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Measurement of umbilical cord blood flow by local thermodilution
Measurement
of umbilical cord
blood flow by local thermodilution Z.
K.
J.
HODR,
M.D.
V.
GANZ,
M.D.
A.
FRON&K,
Prague,
STEMBERA,
M.D.
M.D.
Czechoslovakia
peared the research on the fetoplacental circuit which, although relying upon indirect determination of blood flow, became the classic foundation for subsequent work in this field.2j 3, 4 A methodological advance which allowed direct measurement of umbilical cord blood flow involved the use of plethysmography.5-7 More recently introduced approaches to determination of fetoplacental blood flow have involved flowmeters, either directly or indirectly.“-I’
E N D E A v o R s to elucidate fetal physiology and pathophysiology remain the center of interest for obstetricians throughout the world. Since asphyxia is still one of the most frequent causes of perinatal death, obstetric concern has focused upon fetal gas and energy metabolism. The first phases of our research in this field were limited to the determination of the concentration of various substances (oxygen, carbon dioxide, lactic acid, pyruvic acid, etc.) in the umbilical artery and vein, and comparison of these values with those in the mother. Further progress, however, was dependent upon a means of determining the quantitative rates of fetal uptake or output of substances in question. This required not only the knowledge of the concentrations, but also of the blood flow in the fetoplacental circuit. The fetoplacental circulation was the subject of physiological research even in the last century, as shown by the work of Cohnstein and Zuntzl in 1884, who attempted to measure blood flow in the fetoplacental circuit in the animal, using a flowmeter. The lack of technical progress at that time made their results dubious, and they remain chiefly of historical interest. Only 30 years ago ap-
Mef hod We have used the method of local thermodilution, as described by FronEk and Ganz” for determining umbilical cord blood flow. The catheter is replaced by two needles; through one is injected the indicator against the direction of blood flow and in the second is a thermistor to detect changes in temperature. The indicator solution used is 2 ml. of 5 per cent glucose at a temperature of about 20’ C. The needle containing the thermistor is introduced into the umbilical vein 3 to 5 cm. from the first. Changes in resistance in the thermistor are measured using an A-C bridge and registered on the Elema mingograph in the form of a thermodilution curve (Fig. 1). Calibration of resistance changes is carried out using resistance connected in series with the thermistor. Resistances are converted to temperature changes employing the curves obtained from
From the Institute for the Care of Mother and Child, Prague-Podoli, and Znstitute for Cardiovascular Research, Prague-Kr?.
531
532
Stembera
Octohc~l-
et al.
Am.
m x 60 x r (tl, - t,) Axf
m .x Volume
of injected
indicator
RESISTANCE BRIDGE
Speed of recording paper in millimeters per second. tt, = Initial temperature of blood, determined by thermistor introduced in second, detector needle. ti = Temperature of indicator solution. 1.08 = Constant, given by specific gravidity and specific heat of blood and indicator solution. A = Area under dilution curve, measured by planimetry. f = Factor relating temperature change in degrees to 1 mm. deflection of curve.
x 1.08
in millimeters.
RECORDING UNIT
Results The accuracy of this approach was checked using an isolated segment of human umbilical cord. The segment was joined to a rubber tube through which flowed water at about 37’ C. Proximal to the cord was a flowmeter measuring and governing the rate of fluid flow and distal to it was the clamp by means of which the pressure within the cord was raised to 30 mm. Hg. Various dilution curves obtained using this model were inversely proportional to the rate of flow (Fig. 3 and Table I). In Table I are given the absolute values of flow with the corresponding measured flow values as determined by local thermodilution. Table I contains estimates of variances (Sf ) and stan-
t
THERMISTOR Fig. 1. registration umbilical
Schematic systems cord blood
diagram of measuring and set up for determination of flow by local thermodilution.
18000 -
Fig.
2. Relationship
I
I
I
I
I
39
38
37
36
35
between
thermistor
resistance
15, 1964 & (:ynw.
r =
the thermistor alone at various temperatures (Fig. 2). The area under the curve is determined planimetrically. Blood flow in the umbilical vein is calculated by equation (1) : (1) F1ow =-
J. Ohs.
and
temperature
I
34 OC of fluid.
Volume Numht,r
Umbilical
90 4
dard deviations (Sr) for each average flow value. It can be seen, that with increasing values of flow the estimates of standard deviations increase, practically linearly. We have, therefore, used a logarithmic transformation with which variances of the log flow do not change with the actual flow values, and we have, then, calculated the estimated variance of log flow according to equation
12) : - ql)” 2 n, q, = Log actual fllow (qx = log Pi ). yii =L Log measured flow xi, (y,, = log using local thermodilution XII) method. III = the number of measurements of PI.
