Effects of biorhythms on blood flow distribution in the pregnant uterus (Macaca mulatta)

Effects of biorhythms on blood flow distribution in the pregnant uterus (Macaca mulatta) GUY

M.

HARBERT.

BARBARA

Y.

KENNETH

R. SPISSO,

Chnllottrslrill~J,

CROFT,

JR.,

M.D. PH.D.

M.D.

Virginia

Ten rhesus monkeys were studied between 143 and 161 days of gestation. The unaneathetised animals were confined to restraining chairs and maintained in a controlfed environment with continuous monitoring of spontaneous uterine dynamics. Forty-five determinations of btood ffow distribution were made using radionuclide-labeled microspheres. The microsphere injections confirmed a circadian pattern of uterine blood flow. Th&e biorhythms are related to at&rations in distribution of blood flow to ptacental and nonplacentai portions of the uterus. MyomWal blood Row is highest during the period of IigM. It is correlated positively to aortic blood pressure (r = 0.490, p < 0.05) and inversely to uterine artery blood flow (r = -0.508, p < 0.05). Placental blood flow is highest during the period of darkness. The flow rates are significantly correlated to intfa-amniotic pressure (r = -0.602, p < 0.05) but not to aortic blood pressure (r = -0.185, p > 0.05). The data indicate that blood flow distribution in the primate uterus is modulated by factors in addition to physiologic pressure-flow relationships. Extrapolation to the human has potential significance. (AM. J. OBSTET. GYNECOL. 135:82&I, 1979.)

specific bioPREVIOUS STUDIES have demonstrated rhythms in the dynamics of the nonhuman primate uterus.’ When recorded continuously during the last half of pregnancy, intra-amniotic pressure, aortic blood pressure, and uterine artery blood flow, as measured by the area under the respective pressure and changes which follow time flow curves, undergo Courses that are repetitive and predictable and vary from hour to hour in diurnal patterns2 These patterns conform to highly significant Fourier curves synchronized by the light-dark cycle. Highest hourly average values of intra-amniotic pressure are recorded during the period of light and lowest values during the period of darkness. These variations result from detectable changes in baseline resting pressure, as well as from alterations in the amplitude, duration, and frequency

From the Department of Obst&cs and Gyvwcology and the Dirkion of hluclear Med@ine, Depatimrnt oj Radiology, C’nirwsity of Virginia School yf Mrdicine. Supported in part b Rewwch Grant No. HD-02798, Nationnl In.rtitutes of Health, [‘nited State.7Public Hralth Serrke.

of the contractile complexes. Aortic blood pressure also varies in a diurnal pattern. Lowest values are recorded during the period of darkness and the early morning hours. Subsequently, variations in both systolic and diastolic blood pressures between uterine contractions as well as increases resulting from contraction produce maximal values in the late afternoon during the period of light or in the early evening hours during the initial period of darkness. In general, uterine artery blood flow varies inversely to the average values of intra-amniotic pressure. Highest flow rates occur during the hours of darkness when intra-amniotic pressure is lowest and decrease as average pressure values approach their apogee during the period of light. In late gestation, the highest and lowest hourly average values of uterine artery blood flow may be as much as 32% above and below the mean flow f6r the day. These changes in uterine artery blood flow may be modified by the circadian changes in aortic blood pressure. The present study was undertaken to investigate the relationships between these biorhythms in spontaneous uterine dynamics and blood flow distribution to placental and nonplacental portions of the uterus.

Prewnted by inuitation at the One Hundred and Second Annual Meeting of the American Gynecological Society, Hot Springs, Virginia, May 16-l 9, 1979. Reprint requests: Dr. Guy M. Harbert, Jr., Box 387, I’nizJersity Hospital, Chariottt~.~~~ille,Virginia 22908.

828

Pregnant rhesus monkeys conditioned to restraining chairs and weighing between 6.0 and 8.5 kilograms OOOZ-937817Y/230828+

15$01..50/0 0

1979 The C. V. Mosby Co.

Volume 135 Number 7

Table

Effects of biorhythms on uterine blood flow

I. Percent crossover

between nuclides used in calculating

18.9% 3.59 x IO--5% 3.1% 1.34 x 10.2%

51.0% 2.80 x 1O-3% 5.1% 5.2 x lo-*%

were selected from our primate colony. Duration of gestation ranged from 143 to 161 days. Dates of conception were known within 3 days. Animal preparation. Intra-amniotic and aortic blood pressures were monitored through open-end, Buidfilled catheters, connected by way of pressure transducers to a recording polygraph. Direct measurement of uterine artery blood flow was achieved by use of an electrotnagnetic flow meter. The flow probe was placed on the uterine vessel contralateral to the femoral arterr that was catheterized for blood pressure measurements. Sensor placement, c,alibration, and maintenance were accomplished by previously established techniques.2 Another catheter was threaded beside the blood pressure catheter into the left ventricle. A catheter was also placed in the ovarian vein or threaded from the femoral vein into the inferior vena cava to a level approximating t!he ovarian and renal veins. Position of the catheter tips was confirmed by noting the typical wide pulse pressure of ventricular contractions recorded from the arterial catheter and by palpation of the venous catheter and/or by roentgenography. Catheter positions were reconfirmed by direct visualization at the time of necropsy. As soon as the operation was completed, the animal was placed in a restraining chair and allowed to recover from anesthesia in the recumbent position over an 1% to 24-hour period. Then the monkey was placed in an upright, sitting position and maintained in this posture throughout the study period. During both the recovery and experimental periods, the monkeys were kept in a controlled emironment with 12-hour cycles of dark and constant-intensity light. Intra-amniotic pressure, aortic blood pressure, and uterine artery blood flow were monitored continuousl) in the unanest hetized animal throughout the experimental procedures. On-line electronic integration of the area under the polygraph curves was used to facilitate quantitative evaluation of the changes occurring in the patterns of the pressure and Row signals. Mean pressure and flow were determined electronically with

38.2% 31.9% 33.7% 17.5% Large

829

the true counts per minute

41.8% 32.7% 2.04 x 1O-2% 0.79% Small

4 1.6% 38.7% 2.9% 28.0%

Large

Medium Small

Small

the use of a l-second time constant. Average values of mean pressure and flow were calculated by dividing total integrated values by a specihed time interval.* Blood flow distribution. Radioactive polystyrene beads with a mean diameter of 50 p ? 10 SD were employed for measurements of blood flow distribution. The first studies were performed in five monkeys using microspheres with four different nuclide labelings: ceand ytterbirium- 14 1, chromium-5 I , strontium-%, um-169. Five nuclides-ceriuml-11, chromium-5 1, niobium-95, scandium-46 and strontium-M-were used in later studies. Specific activity of the chromium-51 microspheres was 35 mCi/gm. All other radioactive microspheres had a specific activity of 10 mCi/gm. Administration and analysis procedures were adapted from the methodology employed by Rosenfeld and associates.:’ The microspheres were placed in preweighed counting vials containing a magnetic stirring rod, 10 ml of 10% Dextran, and 2 drops of Tween-20. While this suspension was thoroughly mixed on a magnetic stirrer, four 0.1 ml samples of suspension were removed, placed in preweighed counting vials, weighed, and counted. The mean counts per minute per gram of suspension were calculated. The suspended microspheres were infused through the ventricular catheter over 20 to 50 seconds by air displacement. The catheter was then flushed with 10 ml of isotonic saline. Starting between 60 hours and 5 days following surgery, the individual nuclides were injected at essentially equally spaced intervals designed to correspond with different phases of the circadian patterns of uterine dynamics observed during the last half of pregnancy.* The time chosen for the first injection and the order of microsphere injection were designed so that the same isotope was not given to two animals at the same time of the day. After each injection, the radioactivity remaining in the administrative unit was redetermined and the entire unit reweighed. The approximate number of microspheres infused ranged from 17.6 ? 2.5 x 10” (SEM) (“‘Cr) to 49.4 + 6.7 X IO-’ (SEM) (“SC). The quantity of each microsphere injected was chosen to

