Acta Obstet Gynecol Scand 58: 353 - 360, 1979
AMNIOTIC FLUID CELL EXFOLIATION IN EARLY HUMAN PREGNANCY Staffan Bergstrom From the Departments of Obstetrics and Gynecology and Human Anatomy, University Hospital, Uppsala, and Department of Obstetrics and Gynecology, Central Hospital, Eskilstuna, Sweden
Abstract. An ultrasctructural study of amniotic epithelium and fetal periderm was undertaken in order to investigate the exfoliative capacity in these tissue surfaces in early human pregnancy. Contrary to earlier reports, no exfoliation could be detected from amniotic epithelium, while a heavy detachment of cells and cell fragments was observed from fetal skin. The importance of meticulous tissue preparation is obvious; detachment phenomena are easily produced artifactually by tissue scrapings and improper tissue preparation for morphological investigations.
Since the discovery of the cells in amniotic fluid by Daniel (l), few studies on amniotic fluid have focussed on the clinical applications of amniotic fluid cytology. Not until half a century after Daniel’s paper was an attempt made to apply these cytological findings in the diagnosis of ruptured membranes (2). A decade later antenatal sex determination on the basis of amniotic fluid cytology was described by Rosa and Fanard (3). This achieved a breakthrough for cytodiagnosis by amniocentesis and a large number of publications have since described the antenatal diagnosis of several sex-linked inherited diseases (for review, see Fuchs and Cederquist (4)). Amniotic fluid cells constitute the earliest easily available tissue sample from the fetus, carrying significant information on its chromosomal features. In early pregnancy, cytodiagnosis for genetic counselling is of primary concern, while in late pregnancy cytodiagnosis is used almost exclusively for determination of fetal maturity. Prematurity still plays an important role in neonatal mortality. According to a recent study (5) in the U S , more than 1 per cent of all neonates born alive develop RDS, which still has a lethal outcome in about 30 per cent of cases. Cytodiagnosis, if reliable, would be a worhtwhile alternative to biochemical monitoring in testing for fetal maturity in risk cases - e.g. with meconium contamination - where the L/S ratio is less well related to maturity. In an attempt to settle the question of
reliability of cytodiagnosis in the determination of fetal maturity, Morrison and co-workers (6, 7), carried out critical studies on the methodology of amniOtiC fluid cytodiagnosis. By Carefully evaluating various steps in the cell preparation they demonstrated that a number of crucial details could explain the variation in reliability observed by several investigators utilizing the Nile Blue sulphate technique. The cell pattern as an expression of fetal maturity accordingly became more important than before. The origin of the maturity-typical cell pattern is unknown and little is understood about the behaviour of tissue surfaces facing the amniotic cavity and their potential contribution to amniotic fluid cytology. The purpose of the present study was to investigate by scanning electron microscopy (SEM) the existence of exfoliative properties in the major components of the surface facing the amniotic fluid, the amniotic epithelium and the fetal periderm. In order to obtain extensive biopsies, the study was limited to early pregnancy.
MATERIAL AND METHODS Amniotic sacs from 16-19 week pregnancies interrupted by hysterotomy for legal abortion were collected from 10 women and in the operation room immediately immersed in 2.5 per cent glutaraldehyde in Soerensen’s phosphate buffer, pH 7.4. Small specimens of reflected and placental amnion and from fetal periderm were cut and immersed in aliquots of glutaraldehyde, in which they were kept until further processing. After at least a few days in glutaraldehyde the specimens were rinsed in the same buffer, postfixed in 1 per cent OsO, and subsequently rinsed in three consecutive baths of redistilled water. Preparation for SEM was then carried out by freezing each specimen in a minute amount of redistilled water by dropping it in a quenching medium of iso-penthane cooled by liquid nitrogen (-196OC). Ensuing drying was then carried out at the temperature of dry ice for 2-3 days. Orientation and mounting on SEM stubs were then carried out before sputter coating with gold took place. Observations were made in a Jeol JSM-U3 scanning electron microscope run at 15-20 kV. Acta Obstet Gynecol Scand 58 (1979)
354
S.BergstrOm
Fig. 1. Amniotic epithelium, 19th week. The bulging, microvillous appearance of the epithelial cells is typical. Note regular polygonal strands constituting intercellular borders. No exfoliation. SEM. Magn. 2 OOO x
.
