British Journal of Obstetrics and Gynaecology October 1975. Vol82. pp 790-793
THE DISTRIBUTION OF PLACENTAL LACTOGEN IN THE HUMAN TERM PLACENTA BY
GILLIAN GAU AND
T. CHARD Departments of Obstetrics, Gynaecology and Reproductive Physiology St. Bartholomew's Hospital Medical College and the London Hospital Medical College and Department of Histopathology, Wycombe General Hospital Summary Serial frozen sections of human term placentae were examined by quantitative morphology and, after extraction, by a radioimmunoassay for human placental lactogen (HPL). The total quantity of HPL in the placenta was estimated as 720 mg. The amount of HPL was related to the amount of trophoblast in any area, but otherwise did not vary in different parts of the placenta. A semi-logarithmic relationship was observed between the amount of HPL and the percentage trophoblast in a section. It is suggested that the amount of HPL in a given area of the placenta is related to the blood-flow in that area.
IMMUNOHISTOCHEMICAL studies have demonstrated that human placental lactogen (HPL) is localized to the syncytiotrophoblast of the placenta (Currie et al., 1966; Ikonicoff and Cedard, 1973; Beck et al., 1969; Sciarra et al., 1963). However, there is no information as to whether or not the trophoblast in different areas of the placenta is equally active in the synthesis of HPL. In the present studies, using quantitative morphology and radioimmunoassay the distribution of HPL in the term placenta has been studied, and the quantitative relationship to the amount of trophoblast has been examined.
taken from the centre and periphery of the placenta, and then divided horizontally into three or four blocks (Fig. 1). The second consisted of full thickness blocks taken from the centre and periphery of a central maternal cotyledon and a peripheral maternal cotyledon (Fig. 2). The blocks were immediately frozen in liquid nitrogen and 10 micron sections cut on a Slee cryostat. For blocks taken in the horizontal plane (Fig. 1) sections were cut at 1 mm intervals from the maternal to the fetal surface. For blocks taken in the vertical plane (Fig. 2), sections were cut at 0.1 mm intervals in the centre of the block. Two successive sections were cut at each level; one was stained for morphometric examination with van Gieson's trichrome and the other was transferred to a preweighed coverslip which was then reweighed and reserved for hormone analysis. For morphometry five fields from each section were projected onto a grid of equidistant points; a total of 840 points was counted for each
AND METHODS MATERIALS Fresh placentae were collected at the time of normal delivery. After removal of membranes and umbilical cord each placenta was weighed, measured in three dimensions and any macroscopic abnormality noted. Two types of block were taken. The first consisted of full thickness segments with a face area of 1.5 x 1 . 5 cm
790
HPL DISTRIBUTION
pH 7.4,containing 2 per cent horse serum then exposed to ultrasonic radiation for four successive periods of 30 seconds (MSE Ultrasonic Disintegrator No. 3000 fitted with titanium probe). Placental lactogen levels in the supernatant were estimated by radioimmunoassay, and the results expressed as ng per pg placental tissue.
-
plane of sections
FIG.1 Full thickness segments (shaded areas) were taken from a central and marginal cotyledon. Each segment was divided into three or four blocks in the horizontal plane, and serial sections taken in this plane. maternal surface
I
plane of sections
FIG.2 Full thickness blocks (shaded areas) were taken from the centre and periphery of a central and marginal cotyledon. The blocks were sectioned in the vertical plane.
section. The result was expressed as the percentage of the total points counted which coincided with trophoblast. For hormone estimations the weighed frozen section and coverslip were placed in a vessel containing 2 ml of 0.05 M phosphate buffer, 20
RESULTS For sections taken in the horizontal plane (i.e. from maternal to fetal surface, Fig. l), the percentage trophoblast was progressively reduced in the area adjacent to the chorionic plate (Fig. 3). The amount of HPL showed a semilogarithmic relationship to the amount of trophoblast present in a given section (Fig. 4). In other words, areas with a relatively small amount of trophoblast (i.e. within 4 to 5 mm of the chorionic plate) contained proportionately less HPL than areas with abundant trophoblast. There was no difference in the percentage trophoblast or HPL content between central and peripheral areas of the placenta, or between central and peripheral areas of a given cotyledon. The mean HPL content was 1.6 mg per g of placental tissue. DISCUSSION This study represents the first attempt to demonstrate a quantitative relationship between placental morphology and hormone content.
-
%
Trophoblast 10
Maternal ____)
79 1
Fetal Surface Maternal-
Central cotyledon
Fetal Surface Peripheral cotyledon
FIG.3 The percentage of trophoblast, as assessed by morphometry, in serial horizontal sections through a central cotyledon (on the left) and a marginal cotyledon (on the right). Note that the amount of trophoblast decreases in areas adjacent to the fetal surface (chorionic plate).