830

Harbert, Croft, and Spisso

Table IIA. Values of uterine during uterine relaxation

dynamics and blood HOH.distribution .M-304 (6.8

U/winP

blood j&m

(mlhin.}:

Cervix Myometrium Endometrium Placentas Primal-y Secondary Fetus (ml/min./kg) Fetus and uterus (ml/min/kg) (Lrdiac output (mUmin.) Uterine artery blood flow (mlimin.) Intra-amniotic pressure (mm Hg) Aortir blood pressure (mm Hg)

Measurements

kg)

Light

Injection orderTime (hr.)

measurctl:

.A-309 Dark

2 15OY 92.3 2.9 11.1 1.8 76.7 50.2 26.5 23 I .9

98.6 :+.,i 11.9 I .6 81.6 44.9 36,‘i 247.7

4 0304 99.7 3.5 12.1 I.6 82.5 3i.8 44.7 250.5

147.4

149.7

1.59.9

161.7

27.7

29.9

31.8

32.1

-

15.2

15.1

lS.6

IS.9

15.4

115.0

116.0

97.7

98.6

126. I

3

produce approximately equal count rates. Mean of the total counts per minute infused ranged from 40.8 ? .i.9 x 10” (SF&) (““Nb) to 49.9 ? 4.5 x 10” (SERI) (l”!‘y,)), Starting approximately 15 seconds prior to microsphere infusion and continuing for approximatel! 30 seconds after Hushing the intraventricular catheter, reference samples were drawn into preweighed counting vials from the venous catheter and each femoral artery catheter at a rate of 1.29 mlimin, using a Harvard pump. Following infusion of the last suspension of microspheres. the animal was reanesthetized with sodium pentobarbital and the uterus rernoved intact under operative hemostasis and weighed. To prevent spillage of the microspheres lodged between placental villi, the uterus and contents were frozen intact. Fixation of the tissues was accomplished by allowing the specimen to thaw in 10% buffered formalin at 4” C. The uterus was then opened, and the fetus was retnoved and weighed. The cervix ruas dissected from the uterus, and the cndomctrium and myometrium were separated by sharp and blunt dissection. Tbe primary placenta, to which the umbilical cord was attached. and the secondary placenta were separated from the uterine wall and the underlying endometrium was separated from the myometrium by sharp dissection and included with the placental specimens. The efficiency of the dissections was confirmed by histologic examination. The myometrium. cervix, endometrium, and placentas were weighed. The tissues were minced and the entire portions of each tissue were placed in preweighed count-

(7.6 kg)

Light

1 1456 90.9 3.0 I l.:$ 1.6 75.0 32.6 42.4 228.2

0259

lnatle

Dark

1500 !#3.4 33 I:43 2.9 ix.9 33 .X 45.1 240.9

I505 97.9 3.2 13.t; 2.9 78 2 46.2 32.0 239.7

Or13 104.3 3.3 15.0 2.1 x3.9 45.6 X4.3 255.3

2 03 18 103.2 3.4 14.9 2.2 82.7 44.1 X3.6 252.7

166.7 1.288.2

165.9 I ,:w 1.2

176.7 I ,094.u

174.9 1.105.7

-

-

15.2

l4..5

13.8

124.Y

122.0

121.0

3

4

I

ing vials measuring 10 bv 5.5 mm. Specific eftiyrt was made to confine ali the specimens to the lower 4 cm of each vial. Absence of detectable radioactivit,- on the dissection surface after completion of’ the separations indicated there was no significant loss of microspheres from the tissues during handling. Based on rhe number of nuclides used, the weighed samples \vcre counted in four or five channels in a Beckman Riogamma automatic gamma counter. The sets of standards for each nuclide were counted at the same time as the samples. A computer program was written to separate the effects of percentage crossover (Table I) for the nuclicles which were counted using the solution of )I cqua’ions in 11unknowns. The true counts in all the samples were calculated using the computer program. Individual tiwrt’ flows were calculated by solution of the equation: Tissue flow (mlimin)

=

total counts per minute for the tissue mean counts per minute in the arterial samples )i withdrawal Qrdiat:

speed (mlimin).

output was obtained by solution of the equation:

(Cardiac output

(mlimin)

=

total microspheres infused mean microspheres in rhe arterial samples x withdrawal Fieswt3 Fort.y-five determinations wzere made in IO tnonkcw.

speed (mlimin).

of blood flow distribution -Thirty-se\en microsphere

Effects of biorhythms on uterine blood flow

Table IIB. Values of uterine dynamics and blood flow distribution uterine relaxation and subsequent uterine contractions T

Iv-315 (7.3 kg)

measured:

Measurements

831

made during

IV-413 (8.1 kg)

I

Light

Injection order Time (hr.) Uterine hloodJtou~ (mllmin):

Cervix Myometriurn Endometrium Placentas Primar? Secondary Fetus (ml/n&kg) Fetus and uterus (ml/minlk@ Cardiac outpu’ (ml/min) Uterine artery blood flow (ml/min) lntra-amniotic pressure (mm W Aortic blood pressure (mm Hg)

3 1223 105.5 1.2 12.8

1.1 90.4 56.5 33.9 247.6

369.1 1.103.0

4 1243 81.4 2.8 14.6 2.6 61.4 33.7 27.7

191.1

1 0003 106.7 2.0 15.6 1.8 87.4 47.6 39.8 250.4

130.5

171.0

1.118.4

1,034.o

2 0009 88.7 1.4 14.6

1 1243 111.4 3.9

0

124; 108.2 2.4 15.4 1.2 89.7 51.1 38.6 251.0

71.6 29.8 41.8 208.2

13.6 1.3 92.4 52.7 39.7 258.5

142.2 1.048.2

1.098.2

167.8 1.107.8

1.1

172.8

3 1254 96.1 2.4

4 1258 87.4 1.8

13.3

13.9

2.4 78.1 41.8 36.3 222.9

2.7 69.0 40.5 28.5 202.8

149.1 1.154.0

135.6 1,183.3

28.3

22.1

33.7

30.1

16.8

32.8

15.0

37.5

15.0

20.0

22.4

45.4

110.1

98.3

101.9

101.3

112.0

122.0

127.1

106.0

injections were made during uterine diastole and eight during uterine contraction. Continuous measurements of intra-amniotic pressure and aortic blood pressure were accomplished in all 10 animals. Satisfactory continuous measurement of uterine artery blood flow was obtained in eight animals. Postmortem examination confirmed the presence of the microsphere injection catheter in the left ventricle in seven animals and in the ascending aorta in three. Cardiac output was not calculated in these three animals because of uncertairq as to catheter location at the time of’ injection. Continued patency of both femoral artery catheters permrtted withdrabval of bilateral arterial reference samples in seven animals. The mean difference in total radioactivity between the two samples was I .8% ? 0.9%. Venous blood samples were obtained during microsphere injections from the catheter placed in the vena cava in six animals and from the ovarian vein in two. Radioactivity in these samples ranged from undetectable in vcna caval samplings to 0.8% of the arterial counts in the ovarian vein samples. Evaluation of methodology. Initial experiments were performed to determine the reproducibility of the microsphere injection technique. In two animals microspheres were injected during periods of uterine diastole on two occasions occurring one or two contractions apa-rt (Table II,\). The times for injection were chosen to approximate the occurrence of the nadir (0200 to 0400 hours) and the apogee (1100 to 1600 hours) of the diurnal patterns of uterine activity. The average values of uterine artery blood flow and intra-amniotic and aortic blood pressures were cal-

