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Scanning electron microscopy of human placental villi from early and late in gestation
Scanning electron microscopy of human placental villi from early and late in gestation BARRY
F.
KING,
DAVID
N.
MENTON,
St. Louis,
PH.D. PH.D.
Missouri
Human placental uilli from term placentas and from 10 to 14 week placentas were examined in the scanning electron microscope (SEM) after critical point drying. When prepared by this method, the syncytial surface appears covered with slender microvilli similar to those seen in transmission electron micrographs. We find no support in our observations for the suggestion that the trophoblastic surface is a mosaic of microvillous and nonmicrovillous areas or that substantial areas lack microvilli. In addition, the villi often show circumferentially oriented furrows. SEM’s of the early placentas showed numer-ous syncytial sprouts, most of which represent stages in the formation of new free or terminal villi.
THE TRAN s FER of substances between the maternal blood in the intervillous space and the fetal placental capillaries is across the syncytial trophoblast covering the chorionic villi. Many substances which are transferred do so by the mechanism of simple diffusion, the rate of which is proportional to a number of factors including the area of exchange, in accordance with Fick’s equation. It is, therefore, important to have a thorough knowledge of the structure of the placental surface, especially of any structural features which may amplify surface area. Several light microscopic and transmission electron microscopic studies have suggested that there are regional variations in surface morphology such that the surface of a chorionic villus is a mosaic of microvillous and nonmicrovillous areas.l These regional variations are thought From the Department of Anatomy, University School of Medicine.
by some to represent functionally different areas of the placenta. The SEM is a useful instrument for examining large expanses of surface area at relatively high resolution. Critical point drying is an important prerequisite for obtaining specimens free from surface distortional forces unavoidable with other techniques.’ In this study we have used scanning electron microscopy of critical point dried placental villi to examine two questions: ( 1) Are regional differences in placental surface structure observable in the SEM and, if so, what if any is the pattern? (2) Are there changes in the microscopic appearance of the placental surface between early and late gestation? Materials
Washington
investigation was supported in part by 5 ROI HD06734 from the National Institute of Child Health and Human Development and Health Sciences Advancement Award F-304-RF06115 to Washington University. Grant
Revised Accepted
for
publication
October October
April
methods
Tissue from placentas at 10 to 14 weeks’ gestation was obtained from legal abortions by means of vacuum aspiration (three placentas) and at term from normal deliveries or uncomplicated cesarean sections (six placentas). Small pieces of villi were rinsed in normal saline or balanced salt solutions and fixed in 2 per cent paraformaldehyde-2.5 per cent glutaraldehyde in O.lM cacodylate buffer. The tissue was rinsed in buffer until secondary fixation in 2 per cent OsOl in O.lM cacodylate buffer, rapidly dehydrated in ethanol, then transferred to acetone. The tissue was placed, under acetone, in a critical point drying apparatus (Parr Instrument Co., Moline, Ill.) and dried out of liquid carbon
This
Received
and
22, 1974.
30, 1974. 30, 1974.
Reprint requests: Dr. Barry F. King, Department of Anatomy, Washington University School of Medicine, 660 S. Euclid Ave.. St. Louis, Missouri 63110.
824
Volumr Number
122 7
Placental
Fig. 1. SEM of a branched terminal villus in the term is covered with microvilli, giving the surface a “velvety” furrows in the surface. (x990.)
Fig. 2. Micrograph showing the microvillous surface term the microvilli appear to be smaller in diameter the microvilli are sometimes bulbous. (~8,400.)
placenta. Virtually appearance. Note
of the trophoblast than at 12 weeks
the entire surface also the frequent
at term. Note that (cf. Fig. 4). The tips
at of
villi
825
826
King
and
Menton
Fig. 3. Micrograph showing Nate the larger stem villi and or creases on the surface are
the morphology of placental the small syncytial sprouts also evident, especially at
dioxide. Pieces of dried tissue were attached to metal scanning studs with a colloidal silver solution, coated with chromium in a varuum evaporator, and esamined in a Cambridge Stereoscan SEM. Results
Fig. I shows the typical appearance of free chorionic villi from term placentas. At this relatively low magnification, the microvillous surface gives the villi a “velvety” appearance. Circumferentially oriented furrows or grooves are also present on most terminal villi (Fig. 1 1. At higher magnification, individual microvilli on the surface can be clearly discerned (Fig. 2). The microvilli are slender filiforrn proc.esses and appear to be rather evenly distributed over thr surface of the syncytium. Some of the microvilli have small bulbous terminal dilations; others appear to branch. There are occasional long surface projections. Only rarely have relatively smooth or nonmicrovillous areas been observed on the surface, and we believe these may represent areas where solvent from the silver paint used to attach the tissue to the scanning studs was “wicked” up the villi and subsequently air dried, causing distortion. SEM’s of 10 to 14 week chorionic villi often show relatively thick (i.e., large diameter) villous branches.
villi at 10 to 14 weeks’ gestation. at various stages of formation. Furrows the bases of larger villi. (x340.)
