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Origin of the cells in the liquor amnii
Origin of the cells in the liquor amnii H.
J.
Groningen,
HUISJES, The
M.D.
Netherlands
The cytologic features of the amniotic fluid were studied. Four epithelial cell types are ordinarily present, namely, large eosinojhilic cells, large cyanophilic cells, small round cyanophilic cells, and polygonal sosinophilic cells. The first three cell forms may be nucleate or anucleate, the fourth is always anucleate. The polygonal cells occur after the thirty-eighth week only. The origin of the cells was established by comparing them with scrafiings of fetal surfaces. The cells appeared to be shed essentially by the fetal buccal mucosa, vaginal epithslium, and skin, and there is probably a contribution from the urine. The source of the small blue cells and the polygonal cells in particular is discussed.
A M 0 N G T H E M 0 R E COnfUSingaspects of the cytologic examination of amniotic fluid is the question of the origin of the cells. Many diverse assumptions and conclusions are dealt with in the literature. After a classification of the cell forms present, their respective sourceswere systematically looked for. Material
and
methods
One hundred and eight samples of amniotic fluid from 75 gravidas were examined. Of these, 83 originated from patients with rhesus sensitization and 18 from normal pregnant women, In 5 patients, a diagnosis was made of pre-eclampsia or renal disease. In addition, there was one patient with hydramnios and one with anencephaly. The duration of pregnancy varied from 25 through 43 weeks. The liquor amnii was furnished by amniocentesis, transabdominal or at the time of cesareansection, and by vaginal puncture of the bulging membranes. The material was screened systematically according to criteria enumerated elsewhere.1 For each sample a cytogram was made based on an examination of 400 cells. An additional 100 specimenswere screened without From the Department Gynecology, University
of Obstetrics Hospital.
and
1222
making a cytogram. Afterward, the cells were morphologically compared with those in smears from buccal mucosa, vulva, skin, amnion, and umbilical cord of the fetus, and from newborn male infants’ urine. Urine from female infants is unsuitable becauseof strong contamination with vulvar cells. All slides were stained by the Harris-Shorr method. The size of the cells was measured by means of a screw micrometer. The long and the short side of an imaginary rectangle snugly enclosing the cell or the nucleus were determined. The measurement was performed on 100 cells from each class. Results
The cell population appeared to be made up usually from the following cell forms. la. Nucleate eosinophik cells (52.5 f. 13.8 x 36.1 + 10.5 p). Cytoplasm is red to orange, sometimes containing dark punctiform inclusion bodies. The nucleus is brown or dark red (6.0 +- 1.2 x 4.4 + 0.8 p). Of the 100 cells measured, 33 had a pyknotic nucleus. lb. Anucleate eosinophdic cells (50.9 + 13.0 x 34.1 It 7.9 /A). Often a nuclear ghost is visible. 2a. Nucleate cyanopbilic cells (49.0 + 11.6 x 30.2 f 7.1 u). Cytoplasm is light blue.
Volums 106 Number 8
Fig. 1. Large cyanophilic
Cells
cells and anucleate small cyanophilic
The nucleus is brown or dark violet (7.4 ? 2.7 x 4.9 + 0.9 p) . Of the 100 cells measured 14 were karyopyknotic. 2b. Anucleate cyanophilic cells (44.0 + 10.9 x 30.8 + 8.1 p). Cytoplasm is light blue and often contains a nuclear ghost (Fig. 1). 3a. Small nucleate cyanophilic celk (21.0 +_6.2 x 15.8 +- 4.6 EL).These cells are mostly more or Iess round, in contrast with the above categories which have a more angular outline (Fig. 2). There is no indication of a cylindricaI form. Often the cytoplasm, which stains dark blue, contains one or several vacuoles. The nucleus is brown and vesicular (7.0 f 1.5 x 5.3 + 1.0 p). There is a propensity to form clusters in which the cells are sometimes connected by a broad intercellular bridge. 3b. SmaIl am&ate cyanophilic cells (23.1 2 6.6 x 16.5 k 1.6 p) with rounded contour (Fig. I). Cytoplasm is light bIue and rareIy vacuolated. There are no nuclear ghosts. It is not always easy to distinguish these cells from the anucleate cyanophihc cells described above. 4. Polygonal cells (35.0 f 6.1 x 29.6 f 1.4 p). The outline approximates a regular pentagon or heptagon (Fig. 3). The cytoplasm is transparent orange with a veined or granular aspect. In the center often an annular structure is visible, probably indicating the former nuclear site; the nucleus
cells. Amniotic
in liquor
amnii
1223
fluid. (x800.)