(2) s? (log)
Whw-e
=
211
(Yil
On the basis of this estimate we determined the tolerance limits for the log of the measured values and by antilogs we determined the limits presented in Fig. 4. These tolerance limits were calculated to include 95 per cent to 99 per cent of the measurements with a confidence coefficient of 0.95.
Fig. 3. Local
thermodilution
curves
obtained
at various
cord blood
flow measurement
533
These tolerance limits indicate that the actual flow is, for example, 300 ml., 95 per cent of the individual measurements will lie between 274 and 328 ml. with a confidence coefficient of 0.95. We have carried out blood flow measurements in the umbilical vein of the human newborn using this method, and the results obtained in the first 6 cases are presented in Table II. Further data on the human will be summarized in another report. Comment
Two main difficulties long prevented adequate studies of fetoplacental circuIation in man: 1. Access to the fetus such as to insure maintenance of physiological intrauterine conditions. 2. Availability of a reliable method usable in the human and permitting objective registration of blood flow. The first requirement mentioned, obtain-
rates
of flow,
in model
experiment.
534
Stembera
et al.
ing access to the fetus and umbilical cord in such a manner as to preserve as far as possible physiological conditions within the uterus, has been resolved in the same way by all workers in this field; measurements were carried out with the fetus delivered and left attached to the pulsating cord, during the period when the placenta was still firmly in place on the uterine wall. Here, however, a marked species difference betwc!en the human and the sheep arises: while
ml measured 500 I of flow
in the sheep, the placenta is normally adherent to the uterine wall for several hours following birth, in the human, as confirmed by Warnekros,13 Weibel,14 Burton-Browne,l’ Leinzinger,l” MacPherson and Wilson,” Gaz6rek and KFikal,lS Stembera and Hodr,l” only a few minutes intervene between the birth of the infant and placental detachment from its bed in the uterine wall. Only Assal?” measured blood flow in human fetuses in utero during therapeutic abortion
values
400 --
L-.A
100
200
400 absolute
500 ml values of flow
Fig. 4. Tolerance limits of 95 per cent and 99 per cent for the measured values of flow determined local thermodilution method (95 per cent, full line, and 99 per cent, interrupted line).
by
Umbilical
Table I. Measured statistical data . -.. -_____Absolute values
flow values determined
Pi
1 2 3 4
505 495 427 435 346 362 292 304 224 159 88 60
i 7 8 9 IO 11 12
Measured by local
flow values thermodilution
after
determined method
XI) ’
I
506 529 410 441 357 374 319 320 220 161 89 60
Table II. Umbilical minutes
by local thermodilution
2 524 477 423 441 347 346 298 308 224 159 83 60
I
3 524 493 416 419 319 357 304 308 212 152 91 62
I
4 524 529 404 433 328 374 291 316 204 158 85 61
I
5
I
6
-
-
520 430 406 347 346 305 220 82 66
-337 346 -
Mean flow value XL 519 509 417 428 339 357 303 311 216 157 86 62
No.
Birth 804 818 828 934 936 939
No.
flow
method
measurement
535
and
Number of neasure-
mentc n, 4 5
weight
(grams)
2,900 4,100 3,030 3,050 3,550 3,250
from birth to start of measurement (sec.) 22 24 20 23 20 23
(at 3 to 6 months gestation), during uterotomy when preserving fetal life was not of concern. In previous work Stembera and HodP, 21, 22 we found that within the first 2 minutes following birth several physiological parameters dependent upon fetoplacental circulation remain the same as under intrauterine conditions (volume per cent oxygen content in umbilical vessel blood, rate of disappearance of various substances into the placenta, placental circulation time, etc.). These considerations dictated the fundamental requirements of method : 1. A technically accurate method of direct measurement. 2. One involving no longer either to mother or infant.