culated from the electronic integrations beginning 30 seconds prior to injection and continuing for 30 seconds after flushing the intraventricular catheter. Under similar measurement conditions, most repeat determinations were within 2% of the average of the two injections. However, microsphere distribution to the primary placenta differed from the average by as much as 2 1.3% and that to the secondary placenta by as much as 23.1%. Table IIB compares results of injections made during uterine relaxation and during subsequent contractions of various magnitudes in two other monkeys. Three injections were made during uterine diastole and five injections were initiated during the ascending limb of the contraction. Because the injection interval varied in length and each uterine contraction varied in duration and intensity, some injections terminated at the peak of the contraction while others extended beyond the peak to the initial portions of relaxation. Uterine and placental blood flow decreased as intraamniotic pressure increased (M-413). Distribution of the microspheres was more variable when the injections were made during contraction. Comparisons of uterine dynamics and of the changes in uterine blood flow were also made according to the periods of light and darkness (M-315). During a contraction in the period of light, the reduction in uterine blood flow was 22.9% and that in uterine artery blood flow was 22.0%, accompanying a 95.2% increase in intra-amniotic pressure. In the period of darkness uterine blood flow decreased 16.9% and uterine artery blood How 10.7% although intra-amniotic pressure increased 150% during

832

Harbert, Croft, and Spisso

Table III. Mean values of uterine dynamics expressed as actual values, percent cardiac out put, per~nt uterine blood flop. and uterinr vascular resistance talculatcd from the tnic rosphcrc data mcasurcd dur-ing relaxation in the periods of light and dark (mean tissue \\eighls are listed) Actual values (mllmin) (mean t SEM) Variable

Uterine blood flow Cervix

Light 92.4 t 1.6 (IW) 3.3 t 0.2

(19) Myometrium Endometrium PlacfWas:

13.94 + 0.4 (1% 2.1 r 0.2

(19

Primary

72.9 -+ 2.5 (19) 38.5 -+ 2.7

Secondary

35.2 2 2.5

Fetus (per kg)

251.3 t 8.0

(16) (16)

Fetus and uterus (per kg)

Uterine artery blood flow Cardiac output

Intra-amniotic pressure Aortic blood pressure

(1% 170.1 2 6.0 (19) 32.0 2 1.3

(16)

1,296.3* + 61.4 (15)

1.5.5s+ 0.5 (1% 114.Y rt 1.7 (19)

Dark 9s.2* ? 1.9 (18) 3.6 k 0.4

Cardiac output (%) (mean ? SSM) Light

Dark

7.3 ?I 0.3

8.7’” + 0.4 (14) 0.3 + 0.03 (14) 1.1 + 0.07 (14) 0.2 t (1.02 (14) 7.1*:? 0.3 (14) 3.8 k 0.3 (12j 3.3 + 0.2

(15) 0.3 ” 0.03

(18)

(15)

12.5 + 0.5 (18) 2.1 i- 0.2

1.1 r 0.04 (1.5) 0.2 ‘- 0.02 (15) 5.8 2 0.3 (15) 3.1 t 0.3 (13) 2.8 k 0.2

(18) 80.0* k 1.9

(18) 43.4 ir 2.9

(15) 36.6 + 2.6

(15) 257.1 r 4.7 (18) 175.4 + 3.6

(18) 36.9* 2 1.7 (15) 1,138.2 + 48.5 (14)

13.5 2 0.6 (18) 100.9 ? 2.8

(13) 20.7 + 1.1

(12) 14.1 -e 0.7

(15) 2.8 -t 0.2 (12) -

(13 22.3 -+ 0.8 (14) 15.3 -t 03 (14) 2.8 k 0.2 (11)

-

-

-

-

(18)

*Student’s t test: p < 0.05. tNumber of determinations. a contraction. A greater proportionate fall was noted in total uterine and uterine artery blood flows determined from measurements obtained during the period ot light compared to those obtained during the period of darkness despite a 54.8% difference in intra-amniotic pressure measurements. The reduction in total uterine blood Row and that distributed to the plac?ntas was likewise disparate, with a greater propo&nate decrease seen in placental blood flow. The linear-regression correlation coefficients obtained by comparison of intra-amniotic pressure to total uterine blood flow and placental How were -0.868 and -0.852, respectively. The correlation coefficient obtained by comparison of aortic blood pressure measurements obtained during the time of injection to total uterine blood How was -0.442 and to placental blood flow was -0.433. To further evaluate the methodology, blood flow measured by the How probe on a single uterine artery was compared to the total uterine blood How determined by the microsphere technique. The regression lines and correlation coefficients measured in six animals are illustrated in Fig. 1. The correlation coefficients ranged from 0.778 (M-705) to 0.959 (M-310). Uterine artery blood flow ranged from 26.4% (M-705)

to 48.0%’ (M-610) of total uterine blood How. For a given monkey, the ratio of total uterine blood flow to uterine artery blood How was consistent with the maximal difference between ratios ranging from 0.04 (M-304) to 0.16 (M-305). Evaluathn of bicdqdums. The data obtained from the 37 injections made during uterine diastole are collated in Table III. The mean values in milliliters per minute and millimeters of mercury of the 19 measurements made during the period of light are compared to the 18 determinations made during the period of darkness. Mean values statistically greater during the period of light are: intra-amniotic pressure, 15.7%; aortic blood pressure, 13.8%; cardiac output, 13.X%, and myometrial blood How, 11.7%. During the period of darkness statistically greater mean values of uterine arEery (9.6%), total uterine (6.2%), and placental (9.6%,) blood flows were recorded. The blood How distributional fractions to placental and nonplacental uterine tissues in two monkeys found at necropsy to have single placentas were similar to those recorded in monkeys with bidiscoid placentas. Evaluation of the data based on blood flow per gram of tissue did not alter the statistical relationships. Despite a statistically

Volume

Effects of biorhythms on uterine blood flow

135

Number 7

Uterine bloodjow (76) (mean 2 SEM) Light

Dark

3.6 -e 0.3 (19)

3.8 ” 0.4

(18)

15.2* f 0.5

12.8 f 0.5

(19) 2.3 t 0.2

(18)

(19)

78.8 k 0.8 (19) 42.4 2 2.6

(16)

37.1 k 2.3

(16) -

2.2 2 0.2

(18)

81.3* f 0.7

(18)

43.3 2 2.6 (15) 38.1 t 2.4 (15)

Vascular resistance (mtan

Dark

1.08* + 0.03

0.90 iz 0.05

(16)

3.10 ‘- 0.38

(16)

-

3.21* k 0.19

(16) -

-

-

-

significant difference in total uterine blood flow, blood flow per kilogram to the fetus and to the uterus and its contents did not differ significantly between the periods of light and dark. Based on weight at the time of surgical removal, blood flow to the uterus and its contents, including amniotic fluid, was 155.2 mliminl kg * 5.9 (SEM). Distribution of cardiac output to the component tissues of the uterus did not differ statistically between the periods of light and dark except for the portion going to the placentas. Uterine blood flow distributed to the myometrium, on the average, was 18.7% greater when measured during the period of light although the distribution to the placentas was 3.1%’ less. Vascular resistance to the uterus and placentas was calculated by the formula: aortic blood pressure - intra-amniotic pressure + blood flow; that to the other uterine tissues was determined by the formula: aortic blood pressure + blood Bow.* On the average, total *Limitations of equipment precluded simultaneous measurement of venous pressure in the present experiments. However, previous continuous recordings of inferior vena caval pressures throughout the day have not displayed detectable changes during uterine tiiastole.