Fig. 2. Small nucleate cyanophilic cells and one anucleate large cell. The 2 small dots probably are leukocytes. Note the intercellular bridges. Amniotic fluid. (x800.)
itself is always absent. There is a marked propensity to clustering or membrane-forming. It is extremely rare for these cells to occur before the thirty-eighth week of pregnancy. Two less reguIarIy occurring ceil forms may be described. They include vaguely delineated pale blue conglomerations with a regular pattern of more or lessunstained oval patches (Fig. 4) . These patches sometimes are found filled in with violet nuclear structures. There are also light blue angular stratified epithelial cells, often showing a ragged outline. A glassy, unstructured cyto-
1224
Huisjes
Amer.
Fig. 3. Polygonal cells in amniotic central annular marking. (x800.)
fluid sediment. Note transparency,
angular
April 15, 1970 J. Obstet. Gynec.
outline,
and
actly; they had diameters of about 50 x 30
Fig. 4. Conglomeration of degenerating sumably from amnion. Amniotic fluid.
cells, pre(x800.)
plasm is characteristic. Sometimes a nuclear ghost is visible. The cells tend to be lying in membranes (Fig. 5). In the smearsfrom the fetal surfaces the following cell forms were found: Buccal mucosa. Largish epithelial cells, eosinophilic or cyanophilic (about equal amounts of each). Eosinophilic cells more often are karyopyknotic than are the cyanophilic cells, which mostly contain a finely granulated vesicular nucleus. Very often there is a dark brown punctiform inclusion body. These cells were not measured ex-
/JVaginal vestibule. Essentially cyanophilic “intermediate” cells with a vesicular or prepyknotic nucleus. Further eosinophilic “superficial” cells and some “parabasal” cells. Urine. Apart from leukocytes and large cyanophilic or eosinophilic epithehal cells, a typical part of the cellular contents is formed by round, dark blue cells (25.0 +8.8 x 18.0 + 3.9 CL)with a central or parietal nucleus (8.7 + 1.2 x 5.8 + 0.9 p). These cells sometimesare lying in clusters. Epidermis. Slides from premature as well as mature newborns almost exclusively contain polygonal cells (Fig. 6). Am&n. Especially from the reflected amnion, scarcely any cells could be scraped with a wooden spatula. The placental amnion yielded inconstant amounts of oblong or club-shaped cells (Fig. 7). They were cyanophilic and had basal processes,often as long as the cell body itself. Often a “brush border” could be seen. The cytoplasm was diffusely vacuolated. The irregular nucleus was brown and apically situated, In addition to these intact cells there were degenerating ones with their nucleus fading. Preparations of the whole amnion spread on a glassslide showed the well-known irregular honeycomb
Volume Number
106 8
Fig. 5. Three
Cells
large nucleate
cells and sheet of cells from umbilical
in
liquor
cord. Amniotic
am&
1225
fluid.
(x800.)
Fig. 6. Epidermal
cells from fetus at 32 weeks. Contact preparation
pattern. The occurrence of stretches of degenerating cells with pale cytoplasm was notable. Umbilical cord. Almost exclusively pale blue structureless squarnes were seen, mostly forming membranes. Most of the cells were anucleate, sometimes a nuclear ghost or a lightly stained vesicular nucleus could be observed (Fig. 8) .
Results
on adhesive slide. (x800.)
of comparisons
Comparing the results of the cytologic analysis of the liquor on one side and the fetal
surfaces
on the other
leads to the fol-
lowing conclusions: Eosinophilic cells can only originate from the mouth, as no other source could be demonstrated. For the same reason the cyanophilic
cells have
to spring
from
the vulva
1226
Huisies
Amer.
April 15, 1970 J. Obstet. Gynec.
much alike the fields of degenerating cells of the intact amnion. The pale structureless cells and cell membranes are identical with the cells coming off the umbilical cord.