16.4 25.5 13.9 15.3 11.5 13.4 15.1 9.3 10.7 3.7 4.0 2.9
271 653 193 235 209 180 227 87 115 13 16 8
; 6 6 4 5 5 4 5 5
blood flow in human newborn infants during measured by local thermodilution method
birth,
Time
Case
blood
-__
Of Pow i
cord
the first 2
of
measurements (per local thermodiiution curves) 7 6 6 8 6 7
Umbilical
blood /
__I
207 340 231 250 235 266
fkxo
-
ml./min./Kg. 71 83 77 81 71 ____--__ 74
3. A method whose total period of preparation, from the moment of access to the umbilical cord following delivery of the infant, takes only 20 to 30 seconds. 4. A method yielding reliable data within 60 to 90 seconds of actual measurement. Of currently employed methods for blood flow determination, local thermodilution proved most satisfactory in fulfilling the requirements outlined above. Some practice was necessary under clinical conditions in carrying out preparatory maneuvers (introduction of thermistor-needle, adjustment of resistance bridge to zero with resistances cancelling out blood temperature effects, injection of indicator solution, and registration of dilution curve) before we were able to achieve the first measurement of blood flow
536
Stembera
October Am. J. Obst.
et al.
on the average 20 seconds after the moment of complete birth of the infant. Repeated injections of indicator solution can be made at about 8 to 10 second intervals, so that usually about 6 to 8 thermodilution curves can be recorded during the first 90 seconds following the birth.
15, 1964 & Gynec.
cord joined to a flow system. There was good agreement between flow values measured by flowmeter and those calculated from local dilution curves, in the range 60 to 500 ml. per minute. This method has proved feasible and reliable when used to determine blood flow in the pulsating human umbilical cord immediately after birth.
Conclusion A method for determining blood flow in the human umbilical cord is described. The local thermodilution method was used and first tested in vitro using isolated umbilical
by
The statistical calculations Ing J. Vondr6c’ek from
Institute of the ences, Prague.
Czechoslovak
were carried the Mathematical Academy
of
out Sci-
REFERENCES
Cohnstein, J., and Zuntz, N.: Pfliig. Arch. Ges. Physial. 34: 173, 1884. 2. Barcroft, J., Flexner, L. B., and McClurkin, T.: J. Physiol. 82: 498, 1934. 3. Barcroft, J., Kennedy, J. A., and Mason, M. F.: J. Physiol. 95: 269, 1939. 4. Haselhorst, G.: Ztschr. f. Geburtsh. u. Gyn6k. 96: 487, 1929. 5. Barcroft, J., and Torrens, D. S.: J. Physiol. 105: 22, 1946. K. E., and Greenfield, A. D. M.: 6. Cooper, J. Physiol. 108: 167, 1949. 7. Acheson, G. H., Dawes, G. S., and Mott, J. C.: J. Physiol. 135: 623, 1957. G. S., Mott, J. C., and Vane, J. R.: 8. Dawes, J. Physiol. 121: 72, 1953. G. S., and Mott, J. C.: J. Physiol. 9. Dawes. 146: 295, 1959. A.: Nord. med. 43: 221, 1950. IO. Odeblad, Kolin, A., Assali, N., Herold, G., and Jensen, 11. R.: Proc. of the Nat. Acad. Sci. 43: 527-540, 1957. FronPk, A., and Ganz, V.: Circulation Res. 12. 8: 175, 1960. 1.
13. 14. 15. 16. 17. 18. 19.
20. 21. 22.
Warnekros, K.: Arch. Gynlk. 109: 266, 1918. Weibel, W.: Arch. GynLk. 111: 413, 1919. Burton-Browne: J. Obst. & Gynaec. Brit. Emp. 56: 847, 1949. Leinzinger, E.: Arch. GynHk. 187: 154, 1955. MacPherson, J., and Wilson, K. J.: J. Obst. & Gynaec. Brit. Emp. 63: 321, 1956. Gazgrek, F., Kiikal, Z.: I%. Gynlk. 23: 442, 1958. Stembera, Z. K., and Hodr, J.: “GynHkologentagung 1959,” Leipzig, 1962, VEB Georg Thieme, pp. 81-93. Assali, N. S., Rauramo, L., and Peltonen, T.: AM. J. OBST. & GYNEC. 79: 86, 1960. Stembera, Z. K., and Hodr, J.: Cesk Physiol. 11: 482, 1962,. Stembera, Z. K., and Hodr, J.: Cesk Physiol. 11: 483, 1962.