Tissue weight (grams) (mean 2 SEM)

(18)

33.21 -t 4.20

(18)

8.25 2 0.38

(18)

55.74 -+ 5.67

(18)

1.15 k 0.07

(18)

2.33 -t 0.33 (15) 2.53 2 0.22 (15) -

-

+ SEM)

Light

(19) 36.21 r 1.98 (19) 8.30 * 0.28 (19) 59.80 k 5.13 (1% 1.38* t 0.05 (19) 2.71 e 0.22

833

2.49 ‘-’ 0.22 (15)

15.5 2 0.7 (10) 44.8 + 3.1 (10) 14.9 2 1.1 (10) 104.6 + 6.2 (10) 66.5 t 4.1 (10) 50.0 2 5.7 (10) 368.2 t- 16.1 (10) 546.7 ‘- 26.4 (10) -

uterine vascular resistance was 16.6% greater; vascular resistance to the placentas, 16.6% greater; and uterine artery resistance, 22.4% greater during the period of light than in the period of darkness. Calculation of vascular resistance by multiplying proportionate individual tissue flows by uterine artery blood flow did not alter the statistical relationships. The average values of intra-amniotic pressure, aortic blood pressure, and uterine artery blood flow measured during each hour of the day and during microsphere infusion in a monkey injected with four different nuclides are depicted in Fig. 2. The circadian biorhythms of hourly average values in uterine dynamics are illustrated. Intra-amniotic and aortic blood pressure measurements obtained during the microsphere injections display similar diurnal variations. Uterine artery and total uterine (microsphere) blood flows shared reciprocal relationships to the pressure values. Fig. 3 presents measurements obtained in a monkey that received five nuclide injections over a 24-hour period. Values varied above and below the mean of the five determinations, and the biorhythms of intraamniotic pressure, aortic blood pressure, and uterine artery blood flow to the periods of light and dark per-

834

Harbert, Croft, and Spisso

UABF

UEtFml/min

Fig. 1. Linear correlation coefficients and regression lines calculated for comparisons between blood flow measured by a flow probe on a single uterine artery and total uterine blood flow measured by the microsphere technique. Three monkeys received injections of four different nuclide microspheres and three animals received five different microspheres. sisted. The individual determinations of’htood flow distribution within the uterus likewise showed a variation above and below the mean of the five determinations. In general, highest values of’cervicat, endometrial, and rnyometriat blood flows were observed for in,jections made during the period of’light white total uterine and placental blood flows were greatest during the period of’ ctarkness. In order to further evaluate the observed differences, the measurements recorded in three monkevs that received five different nuctide-labeled microspheres in.jetted during uterine diastole were subjected to the analysis of variance. There were no significant diff’erences between monkeys. The analyses confirmed statistically significant dif‘ferences between the five periods of microsphere in.jections for uterine artery blood How, intra-amniotic pressure, and aortic blood pressure. In addition, the F ratios obtained for the variation with in-@ions of cardiac output (F4, x = 4.34) and placental blood How (F4, x = 3.97) were statistically significant. The F ratio obtained for the variation of’ myometrial blood How (3.52) approaches the 3.84 required for statistical significance at the 95% confidence level. The variations in cervical and endometrial blood flows were not statistically significant. Both tnyometriat (F,, S = 4.12) and placental (E;,, = 3.96) blood Hews showed significant variation in the proportion of cardiac output received. The percentage of uterine blood How supplied to the myometrium varied significantly with the injections (F,, 8 = 3.85), white the F ratio (2.43) obtained f’or percent of uterine blood

flow supplied to the placentaa \!as tool Ggtlillc,irlt. I tic I; ratios calculated t.c)r \asc~&~r resistantc‘ illdic~;ilc~ that only lotat utcritic rcsistanc(’ basc~tl OII the, mi( i-o~ptlt*r~~ nieasurenien1b (5.10). rltt+lir artcry blood 110~ (7. l6), arid rcsistanre to the ptacenlas C,~..G~)varic~ti significantly ket\vcrtt it!jections performed (luring IIIC: periods of‘ light and those performed during thy pet-iads of’ darkness. Since the p~cx~~e of’ only li)ur OI- live equally sprrcd measuremettts does not permit valid analysis of‘ \r;triante t;)r Fourier terms.’ the data were normalized by expressing each value a\ the ratio of the I~XIII f’or the total number ot measurements performed during periods of uterine retaxarion in a given animal. The normalized data f’oi- blood ftow c)htairied ft-om tilt, six allimats, each receiving four or five nuctide injectiotts ovet a 24-hour pel.ioct, are depicted in Fig. 4. In grnrral, the values ot’ total uterine blood flow sho\\~ed reciprocal relationships LO the amniotic Huid pressures, \%ith vatues 7.5’1 abo\,e the mean being obtained du~kg the period of darkness and values 7. 1% beton the mean being recorded during the period of tight. None of the normalized values of blood H O W to the cndometrium was statistically dif’ferertt f’rom the mean tar the 24hour period. Blood flow to the myometrirtm was tiighest during the period of’ tight and lowest during the period of darkness. Statistically significant values range from 13.0% below to IO.O’i, above Lhe mean. Placeiltat blood ficn\ showrtt less variation but did demonstrate statistically significant value5 !).7’% above the mean during the period of darkness and 9.5% below the mean during the pt+ocl of tight. III Fig. .i r he total placental blood flow is sho!vn, as well as the distribution to the primary and secondary placentas, obtained during microsphere injections in two monkeys. 1n both animals, on some occasitms the primary placenta received a greater proportion of uterine blood flow Chile on other occasions the secondary placenta received a higher proportion. These diff’erences showed no consisteltt relationships to the periods of tight and dark. The correlation coefficients obtained by comparison between primary and secondal y placentat blood H O W in individual animals ranged from 0.164 to 0.935. Regardless of’ which placental disc received the greater or lesser proportionate How, total placental blood How remained fairly constant within the limits ofthe diurnal variations noted. In an effort to elucidate other f’actors that might influence blood Now distribution throughout a 24-hour period, linear correlations were determined between the measurements of aortic blood pressure, intra-amniotic pressure, uterine artery blood How, and the mit-

Volume Number

Effects of biorhythms on uterine blood flow

135 7

UBF

120_ i. : 1.. 0 110

ml/min

n

..

40

UABF

m

30

0 q

rrc

20 F[

”

ABP

IAP

12ixl

24bo

Time- hours Fig. 2. Illustration of the circadian variation in average hourly values of intra-amniotic pressure (IAP), aortic blood pressure (ABP), and uterine artery blood flow (UABFJ measured over 24 hours in an unanesthetized monkey at 15 1 days’ gestation. The isolated individual points on the plots for ZAP, ABP, and UABF are the integrated electronic averages obtained for a period starting 30 seconds prior to microsphere infusion and continuing for 30 seconds after flushing the intraventricular catheter. Points for total uterine blood flow (UBF) were calculated from the microsphere data. The stippled background areas represent the periods of darkness.

8

8 I 6

Endmelrial Blood Flow (ml/min)

Fig. 3. Bar graphs depicting values of intra-amniotic pressure, aortic blood pressure, and uterine artery blood flow obtained at the time of injection in an animal that received five nuclides. Measurements of uterine, cervical, endometrial, myometrial, and placental blood flows were calculated from the microsphere counts. The shaded bars represent measurements obtained during periods of darkness.

835

836

Harberl, Croft, and Spisso

Time- hours

Fig. 4. Composite of normalized values of intra-amniotic pressure, aortic blood pressure, and total uterine blood Row and its distribution calculated from animals that received four nuclides (open symbols) and animals that received five (closed symbols). Each point depicts the mean and standard deviation of three determinations. Values that differ statistically from the mean of the four or five respective injections are marked by asterisks.