7. Amnion cells. Scraping from placental at term. Note basal processes and degenerating cell at center. (x800.)
Fig. amnion
Fig. 8. Sheet of cells from umbilical ing. (X500.)
cord. Scrap-
and the oral cavity. It can be expected that in girls these cells are present in the amniotic fluid in great numbers. This has, in fact, been demonstrated.ll 2~ 3 The round blue cells probably originate from the urinary epithelium, as the difference in aspect between these cells and those from the placental amnion, as well as the restricted cellular yield of the reflected amnion tell against their amniotic origin; on the other hand, they bear much likeness with the small round cells from the urine. Polygonal cells are shed by the epidermis. The pale blue conglomerations are very
Comment Daniel,* to the best of my knowledge the first to publish on this subject, discerned epidermal cells and cells from the amnion; he did not elucidate this assumption. Rosa and Fanard* were the first investigators who systematically searched for the origin of the cells. From the fetal epidermis cells, staining yellowthey got anucleate brown by Papanicolaou’s method. The amount of these cells in the liquor depended on the degree of the skin being covered with vernix. They got further characteristic cell types a.0. from the fetal mouth, the urine, and the vulva. As regards the contribution of the amniotic epithelium the authors state : “Normally the amnion, . . . does not desquamate and consequently provides no elements toward the formation of an amniotic sediment.” Langreder5 also discerned cutaneous cells and cells from the buccal, vaginal, and urinary mucosa and from the glans penis. He did not mention the amnion as a possible source. On the basis of histologic and cytologic studies Sachs, Serr, and Danon reached the conclusion that cornified cells originate from the skin and nucleate cells mainly from the oral mucosa and the vaginal epithelium. Van Leeuwen, Jacoby, and Charles’ divided liquor cells in fetal and amniotic cells. The first category was not indicated more precisely (only the fetal skin had been examined), the latter were determined according to morphologic likeness with cells in smears and histologic preparations of the amnion. Brosens and Gordon*3 Q applied the Nile blue sulfate staining. In the last weeks of pregnancy they found chiefly lipid-laden, orange-staining cells in the amniotic fluid. Histologic examination of the fetal skin gave them the conviction that these cells are secreted by the sebaceous glands which were
Volume Number
106 8
Cells
equally stained orange by Nile blue sulfate. In addition, scraping of skin areas rich in sebaceous glands yielded more orange cells than other areas. SharplO recently doubted the conclusions of Brosens and Gordon that all orange cells originate from the sebaceous glands. He thought some elements were indeed secreted by them, but these were distinguishable from polygonal cells which stain orange as a consequence of adhering lipid material and were directly shed by the vernix caseosa. By now there should be no doubt as to the origin of the large eosinophilic and cyanophilic cells. General agreement exists about their descent from the fetal mouth and vulva. For the anucleate variety of both cell types probably degenerative processes are responsible, as these anucleate cells are only sporadically encountered in smears from the buccal mucosa and the vaginal vestibule. The orange cells seen after the thirtyeighth week of pregnancy in Nile blue sulfate-stained slides of amniotic fluid are by morphology the same as the polygonal cells found in preparations stained by the HarrisShorr procedure. We do not think, however, their cytoplasm contains lipid. The cell body stains blue with Nile blue sulfate: it makes the impression that the orange droplets and patches of fat are not situated within the cell, but upon it. Though this is difficult to prove, a strong indication for it is the presence of free fat globules between the cells. It is unlikely, therefore, that polygonal cells originate from sebaceous glands. First, as Sharp pointed out, the sebaceous glands are holocrine glands and it would be highly improbable for them to excrete intact cells in very large numbers. Second, the polygonal cell morphologically is an epidermal cell (by pressing an adhesive glass slide upon the
in
liquor
amnii
1227
skin one gets closed fields of them). The fact that sebaceous glands in a histologic section of fetal skin are stained orange by Nile blue sulfate can be easily explained by their containing large amounts of fat. Using the Harris-Shorr staining method we were not able to corroborate the finding of Brosens and Gordon that more polygonal cells can be scraped from sebaceous gland containing areas of skin. In our experience all areas yield equal amounts of them. This points to their really demonstrating fat and non-fat-laden cells. What exactly is the reason for the sudden appearance of polygonal cells about the thirty-eighth week of pregnancy is unknown. The disappearance to a large extent of vernix caseosa about this time, however, is well known. This might be the underlying phenomenon in the sense of the removal of a hindrance to desquamation. The physiology of this phenomenon is a very interesting problem of which nothing seems to be known as yet. Another issue of discussion is the origin of the small blue cells. The general assumption is now that they are amnion cells. On purely morphologic considerations we think this has to be doubted. The more so as for the same reason the origin from the urine is highly probable. BournelI thought it unlikely that the intact amnion sheds its epithelial cells, as did Rosa and Fanard.z On the other hand, in our investigation degenerating amnion cells could be demonstrated, which corroborates the opinion of Bourne on the possibility of desquamation of dead amniotic epithelium. What is the exact source of the urinary cells remains conjectural; maybe they are cover cells of the transitional epithelium, or renal tubular cells, which are reported to be excreted with the fetal urine in large numbers.lz
REFERENCES
1.
