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Permeability of chorion laeve in cases of normal and retarded fetal growth
Permeability
of chorion laeve in cases of normal
and retarded fetal growth W. B.
M.
0.
MOORE,
S. WARD,
Manchester,
F.R.C.O.G.
H.N.C.
England
The in vitro permeability of the human placenta to sucrose and urea is described. The conclusion from these membrane transport studies is that, ger unit surface area of chorion laeve, there is no significant difference in fiermeability between placentas from cases of normal and retarded fetal growth.
THE TERM “placental insufficiency” is used by obstetricians as a plausible explanation for cases of intrauterine growth retardation. While there is no doubt about the impairment with regard to fetal nutrition and oxygen supply, it is not clear whether the impairment is due to changes within the placenta, a reduction in blood flow to the placenta, or a combination of both. In order to determine in vivo the amount of a substance transferred to the fetus per unit time, it is necessary to measure the rate of blood flow on each side of the placenta and the arteriovenous difference of the particular substance. Because there are no satisfactory methods for making these measurements, investigations have been directed toward in vitro methods which permit quantitative studies of the transfer and utilization of solutes by the membranous portion of the placenta. In this project, the in vitro permeability of the human chorion laeve to sucrose and
From the Department Gynecology, University
urea is described for two groups ( 1) cases of normal fetal growth, cases of retarded fetal growth. Material
by Grant No. Research
Council. Received
for
Accepted
for publication
publication
April 25, 1972. June
and
(2)
methods
The method of Battaglia and associates’ for in vitro investigation of chorion as a membrane system provides a quantitative description of the permeability of the placental membrane. The tissue referred to as chorion laeve and amnion represents a piece of intact membranous portion of the placenta taken between 2 and 6 cm. from the edge of the cake and obtained within 5 minutes of dclivery, The chorion laeve was not studied as an isolated tissue layer because, in some cases of premature delivery, stripping off the amnion is difficult and may damage the chorion. The method provides for the mounting of tissue as a vertical membrane which separates two fluid chambers, each containing 10 ml. of the bicarbonate-buffered solution of Gel and Gey? with 50 mg. per cent glucose added. Where possible, three pieces of membrane were studied simultaneously from each plaIn each experiment, sucrose, 0.3 centa. mmole, and urea, 0.2 mmole, were added to the solution in contact with the maternal surface so that a permeability constant could be obtained for the same tissue to two solutes. The in vitro incubations were carried out in a constant temperature water bath at 37’ C. In each experiment, samples were taken
of Obstetrics and of Manchester.
This project was supported G968/170 from the Medical
and
of cases:
14, 1972.
Reprint requests: Dr. W. M. 0. Moore, St. Mary’s Hospital, Whitworth Park, Manchester, Ml3 O/H, England. 904
Volllme Number
114 7
Permeability
from the solution in contact with the fetal and maternal surfaces of the membrane prior to the commencement and at 30 minute intervals throughout the 120 minutes of the incubation, and their sucrose and urea contents were measured. The permeability constant of the membrane to each solute was determined, the calculations being those described by Battaglia and Hellegers.3 A total of 24 placentas were studied, 16 from cases of normal fetal growth and 8 from cases of retarded fetal growth. Birth weights were related to gestational maturity at delivery, and comparison with the data of Butler and Alberman distinguished those within the normal range from those small for gestational age, the latter being below their tenth percentile. Histologic examination of the membranes was carried out in all cases. With the use of an ocular micrometer, the amount of decidua attached to the chorion was found to be similar in both groups. The permeability constants refer to the total thickness of the membranes, with no correction made for attached decidua. Results The complete data for one in vitro incubation are illustrated in Table I. For each membrane studied, concentration gradients were calculated at 30 minute intervals, as follows: (Cm - Cf)T,
+ (Cm - Cf)T, 2 = Gradient T1 - T,
where (Cm - Cf) T, = the concentration difference between both sides of the membrane at the beginning of a 30 minute interval and (Cm - Cf) T, = the concentration difference between both sides at the end of a 30 minute interval. The permeability constant of the membrane represented by the quantity transferred per unit concentration gradient is expressed in units of clearance (microliters per minute). Over the 120 minutes of incubation, the permeability constants for each 30 minute period showed a tendency to increase as the incubation continued. The results for all ex-
of chorion
laeve
905
periments are given in Table II. The permeability constants given in this table refer to the mean value calculated from the four permeability constants for each 30 minute period. Previous studies32 5 have shown that the transfer of sucrose and urea across the chorion laeve is proportional to the concentration gradient consistent with Fick’s law governing transfer by simple diffusion. By rearranging the Fick equation:
dQ ix.4 dc Z-ZC, - C, d so that the membrane becomes part of the constant, it is possible to use the equation to compare the permeability characteristics of different membranes to similar solutes.” Larger
values
for
y
indicate
more
rapid
diffusion. Analysis of the results for urea show that the differences in the permeability constants between individual placentas in both the “normal” and “retarded” groups were highly significant (p < 0.005)) but the difference in permeability constants between the “normal” and “retarded” groups was not significant (0.1 > p > 0.05). Analysis of the results for sucrose showed that the differences in the permeability constants between individual placentas in both the “normal” and “retarded” groups were highly significant (p < 0.005)) but the difference in permeaability constants between the “normal” groups was not signifiand “retarded” cant (0.25 > p > 0.1). It cannot be concluded, therefore, that there is a significant difference in the permeability constant between the “normal” and “retarded” groups. Comment As discussed by Seeds,c the bulk of transfer of nutrients from mother to fetus probably takes place across the chorion frondosum. The absence of a perfusing circulation must diminish the amount of transfer across those portions of chorion laeve that are distant
906
Moore
December 1, 1972 Am. J. Obstet. Gynecol.