Fig. 2. Survey over amniotic epithelium, showing a few poorly microvillous epithelial cells, obvioiisly in a different phase of development from the cells adjacent to them. SEM. Magn. 1 100 X . Acta Obstet Gynecol Scand 58 (1979)
Amniotic fluid cell exfoliation
355
Fig. 3. Close-up of Fig. 2, demonstrating strikingly different surfaces of adjacent amniotic epithelial cells. SEM. Magn. 5 200 x .
Fig. 4. In most amniotic epithelial cells small holes in the three-celljunctions are seen. Their function remains unclear but they have been regarded as a morphological correlate of the extensive turnover of amniotic fluid. SEM. Magn. 5 200 x . Acta Obstet Gynecol Scand 58 (1979)
356
S. BergstrOm
Fig. 5. Close-up of intercellular canal between amniotic epithelial cells. Detail of Fig. 4. SEM. Magn. 43 OOO x .
RESULTS
Amnion. Both in reflected and in placental amnion the epithelium showed a homogeneous appearance (Fig 1) with somewhat convex, polygonal cell surfaces, densely populated by microvilli evenly distributed over the apical surface of the epithelium. In some instances smooth, non-microvillous surfaces were encountered (Figs 2-3) but there were no signs of any detachment of cells from the amniotic epithelium. In many areas intercellular spaces were conspicious in the three-cell junctions of intercellular borders (Figs 4-5). Periderm. In contrast to the inactive appearance of the amniotic epithelium, the outermost layer of fetal skin displayed a marked activity of detachment of cells and cell fragments. A series of events could be established, elucidating the process whereby periderma1 elements were liberated to the amniotic fluid. Within a single cell surface various stages of detachment could be visualized and followed in detail. The first visible event was a slight elevation of a limited portion of the apical surface area concomitant with the appearance on its surface of microvillous lining surrounded by a smooth area of non-microvillous Acta Obstet Gynecol Scand 58 (1979)
surface (Fig 6). The second event was the emergence of minor indentations around the slightly elevated portion, first like single holes, later like minute spaces joining to form confluent larger spaces. The third event was a more pronounced protrusion of the elevated part by a gradual pinching-off effect at its base (Fig 6). The protrusion was thereby rendered less microvillous and sometimes displayed indentations also on its surface. The fourth event was the very moment of cell detachment, captured at the instant of exfoliation (Figs 7-8). Some areas were particularly rich in the detachment stages, suggesting that at a given time certain areas but not others undergo exfoliation. The cells liberated were ovoid or round in shape and covered with a thin lining of short microvilli. The stalks connecting the detaching cells to the underlying peridermal surface were fairly homogenous, around 1-2 p in diameter. The fifth event was the disappearance of the cell and the remaining stalk (Fig 9). This presumably occurred immediately before a distinct peridermal membrane reaction to the detachment, visible as a withdrawal of the stalk, with the formation of a crater around the stalk (Fig 9-10).
Amniotic fluid cell exfoliation
351
Fig. 6. Fetal skin surface, 19th week. A general tendency to exfoliation of cell fragments from each peridermal cell is apparent. Note various stages in the exfoliation process. The first stage is typically a barely visible elevation of a part of the cell surface, characteristically microvillous (arrows). A more and more pronounced protrusion of the elevated part is seen prior to its final detachment from the surface. The indentations (I) are seen in several fragments. SEM. Magn. 1 200 x .
Fig. 7. Exfoliation of cell fragment captured just at the detachment stage (arrows). Note stalks and microvillous surfaces of exfoiiating fragments. SEM. Magn. 1 700 x , Acta Obstet Gynecol Scand 58 (1979)
358
S. Bergstrom
Fig. 8. Detail of Fig. 7. The thin stalk stemming from the mother cell is obviously stretched for a while before detachment is finished. SEM. Magn. 5 OOO x .