Off these larger villi arise smaller order villi and finally numerous small protrusions which represent synrytial sprouts (Fig. 3). The sprouts vary in morphology from small mounds to lomg digitiform protrusions u,hich may have a bulbous end. As in the free c:horionic villi of the term placenta, the placental surface of these younger stages looks “velvety” at intermediate ma,~nifications. At higher magnification it is clear that numerous mirrovilli rover the surface (Fig. 4). The microvilli arc of variable length, and some have bulbous ends: they appear to be slightly larger in diameter than those observed on the terminal villi of the mature placenta. Furrows or folds are also common features of tllc placental surface at this age, particularly at the bases ol villi. Comment
Previous SEM studies of villi of the term placenta have presented varying conclusions as to the surface topography of the zyncytial trophoblast. In some. instances micrographs suggest the presence of microvilli; in other instances thy): suggest a nearly smooth surface? ’ Transmission electron micrographs, on the other hand , generally hhow a surface covered with slender microvilli. We believe that the inconsistenc.i::s are due primarily to varying methods of
Volume Number
Placental
122 7
Fig. 4. Micrograph are somewhat larger projections is shown.
showing microvillous surface of trophoblast in diameter than at term (cf. Fig. 2). One (~8,400.)
specimen preparation for SEM and that the use of critical point drying to minimize surface distortional forces gives the more accurate picture of the placental surface and one which is entirely compatible with transmission electron microscopic results. Our results indicate that the placental surface is virtually everywhere covered with slender mirro\illi. We can find no support in our observations for the suggestion that the trophoblastic surface is a mosaic of microvillous and nonmicrovillous areas or that substantial areas lack microvilli.‘. ’ There have been several SEM studies of placental villi from early in gestation.“, Ii Bergstrom,” using a variety of methods including critical point drying, showed the surface of villi from 12 weeks of gestation to be almost completely covered by microvilli; we agree with his observations and would extend them to apply to the surfaces of villi at term. The microvillous border (the ‘%brush” border of the light microscopists) is a common specialization of the free surface in absorptive epithelia and the usual interpretation of the functional significance of microvilli is that they greatly increase the area of membrane exposed to substances that are to be absorbed.” It has been estimated, for example, that
villi
827
at 10 to 14 weeks. Microvilli of the occasional long surface
microvilli on the surface of intestinal absorptive cells may result in a 24-fold increase in surface area over that of a smooth surface.” Undoubtedly this surface specialization greatly increases the absorptive area of the chorionic villi as well. Variations in structure among microvilli on the syncytial surface at different times throughout gestation have been described by transmission electron microscopy.“’ We have observed examples of the rlavate type, which terminate in slight bulbous dilations, and the branched type. The significance of the furrows or grooves present on the villi is unknown. They may represent flexure lines brought about by the bending of villi. On the other hand, some of these may be related to the periodic surface elevations on early placentas described by light and transmission electron microscopy.“’ Our observations of 10 to 14 week specimens showed numerous syncytial sprouts, most of which represent stages in the forrnation of new free or terminal villi. The structures we have described are compatible with light and transmission electron microscopic observations of syncytial sprouts,“) and the SEM observations of Bergstrom.”
REFERENCES
1.
Ludwig, placenta
K. in
S.: The relation
morphologic structure of to its exchange function,
the in
Longo, L. D., Gas Exchange
and Bartels, and Blood
H., editors: Respiratory Flow in the Placenta,
828
2.
3. 4.
King
and
Menton
Bethesda, Md., 1972, U. S. Dept. Health, Education and Welfare, pp. 13-21. Porter, K. R., Kelley, D., and Andrew, P. M.: The preparation of cultured cells and soft tissues for scanning electron microscopy, in Proc. Fifth Ann. Stereoscan Colloquium, Morton Grove, Ill., 1972, Kent Cambridge Scientific Co. Ludwig, H.: AM. J. OBSTET. GYNECOL. 111: 328, 1971. Sheppard, B. L., and Bonnar, J.: J. Obstet. Gynaecol. Br. Commonw. 81: 20, 1974.