and Ward
Table I. Data of urea permeability for a typical chorion laeve and amnion as one tissue Time (min.)
Concentration on maternal side (pmole/ml.)
Concentration on fetal side (,umole/ml.)
20.29 17.93 17.24 16.67 16.25
0 0.29 0.90 1.49 1.97
0 30 60 90 120 Mean
Concentration gradient (ymole/ml.) 18.97 16.99 15.76 14.73
Table II. Urea and sucrose permeability constants for placentas from cases of normal and retarded fetal growth
(Zii)
(5%)
Yi$$
~ “gij!y
Cases of normal fetal growth 26 36 40 40 39 37 34 35 39
765 2,126 3,374 3,572 3,317 2,750 1,871 2,190 3,487
38 40
3,572 3,856
40
3,997
37
2,820
39
3,150
33
1,760
35
2,300
Cases of retarded 38 2,509 2,552 ;; 2,071 38
2,525
37
2,119
36
1,531
37
1,616
38
1,765
7.17 7.20, 15.47 16.61 14.64
7.27
10.55, 7.58 19.47, 20.35, 16.68 15.98, 13.89 10.24, 13.32, 8.58 16.64, 14.56, 18.37 9.32, 8.10, 13.50 10.96, 13.77, 11.84 13.42, 15.11, 11.60 6.56, 11.90, 14.42 fetal growth 15.41 8.53 14.42, 13.68, 12.21 9.30, 10.37, 12.60 5.25, 6.18, 5.93 5.41, 4.69, 4.48 12.84, 13.15, 10.41 13.80, 8.36, 12.32
2.33 2.11, 3.38 6.96
single
2.40
3.28, 2.24, 2.32, 4.97,
5.37, 5.82, 2.20 3.44
4.58 7.25
5.48, 1.82,
7.12, 3.41,
5.04 2.1.9
4.50,
3.22,
4.09
2.54,
3.27,
2.68
4.60,
5.65,
3.43
2.67,
3.04,
4.22
5.85 3.86 4.10,
3.89,
4.55
2.52,
2.56,
2.95
0.99,
1.46,
1.21
1.45,
1.27,
0.92
3.35,
3.67,
2.86
4.03,
2.05,
2.51
2.99
in vitro
incubation
Quantit~~ transferred (pmole/m;n. 0.097 0.147 0.159 0.154
of
)
Permeability constant (pl/min.) 5.11 8.65 10.09 10.45 8.58
from the placental cake. Because the most significant exchange across the chorion laevc is likely to occur in those areas adjacent to the placental cake, the tissues studied were taken from there. Factors influencing diffusion of solutes across the chorion include concentration difference, perfusion of the eschanging surface area, size of the fetal capillary bed, and thickness of the membrane. The conclusion from these membrane transport studies is that per unit surface area of chorion laeve and amnion there is considerable variability in the permeability of individual placentas, but there is no significant difference between placentas from cases of normal and retarded fetal growth. Histologic examination of the membranes from the growth-retarded cases showed no clear abnormality. To date, no chemical abnormality has been demonstrated in such placentas.7 Aherne and DunniP report that fetal capillary surface area corresponds closely to the villous surface area. In cases with growthretarded infants, they found a mean placental volume of 350 ml. at term (normal = 488 ml.) with a mean villous surface area of 6.4 sq. M. (normal = 11.0 square meters). Thomson and associate9 have demonstrated that the small-for-dates baby does not have, on the average, a relatively small placenta when it is born, and they point out it is not permissible to assume, without further evidence, that the functional adequacy of the placenta is proportional to its size. Duncan and LewiP showed in rabbits that larger fetuses had larger placentas but also had both absolutely and relatively higher maternal placental blood flows. In other
Volume Number
114 1
words,
the correlation between fetal size and placental blood flow was not wholly explained by placental size. Investigation of causes of intrauterine growth retardation should inelude, in addition to the factors already listed, studies on the role of metabolic activity within the placenta.