DISCUSSION
The cell content of amniotic fluid in early pregnancy is sparse. In various studies the fluid has been described as virtually acellular prior to the 14th week (8) and even as late as the 25th week (9). Although general agreement exists regarding the scarcity of cells prior to the 16th week (lo), there is evidence that cells and cell fragments make a significant contribution to the amniotic fluid during this period, as shown in the present study. Detached cells are regarded as viable at the moment of exfoliation but are presumably degenerating rapidly due to the presence of nuclear pycnosis and membrane-bound structures similar to lysosomes (10). Amniotic fluid cells obtainable by amniocentesis in early pregnancy are of two types. The first, most prevalent one is characterised by a dense microvillous lining on a round or oval outline covering a cytoplasm laden with a glycogen deposit, which is heavier in younger specimens. The second type has obviously phagocytotic capacity due to the contents of engulfed material (10). The first type of cell, as described by Hoyes (lo), is presumably identical to Acto Obstet Gynecoi Scand 58 (1979)
the cell exfoliated from the periderm as observed by SEM in the present study. The surface morphology of tissues facing the amniotic cavity has been subject to several investigations since the classical studies of Bowen (11). The “blad. der cells” discovered in the light microscope got an ultrastructural interpretation later and were found tc appear during a limited period of human pregnancy. from around the fourth up to the sixth month (12), As peridermal derivates of apparent functiona significance, considering the extensive contributior of cell material from the fetus to the amniotic fluid these “bladder cells” have attracted astonishingly lit tle attention. Since they display an abundance o glycogen granules their presumed degeneration i: believed to represent a contribution of glucose to am niotic fluid (10). It is not clear whether there is a cir culation of glucose between peridermal cells and am niotic fluid. It has been suggested that the significan amounts of glucose in amniotic fluid - around 200 m; per liter (13) - are taken up partly by the outermos peridermal cells to form the glycogen deposit visibl in the electron microscope (14). The steady state ii amniotic fluid glucose concentration is obvious1
Amniotic fluid cell exfoliation
359
Fig. 9. Two recently detached “mother areas” (arrows) showing remnants of the stalk connecting the detached cell fragment and the mother cell. Note also the microvillous areas, presumably representing the cell membrane preparing itself for ensuing exfoliation. SEM. Magn. 1 700 x .
Fig. 10. Detail of Fig. 9, demonstrating the microvillous appearance of the cell membrane adjacent to the recently detached area. To the left, microvillous protrusions presumably loosely connected to underlying periderm and prepared to be detached to the amniotic fluid. SEM. Magn. 5 200 x . Acta Obstet Gynecol Scand 58 (1979)
360
S. BergstrOm
maintained despite the extensive detachment of cell material demonstrated by SEM. Hence, this abundant contribution of glycogen to the fluid should involve a similar output of glycogen or glucose from the fluid, either by reabsorption across the periderm or by alternate clearance routes. Amniotic epithelium was found by SEM not to contribute at all to the cell contents of amniotic fluid. This is contrary to the belief of several earlier investigators (8, 15, 16, 17, 18, 19). Most of these studies drew conclusions from scrapings of amniotic epithelium, a method obviously open to the criticism of artefactual detachment of tissue fragments from non-exfoliating surfaces (20). The striking difference in the present study in appearance of amniotic epithelium and periderm, respectively, by SEM investigations on intact tissue surfaces underscores the risk of morphological conclusions from correlate studies involving delicate events that are easily deranged and interfered with by improper methodology. To conclude, by SEM of intact surfaces of amniotic epithelium and fetal periderm in early pregnancy it was shown that an extensive contribution was obvious from the latter to amniotic fluid, while virtually no detachment of cell material took place from the former, contrary to earlier reports.
ACKNOWLEDGEMENTS Supported by grants from the Swedish Medical Research Council (B 78-17X), Stiftelsen Allmanna BarnbBrdshusets Minnesfond and Prenatalforskningsnamnden.