792
GAU AND CHARD
2.7
2.7
1.65
1.65
1.0
1.0
ng HPLl
ng HPLl
pg Placenta
~rgPlacenta
a 6
0.6
a 37
0.37
0.22
0.22 ~~
4
8
12
16
20
B Trophoblast
4
8
12
16
20
B Trophoblast
FIG.4 The relationship between the percentage trophoblast observed in sections of term placenta, and the HPL content of adjacent sections.
Previous investigations have been based mainly on immunohistochemical techniques using material which has been chemically fixed and embedded in paraffin wax. This procedure can lead to substantial or total loss of contained hormone (Gau, unpublished observations), a problem which is obviated by the use of frozen sections. The present studies provide original data in three areas: on the total HPL content of the placenta; on the distribution of HPL in different parts of the placenta; and on the relationship between trophoblast and HPL content. The total amount of HPL in the placenta, as calculated from the present results and assuming a mean stripped placental weight of 450 g at term, is approximately 720 mg. This figure is considerably higher than that suggested by previous workers (Josimovich and Atwood, 1964: 22.9 mg; Suwa and Friesen, 1969: 138.6 mg), a discrepancy which can be attributed to the fact that in the present studies the hormone was extracted by a highly efficient method (ultrasonication) from a very small volume of tissue. Thus the total HPL in the placenta at any time is approximately equivalent to the amount which it secretes during a
24-hour period (c. 1 -03 g) (Kaplan et al., 1968). The overall distribution of HPL in the placenta has not previously been the subject of specific investigation. In this study it has been shown that the quantity of hormone is related to the amount of trophoblast, but otherwise does not vary in different parts of the organ. Thus, there is no evidence for functional subspecialization, for instance, between the centre and the periphery: in terms of content, the whole placenta appears to be active in the synthesis of HPL. In view of earlier demonstrations that HPL is limited to the syncytiotrophoblast (Currie et al, 1966), it is not surprising to find that the amount of HPL is related to the percentage of trophoblast in a given section. However, the observation that the relationship is not a simple linear function, but rather is semi-logarithmic, has not previously been made. Perhaps the most likely explanation is that the relationship is secondary to the rate of blood-flow in a given area. The amount of trophoblast decreases towards the fetal surface of the placenta, i.e. adjacent to the chorionic plate and studies by Lemtis (1966) have shown that there is a reduced blood flow in this area. There is good evidence
HPL DISTRIBUTION
793
fetal surface sparse trophoblast I low blood flow
abundant
’ trophoblast high blood flow
maternal surface FIG.5 Schematic presentation of a hypothesis linking hormone content to blood-flow in the human term placenta. Both blood-flow and trophoblast are reduced in areas adjacent to the fetal surface, and the hormone content of the areas relative to the amount of trophoblast is less than at other sites. These findings might be explained if the hormone content of the trophoblast is a direct function of the rate of bloodflow (see text).
that the rate of synthesis of placental hormones is primarily controlled by the rate of blood-flow past the trophoblast cells (McNeilly et al., 1975); thus an area of reduced blood-flow would be associated with reduced synthesis and content (Fig. 5). This concept has important implications with respect to the changes in hormone levels which may accompany placental pathology.
ACKNOWLEDGEMENT These studies were supported by a grant from the Special Trustees of St. Bartholomew’s Hospital. REFERENCES Beck, J. S., Gordon, R. C., Donald, D., and Melvin, J. M. 0. (1969): Journal of Pathology, 97, 545.
Curie, A. R., Beck, J. S., Ellis, S., and Read, C. H. (1 966) : Journal of Pathofogy and Bacteriology, 92, 395. Ikonicoff, L. de, and Cedard, L. (1973): American Journal of Obstetrics and Gynecology, 116, 1124. Josimovich, J. B., and Atwood, B. L. (1964): American Journal of Obstetrics and Gynecology, 88, 867. Kaplan, S . L., Gurpide, E., Sciarra, J. J., and Grumbach, M. M. (1968): Journal of Clinical Endocrinology and Metabolism, 28, 1450. Lemtis, H. (1966). Quoted by Thomsen, K., and Hiersche, H.-D. (1969). In Fetus and Placenta. (Edited by A. Klopper and E. Diczfalusy). Oxford, Blackwell, p. 77. McNeilly, A. S., Gardner, J. S., Bradford, D., and Chard, T. (1975). In preparation. Sciarra, J. J., Kaplan, S. L., and Grumbach, M. M. (1963): Nature, 199, 1005. Suwa, S., and Friesen, H. G. (1969): Endocrinology, 85, 1037.