Huisjes, H. J.: Assen,
the
Amniotic
Netherlands,
82 Company.
1968,
Fluid Cytology, Van Gorcum
2.
Rosa, P. A., and Fanard, Sexol. 4: 160, 1951. 3. Huisjes, H. J.: Acta Cytol.
A.
E.:
12: 42,
Int. 1968.
J.
1228
4. 5. 6. 7. 8.
Huisjes
Gym%. Obst&. 1: 466, Daniel, Cl.: Ann. 1904. (Series 2.) Langreder, W.: Z. Geburtsh. Gynaek. 136: 136, 1952. Sachs, L., Serr, D. M., and Danon, M.: Science 123: 548. 1956. Van Leeuwen, L., Jacoby, H., and Charles, D.: Acta Cytol. 9: 442, 1965. Brosens, I., and Gordon, H.: J. Obstet. Gynsec. Brit. Comm. 72: 342, 1965.
Amer.
9. 10. 11. 12.
April 15, 1970 J. Obstet. Gynec.
Brosens, I., and Gordon, H.: J. Obstet. Gynsec. Brit. Comm. 73: 88, 1966. Sharp, F.: J. Obstet. Gynaec. Brit. Comm. 75: 812, 1968. Bourne,G.: The Human Amnion and Chorion, London, 1962, Lloyd-Luke, Ltd. Cruickshank, G., and Edmond, E.: Brit. Med. J. 4: 705, 1967.
J.
Groningen,
HUISJES, The
M.D.
Netherlands
The cytologic features of the amniotic fluid were studied. Four epithelial cell types are ordinarily present, namely, large eosinojhilic cells, large cyanophilic cells, small round cyanophilic cells, and polygonal sosinophilic cells. The first three cell forms may be nucleate or anucleate, the fourth is always anucleate. The polygonal cells occur after the thirty-eighth week only. The origin of the cells was established by comparing them with scrafiings of fetal surfaces. The cells appeared to be shed essentially by the fetal buccal mucosa, vaginal epithslium, and skin, and there is probably a contribution from the urine. The source of the small blue cells and the polygonal cells in particular is discussed.
A M 0 N G T H E M 0 R E COnfUSingaspects of the cytologic examination of amniotic fluid is the question of the origin of the cells. Many diverse assumptions and conclusions are dealt with in the literature. After a classification of the cell forms present, their respective sourceswere systematically looked for. Material
and
methods
One hundred and eight samples of amniotic fluid from 75 gravidas were examined. Of these, 83 originated from patients with rhesus sensitization and 18 from normal pregnant women, In 5 patients, a diagnosis was made of pre-eclampsia or renal disease. In addition, there was one patient with hydramnios and one with anencephaly. The duration of pregnancy varied from 25 through 43 weeks. The liquor amnii was furnished by amniocentesis, transabdominal or at the time of cesareansection, and by vaginal puncture of the bulging membranes. The material was screened systematically according to criteria enumerated elsewhere.1 For each sample a cytogram was made based on an examination of 400 cells. An additional 100 specimenswere screened without From the Department Gynecology, University
of Obstetrics Hospital.