REFERENCES 1 . Daniel, M. C.: Recherches sur la cytologie du liquide amniotique. Ann Gynircol ObstCt. 1:466, 1904. 2. Bourgeois, G. A.: The identification of fetal squames and the diagnosis of ruptured membranes by vaginal smears. Am J Obstet Gynecol 44:80, 1952. 3. Rosa, P. A. & Fanard, A. E.: A new method of prenatal diagnosis of sex. Int J Sexol 4:160, 1951. 4. Fuchs, F. & Cederqvist, L.: Prenatal diagnosis based upon amniotic fluid cells. In Amniotic fluid - research and clinical application (eds. D. V. I. Fairweather and T. K. A. B. Eskes), p. 262. Amsterdam, Excerpta Medica, 1973. 5. Driscoll, J. M. & Mellins, R. B.: Idiopathic respiratory distress syndrome. Am Thoracic SOC1:5, 1973.
Acta Obstet Gynecol Scand 58 (1979)
6. Morrison, J. C., Morrison, D. L., Lovett, F. A., Whybrew, W. D., Bucovaz, E. T., Wiser, W. L. &Fish, S. A.: Nile blue staining of cells in amniotic fluid for fetal maturity. Part I: A reappraisal. Obstet Gynecol 44:355, 1974. 7. Morrison, J . C., Morrison, D. L., Lovett, F. A., Whybrew, W. D., Bucovaz, E. T., Wiser, W. L. & Fish, S. A.: Nile blue staining of cells in amniotic fluid for fetal maturity. Part 11: In complicated obstetric cases. Obstet Gynecol 44:362, 1974. 8. Wachtel, E., Gordon, H. & Olsen, E.: Cytology of amniotic fluid. J Obstet Gynaecol Brit Commonw 76:596, 1969. 9. Marianowski, L. & Wacker-Pujdak, B: Cytology of amniotic fluid in different periods of pregnancy. Matern Med Pol 5:203, 1973. 10. Hoyes, A. D.: Ultrastructure of the cells of the amniotic fluid. J Obstet Gynaecol Brit Commonw 75:164, 1968. 1 1 . Bowen, J. T.: The epitrichial layer of the human epidermis. Anat Anz 4:412, 1889. 12. Hoyes, A. D.: Electron microscopy of the surface layer (periderm) of human fetal skin. J Anat 103:321, 1968. 13. de Miguel Adrihn, J. M., Gonzalez Batros, C., Garcia Perez, M. C., SBez-Benito, J. & G6mez Calatayud, M.: Bioquimica y citologia del liquid0 amniotic0 en el tercer trimestre de la gestaci6n. Acta Obstet Ginecol HispLusit 20:313, 1972. 14. Serri, F. & Montagna, W.: The structure and futlction of the epidermis. Pediat Clin N Am 8:917, 1961. 15. van Leeuwen, L., Jacoby, H. &Charles, D.: Exfoliative cytology of amniotic fluid. Acta Cytol (Baltimore) 9.442, 1965. 16. Votta, R. A, Bobrow de Gagneten, C., Parada, 0. & Giuleitti, M.: Cytologic study of amniotic fluid in pregnancy. Am J Obstet Gynecol 102:571, 1968. 17. Casadei, R., D’Ablaing, G., Kaplan, B. J. & Schwinn, C. P.: A cytologic study of amniotic fluid. Acta Cytol 17:289, 1973. 18. Churchouse, M. J. & Langstone, V. E.: The occurrence and origin of “satellite cells” in amniotic fluid (preliminary report). Acta Cytol 14:609, 1970. 19. Morris, H. H. B. & Bennett, M. J.: The classification and origin of amniotic fluid cells. Acta Cytol 18:149, 1974. 20. BergstrBm, S.: Ultrastructure of cell detachment from the human fetus in early pregnancy. Acta Obstet Gynecol Scand, in press, 1979.