Nabrezi K. Marxe Prague-Pod&i C.zechoslovakia
157
of umbilical cord
blood flow by local thermodilution Z.
K.
J.
HODR,
M.D.
V.
GANZ,
M.D.
A.
FRON&K,
Prague,
STEMBERA,
M.D.
M.D.
Czechoslovakia
peared the research on the fetoplacental circuit which, although relying upon indirect determination of blood flow, became the classic foundation for subsequent work in this field.2j 3, 4 A methodological advance which allowed direct measurement of umbilical cord blood flow involved the use of plethysmography.5-7 More recently introduced approaches to determination of fetoplacental blood flow have involved flowmeters, either directly or indirectly.“-I’
E N D E A v o R s to elucidate fetal physiology and pathophysiology remain the center of interest for obstetricians throughout the world. Since asphyxia is still one of the most frequent causes of perinatal death, obstetric concern has focused upon fetal gas and energy metabolism. The first phases of our research in this field were limited to the determination of the concentration of various substances (oxygen, carbon dioxide, lactic acid, pyruvic acid, etc.) in the umbilical artery and vein, and comparison of these values with those in the mother. Further progress, however, was dependent upon a means of determining the quantitative rates of fetal uptake or output of substances in question. This required not only the knowledge of the concentrations, but also of the blood flow in the fetoplacental circuit. The fetoplacental circulation was the subject of physiological research even in the last century, as shown by the work of Cohnstein and Zuntzl in 1884, who attempted to measure blood flow in the fetoplacental circuit in the animal, using a flowmeter. The lack of technical progress at that time made their results dubious, and they remain chiefly of historical interest. Only 30 years ago ap-
Mef hod We have used the method of local thermodilution, as described by FronEk and Ganz” for determining umbilical cord blood flow. The catheter is replaced by two needles; through one is injected the indicator against the direction of blood flow and in the second is a thermistor to detect changes in temperature. The indicator solution used is 2 ml. of 5 per cent glucose at a temperature of about 20’ C. The needle containing the thermistor is introduced into the umbilical vein 3 to 5 cm. from the first. Changes in resistance in the thermistor are measured using an A-C bridge and registered on the Elema mingograph in the form of a thermodilution curve (Fig. 1). Calibration of resistance changes is carried out using resistance connected in series with the thermistor. Resistances are converted to temperature changes employing the curves obtained from
From the Institute for the Care of Mother and Child, Prague-Podoli, and Znstitute for Cardiovascular Research, Prague-Kr?.
531
532
Stembera
Octohc~l-
et al.
Am.
m x 60 x r (tl, - t,) Axf
m .x Volume
of injected
indicator
RESISTANCE BRIDGE
Speed of recording paper in millimeters per second. tt, = Initial temperature of blood, determined by thermistor introduced in second, detector needle. ti = Temperature of indicator solution. 1.08 = Constant, given by specific gravidity and specific heat of blood and indicator solution. A = Area under dilution curve, measured by planimetry. f = Factor relating temperature change in degrees to 1 mm. deflection of curve.
x 1.08
in millimeters.
RECORDING UNIT
Results The accuracy of this approach was checked using an isolated segment of human umbilical cord. The segment was joined to a rubber tube through which flowed water at about 37’ C. Proximal to the cord was a flowmeter measuring and governing the rate of fluid flow and distal to it was the clamp by means of which the pressure within the cord was raised to 30 mm. Hg. Various dilution curves obtained using this model were inversely proportional to the rate of flow (Fig. 3 and Table I). In Table I are given the absolute values of flow with the corresponding measured flow values as determined by local thermodilution. Table I contains estimates of variances (Sf ) and stan-
t
THERMISTOR Fig. 1. registration umbilical
Schematic systems cord blood
diagram of measuring and set up for determination of flow by local thermodilution.
18000 -
Fig.
2. Relationship
I
I
I
I
I
39
38
37
36
35
between
thermistor
resistance
15, 1964 & (:ynw.
r =
the thermistor alone at various temperatures (Fig. 2). The area under the curve is determined planimetrically. Blood flow in the umbilical vein is calculated by equation (1) : (1) F1ow =-
J. Ohs.
and
temperature
I
34 OC of fluid.
Volume Numht,r
Umbilical
90 4
dard deviations (Sr) for each average flow value. It can be seen, that with increasing values of flow the estimates of standard deviations increase, practically linearly. We have, therefore, used a logarithmic transformation with which variances of the log flow do not change with the actual flow values, and we have, then, calculated the estimated variance of log flow according to equation
12) : - ql)” 2 n, q, = Log actual fllow (qx = log Pi ). yii =L Log measured flow xi, (y,, = log using local thermodilution XII) method. III = the number of measurements of PI.