and
1222
making a cytogram. Afterward, the cells were morphologically compared with those in smears from buccal mucosa, vulva, skin, amnion, and umbilical cord of the fetus, and from newborn male infants’ urine. Urine from female infants is unsuitable becauseof strong contamination with vulvar cells. All slides were stained by the Harris-Shorr method. The size of the cells was measured by means of a screw micrometer. The long and the short side of an imaginary rectangle snugly enclosing the cell or the nucleus were determined. The measurement was performed on 100 cells from each class. Results
The cell population appeared to be made up usually from the following cell forms. la. Nucleate eosinophik cells (52.5 f. 13.8 x 36.1 + 10.5 p). Cytoplasm is red to orange, sometimes containing dark punctiform inclusion bodies. The nucleus is brown or dark red (6.0 +- 1.2 x 4.4 + 0.8 p). Of the 100 cells measured, 33 had a pyknotic nucleus. lb. Anucleate eosinophdic cells (50.9 + 13.0 x 34.1 It 7.9 /A). Often a nuclear ghost is visible. 2a. Nucleate cyanopbilic cells (49.0 + 11.6 x 30.2 f 7.1 u). Cytoplasm is light blue.
Volums 106 Number 8
Fig. 1. Large cyanophilic
Cells
cells and anucleate small cyanophilic
The nucleus is brown or dark violet (7.4 ? 2.7 x 4.9 + 0.9 p) . Of the 100 cells measured 14 were karyopyknotic. 2b. Anucleate cyanophilic cells (44.0 + 10.9 x 30.8 + 8.1 p). Cytoplasm is light blue and often contains a nuclear ghost (Fig. 1). 3a. Small nucleate cyanophilic celk (21.0 +_6.2 x 15.8 +- 4.6 EL).These cells are mostly more or Iess round, in contrast with the above categories which have a more angular outline (Fig. 2). There is no indication of a cylindricaI form. Often the cytoplasm, which stains dark blue, contains one or several vacuoles. The nucleus is brown and vesicular (7.0 f 1.5 x 5.3 + 1.0 p). There is a propensity to form clusters in which the cells are sometimes connected by a broad intercellular bridge. 3b. SmaIl am&ate cyanophilic cells (23.1 2 6.6 x 16.5 k 1.6 p) with rounded contour (Fig. I). Cytoplasm is light bIue and rareIy vacuolated. There are no nuclear ghosts. It is not always easy to distinguish these cells from the anucleate cyanophihc cells described above. 4. Polygonal cells (35.0 f 6.1 x 29.6 f 1.4 p). The outline approximates a regular pentagon or heptagon (Fig. 3). The cytoplasm is transparent orange with a veined or granular aspect. In the center often an annular structure is visible, probably indicating the former nuclear site; the nucleus
cells. Amniotic
in liquor
amnii
1223
fluid. (x800.)
Fig. 2. Small nucleate cyanophilic cells and one anucleate large cell. The 2 small dots probably are leukocytes. Note the intercellular bridges. Amniotic fluid. (x800.)
itself is always absent. There is a marked propensity to clustering or membrane-forming. It is extremely rare for these cells to occur before the thirty-eighth week of pregnancy. Two less reguIarIy occurring ceil forms may be described. They include vaguely delineated pale blue conglomerations with a regular pattern of more or lessunstained oval patches (Fig. 4) . These patches sometimes are found filled in with violet nuclear structures. There are also light blue angular stratified epithelial cells, often showing a ragged outline. A glassy, unstructured cyto-
1224
Huisjes
Amer.
Fig. 3. Polygonal cells in amniotic central annular marking. (x800.)
fluid sediment. Note transparency,
angular
April 15, 1970 J. Obstet. Gynec.
outline,
and
actly; they had diameters of about 50 x 30
Fig. 4. Conglomeration of degenerating sumably from amnion. Amniotic fluid.
cells, pre(x800.)