Submitted for publication March 8, 1978 Staffan Bergstrdm, M.D. Department of Obstetrics and Gynecology Central hospital S-631 88 Eskilstuna Sweden
AMNIOTIC FLUID CELL EXFOLIATION IN EARLY HUMAN PREGNANCY Staffan Bergstrom From the Departments of Obstetrics and Gynecology and Human Anatomy, University Hospital, Uppsala, and Department of Obstetrics and Gynecology, Central Hospital, Eskilstuna, Sweden
Abstract. An ultrasctructural study of amniotic epithelium and fetal periderm was undertaken in order to investigate the exfoliative capacity in these tissue surfaces in early human pregnancy. Contrary to earlier reports, no exfoliation could be detected from amniotic epithelium, while a heavy detachment of cells and cell fragments was observed from fetal skin. The importance of meticulous tissue preparation is obvious; detachment phenomena are easily produced artifactually by tissue scrapings and improper tissue preparation for morphological investigations.
Since the discovery of the cells in amniotic fluid by Daniel (l), few studies on amniotic fluid have focussed on the clinical applications of amniotic fluid cytology. Not until half a century after Daniel’s paper was an attempt made to apply these cytological findings in the diagnosis of ruptured membranes (2). A decade later antenatal sex determination on the basis of amniotic fluid cytology was described by Rosa and Fanard (3). This achieved a breakthrough for cytodiagnosis by amniocentesis and a large number of publications have since described the antenatal diagnosis of several sex-linked inherited diseases (for review, see Fuchs and Cederquist (4)). Amniotic fluid cells constitute the earliest easily available tissue sample from the fetus, carrying significant information on its chromosomal features. In early pregnancy, cytodiagnosis for genetic counselling is of primary concern, while in late pregnancy cytodiagnosis is used almost exclusively for determination of fetal maturity. Prematurity still plays an important role in neonatal mortality. According to a recent study (5) in the U S , more than 1 per cent of all neonates born alive develop RDS, which still has a lethal outcome in about 30 per cent of cases. Cytodiagnosis, if reliable, would be a worhtwhile alternative to biochemical monitoring in testing for fetal maturity in risk cases - e.g. with meconium contamination - where the L/S ratio is less well related to maturity. In an attempt to settle the question of
reliability of cytodiagnosis in the determination of fetal maturity, Morrison and co-workers (6, 7), carried out critical studies on the methodology of amniOtiC fluid cytodiagnosis. By Carefully evaluating various steps in the cell preparation they demonstrated that a number of crucial details could explain the variation in reliability observed by several investigators utilizing the Nile Blue sulphate technique. The cell pattern as an expression of fetal maturity accordingly became more important than before. The origin of the maturity-typical cell pattern is unknown and little is understood about the behaviour of tissue surfaces facing the amniotic cavity and their potential contribution to amniotic fluid cytology. The purpose of the present study was to investigate by scanning electron microscopy (SEM) the existence of exfoliative properties in the major components of the surface facing the amniotic fluid, the amniotic epithelium and the fetal periderm. In order to obtain extensive biopsies, the study was limited to early pregnancy.
MATERIAL AND METHODS Amniotic sacs from 16-19 week pregnancies interrupted by hysterotomy for legal abortion were collected from 10 women and in the operation room immediately immersed in 2.5 per cent glutaraldehyde in Soerensen’s phosphate buffer, pH 7.4. Small specimens of reflected and placental amnion and from fetal periderm were cut and immersed in aliquots of glutaraldehyde, in which they were kept until further processing. After at least a few days in glutaraldehyde the specimens were rinsed in the same buffer, postfixed in 1 per cent OsO, and subsequently rinsed in three consecutive baths of redistilled water. Preparation for SEM was then carried out by freezing each specimen in a minute amount of redistilled water by dropping it in a quenching medium of iso-penthane cooled by liquid nitrogen (-196OC). Ensuing drying was then carried out at the temperature of dry ice for 2-3 days. Orientation and mounting on SEM stubs were then carried out before sputter coating with gold took place. Observations were made in a Jeol JSM-U3 scanning electron microscope run at 15-20 kV. Acta Obstet Gynecol Scand 58 (1979)
354
S.BergstrOm
Fig. 1. Amniotic epithelium, 19th week. The bulging, microvillous appearance of the epithelial cells is typical. Note regular polygonal strands constituting intercellular borders. No exfoliation. SEM. Magn. 2 OOO x
.