REFERENCES 1.
2. 3. 4.
5.
Battaglia, F. C., Hellegers, A. E., Meschia, G., and Barron. D. H.: Nature 196: 1061. 1962. Gey, G. 0.1 and Gey, M. K.: Am. J. Cancer 27: 45, 1936. Battaglia, F. C., and Hellegers, A. E.: AM. T. OBSTET. GYNECOL. 89: 771. 1964. Butler, N. R., and Alberman, E. D.: The Second Report of the 1958 British Perinatal Mortality Survey, Edinburgh, 1969, E. & S. Livingstone, Ltd.. D. 331. Moore, W.’ M. ’ 6., Hellegers, A. E., and Battaglia, F. C.: AM. J. OBSTET. GYNECOL. 96: 951, 1966.
Permeability
of chorion
laeve
907
The statistical analysis was carried out by Mr. A. C. C. Gibbs, Department of Social and Preventive Medicine, University of Manchester, who advised on the presentation of results.
6.
7. 8. 9.
10.
Seeds, A. E.: In Barnes, A. C., editor: Intrauterine Development, Philadelphia, 1968, Lea & Febiger, Publishers, pp. 103 and 111. Wiiick, M.: J. Pediatr. 71: 390, 1967. Aherne, W., and Dunnill, M. S.: J. Path. Bact. 91: 123, 1966. Thomson, A. M., Billewicz, W. Z., and Hytten, F. E.: J. Obset. Gynaecol. Br. Commonw. 76: 865, 1969. Duncan, S. L. B., and Lewis, B. V.: J. Physiol. 202: 471, 1969.
of chorion laeve in cases of normal
and retarded fetal growth W. B.
M.
0.
MOORE,
S. WARD,
Manchester,
F.R.C.O.G.
H.N.C.
England
The in vitro permeability of the human placenta to sucrose and urea is described. The conclusion from these membrane transport studies is that, ger unit surface area of chorion laeve, there is no significant difference in fiermeability between placentas from cases of normal and retarded fetal growth.
THE TERM “placental insufficiency” is used by obstetricians as a plausible explanation for cases of intrauterine growth retardation. While there is no doubt about the impairment with regard to fetal nutrition and oxygen supply, it is not clear whether the impairment is due to changes within the placenta, a reduction in blood flow to the placenta, or a combination of both. In order to determine in vivo the amount of a substance transferred to the fetus per unit time, it is necessary to measure the rate of blood flow on each side of the placenta and the arteriovenous difference of the particular substance. Because there are no satisfactory methods for making these measurements, investigations have been directed toward in vitro methods which permit quantitative studies of the transfer and utilization of solutes by the membranous portion of the placenta. In this project, the in vitro permeability of the human chorion laeve to sucrose and
From the Department Gynecology, University
urea is described for two groups ( 1) cases of normal fetal growth, cases of retarded fetal growth. Material
by Grant No. Research
Council. Received
for
Accepted
for publication
publication
April 25, 1972. June
and
(2)
methods
The method of Battaglia and associates’ for in vitro investigation of chorion as a membrane system provides a quantitative description of the permeability of the placental membrane. The tissue referred to as chorion laeve and amnion represents a piece of intact membranous portion of the placenta taken between 2 and 6 cm. from the edge of the cake and obtained within 5 minutes of dclivery, The chorion laeve was not studied as an isolated tissue layer because, in some cases of premature delivery, stripping off the amnion is difficult and may damage the chorion. The method provides for the mounting of tissue as a vertical membrane which separates two fluid chambers, each containing 10 ml. of the bicarbonate-buffered solution of Gel and Gey? with 50 mg. per cent glucose added. Where possible, three pieces of membrane were studied simultaneously from each plaIn each experiment, sucrose, 0.3 centa. mmole, and urea, 0.2 mmole, were added to the solution in contact with the maternal surface so that a permeability constant could be obtained for the same tissue to two solutes. The in vitro incubations were carried out in a constant temperature water bath at 37’ C. In each experiment, samples were taken
of Obstetrics and of Manchester.