plasm is characteristic. Sometimes a nuclear ghost is visible. The cells tend to be lying in membranes (Fig. 5). In the smearsfrom the fetal surfaces the following cell forms were found: Buccal mucosa. Largish epithelial cells, eosinophilic or cyanophilic (about equal amounts of each). Eosinophilic cells more often are karyopyknotic than are the cyanophilic cells, which mostly contain a finely granulated vesicular nucleus. Very often there is a dark brown punctiform inclusion body. These cells were not measured ex-
/JVaginal vestibule. Essentially cyanophilic “intermediate” cells with a vesicular or prepyknotic nucleus. Further eosinophilic “superficial” cells and some “parabasal” cells. Urine. Apart from leukocytes and large cyanophilic or eosinophilic epithehal cells, a typical part of the cellular contents is formed by round, dark blue cells (25.0 +8.8 x 18.0 + 3.9 CL)with a central or parietal nucleus (8.7 + 1.2 x 5.8 + 0.9 p). These cells sometimesare lying in clusters. Epidermis. Slides from premature as well as mature newborns almost exclusively contain polygonal cells (Fig. 6). Am&n. Especially from the reflected amnion, scarcely any cells could be scraped with a wooden spatula. The placental amnion yielded inconstant amounts of oblong or club-shaped cells (Fig. 7). They were cyanophilic and had basal processes,often as long as the cell body itself. Often a “brush border” could be seen. The cytoplasm was diffusely vacuolated. The irregular nucleus was brown and apically situated, In addition to these intact cells there were degenerating ones with their nucleus fading. Preparations of the whole amnion spread on a glassslide showed the well-known irregular honeycomb
Volume Number
106 8
Fig. 5. Three
Cells
large nucleate
cells and sheet of cells from umbilical
in
liquor
cord. Amniotic
am&
1225
fluid.
(x800.)
Fig. 6. Epidermal
cells from fetus at 32 weeks. Contact preparation
pattern. The occurrence of stretches of degenerating cells with pale cytoplasm was notable. Umbilical cord. Almost exclusively pale blue structureless squarnes were seen, mostly forming membranes. Most of the cells were anucleate, sometimes a nuclear ghost or a lightly stained vesicular nucleus could be observed (Fig. 8) .
Results
on adhesive slide. (x800.)
of comparisons
Comparing the results of the cytologic analysis of the liquor on one side and the fetal
surfaces
on the other
leads to the fol-
lowing conclusions: Eosinophilic cells can only originate from the mouth, as no other source could be demonstrated. For the same reason the cyanophilic
cells have
to spring
from
the vulva
1226
Huisies
Amer.
April 15, 1970 J. Obstet. Gynec.
much alike the fields of degenerating cells of the intact amnion. The pale structureless cells and cell membranes are identical with the cells coming off the umbilical cord.
7. Amnion cells. Scraping from placental at term. Note basal processes and degenerating cell at center. (x800.)
Fig. amnion
Fig. 8. Sheet of cells from umbilical ing. (X500.)
cord. Scrap-
and the oral cavity. It can be expected that in girls these cells are present in the amniotic fluid in great numbers. This has, in fact, been demonstrated.ll 2~ 3 The round blue cells probably originate from the urinary epithelium, as the difference in aspect between these cells and those from the placental amnion, as well as the restricted cellular yield of the reflected amnion tell against their amniotic origin; on the other hand, they bear much likeness with the small round cells from the urine. Polygonal cells are shed by the epidermis. The pale blue conglomerations are very
Comment Daniel,* to the best of my knowledge the first to publish on this subject, discerned epidermal cells and cells from the amnion; he did not elucidate this assumption. Rosa and Fanard* were the first investigators who systematically searched for the origin of the cells. From the fetal epidermis cells, staining yellowthey got anucleate brown by Papanicolaou’s method. The amount of these cells in the liquor depended on the degree of the skin being covered with vernix. They got further characteristic cell types a.0. from the fetal mouth, the urine, and the vulva. As regards the contribution of the amniotic epithelium the authors state : “Normally the amnion, . . . does not desquamate and consequently provides no elements toward the formation of an amniotic sediment.” Langreder5 also discerned cutaneous cells and cells from the buccal, vaginal, and urinary mucosa and from the glans penis. He did not mention the amnion as a possible source. On the basis of histologic and cytologic studies Sachs, Serr, and Danon reached the conclusion that cornified cells originate from the skin and nucleate cells mainly from the oral mucosa and the vaginal epithelium. Van Leeuwen, Jacoby, and Charles’ divided liquor cells in fetal and amniotic cells. The first category was not indicated more precisely (only the fetal skin had been examined), the latter were determined according to morphologic likeness with cells in smears and histologic preparations of the amnion. Brosens and Gordon*3 Q applied the Nile blue sulfate staining. In the last weeks of pregnancy they found chiefly lipid-laden, orange-staining cells in the amniotic fluid. Histologic examination of the fetal skin gave them the conviction that these cells are secreted by the sebaceous glands which were