Table IV. Linear correlation coefficients calculated by comparison of values of intra-amniotic pressure, aortic blood pressure, and uterine artery blood tlow with the milliliters of blood flow per minute to placental and nonplacental uterine tissues

I Cervix Myometrium Endometrium Placentas

-0.459* 0.226 -0.202 -0.602*

0.299 0.490* 0.241 -0.185

Uterine urtery blood flow (N = 31)

-0.194 0.508* -0.072 0.652*

*p < 0.05. liliters of blood How per minute distributed to placental and nonplacental uterine tissues. The results are shown in Table IV. There are statistically significant negative correlations between intra-amniotic pressure and blood flow to the cervix and placentas but not to the endometrium or myometrium. Aortic blood pressure has significant correlation only with biood flow to the myometrium. Uterine artery blood flow displayed a greater correlation with placental than with nonplacental tissue Hews.

Comment Use of radioactively labeled microspheres for determination of proportionate as well as actual blood

flow distribution in the rhesus monkey is established.” The present study confirms the variation in uterine blood flow with periods of light and dark and demonstrates that these circadian biorhythms in uterine dynamics engender alterations in distribution of blood How to placental and nonplacental portions of the uterus. On the average, total uterine blood How measured by the microsphere technique is 6.0% less during the period of light than during the period of darkness (Table 111) and shows a circadian pattern that averages as much as 7% above and below the mean throughout a 24-hour period (Fig. 4). This does not approach the variations recorded using an electromagnetic How probe on a single uterine artery.’ The differences are probably the result of asymmetry of the placerital implantation sites and the unequal distribution of uterine artery hlood How. The current findings for arterial inHow are consistent with studies showing that in pregnant rhesus monkeys placental drainage is variable between the two ovarian veins.’ The data further indicate that measurements of uterine artery blood flow in thepregnant nonhuman primate by use of an electromagnetic How probe on a single uterine arter) will give qualitative hut not quantitative information on the flow changes in the entire uterine vascular bed. Many of- the recognized problems inherent from injection of as many as five nuclides in one animal are alleviated through the use of computer capabilities to

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Effects of biorhythms on uterine blood flow

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837

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0

;” 2400

1

I

1

1200

( 2400

O,‘, 24400

I

1200

I

2400

Time-hours

Fig. 5. Illustration of’the variation in total placental blood flow and its distribution to the primary and secondary discs recorded with different nuclide injections in two monkeys. The variations in blood flow to the primary and secondary placentas were unrelated to the periods of light and dark. simultaneously solve five equations in five unknowns. Potential experimental bias resulting from the radiation energies or from the number of microspheres is minimized by randomizing the order and timing of the series of nuclide injections. Similar proportionate flows were recorded for a given period of the day regardless of the energy emission peaks, specific activity, or time of the initial in.jection during the 24-hour period. Likewise, the reproducibility of the results indicates that injection of a total of 1.5 to 2.0 million microspheres did not have a detectable effect on the microcirculation to placental or nonplacental tissues of the uterus. Reliability of the present data is further documented by the close agreement with the values of cardiac OUIput in the pregnant rhesus monkey reported by other investigators.*, ” The proportionate distribution of blood flow to placental and nonplacental uterine tissues recorded during both uterine relaxation and contraction is also in agreement with published results.g The absolute values of total uterine blood flow in the current study approximate the general range measured with antipyrine# and the blood flow reported per kilogram of uterus and contents recorded for another species.‘” However, the values are considerably higher than those reported by Lees and co-workers,g \vho used a combination of proportionate distribution of microspheres to the tissues and dye-dilution measurements for cardiac output to calculate their flows. The discrepancies may be related to the methods of measurement and to the design of the present experiments which were performed in unanesthetized animals. The dynamics of blood flow to the primate uterus are complex. Spiral artery inflow into the intervillous space has been noted to be intermittent during periods of uterine diastole.” The marked variations of blood How distribution IO the primary and secondary placentas

noted between consecutive injections (Tables II and III, Fig. 3) confirmed these observations and suggested that factors controlling blood How to each of the bidiscoid placentas of the rhesus monkey may be acting independently. However, presence of a homeostatic mechanism is suggested by the observation that total placental blood How remains fair11 constant within the circadian variations noted. The presence of compensatory mechanisms controlling placental blood How is further evidenced by the observation that the percentage of cardiac output distributed to the placentas but not to the other portions of the uterus is greater during the period of darkness when cardiac output is lowest. Additional support is given by the correlation coefficients, indicating that blood How to the placenta is predicated more upon intra-amniotic pressure than upon a driving of blood to the uterus by the head of pressure in the aorta (Table IV). The disproportionate decreases in blood How recorded during uterine contraction (Table IIB) also demonstrate a possible homeostatic mechanism for placental blood How. However, the variability of blood How distribution that may result from uterine contraction in relationship to timing of injection was felt to preclude use of contraction data in evaluation of spontaneous changes that occur as a result of inherent biorhythms. Blood flow to cervical and endometrial tissues did not vary significantly, whether elevated in terms of actual values, percent cardiac output, or percent of uterine blood How (Tables IIA and III). Myometrial blood How was higher during the period of light than during the period of darkness and the percentage of cardiac output and uterine blood How supplied to the myometrium varied significantly between the periods of microsphere injection. The correlation of myometrial blood How to aortic blood pressure (Table IV) suggests that the increase in blood pressure during the period of

838

Harbert, Croft, and

light

Illa)’

Ile primaril!

mvomelrial

Spisso

responsible

l’or

the

changes

in

Hfnv.

did not show an increase in the studs How during periods of relaxation when Ineasurements were made within a matter of’ minutes (Table IIA). This diff’erence may result from the lesser magnitude of contraction occurring between measurements and the temporal relationship of’ Inicrosphcre injection during diascf)le to completifm of the cc)ntl.action-i-el;ixatioli conlplcs. An afternale cxplanation may relate to the timing of the studies. The I l.Z\/r, increase in m~ometrial blood How observed during the period ot‘lighc compared IO rhe period of’darkIICSS (l‘abfc III) and the 13.(i’,;i difference recorded bctwcn maximal and minimal bloftd flow rates (Fig. 4) cilf~o~lipaas the 14.‘L%’ increase in mean m~ometrial blood flow noted during relaxation in studies!’ which were perfhrmed several hours at‘ter the control values \vcre obtained. Blood How is the quotient of pressure and resistance. The lack of‘correlation of placental blood How to aortic Mood pressure (Table IV) necessitates rpsistanre mechanisms that negate the head of pressure in the aorta. There is a statistically significant change in placental vascular resistance between the periods of light and darkness (Table III). The mechanism and site of this resistance are uncertain. Anatomic studies have shown that ti-ophoblast invades the vessel \\.alls of’ the primate utcrus’S and decreases resistance to placental blood flow. Experiments performed in rhesus monke);s b) cannulatifm and measurement of the pressure gradient between the spiral artery and the intervillous space revealed resistance across those vessels to range from I .O IO 2.6 m m Hg ml-’ min.‘? According to those data a major reduction in pressure of blood flowing to the placentas occurs proximal to the endometrial portion of‘ the coiled uteropfacental artery. ‘I‘he

present

m?ometrial wnsecutiw

In

addition

to

intra-amniotic

pressure

changes,

rc-

sistance to placental blood How could reHect local nl~olnetrial action and \.asoconstrictiol1 and involve ncurohormonal factors. It is known that there is a relationship of the circadian patterns of spontaneous uterine activity that can be abolished with alpha-adrenergic blockers.”