Fig. 2. Survey over amniotic epithelium, showing a few poorly microvillous epithelial cells, obvioiisly in a different phase of development from the cells adjacent to them. SEM. Magn. 1 100 X . Acta Obstet Gynecol Scand 58 (1979)
Amniotic fluid cell exfoliation
355
Fig. 3. Close-up of Fig. 2, demonstrating strikingly different surfaces of adjacent amniotic epithelial cells. SEM. Magn. 5 200 x .
Fig. 4. In most amniotic epithelial cells small holes in the three-celljunctions are seen. Their function remains unclear but they have been regarded as a morphological correlate of the extensive turnover of amniotic fluid. SEM. Magn. 5 200 x . Acta Obstet Gynecol Scand 58 (1979)
356
S. BergstrOm
Fig. 5. Close-up of intercellular canal between amniotic epithelial cells. Detail of Fig. 4. SEM. Magn. 43 OOO x .
RESULTS
Amnion. Both in reflected and in placental amnion the epithelium showed a homogeneous appearance (Fig 1) with somewhat convex, polygonal cell surfaces, densely populated by microvilli evenly distributed over the apical surface of the epithelium. In some instances smooth, non-microvillous surfaces were encountered (Figs 2-3) but there were no signs of any detachment of cells from the amniotic epithelium. In many areas intercellular spaces were conspicious in the three-cell junctions of intercellular borders (Figs 4-5). Periderm. In contrast to the inactive appearance of the amniotic epithelium, the outermost layer of fetal skin displayed a marked activity of detachment of cells and cell fragments. A series of events could be established, elucidating the process whereby periderma1 elements were liberated to the amniotic fluid. Within a single cell surface various stages of detachment could be visualized and followed in detail. The first visible event was a slight elevation of a limited portion of the apical surface area concomitant with the appearance on its surface of microvillous lining surrounded by a smooth area of non-microvillous Acta Obstet Gynecol Scand 58 (1979)
surface (Fig 6). The second event was the emergence of minor indentations around the slightly elevated portion, first like single holes, later like minute spaces joining to form confluent larger spaces. The third event was a more pronounced protrusion of the elevated part by a gradual pinching-off effect at its base (Fig 6). The protrusion was thereby rendered less microvillous and sometimes displayed indentations also on its surface. The fourth event was the very moment of cell detachment, captured at the instant of exfoliation (Figs 7-8). Some areas were particularly rich in the detachment stages, suggesting that at a given time certain areas but not others undergo exfoliation. The cells liberated were ovoid or round in shape and covered with a thin lining of short microvilli. The stalks connecting the detaching cells to the underlying peridermal surface were fairly homogenous, around 1-2 p in diameter. The fifth event was the disappearance of the cell and the remaining stalk (Fig 9). This presumably occurred immediately before a distinct peridermal membrane reaction to the detachment, visible as a withdrawal of the stalk, with the formation of a crater around the stalk (Fig 9-10).
Amniotic fluid cell exfoliation
351
Fig. 6. Fetal skin surface, 19th week. A general tendency to exfoliation of cell fragments from each peridermal cell is apparent. Note various stages in the exfoliation process. The first stage is typically a barely visible elevation of a part of the cell surface, characteristically microvillous (arrows). A more and more pronounced protrusion of the elevated part is seen prior to its final detachment from the surface. The indentations (I) are seen in several fragments. SEM. Magn. 1 200 x .
Fig. 7. Exfoliation of cell fragment captured just at the detachment stage (arrows). Note stalks and microvillous surfaces of exfoiiating fragments. SEM. Magn. 1 700 x , Acta Obstet Gynecol Scand 58 (1979)
358
S. Bergstrom
Fig. 8. Detail of Fig. 7. The thin stalk stemming from the mother cell is obviously stretched for a while before detachment is finished. SEM. Magn. 5 OOO x .