Postgraduate
international
courses
5. 6. 7. 8. 9. 10.
on
Dempsey, E. W., and Luse, S.: J. Anat. 108: 545, 1971. Bergstrom, S.: Obstet. Gynecol. 38: 513, 1971. Schiebler, T. H., and Kaufmann, P.: 2. Zrllfclrsch. 102: 242, 1969. Fawcett, D. W.: J. Histochem. Cytochem. 13: 75, 1965. Palay, S. L., and Karlin, I,. J.: J. Biochem. Biophys. Cvtol. 5: 363. 1959. Boyd, J. D., ‘and Hamilton, W. J.: The Human Placenta, Cambridge, 1970, W. Heffer & Sons, Ltd.
perinatal
medicine
and
gynaecology
Two postgraduate international courses on perinatal medicine and gynaecology will 1)~ held in Mexico City, August 25-29, 1975. The first course is entitled “Perinatal Medicine.” The chairman is Dr. E. Saling (Berlin), and the co-chairman is Dr. C. Santiago-Pineda (Mexico). The second course is entitled “Diagnostic Methods in Gynaecology.” The chairman is Dr. 0. K&r (Basle), and the co-chairman is Dr. R. Reyes Guerrero (Mexico). These courses arr co-s ponsored by ISSSTIl (Social Security Institute for the Mexican Government Enlployees), the Free University of Berlin, and Basle University. Guest Professors include Drs. Amiel Tison (Paris), Beard (London), Caldeyro-Barcia (Montevideo), Gluck (San Diego), Hammacher (Basle), Hull (London), Kratochwil (Vienna), Richart (New York), Sureau (Paris), and Wood (Melbourne). Mexican professors include Drs. Alvarez Bravo, Castelazo Ayala, De Wit Greene, Gutierrez Najar, Jurado Garcia, Kumate Rodriguez, Lowenherg Favela, MacGregor, Quilionps, and Tommasi Navarrete. Lectures, panel discussions, seminars, and formal and informal group disc-lrssionr arc planned, together with practical d
F.
KING,
DAVID
N.
MENTON,
St. Louis,
PH.D. PH.D.
Missouri
Human placental uilli from term placentas and from 10 to 14 week placentas were examined in the scanning electron microscope (SEM) after critical point drying. When prepared by this method, the syncytial surface appears covered with slender microvilli similar to those seen in transmission electron micrographs. We find no support in our observations for the suggestion that the trophoblastic surface is a mosaic of microvillous and nonmicrovillous areas or that substantial areas lack microvilli. In addition, the villi often show circumferentially oriented furrows. SEM’s of the early placentas showed numer-ous syncytial sprouts, most of which represent stages in the formation of new free or terminal villi.
THE TRAN s FER of substances between the maternal blood in the intervillous space and the fetal placental capillaries is across the syncytial trophoblast covering the chorionic villi. Many substances which are transferred do so by the mechanism of simple diffusion, the rate of which is proportional to a number of factors including the area of exchange, in accordance with Fick’s equation. It is, therefore, important to have a thorough knowledge of the structure of the placental surface, especially of any structural features which may amplify surface area. Several light microscopic and transmission electron microscopic studies have suggested that there are regional variations in surface morphology such that the surface of a chorionic villus is a mosaic of microvillous and nonmicrovillous areas.l These regional variations are thought From the Department of Anatomy, University School of Medicine.
by some to represent functionally different areas of the placenta. The SEM is a useful instrument for examining large expanses of surface area at relatively high resolution. Critical point drying is an important prerequisite for obtaining specimens free from surface distortional forces unavoidable with other techniques.’ In this study we have used scanning electron microscopy of critical point dried placental villi to examine two questions: ( 1) Are regional differences in placental surface structure observable in the SEM and, if so, what if any is the pattern? (2) Are there changes in the microscopic appearance of the placental surface between early and late gestation? Materials
Washington
investigation was supported in part by 5 ROI HD06734 from the National Institute of Child Health and Human Development and Health Sciences Advancement Award F-304-RF06115 to Washington University. Grant
Revised Accepted
for
publication
October October
April
methods
Tissue from placentas at 10 to 14 weeks’ gestation was obtained from legal abortions by means of vacuum aspiration (three placentas) and at term from normal deliveries or uncomplicated cesarean sections (six placentas). Small pieces of villi were rinsed in normal saline or balanced salt solutions and fixed in 2 per cent paraformaldehyde-2.5 per cent glutaraldehyde in O.lM cacodylate buffer. The tissue was rinsed in buffer until secondary fixation in 2 per cent OsOl in O.lM cacodylate buffer, rapidly dehydrated in ethanol, then transferred to acetone. The tissue was placed, under acetone, in a critical point drying apparatus (Parr Instrument Co., Moline, Ill.) and dried out of liquid carbon
This
Received
and
22, 1974.