Resistance to m~omerrial blood 110~ flit1 IIO~ ~hc,\\ ‘I statistically sigtiificanr c,harrgc clrspitc~ si,gniiic :11i1 iiicreases in aortic blood prcssurf‘. carcliac ourput, Clued ititra-amniotic pressure (?‘able I I I). suggvqing InIIltipfe factors al’e opwative. Sttldies iti 0tlit.r ipwic:s itldi\~;iwil;lI~~i~t. fliv cate that while the n~~f~eIltlf~ilietrial plars marLed dilatation fiui-ing Iarc, 1” egriai~c b, it remains imeills

responsi\~e in other

to aclre~lcrgicspwies

liavf,

also

stini~tlatiftn.‘~ sllowIl

L.spvl--

tl1;1t 1’~rro~cIl

stimulation leads to 2 i-k in Hood Ho\\- io I1lc m~fm~ctrium, endomctr-ium, and plxenta with fiistrit)ution ot tdooft flo\v to the m~oIuctiium 2nd f.c~~-is Ixitlg !;Ib\ estrogen while the pl;icent;d IION is L’;Ivo~-cdb! vored progesterone.”

(:ircadiari

\~ari;itiorl

in

t5ti ctgcn

,11ld

progesterone in the pregnant 11onl~u11la11 prinl;l(e tna! contribute to the variations seen in the presc’n~ espcrimerits. Allei-atifms in c;~techfhniiri~ scwsitivit\ iu turn are related to progesterone wrsus estrogen dominance, suggesting that the +twoid hormones infiwncr either catediolamiile receptor coIlc.entratifms or the coupling of receptors to the cspressioil of wsomotftr function.“’ Additional substances that nla~ hz ilk\ ol\ed are prostaglandins. Infusion of norepiuephrine has been demonstrated to cause release of prosLaglandins f‘rflm several organs.” Other reports ha\,e indicatefl that prostaglaudins ma) influence responses of. s!mpathetic nerve stimujatifm. In general, proslaglaudins no1 only have a direct action on vascular- smooth muscle but also facilitate adrenergic \asoconstrif:tor responses” and may be involved in mediating the uterine \asodilatation produced by estrogens.‘! Although the precise controlling mechanisms must be elucidated, the studies suggest that the effect of these biorh$ms should be considered in interpretation of “stead\.-state” conditions. E:xtrapolation of‘ Ihe present find&s to the human ma!’ have clinical significance in explaining the high percentage of‘ Ualse positive tests encountered on evaluation of the effect of uterine activity on intrauteritie t‘elal heal-t rate patterns. The causative medianisnls nla)’ have a direct etf’ect on intrauterine environmcm in conditions such as pregnancy-induced hypertension.

REFERENCES 1. Harbert, G. M., Cornell, G. W., and Thornton,

W. N.: Diurnal variation of spontaneous uterine activity in nonpregnant primates (Wrcncn mulntfrr), Science 170:82, 1970. 2. Harbert, G. M.: Biorhythms of the pregnant uterus (,kkncn ~wlrtltn), AN J. OBSTET. GYNECOL. 129:401, 1977.

3. Rosenfeld, C. R., Killatn, A. P., Battaglia, F. C., Makowski, E. L., and Meschia, G.: Effect of estradiol-17-beta on-the magnitude and distribution of uterine blood flow in nonpregnant, oophorectomized ewes, Pediatr. Res. 7: 139. 1973. 4. Makowski. E. L., Meschia, G., Droegemueller, W ’., and Battaglia, F. C.: Measurement of umbilical arterial blood

Effects of biorhythms on uterine blood flow

flop to the sheep placenta and fetus in utero, Circ. Res. 23:62:3, 1968. 5. Bliss, C. L.: Statistics in Biology, Ne\v York, 1970, vol. 2.

McGraw-Hill Book Company, Inc.. p. 219. 6. Hoffbrand, B. I., and Forsyth, R. P.: Validity studies of the radioactive microsphere method for the study of the distribution of cardiac output, organ blood Horn, and resistance in the conscious rhesus monkey, Cardiovasc. Res. 3:426, 1969. 7. Battaglia, F. C.. Makulvski. E. I.., and Meschia, G.: Physiologic study of the uterine venous drainage of the pregnant rhesus monkey, Yale J, Biol. Aled. 42:218, 1969 1970. 8. Peterson, .E. N\‘.,and Behrman, R. E.: Changes in the cardiac output and uterine blood flow in the pregnant MNC~ICO mtlntk

Ahf.

J. OBSTET. GYNECOL. 104:988,

1969.

9 Lees, 11. H., Hill, J. D., Ochsner, A. J., Thomas, C. L., and Navy, M. J.: Maternal placental and myometrial blood Ilo\\, of the rhesus monkey during uterine contractions, hf. J. OBSTET. GYNECOI.. 110:68, 1971. 10. Makowski, E. L., Meschia, G., Droegemueller, ‘II’., and Battaglia, F. C.: Distribution of uterine blood How in the pregnant sheep, A&r. 1. OBSTET. GYNECOL. 101:409, 1968. 11. Ma&n, C. B.,‘McGa;ghey, H. S., Kaiser, I. H., Donner, M. LV.. and RamseF. E. M.: Intermittent functionine” of the uteroplacental arteries, Anr. J. OBSTET. GYNECOI.. 90:8 19, 1964. 12. hloll. W., Q’allcnburg, H. C. S., Kastendieck. E., and Vos-

Discussion1 Denver, Colorado. Those who have had previous investigative experience with nonhuman primates can immediately appreciate the problems which may have been encountered in a project of this magnitude. Dr. Harbert and his co-investigators have successfully developed an experimental model utilizing unanesthetized pregnant rhesus monkeys confined to restraining chairs and maintained in a controlled environment. The relationship of the biorhythms in spontaneous activity of the uterus to uterine hemodynamics W:IIS then studied postoperatively according to a well-designed protocol. Total uterine blood H O W as well as its distribution and cardiac output: was measured according to the radioactive microsphere technique. It is interesting to note that following the injection of a total of 2.5 million microspheres the investigators were unable to detect an) change in the microcirculation of the uterus. A similar observation was made in a different species, the pregnant ovine, in our laborator),. No demonstrable effect on the uterint microcirculation was observed in sheep after infusion sofa total of 3.9 million microspheres into the fetal side of the placenta or 2.1 million tnicrospheres into the maternal circulation.‘, 2 Near term gestation, the authors observed in the rhesus monkey an average uterine blood flow of 175 ml/min . kg. iiithough this is in agreement. with other studies employing a different technique, it is, however, considerabl! less than the uterine blood flow of 250 ml/min . kg in sheep at a comparable gestational age. In the event the measurement of uterine blood flow in the near-term DR. EDGAR L. MAKOWSKI,

839

lar, M.: The flop resistance of the spiral artery and the related intervillous space in the rhesus monkey placenta, Pfluegers Arch. 377:225, 1978. 13. Harbert. G. M., and Zuspan, F. P.: Relationship between catecholamines and the periodicit? of spontaneous uter ine activity in a nonpregnant primate iibfocacct mulntta), AM. 1. OBSTET. GYNECOL. 129:51, 1977. 14. Anderson, S. G., Still, J. G., and Greiss, F. C.: Differential

reactivity of the gravid uterine vasculatures: Effects of norepinephrine, AM. J. OBSTET. GYXECOL. 129:293, 1977.

15. Greiss, F. C., and .4nderson, S. G.: Eff’ect of ovarian hormones on the uterine vascular bed. AM. J. OBSTET. GYNECOl.. 107:829, 1970. 16. Sliller, M. D., and Marshall. J. M.: Uterine response to nerve stimulation: relation to hormonal status and catecholamines, Am. J. Physiol. 209:%9. 1965. 17. Hedqvist, P.: Autonomic neurotransmission, in vol. 1, Ramwell, P. it’., editor: The Prostaglandins, Nelv York, 1973, Plenum Press, p. 101. 18 Brady. M. J., and Kadowitz, P. J.: Prostaglandins as modulators of the autonomic nervous system. Fed. Proc. 33:48, 1971. 19. Navy, M. J., Thomas, C. L., and Lees, M. H.: Uterine contractiliti and regional blood flow responses to oxytotin and prostaglandin EP in pregnant rhesus monkeys, Aar. J. OBSTET. GYNECOL.