DISCUSSION
The cell content of amniotic fluid in early pregnancy is sparse. In various studies the fluid has been described as virtually acellular prior to the 14th week (8) and even as late as the 25th week (9). Although general agreement exists regarding the scarcity of cells prior to the 16th week (lo), there is evidence that cells and cell fragments make a significant contribution to the amniotic fluid during this period, as shown in the present study. Detached cells are regarded as viable at the moment of exfoliation but are presumably degenerating rapidly due to the presence of nuclear pycnosis and membrane-bound structures similar to lysosomes (10). Amniotic fluid cells obtainable by amniocentesis in early pregnancy are of two types. The first, most prevalent one is characterised by a dense microvillous lining on a round or oval outline covering a cytoplasm laden with a glycogen deposit, which is heavier in younger specimens. The second type has obviously phagocytotic capacity due to the contents of engulfed material (10). The first type of cell, as described by Hoyes (lo), is presumably identical to Acto Obstet Gynecoi Scand 58 (1979)
the cell exfoliated from the periderm as observed by SEM in the present study. The surface morphology of tissues facing the amniotic cavity has been subject to several investigations since the classical studies of Bowen (11). The “blad. der cells” discovered in the light microscope got an ultrastructural interpretation later and were found tc appear during a limited period of human pregnancy. from around the fourth up to the sixth month (12), As peridermal derivates of apparent functiona significance, considering the extensive contributior of cell material from the fetus to the amniotic fluid these “bladder cells” have attracted astonishingly lit tle attention. Since they display an abundance o glycogen granules their presumed degeneration i: believed to represent a contribution of glucose to am niotic fluid (10). It is not clear whether there is a cir culation of glucose between peridermal cells and am niotic fluid. It has been suggested that the significan amounts of glucose in amniotic fluid - around 200 m; per liter (13) - are taken up partly by the outermos peridermal cells to form the glycogen deposit visibl in the electron microscope (14). The steady state ii amniotic fluid glucose concentration is obvious1
Amniotic fluid cell exfoliation
359
Fig. 9. Two recently detached “mother areas” (arrows) showing remnants of the stalk connecting the detached cell fragment and the mother cell. Note also the microvillous areas, presumably representing the cell membrane preparing itself for ensuing exfoliation. SEM. Magn. 1 700 x .
Fig. 10. Detail of Fig. 9, demonstrating the microvillous appearance of the cell membrane adjacent to the recently detached area. To the left, microvillous protrusions presumably loosely connected to underlying periderm and prepared to be detached to the amniotic fluid. SEM. Magn. 5 200 x . Acta Obstet Gynecol Scand 58 (1979)
360
S. BergstrOm
maintained despite the extensive detachment of cell material demonstrated by SEM. Hence, this abundant contribution of glycogen to the fluid should involve a similar output of glycogen or glucose from the fluid, either by reabsorption across the periderm or by alternate clearance routes. Amniotic epithelium was found by SEM not to contribute at all to the cell contents of amniotic fluid. This is contrary to the belief of several earlier investigators (8, 15, 16, 17, 18, 19). Most of these studies drew conclusions from scrapings of amniotic epithelium, a method obviously open to the criticism of artefactual detachment of tissue fragments from non-exfoliating surfaces (20). The striking difference in the present study in appearance of amniotic epithelium and periderm, respectively, by SEM investigations on intact tissue surfaces underscores the risk of morphological conclusions from correlate studies involving delicate events that are easily deranged and interfered with by improper methodology. To conclude, by SEM of intact surfaces of amniotic epithelium and fetal periderm in early pregnancy it was shown that an extensive contribution was obvious from the latter to amniotic fluid, while virtually no detachment of cell material took place from the former, contrary to earlier reports.
ACKNOWLEDGEMENTS Supported by grants from the Swedish Medical Research Council (B 78-17X), Stiftelsen Allmanna BarnbBrdshusets Minnesfond and Prenatalforskningsnamnden.
