting an increase in placental vascular resistance. The fetal endocrine environment changed with time in a direction that could prevent the onset of premature axis activation of the hypothalamic-pituitary-adrenal and premature delivery, in spite of a 507c increase in baseline myometrial activity. It remains to be determined whether changes in baseline norepinephrine are related to alterations in adrenergic receptor sensitivity and could be responsible for the decrease in FHR variability and FIIR accelerations without changes in FHR baseline, which have been reported in human pregnancy complicated with IUGR and placental insufficiency. " I.

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