122:419, 1975.

rhesus monkey reflects normal conditions, it is an important observation and demonstrates that an interspecies difference may be present. This in itselfjustifies the need for additional careful experiments in different species so that valid comparative studies can be made. In their experiments, did the authors see any trend for a decrease or increase in absolute uterine blood How following surgery, regardless of any circadian rhythm? During periods of light, the investigators noted a significant increase in the mean of the absolute values of cardiac output, aortic blood pressure, intra-amniotic pressure, total uterine and placental vascular resistance, and myometrial flow with a simultaneous decrease in the mean values of absolute uterine, uterine arterial, and placental blood flows. With a significant decrease in absolute uterine blood flow, the authors were unable to demonstrate any significant difference in uterine blood flow per kilogram of uterine weight and its contents between periods of light and darkness. I am somewhat perplexed about this observation and wonder if the authors compared the same animal between periods of light and darkness and analyzed their data by a paired Student’s t test. Although the authors noted a correlation in the increase of myometrial blood flow with an increase in aortic blood pressure du.ring periods of light, their observations suggest that the increase in blood pressure is not primarily responsible for changes in myometrial flow and that multiple factors may be operative. Perhaps an additional explanation for the increase in myometrial flow is a redistribution of uterine blood

840

Harbert, Croft, and Spisso

flow due to a significant increase in placental vasculat resistance without any significant change in m!~ometriai vascuiar resistance. Another important observation made b\ the authors is titat total placental blood flow is predicated more upon itttra-amniotic. pressure than on the aorlic pressure head. However, the distribution of placental. Ho\\ to the primary and secondary placentas showed considerable variations within periods of light and darkness. Since total placental blood W O W remained rathct constant during the circadian rhythms. the authors suggested that a homeostatic mechanism may be operative in controlling blood HOM Lo each of the bidiscoid placentas. Acute expt.riments itt our laboratory on nonhuman primates have shown a marked variation itt placcttral blood BOW distribution due to the obstruction of att ovarian vein draining the placental sites. Did the authors note an)- such obstrucrivc mechanism in rhcit experiments? Finally do the authors have any data on thy reiationship 01‘the biorhythms in spontaneous activity of tht nonpregnattt uterus to uterine htmodynatnics in tltr rhesus monkey? If’ so, is the magnitude of’ uterine hemodynamic c,hanges greater in the nottprc,~tiatll than in the pregnant nonhuman primates? Dr. Harbert and his co-investigators should be congratulated f’or their valuable experimental data and ettcotiraged to eiuciclatc the mechanisms \Vhich regulate uterine blood Row and its distribution in nonhuman primates. REFERENCES I

2.

Makowski, E. L., Meschia, G., Droegemueller, W., and Balraglia. F. (1.: Mcaasurement of umbilical arterial blood fiou ro the sheep placenta and f’etus in utero, Circ. Res. 23:623, 1068. Kosenf’cld, (:. R., Morris, F. H., Jr., Makowski, E. L., Meschia, G., and Battaglia, F. C.; Circulatory changes in the reproductive tissues of ewes during pregnancy. Cynecol. 1n\wt. 5252, 1974.

DR. FRANK C. GREISS,JR., N’inston-Salem, North Carolina. Those of us who work with sheep appreciate their phlegmatic if’ not downright stupid nature since it seems to tninimiye much variability during conscious experiments. The&ore, it might also seem appropriate for us to consider detection of such subtle cardiovascular changes as those due to circadian rhythms in such a capricious mammal as the awake monkey as approaching the impossible. However, it would appear to this observer that Dr. Harbert has achieved this goal by combining a carefully planned experimental protocol and impeccably performed experimental techniques with a pervasive bedside manner to quiet his “little patients.” He realized that the postural changes associated with nocturnal activity, that is, lying down, induce significant cardiovascular changes and eliminated this f’actor by keeping the monkeys in a constant

upright position al.tt>t. tttc’\ 11;1tl rc‘( ovct~d l rot11 iurget-!. Dr. flat-bet L. \~ould 2ot1 desc.ribc those ~~rivit.otimental alterationa attettdatit IO tl~~trt~ntittatiotis pc’rfi)t-med during the dark
1101

t he1

c3

In exscttcc. the results she\\ that during the dark cycle, aortic blood pressure (ABP) anti (ardiac output decreased itt equal proportion so chat the total pcriphera1 rcsistattte (TPK) M~S uttchanged. ~Ivoriietrial blood flow (hlBF) and myotrtetrial activity xs t;leasured by intra-amniotic pressure (IAP) decreased with no change in nt)ontet.rial vasculitt- resistance. Finally, piaccntal blood flo\\ (PBF) ittc,reascd approximately 10%. Let us look first at placental vascular resistance. According

to rhc formula

ABP - IAP . Ri + Re = .--___ i,BF , if per-

f’usion pressurc (ABP - IAP) decreased 125’; and PBF increased 10’7 , then placental vasculat. rcsistancc had to decrease about ZOL%.While a local change in intrinsic vascular resistance (Ri) of’ this tnagnitude could occur without being reflected in ‘I‘PK, such a response is \er! unlikei). Thix ot~set~vatioti plus the significant lineal- correlation bclween PBF .tntl I Al’ f’ocus our attention upon the \\a>~ that myotnetriai contractions may affect, PBF. In general these are two. Uterine contractions similarly arlcl irttervilious space presincrease ititt-a-amniotic sures, thus decreasing perfusion pressure and, in turn, PBF. Also \,aacuiar resistance ma) be increased by extrinsic. cotnprcssion of blood vessels by surrounding tnyometrium (Kc). Evaluation of the magnitude of this type 01. vascular resistance has been vet-y difficult. Howc~cr. since the mcrhod prrsently used to calculate placental vascular resistance ttscs the above formula, thus takittg into account changes in perf‘usion pressure induced hi myometrial activity, 1 am left with the conclusion that the extrinsic resistance is a more significant factor than I had f’ormcrlv believed. It would appeal thercf’ot-e that the circadian patterns in PBF presentlydescribed are primarily the result of‘ variations in ntyomctrial activity. Dr. Harbert has previously S~MJWII that titcse patterns correlate \\,ith and can be ahcrecl by changes in ttorepinephrinc excretion patterns in nonpregnant tnonkeys. 1 would be interested to know jvhether similar ratechoiamittc patterns oc( ur during pregnant\ Second: we must consider the changes in tn)ometrial vascular resistance, We believe these to be determined by the formula

ABP Ri t Re =MB~.