Volume Number
106 8
Cells
equally stained orange by Nile blue sulfate. In addition, scraping of skin areas rich in sebaceous glands yielded more orange cells than other areas. SharplO recently doubted the conclusions of Brosens and Gordon that all orange cells originate from the sebaceous glands. He thought some elements were indeed secreted by them, but these were distinguishable from polygonal cells which stain orange as a consequence of adhering lipid material and were directly shed by the vernix caseosa. By now there should be no doubt as to the origin of the large eosinophilic and cyanophilic cells. General agreement exists about their descent from the fetal mouth and vulva. For the anucleate variety of both cell types probably degenerative processes are responsible, as these anucleate cells are only sporadically encountered in smears from the buccal mucosa and the vaginal vestibule. The orange cells seen after the thirtyeighth week of pregnancy in Nile blue sulfate-stained slides of amniotic fluid are by morphology the same as the polygonal cells found in preparations stained by the HarrisShorr procedure. We do not think, however, their cytoplasm contains lipid. The cell body stains blue with Nile blue sulfate: it makes the impression that the orange droplets and patches of fat are not situated within the cell, but upon it. Though this is difficult to prove, a strong indication for it is the presence of free fat globules between the cells. It is unlikely, therefore, that polygonal cells originate from sebaceous glands. First, as Sharp pointed out, the sebaceous glands are holocrine glands and it would be highly improbable for them to excrete intact cells in very large numbers. Second, the polygonal cell morphologically is an epidermal cell (by pressing an adhesive glass slide upon the
in
liquor
amnii
1227
skin one gets closed fields of them). The fact that sebaceous glands in a histologic section of fetal skin are stained orange by Nile blue sulfate can be easily explained by their containing large amounts of fat. Using the Harris-Shorr staining method we were not able to corroborate the finding of Brosens and Gordon that more polygonal cells can be scraped from sebaceous gland containing areas of skin. In our experience all areas yield equal amounts of them. This points to their really demonstrating fat and non-fat-laden cells. What exactly is the reason for the sudden appearance of polygonal cells about the thirty-eighth week of pregnancy is unknown. The disappearance to a large extent of vernix caseosa about this time, however, is well known. This might be the underlying phenomenon in the sense of the removal of a hindrance to desquamation. The physiology of this phenomenon is a very interesting problem of which nothing seems to be known as yet. Another issue of discussion is the origin of the small blue cells. The general assumption is now that they are amnion cells. On purely morphologic considerations we think this has to be doubted. The more so as for the same reason the origin from the urine is highly probable. BournelI thought it unlikely that the intact amnion sheds its epithelial cells, as did Rosa and Fanard.z On the other hand, in our investigation degenerating amnion cells could be demonstrated, which corroborates the opinion of Bourne on the possibility of desquamation of dead amniotic epithelium. What is the exact source of the urinary cells remains conjectural; maybe they are cover cells of the transitional epithelium, or renal tubular cells, which are reported to be excreted with the fetal urine in large numbers.lz
REFERENCES
1.
Huisjes, H. J.: Assen,
the
Amniotic
Netherlands,
82 Company.
1968,
Fluid Cytology, Van Gorcum
2.
Rosa, P. A., and Fanard, Sexol. 4: 160, 1951. 3. Huisjes, H. J.: Acta Cytol.
A.
E.:
12: 42,
Int. 1968.
J.
1228
4. 5. 6. 7. 8.
Huisjes
Gym%. Obst&. 1: 466, Daniel, Cl.: Ann. 1904. (Series 2.) Langreder, W.: Z. Geburtsh. Gynaek. 136: 136, 1952. Sachs, L., Serr, D. M., and Danon, M.: Science 123: 548. 1956. Van Leeuwen, L., Jacoby, H., and Charles, D.: Acta Cytol. 9: 442, 1965. Brosens, I., and Gordon, H.: J. Obstet. Gynsec. Brit. Comm. 72: 342, 1965.
Amer.
9. 10. 11. 12.
April 15, 1970 J. Obstet. Gynec.
Brosens, I., and Gordon, H.: J. Obstet. Gynsec. Brit. Comm. 73: 88, 1966. Sharp, F.: J. Obstet. Gynaec. Brit. Comm. 75: 812, 1968. Bourne,G.: The Human Amnion and Chorion, London, 1962, Lloyd-Luke, Ltd. Cruickshank, G., and Edmond, E.: Brit. Med. J. 4: 705, 1967.