30, 1974. 30, 1974.
Reprint requests: Dr. Barry F. King, Department of Anatomy, Washington University School of Medicine, 660 S. Euclid Ave.. St. Louis, Missouri 63110.
824
Volumr Number
122 7
Placental
Fig. 1. SEM of a branched terminal villus in the term is covered with microvilli, giving the surface a “velvety” furrows in the surface. (x990.)
Fig. 2. Micrograph showing the microvillous surface term the microvilli appear to be smaller in diameter the microvilli are sometimes bulbous. (~8,400.)
placenta. Virtually appearance. Note
of the trophoblast than at 12 weeks
the entire surface also the frequent
at term. Note that (cf. Fig. 4). The tips
at of
villi
825
826
King
and
Menton
Fig. 3. Micrograph showing Nate the larger stem villi and or creases on the surface are
the morphology of placental the small syncytial sprouts also evident, especially at
dioxide. Pieces of dried tissue were attached to metal scanning studs with a colloidal silver solution, coated with chromium in a varuum evaporator, and esamined in a Cambridge Stereoscan SEM. Results
Fig. I shows the typical appearance of free chorionic villi from term placentas. At this relatively low magnification, the microvillous surface gives the villi a “velvety” appearance. Circumferentially oriented furrows or grooves are also present on most terminal villi (Fig. 1 1. At higher magnification, individual microvilli on the surface can be clearly discerned (Fig. 2). The microvilli are slender filiforrn proc.esses and appear to be rather evenly distributed over thr surface of the syncytium. Some of the microvilli have small bulbous terminal dilations; others appear to branch. There are occasional long surface projections. Only rarely have relatively smooth or nonmicrovillous areas been observed on the surface, and we believe these may represent areas where solvent from the silver paint used to attach the tissue to the scanning studs was “wicked” up the villi and subsequently air dried, causing distortion. SEM’s of 10 to 14 week chorionic villi often show relatively thick (i.e., large diameter) villous branches.
villi at 10 to 14 weeks’ gestation. at various stages of formation. Furrows the bases of larger villi. (x340.)
Off these larger villi arise smaller order villi and finally numerous small protrusions which represent synrytial sprouts (Fig. 3). The sprouts vary in morphology from small mounds to lomg digitiform protrusions u,hich may have a bulbous end. As in the free c:horionic villi of the term placenta, the placental surface of these younger stages looks “velvety” at intermediate ma,~nifications. At higher magnification it is clear that numerous mirrovilli rover the surface (Fig. 4). The microvilli arc of variable length, and some have bulbous ends: they appear to be slightly larger in diameter than those observed on the terminal villi of the mature placenta. Furrows or folds are also common features of tllc placental surface at this age, particularly at the bases ol villi. Comment
Previous SEM studies of villi of the term placenta have presented varying conclusions as to the surface topography of the zyncytial trophoblast. In some. instances micrographs suggest the presence of microvilli; in other instances thy): suggest a nearly smooth surface? ’ Transmission electron micrographs, on the other hand , generally hhow a surface covered with slender microvilli. We believe that the inconsistenc.i::s are due primarily to varying methods of
Volume Number
Placental
122 7
Fig. 4. Micrograph are somewhat larger projections is shown.
showing microvillous surface of trophoblast in diameter than at term (cf. Fig. 2). One (~8,400.)