(2) s? (log)
Whw-e
=
211
(Yil
On the basis of this estimate we determined the tolerance limits for the log of the measured values and by antilogs we determined the limits presented in Fig. 4. These tolerance limits were calculated to include 95 per cent to 99 per cent of the measurements with a confidence coefficient of 0.95.
Fig. 3. Local
thermodilution
curves
obtained
at various
cord blood
flow measurement
533
These tolerance limits indicate that the actual flow is, for example, 300 ml., 95 per cent of the individual measurements will lie between 274 and 328 ml. with a confidence coefficient of 0.95. We have carried out blood flow measurements in the umbilical vein of the human newborn using this method, and the results obtained in the first 6 cases are presented in Table II. Further data on the human will be summarized in another report. Comment
Two main difficulties long prevented adequate studies of fetoplacental circuIation in man: 1. Access to the fetus such as to insure maintenance of physiological intrauterine conditions. 2. Availability of a reliable method usable in the human and permitting objective registration of blood flow. The first requirement mentioned, obtain-
rates
of flow,
in model
experiment.
534
Stembera
et al.
ing access to the fetus and umbilical cord in such a manner as to preserve as far as possible physiological conditions within the uterus, has been resolved in the same way by all workers in this field; measurements were carried out with the fetus delivered and left attached to the pulsating cord, during the period when the placenta was still firmly in place on the uterine wall. Here, however, a marked species difference betwc!en the human and the sheep arises: while
ml measured 500 I of flow
in the sheep, the placenta is normally adherent to the uterine wall for several hours following birth, in the human, as confirmed by Warnekros,13 Weibel,14 Burton-Browne,l’ Leinzinger,l” MacPherson and Wilson,” Gaz6rek and KFikal,lS Stembera and Hodr,l” only a few minutes intervene between the birth of the infant and placental detachment from its bed in the uterine wall. Only Assal?” measured blood flow in human fetuses in utero during therapeutic abortion
values
400 --
L-.A
100
200
400 absolute
500 ml values of flow
Fig. 4. Tolerance limits of 95 per cent and 99 per cent for the measured values of flow determined local thermodilution method (95 per cent, full line, and 99 per cent, interrupted line).
by
Umbilical
Table I. Measured statistical data . -.. -_____Absolute values
flow values determined
Pi
1 2 3 4
505 495 427 435 346 362 292 304 224 159 88 60
i 7 8 9 IO 11 12
Measured by local
flow values thermodilution
after
determined method
XI) ’
I
506 529 410 441 357 374 319 320 220 161 89 60
Table II. Umbilical minutes
by local thermodilution
2 524 477 423 441 347 346 298 308 224 159 83 60
I
3 524 493 416 419 319 357 304 308 212 152 91 62
I
4 524 529 404 433 328 374 291 316 204 158 85 61
I
5
I
6
-
-
520 430 406 347 346 305 220 82 66
-337 346 -
Mean flow value XL 519 509 417 428 339 357 303 311 216 157 86 62
No.
Birth 804 818 828 934 936 939
No.
flow
method
measurement
535
and
Number of neasure-
mentc n, 4 5
weight
(grams)
2,900 4,100 3,030 3,050 3,550 3,250
from birth to start of measurement (sec.) 22 24 20 23 20 23
(at 3 to 6 months gestation), during uterotomy when preserving fetal life was not of concern. In previous work Stembera and HodP, 21, 22 we found that within the first 2 minutes following birth several physiological parameters dependent upon fetoplacental circulation remain the same as under intrauterine conditions (volume per cent oxygen content in umbilical vessel blood, rate of disappearance of various substances into the placenta, placental circulation time, etc.). These considerations dictated the fundamental requirements of method : 1. A technically accurate method of direct measurement. 2. One involving no longer either to mother or infant.