This project was supported G968/170 from the Medical
and
of cases:
14, 1972.
Reprint requests: Dr. W. M. 0. Moore, St. Mary’s Hospital, Whitworth Park, Manchester, Ml3 O/H, England. 904
Volllme Number
114 7
Permeability
from the solution in contact with the fetal and maternal surfaces of the membrane prior to the commencement and at 30 minute intervals throughout the 120 minutes of the incubation, and their sucrose and urea contents were measured. The permeability constant of the membrane to each solute was determined, the calculations being those described by Battaglia and Hellegers.3 A total of 24 placentas were studied, 16 from cases of normal fetal growth and 8 from cases of retarded fetal growth. Birth weights were related to gestational maturity at delivery, and comparison with the data of Butler and Alberman distinguished those within the normal range from those small for gestational age, the latter being below their tenth percentile. Histologic examination of the membranes was carried out in all cases. With the use of an ocular micrometer, the amount of decidua attached to the chorion was found to be similar in both groups. The permeability constants refer to the total thickness of the membranes, with no correction made for attached decidua. Results The complete data for one in vitro incubation are illustrated in Table I. For each membrane studied, concentration gradients were calculated at 30 minute intervals, as follows: (Cm - Cf)T,
+ (Cm - Cf)T, 2 = Gradient T1 - T,
where (Cm - Cf) T, = the concentration difference between both sides of the membrane at the beginning of a 30 minute interval and (Cm - Cf) T, = the concentration difference between both sides at the end of a 30 minute interval. The permeability constant of the membrane represented by the quantity transferred per unit concentration gradient is expressed in units of clearance (microliters per minute). Over the 120 minutes of incubation, the permeability constants for each 30 minute period showed a tendency to increase as the incubation continued. The results for all ex-
of chorion
laeve
905
periments are given in Table II. The permeability constants given in this table refer to the mean value calculated from the four permeability constants for each 30 minute period. Previous studies32 5 have shown that the transfer of sucrose and urea across the chorion laeve is proportional to the concentration gradient consistent with Fick’s law governing transfer by simple diffusion. By rearranging the Fick equation:
dQ ix.4 dc Z-ZC, - C, d so that the membrane becomes part of the constant, it is possible to use the equation to compare the permeability characteristics of different membranes to similar solutes.” Larger
values
for
y
indicate
more
rapid
diffusion. Analysis of the results for urea show that the differences in the permeability constants between individual placentas in both the “normal” and “retarded” groups were highly significant (p < 0.005)) but the difference in permeability constants between the “normal” and “retarded” groups was not significant (0.1 > p > 0.05). Analysis of the results for sucrose showed that the differences in the permeability constants between individual placentas in both the “normal” and “retarded” groups were highly significant (p < 0.005)) but the difference in permeaability constants between the “normal” groups was not signifiand “retarded” cant (0.25 > p > 0.1). It cannot be concluded, therefore, that there is a significant difference in the permeability constant between the “normal” and “retarded” groups. Comment As discussed by Seeds,c the bulk of transfer of nutrients from mother to fetus probably takes place across the chorion frondosum. The absence of a perfusing circulation must diminish the amount of transfer across those portions of chorion laeve that are distant
906
Moore
December 1, 1972 Am. J. Obstet. Gynecol.
and Ward
Table I. Data of urea permeability for a typical chorion laeve and amnion as one tissue Time (min.)
Concentration on maternal side (pmole/ml.)
Concentration on fetal side (,umole/ml.)