specimen preparation for SEM and that the use of critical point drying to minimize surface distortional forces gives the more accurate picture of the placental surface and one which is entirely compatible with transmission electron microscopic results. Our results indicate that the placental surface is virtually everywhere covered with slender mirro\illi. We can find no support in our observations for the suggestion that the trophoblastic surface is a mosaic of microvillous and nonmicrovillous areas or that substantial areas lack microvilli.‘. ’ There have been several SEM studies of placental villi from early in gestation.“, Ii Bergstrom,” using a variety of methods including critical point drying, showed the surface of villi from 12 weeks of gestation to be almost completely covered by microvilli; we agree with his observations and would extend them to apply to the surfaces of villi at term. The microvillous border (the ‘%brush” border of the light microscopists) is a common specialization of the free surface in absorptive epithelia and the usual interpretation of the functional significance of microvilli is that they greatly increase the area of membrane exposed to substances that are to be absorbed.” It has been estimated, for example, that
villi
827
at 10 to 14 weeks. Microvilli of the occasional long surface
microvilli on the surface of intestinal absorptive cells may result in a 24-fold increase in surface area over that of a smooth surface.” Undoubtedly this surface specialization greatly increases the absorptive area of the chorionic villi as well. Variations in structure among microvilli on the syncytial surface at different times throughout gestation have been described by transmission electron microscopy.“’ We have observed examples of the rlavate type, which terminate in slight bulbous dilations, and the branched type. The significance of the furrows or grooves present on the villi is unknown. They may represent flexure lines brought about by the bending of villi. On the other hand, some of these may be related to the periodic surface elevations on early placentas described by light and transmission electron microscopy.“’ Our observations of 10 to 14 week specimens showed numerous syncytial sprouts, most of which represent stages in the forrnation of new free or terminal villi. The structures we have described are compatible with light and transmission electron microscopic observations of syncytial sprouts,“) and the SEM observations of Bergstrom.”
REFERENCES
1.
Ludwig, placenta
K. in
S.: The relation
morphologic structure of to its exchange function,
the in
Longo, L. D., Gas Exchange
and Bartels, and Blood
H., editors: Respiratory Flow in the Placenta,
828
2.
3. 4.
King
and
Menton
Bethesda, Md., 1972, U. S. Dept. Health, Education and Welfare, pp. 13-21. Porter, K. R., Kelley, D., and Andrew, P. M.: The preparation of cultured cells and soft tissues for scanning electron microscopy, in Proc. Fifth Ann. Stereoscan Colloquium, Morton Grove, Ill., 1972, Kent Cambridge Scientific Co. Ludwig, H.: AM. J. OBSTET. GYNECOL. 111: 328, 1971. Sheppard, B. L., and Bonnar, J.: J. Obstet. Gynaecol. Br. Commonw. 81: 20, 1974.
Postgraduate
international
courses
5. 6. 7. 8. 9. 10.
on
Dempsey, E. W., and Luse, S.: J. Anat. 108: 545, 1971. Bergstrom, S.: Obstet. Gynecol. 38: 513, 1971. Schiebler, T. H., and Kaufmann, P.: 2. Zrllfclrsch. 102: 242, 1969. Fawcett, D. W.: J. Histochem. Cytochem. 13: 75, 1965. Palay, S. L., and Karlin, I,. J.: J. Biochem. Biophys. Cvtol. 5: 363. 1959. Boyd, J. D., ‘and Hamilton, W. J.: The Human Placenta, Cambridge, 1970, W. Heffer & Sons, Ltd.
perinatal
medicine
and
gynaecology
Two postgraduate international courses on perinatal medicine and gynaecology will 1)~ held in Mexico City, August 25-29, 1975. The first course is entitled “Perinatal Medicine.” The chairman is Dr. E. Saling (Berlin), and the co-chairman is Dr. C. Santiago-Pineda (Mexico). The second course is entitled “Diagnostic Methods in Gynaecology.” The chairman is Dr. 0. K&r (Basle), and the co-chairman is Dr. R. Reyes Guerrero (Mexico). These courses arr co-s ponsored by ISSSTIl (Social Security Institute for the Mexican Government Enlployees), the Free University of Berlin, and Basle University. Guest Professors include Drs. Amiel Tison (Paris), Beard (London), Caldeyro-Barcia (Montevideo), Gluck (San Diego), Hammacher (Basle), Hull (London), Kratochwil (Vienna), Richart (New York), Sureau (Paris), and Wood (Melbourne). Mexican professors include Drs. Alvarez Bravo, Castelazo Ayala, De Wit Greene, Gutierrez Najar, Jurado Garcia, Kumate Rodriguez, Lowenherg Favela, MacGregor, Quilionps, and Tommasi Navarrete. Lectures, panel discussions, seminars, and formal and informal group disc-lrssionr arc planned, together with practical d