THE DISTRIBUTION OF PLACENTAL LACTOGEN IN THE HUMAN TERM PLACENTA BY
GILLIAN GAU AND
T. CHARD Departments of Obstetrics, Gynaecology and Reproductive Physiology St. Bartholomew's Hospital Medical College and the London Hospital Medical College and Department of Histopathology, Wycombe General Hospital Summary Serial frozen sections of human term placentae were examined by quantitative morphology and, after extraction, by a radioimmunoassay for human placental lactogen (HPL). The total quantity of HPL in the placenta was estimated as 720 mg. The amount of HPL was related to the amount of trophoblast in any area, but otherwise did not vary in different parts of the placenta. A semi-logarithmic relationship was observed between the amount of HPL and the percentage trophoblast in a section. It is suggested that the amount of HPL in a given area of the placenta is related to the blood-flow in that area.
IMMUNOHISTOCHEMICAL studies have demonstrated that human placental lactogen (HPL) is localized to the syncytiotrophoblast of the placenta (Currie et al., 1966; Ikonicoff and Cedard, 1973; Beck et al., 1969; Sciarra et al., 1963). However, there is no information as to whether or not the trophoblast in different areas of the placenta is equally active in the synthesis of HPL. In the present studies, using quantitative morphology and radioimmunoassay the distribution of HPL in the term placenta has been studied, and the quantitative relationship to the amount of trophoblast has been examined.
taken from the centre and periphery of the placenta, and then divided horizontally into three or four blocks (Fig. 1). The second consisted of full thickness blocks taken from the centre and periphery of a central maternal cotyledon and a peripheral maternal cotyledon (Fig. 2). The blocks were immediately frozen in liquid nitrogen and 10 micron sections cut on a Slee cryostat. For blocks taken in the horizontal plane (Fig. 1) sections were cut at 1 mm intervals from the maternal to the fetal surface. For blocks taken in the vertical plane (Fig. 2), sections were cut at 0.1 mm intervals in the centre of the block. Two successive sections were cut at each level; one was stained for morphometric examination with van Gieson's trichrome and the other was transferred to a preweighed coverslip which was then reweighed and reserved for hormone analysis. For morphometry five fields from each section were projected onto a grid of equidistant points; a total of 840 points was counted for each
AND METHODS MATERIALS Fresh placentae were collected at the time of normal delivery. After removal of membranes and umbilical cord each placenta was weighed, measured in three dimensions and any macroscopic abnormality noted. Two types of block were taken. The first consisted of full thickness segments with a face area of 1.5 x 1 . 5 cm
790
HPL DISTRIBUTION
pH 7.4,containing 2 per cent horse serum then exposed to ultrasonic radiation for four successive periods of 30 seconds (MSE Ultrasonic Disintegrator No. 3000 fitted with titanium probe). Placental lactogen levels in the supernatant were estimated by radioimmunoassay, and the results expressed as ng per pg placental tissue.
-
plane of sections
FIG.1 Full thickness segments (shaded areas) were taken from a central and marginal cotyledon. Each segment was divided into three or four blocks in the horizontal plane, and serial sections taken in this plane. maternal surface
I
plane of sections
FIG.2 Full thickness blocks (shaded areas) were taken from the centre and periphery of a central and marginal cotyledon. The blocks were sectioned in the vertical plane.
section. The result was expressed as the percentage of the total points counted which coincided with trophoblast. For hormone estimations the weighed frozen section and coverslip were placed in a vessel containing 2 ml of 0.05 M phosphate buffer, 20
RESULTS For sections taken in the horizontal plane (i.e. from maternal to fetal surface, Fig. l), the percentage trophoblast was progressively reduced in the area adjacent to the chorionic plate (Fig. 3). The amount of HPL showed a semilogarithmic relationship to the amount of trophoblast present in a given section (Fig. 4). In other words, areas with a relatively small amount of trophoblast (i.e. within 4 to 5 mm of the chorionic plate) contained proportionately less HPL than areas with abundant trophoblast. There was no difference in the percentage trophoblast or HPL content between central and peripheral areas of the placenta, or between central and peripheral areas of a given cotyledon. The mean HPL content was 1.6 mg per g of placental tissue. DISCUSSION This study represents the first attempt to demonstrate a quantitative relationship between placental morphology and hormone content.
-
%
Trophoblast 10
Maternal ____)
79 1
Fetal Surface Maternal-
Central cotyledon
Fetal Surface Peripheral cotyledon
FIG.3 The percentage of trophoblast, as assessed by morphometry, in serial horizontal sections through a central cotyledon (on the left) and a marginal cotyledon (on the right). Note that the amount of trophoblast decreases in areas adjacent to the fetal surface (chorionic plate).