16.4 25.5 13.9 15.3 11.5 13.4 15.1 9.3 10.7 3.7 4.0 2.9
271 653 193 235 209 180 227 87 115 13 16 8
; 6 6 4 5 5 4 5 5
blood flow in human newborn infants during measured by local thermodilution method
birth,
Time
Case
blood
-__
Of Pow i
cord
the first 2
of
measurements (per local thermodiiution curves) 7 6 6 8 6 7
Umbilical
blood /
__I
207 340 231 250 235 266
fkxo
-
ml./min./Kg. 71 83 77 81 71 ____--__ 74
3. A method whose total period of preparation, from the moment of access to the umbilical cord following delivery of the infant, takes only 20 to 30 seconds. 4. A method yielding reliable data within 60 to 90 seconds of actual measurement. Of currently employed methods for blood flow determination, local thermodilution proved most satisfactory in fulfilling the requirements outlined above. Some practice was necessary under clinical conditions in carrying out preparatory maneuvers (introduction of thermistor-needle, adjustment of resistance bridge to zero with resistances cancelling out blood temperature effects, injection of indicator solution, and registration of dilution curve) before we were able to achieve the first measurement of blood flow
536
Stembera
October Am. J. Obst.
et al.
on the average 20 seconds after the moment of complete birth of the infant. Repeated injections of indicator solution can be made at about 8 to 10 second intervals, so that usually about 6 to 8 thermodilution curves can be recorded during the first 90 seconds following the birth.
15, 1964 & Gynec.
cord joined to a flow system. There was good agreement between flow values measured by flowmeter and those calculated from local dilution curves, in the range 60 to 500 ml. per minute. This method has proved feasible and reliable when used to determine blood flow in the pulsating human umbilical cord immediately after birth.
Conclusion A method for determining blood flow in the human umbilical cord is described. The local thermodilution method was used and first tested in vitro using isolated umbilical
by
The statistical calculations Ing J. Vondr6c’ek from
Institute of the ences, Prague.
Czechoslovak
were carried the Mathematical Academy
of
out Sci-
REFERENCES
Cohnstein, J., and Zuntz, N.: Pfliig. Arch. Ges. Physial. 34: 173, 1884. 2. Barcroft, J., Flexner, L. B., and McClurkin, T.: J. Physiol. 82: 498, 1934. 3. Barcroft, J., Kennedy, J. A., and Mason, M. F.: J. Physiol. 95: 269, 1939. 4. Haselhorst, G.: Ztschr. f. Geburtsh. u. Gyn6k. 96: 487, 1929. 5. Barcroft, J., and Torrens, D. S.: J. Physiol. 105: 22, 1946. K. E., and Greenfield, A. D. M.: 6. Cooper, J. Physiol. 108: 167, 1949. 7. Acheson, G. H., Dawes, G. S., and Mott, J. C.: J. Physiol. 135: 623, 1957. G. S., Mott, J. C., and Vane, J. R.: 8. Dawes, J. Physiol. 121: 72, 1953. G. S., and Mott, J. C.: J. Physiol. 9. Dawes. 146: 295, 1959. A.: Nord. med. 43: 221, 1950. IO. Odeblad, Kolin, A., Assali, N., Herold, G., and Jensen, 11. R.: Proc. of the Nat. Acad. Sci. 43: 527-540, 1957. FronPk, A., and Ganz, V.: Circulation Res. 12. 8: 175, 1960. 1.
13. 14. 15. 16. 17. 18. 19.
20. 21. 22.
Warnekros, K.: Arch. Gynlk. 109: 266, 1918. Weibel, W.: Arch. GynLk. 111: 413, 1919. Burton-Browne: J. Obst. & Gynaec. Brit. Emp. 56: 847, 1949. Leinzinger, E.: Arch. GynHk. 187: 154, 1955. MacPherson, J., and Wilson, K. J.: J. Obst. & Gynaec. Brit. Emp. 63: 321, 1956. Gazgrek, F., Kiikal, Z.: I%. Gynlk. 23: 442, 1958. Stembera, Z. K., and Hodr, J.: “GynHkologentagung 1959,” Leipzig, 1962, VEB Georg Thieme, pp. 81-93. Assali, N. S., Rauramo, L., and Peltonen, T.: AM. J. OBST. & GYNEC. 79: 86, 1960. Stembera, Z. K., and Hodr, J.: Cesk Physiol. 11: 482, 1962,. Stembera, Z. K., and Hodr, J.: Cesk Physiol. 11: 483, 1962.
Nabrezi K. Marxe Prague-Pod&i C.zechoslovakia
157