REFERENCES 1 . Daniel, M. C.: Recherches sur la cytologie du liquide amniotique. Ann Gynircol ObstCt. 1:466, 1904. 2. Bourgeois, G. A.: The identification of fetal squames and the diagnosis of ruptured membranes by vaginal smears. Am J Obstet Gynecol 44:80, 1952. 3. Rosa, P. A. & Fanard, A. E.: A new method of prenatal diagnosis of sex. Int J Sexol 4:160, 1951. 4. Fuchs, F. & Cederqvist, L.: Prenatal diagnosis based upon amniotic fluid cells. In Amniotic fluid - research and clinical application (eds. D. V. I. Fairweather and T. K. A. B. Eskes), p. 262. Amsterdam, Excerpta Medica, 1973. 5. Driscoll, J. M. & Mellins, R. B.: Idiopathic respiratory distress syndrome. Am Thoracic SOC1:5, 1973.
Acta Obstet Gynecol Scand 58 (1979)
6. Morrison, J. C., Morrison, D. L., Lovett, F. A., Whybrew, W. D., Bucovaz, E. T., Wiser, W. L. &Fish, S. A.: Nile blue staining of cells in amniotic fluid for fetal maturity. Part I: A reappraisal. Obstet Gynecol 44:355, 1974. 7. Morrison, J . C., Morrison, D. L., Lovett, F. A., Whybrew, W. D., Bucovaz, E. T., Wiser, W. L. & Fish, S. A.: Nile blue staining of cells in amniotic fluid for fetal maturity. Part 11: In complicated obstetric cases. Obstet Gynecol 44:362, 1974. 8. Wachtel, E., Gordon, H. & Olsen, E.: Cytology of amniotic fluid. J Obstet Gynaecol Brit Commonw 76:596, 1969. 9. Marianowski, L. & Wacker-Pujdak, B: Cytology of amniotic fluid in different periods of pregnancy. Matern Med Pol 5:203, 1973. 10. Hoyes, A. D.: Ultrastructure of the cells of the amniotic fluid. J Obstet Gynaecol Brit Commonw 75:164, 1968. 1 1 . Bowen, J. T.: The epitrichial layer of the human epidermis. Anat Anz 4:412, 1889. 12. Hoyes, A. D.: Electron microscopy of the surface layer (periderm) of human fetal skin. J Anat 103:321, 1968. 13. de Miguel Adrihn, J. M., Gonzalez Batros, C., Garcia Perez, M. C., SBez-Benito, J. & G6mez Calatayud, M.: Bioquimica y citologia del liquid0 amniotic0 en el tercer trimestre de la gestaci6n. Acta Obstet Ginecol HispLusit 20:313, 1972. 14. Serri, F. & Montagna, W.: The structure and futlction of the epidermis. Pediat Clin N Am 8:917, 1961. 15. van Leeuwen, L., Jacoby, H. &Charles, D.: Exfoliative cytology of amniotic fluid. Acta Cytol (Baltimore) 9.442, 1965. 16. Votta, R. A, Bobrow de Gagneten, C., Parada, 0. & Giuleitti, M.: Cytologic study of amniotic fluid in pregnancy. Am J Obstet Gynecol 102:571, 1968. 17. Casadei, R., D’Ablaing, G., Kaplan, B. J. & Schwinn, C. P.: A cytologic study of amniotic fluid. Acta Cytol 17:289, 1973. 18. Churchouse, M. J. & Langstone, V. E.: The occurrence and origin of “satellite cells” in amniotic fluid (preliminary report). Acta Cytol 14:609, 1970. 19. Morris, H. H. B. & Bennett, M. J.: The classification and origin of amniotic fluid cells. Acta Cytol 18:149, 1974. 20. BergstrBm, S.: Ultrastructure of cell detachment from the human fetus in early pregnancy. Acta Obstet Gynecol Scand, in press, 1979.
Submitted for publication March 8, 1978 Staffan Bergstrdm, M.D. Department of Obstetrics and Gynecology Central hospital S-631 88 Eskilstuna Sweden