792
GAU AND CHARD
2.7
2.7
1.65
1.65
1.0
1.0
ng HPLl
ng HPLl
pg Placenta
~rgPlacenta
a 6
0.6
a 37
0.37
0.22
0.22 ~~
4
8
12
16
20
B Trophoblast
4
8
12
16
20
B Trophoblast
FIG.4 The relationship between the percentage trophoblast observed in sections of term placenta, and the HPL content of adjacent sections.
Previous investigations have been based mainly on immunohistochemical techniques using material which has been chemically fixed and embedded in paraffin wax. This procedure can lead to substantial or total loss of contained hormone (Gau, unpublished observations), a problem which is obviated by the use of frozen sections. The present studies provide original data in three areas: on the total HPL content of the placenta; on the distribution of HPL in different parts of the placenta; and on the relationship between trophoblast and HPL content. The total amount of HPL in the placenta, as calculated from the present results and assuming a mean stripped placental weight of 450 g at term, is approximately 720 mg. This figure is considerably higher than that suggested by previous workers (Josimovich and Atwood, 1964: 22.9 mg; Suwa and Friesen, 1969: 138.6 mg), a discrepancy which can be attributed to the fact that in the present studies the hormone was extracted by a highly efficient method (ultrasonication) from a very small volume of tissue. Thus the total HPL in the placenta at any time is approximately equivalent to the amount which it secretes during a
24-hour period (c. 1 -03 g) (Kaplan et al., 1968). The overall distribution of HPL in the placenta has not previously been the subject of specific investigation. In this study it has been shown that the quantity of hormone is related to the amount of trophoblast, but otherwise does not vary in different parts of the organ. Thus, there is no evidence for functional subspecialization, for instance, between the centre and the periphery: in terms of content, the whole placenta appears to be active in the synthesis of HPL. In view of earlier demonstrations that HPL is limited to the syncytiotrophoblast (Currie et al, 1966), it is not surprising to find that the amount of HPL is related to the percentage of trophoblast in a given section. However, the observation that the relationship is not a simple linear function, but rather is semi-logarithmic, has not previously been made. Perhaps the most likely explanation is that the relationship is secondary to the rate of blood-flow in a given area. The amount of trophoblast decreases towards the fetal surface of the placenta, i.e. adjacent to the chorionic plate and studies by Lemtis (1966) have shown that there is a reduced blood flow in this area. There is good evidence
HPL DISTRIBUTION
793
fetal surface sparse trophoblast I low blood flow
abundant
’ trophoblast high blood flow
maternal surface FIG.5 Schematic presentation of a hypothesis linking hormone content to blood-flow in the human term placenta. Both blood-flow and trophoblast are reduced in areas adjacent to the fetal surface, and the hormone content of the areas relative to the amount of trophoblast is less than at other sites. These findings might be explained if the hormone content of the trophoblast is a direct function of the rate of bloodflow (see text).
that the rate of synthesis of placental hormones is primarily controlled by the rate of blood-flow past the trophoblast cells (McNeilly et al., 1975); thus an area of reduced blood-flow would be associated with reduced synthesis and content (Fig. 5). This concept has important implications with respect to the changes in hormone levels which may accompany placental pathology.
ACKNOWLEDGEMENT These studies were supported by a grant from the Special Trustees of St. Bartholomew’s Hospital. REFERENCES Beck, J. S., Gordon, R. C., Donald, D., and Melvin, J. M. 0. (1969): Journal of Pathology, 97, 545.
Curie, A. R., Beck, J. S., Ellis, S., and Read, C. H. (1 966) : Journal of Pathofogy and Bacteriology, 92, 395. Ikonicoff, L. de, and Cedard, L. (1973): American Journal of Obstetrics and Gynecology, 116, 1124. Josimovich, J. B., and Atwood, B. L. (1964): American Journal of Obstetrics and Gynecology, 88, 867. Kaplan, S . L., Gurpide, E., Sciarra, J. J., and Grumbach, M. M. (1968): Journal of Clinical Endocrinology and Metabolism, 28, 1450. Lemtis, H. (1966). Quoted by Thomsen, K., and Hiersche, H.-D. (1969). In Fetus and Placenta. (Edited by A. Klopper and E. Diczfalusy). Oxford, Blackwell, p. 77. McNeilly, A. S., Gardner, J. S., Bradford, D., and Chard, T. (1975). In preparation. Sciarra, J. J., Kaplan, S. L., and Grumbach, M. M. (1963): Nature, 199, 1005. Suwa, S., and Friesen, H. G. (1969): Endocrinology, 85, 1037.