Since MBF decreased

10% and ABP decreased 1276, little change in myometriai vascular resistance occurred. However, if’ we look closer at the two components of’ resistance, Ri and Re, it would appear that significant but opposing changes ma) have occurred. Following the reasoning discussed atjo\ e for piacctltai v;rsc~ular rrsist;tnc.r, since

Volume I35 Number

Effects of biorhythms on uterine blood flow

841

7

IAP decrea:,ed significantly, Re decreased similarly. However, as myometrial work decreased, less blooddelivered nutrients were needed and Ri increased accordingly. ‘The net effects of these two resistant changes were offsetting, thus explaining in this case the linear correl.-ltion with ABP. As a perennial studenl. of uterine vascular physiology, I long ago recognized the depths of understanding that can result from carefully performed experiments about basic homeostatic functions. I have been intrigued bl, your findings and I sincerely appreciate the opportumty to comment upon them at this occasion. DR. FREDE,RICK ZUSPAN, Columbus, Ohio. The LIIIanesthetized animal is an excellent model for study. I wonder whether or not you might like to further “tune” your model. Three questions relate to this suggestion. One concerns the position of the mother at night and whether or not you considered having the animal be on its side, even though the monkey is a vertical animal. Second, d.o you really know when the pregnant monkey sleeps in the evening? And is there correlation with your findings? The third point that I would like to make concerns your suggestion about neurohormonal changes concerning homeostasis. We both know that the guinea pig has a depletion of its adrenergic nerves during pregnancy, which means that the amount of norepinephrine might alter myometrial and uterine blood flow. Also did you take any samples of this tissue from the tissue area around the uterus and look at adrenergic nerves by histochemical fluorescence to see whether or not there were alteration:; in amines to confirm this particular finding in the primate? DR. IRWIN .&USER, Bronx, New York. I have a few observations and one or two questions to direct. It cvas stated that these monkeys were all in the upright position during the period of observation. This is interesting since ir demonstrates that these changes of increased placental blood flow do occur with the monkey sitting up in the dark. As Dr. Zuspan indicated, it would be of interest to know what the changes would be if the monkey were turned to a recumbent position, either on its back or on its side. During daylight hours, as was elegantly demonstrated by the studies during uterine contraction, there is a decrease in placental blood flow, which is proportionately greater than I-he decrease in myometrial blood How. This would generate the question of whether there is increased fetal risk due to uterine contractions which occur during daylight. Halberg and: I, about 13 years ago, pointed out that in human beings the time of preference for birth is in the early morning hours. If the present observations are valid for \\omen, labor would be taking place at a time when placental blood How is relatively maximal. The statement is made in the manuscript that the distribution of microspheres to the placenta varies by

approximately 22%--2 I c/cto one placenta and 23% fot another. I wonder whether this is specifically related to the timing of’ the uterine contractions. that is, to the timing of the injection relative to the apogee of uterine contractions. The grou? at the University of Southern California studying tnc fetal side of the placenta reported at a recent meeting of the Society for C) necologic lnvestigation on the evidence of fetal rhythmicity, particularly in corticosteroids and thyroid-stimulating hormone. I wonder if Dr. Harbert would care to comment on any relationship between the fetus and the mother in regulating placental distribution of Hews? Finally, I have two philosophic questions. Other than as a reflection of general rhythmicity, are you willing to say that these observations are of physiologic significance? And what impact does this have on the evaluation of fetal heart rate patterns? DR. MORTIMER ROSEN, Cleveland, Ohio. I continue to follow Dr. Warbert’s work with tremendous interest, and I would like to again compliment him as others have. I think the work is extremely important today for two reasons. As clinicians, 1t.e have little to do that is helpful for patients except to put them at bed rest in the presence of several clinical situations of’ risk to the fetus. This past Sunday I had an opportunity to look around at persons present at a.jogging race I attended. On my left was a woman, advanced in pregnancy, who was obviously to be involved in running about two miles. On my right was a nursing instructor tvho was involved in training pregnant ivomen in exercise and jogging to keep up their physical conditioning during pregnancy. I realized there is very little information that we have about the influence oi rest or ac&ity and exercise on the fetus during pregnanq. My specific question to Dr. Harbert is, would your observations be somewhat more accurate if instead of light and darkness cycles you looked at the behavioral cycles of the studied nonhuman primate? During daylight, the animal is awake to participate in different forms of activity (i.e., both visual and physical activity). But even more specifically, at night, during sleep, there are active phases of sleep (rapid eye movement) and quiet phases of sleep, which also may alter the amount of perfusion and the biorhythms that you are attempting to measure. DR. HARBERT (Closing). In answer to Dr. hlakowski’s questions, I will try to take them in order. With the chronic flow probe preparation we usually start measurements on the third to fifth day after surgery and continue them as long as the How probe remains active. On this basis we have seen an increase in uterine blood flow related to progression of the duration of pregnancy. We did not notice any specific relationship regarding time of surgery and blood How changes with the microsphere techmque. The next question was whether or not the blood

842

Harbert, Croft, and Spisso

flows to the uterus and its contents, which were not statistically different between the light and dark periods despite the other changes, were compared by a paired Student’s t test? The answer is yes and while they approached significance, they were still not statistically different. The third question concerned obstruction of the ovarian \ein. In the two of 10 monkeys that had a catheter in the ovarian vein, we did not pick up any periods of occlusion or other changes; I cannot relate to the variations of placental blood flow. In response to the fourth question, we have not measured blood flow distribution to the uterus in the nonpregnant animals. This would be interesting and it may be needed. But the tissues are so small, the cost and availability of monkeys so prohibitive, that it has not been tried. Dr. Greiss asked how well could I convince the monke) that 1 was not there. The answer is not very well. The situation was thus: The monkey was kept in a room by herself. We did not have the capability of being on the outside to inject the monkey, so whenevel the injection study was made, I was physically present. .4t night this involved setting up the material as much as possible beforehand, then going in and injecting the microspheres as rapidly as possible and leaving. The monkey would wake up, and there could be a startled response. For most injections, there was no increase in blood pressure or pulse rate to indicate such a response. W C attempted to control the procedures for injection and study as much as possible between the periods of night and the periods of day. I would anticipate that a startle response of the monkey at night, or any time, would probably decrease the quantity of change but not necessarily the quality. We would still see the variations noted. I agree with Dr. Greiss’s discussion of the variation and its control, that is, the effect of myometrium and myometrial resistance. It is interesting that he chose to consider the changes of placenta1 blood How in relation to the periods of darkness as an increase in flo~i and a decrease in resistance. One might turn it around and think \vith the same reasoning that perhaps the steady state. if’ there is such a thing as a steady state, may correspond to the period of darkness and the increased resistance occurred in the period of light, causing the

decreased flo\\. ‘I‘his would give us more ilisigllt 11110 positive rather than negati\c, changes. The catec+iolamine patterns during prqqi;inc\ iii the monkey ‘11.e circaclial~ in both scrtltn ant1 ul-itrr f’ractions. Dr. %uspan asked about the positioning of the nlonkey. The monkey is a tree animal. 1 assume it normally sleeps in a sitting position, hut honestly do not kno\\. Obviously the primates here are confined to a sitting position and have been ohser\cd to sleep in that position. LYe have not-because this was another variableattempted to move the monkey from the sitting to the supine or lateral supine position. Perhaps this is something that we need to approach. Likewise, Dr. Luspan, I have not done immunofluorescence studies on tissue catechols. Dr. Kaiser asked about the interrelationship of the maternal and fetal conditions to variations of placental How. U’e have not studied the fetal side. I think this may have a vet-> direct bearing on some of the changes that we see in the intermittency of hlood I-low to the various cotyledons of the placenta. It has hem shown by others (Martin, Ramsey, and co-workers) in radiographic studies of monkeys that just as maternal blood How varies, fetal blood f-low varies. The physiologic significance. I feel, as related in Dr. Greiss’s comments, is primarily that it gives us basic underslanding that may eventually lead to significant clinical applications, such as homeostalic control of placental blood flow that may protect the fetus from sudden changes in maternal blood pressure. The physiologic significance of this on fetal heart rate patterns relates to some observations that indicate there is a difference in fetal heart rate patterns in the so-called nonstress tests, depending on the time of‘ da?. When the tests are performed during the early morning, on a circadian pattern the uterine activity 1s lower and blood ROM. is higher: the results are frequently different f‘rom those of tests performed in the late afternoon when the opposite or l-everse conditions tend to exist. Dr. Rosen’s suggestions, especially those related to rapid eye movement sleep and the various behavior pattern cycles of the monkeys, are interesting aspects. We have not made the studies, but they obviously may have some significance in totally understanding the biorhvthms of uterine tl\ namics.