20.29 17.93 17.24 16.67 16.25
0 0.29 0.90 1.49 1.97
0 30 60 90 120 Mean
Concentration gradient (ymole/ml.) 18.97 16.99 15.76 14.73
Table II. Urea and sucrose permeability constants for placentas from cases of normal and retarded fetal growth
(Zii)
(5%)
Yi$$
~ “gij!y
Cases of normal fetal growth 26 36 40 40 39 37 34 35 39
765 2,126 3,374 3,572 3,317 2,750 1,871 2,190 3,487
38 40
3,572 3,856
40
3,997
37
2,820
39
3,150
33
1,760
35
2,300
Cases of retarded 38 2,509 2,552 ;; 2,071 38
2,525
37
2,119
36
1,531
37
1,616
38
1,765
7.17 7.20, 15.47 16.61 14.64
7.27
10.55, 7.58 19.47, 20.35, 16.68 15.98, 13.89 10.24, 13.32, 8.58 16.64, 14.56, 18.37 9.32, 8.10, 13.50 10.96, 13.77, 11.84 13.42, 15.11, 11.60 6.56, 11.90, 14.42 fetal growth 15.41 8.53 14.42, 13.68, 12.21 9.30, 10.37, 12.60 5.25, 6.18, 5.93 5.41, 4.69, 4.48 12.84, 13.15, 10.41 13.80, 8.36, 12.32
2.33 2.11, 3.38 6.96
single
2.40
3.28, 2.24, 2.32, 4.97,
5.37, 5.82, 2.20 3.44
4.58 7.25
5.48, 1.82,
7.12, 3.41,
5.04 2.1.9
4.50,
3.22,
4.09
2.54,
3.27,
2.68
4.60,
5.65,
3.43
2.67,
3.04,
4.22
5.85 3.86 4.10,
3.89,
4.55
2.52,
2.56,
2.95
0.99,
1.46,
1.21
1.45,
1.27,
0.92
3.35,
3.67,
2.86
4.03,
2.05,
2.51
2.99
in vitro
incubation
Quantit~~ transferred (pmole/m;n. 0.097 0.147 0.159 0.154
of
)
Permeability constant (pl/min.) 5.11 8.65 10.09 10.45 8.58
from the placental cake. Because the most significant exchange across the chorion laevc is likely to occur in those areas adjacent to the placental cake, the tissues studied were taken from there. Factors influencing diffusion of solutes across the chorion include concentration difference, perfusion of the eschanging surface area, size of the fetal capillary bed, and thickness of the membrane. The conclusion from these membrane transport studies is that per unit surface area of chorion laeve and amnion there is considerable variability in the permeability of individual placentas, but there is no significant difference between placentas from cases of normal and retarded fetal growth. Histologic examination of the membranes from the growth-retarded cases showed no clear abnormality. To date, no chemical abnormality has been demonstrated in such placentas.7 Aherne and DunniP report that fetal capillary surface area corresponds closely to the villous surface area. In cases with growthretarded infants, they found a mean placental volume of 350 ml. at term (normal = 488 ml.) with a mean villous surface area of 6.4 sq. M. (normal = 11.0 square meters). Thomson and associate9 have demonstrated that the small-for-dates baby does not have, on the average, a relatively small placenta when it is born, and they point out it is not permissible to assume, without further evidence, that the functional adequacy of the placenta is proportional to its size. Duncan and LewiP showed in rabbits that larger fetuses had larger placentas but also had both absolutely and relatively higher maternal placental blood flows. In other
Volume Number
114 1
words,
the correlation between fetal size and placental blood flow was not wholly explained by placental size. Investigation of causes of intrauterine growth retardation should inelude, in addition to the factors already listed, studies on the role of metabolic activity within the placenta.
REFERENCES 1.
2. 3. 4.
5.
Battaglia, F. C., Hellegers, A. E., Meschia, G., and Barron. D. H.: Nature 196: 1061. 1962. Gey, G. 0.1 and Gey, M. K.: Am. J. Cancer 27: 45, 1936. Battaglia, F. C., and Hellegers, A. E.: AM. T. OBSTET. GYNECOL. 89: 771. 1964. Butler, N. R., and Alberman, E. D.: The Second Report of the 1958 British Perinatal Mortality Survey, Edinburgh, 1969, E. & S. Livingstone, Ltd.. D. 331. Moore, W.’ M. ’ 6., Hellegers, A. E., and Battaglia, F. C.: AM. J. OBSTET. GYNECOL. 96: 951, 1966.
Permeability
of chorion
laeve
907
The statistical analysis was carried out by Mr. A. C. C. Gibbs, Department of Social and Preventive Medicine, University of Manchester, who advised on the presentation of results.
6.
7. 8. 9.
10.
Seeds, A. E.: In Barnes, A. C., editor: Intrauterine Development, Philadelphia, 1968, Lea & Febiger, Publishers, pp. 103 and 111. Wiiick, M.: J. Pediatr. 71: 390, 1967. Aherne, W., and Dunnill, M. S.: J. Path. Bact. 91: 123, 1966. Thomson, A. M., Billewicz, W. Z., and Hytten, F. E.: J. Obset. Gynaecol. Br. Commonw. 76: 865, 1969. Duncan, S. L. B., and Lewis, B. V.: J. Physiol. 202: 471, 1969.