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Incidence of tetraploidy as related to amniotic fluid cell types
Incidence of tetraploidy as related to amniotic fluid cell types T.
R.
TEGENKAMP,
CHARLES Westeruille,
H.
PH.D. HUX,
B.A.
Ohio
Human amniotic fluid cells obtained by amniocentesis performed on 7 women during the thirteenth to sixteenth weeks of pregnancy were cultured. Four of the specimens were used for the isolation of the fibroblast-like cells. Epithelial cells were isolated from the 3 remaining specimens. Higher frequencies of tetraploidy were noted in the fibroblast-like cell cultures when harvested, Two different concentrations of demecolcine+ were utilized prior to harvesting. If tetraploid cells originated predominantly from the fibroblast-like cells, then variances in tetragloidy among amniotic @id specimens could be explained, inasmuch as cell type ratios vary from specimen to specimen. Metabolic variances between the cell types have previously been reported. This investigation shows there is a diflerence in chromosomal analysis (tetraQloid percentage) as related to cell type.
AT L E AS T TWO morphologically distinct cell types, fibroblast-like and epithelium-like cells, have been found and described in cultured amniotic fluid cells.*, ’ Melancon and associates3 have described distinct differences in enzyme activity in these two cell types. They concluded that if amniotic fluid cells are to be utilized for intrauterine detection of genetic disorders an understanding of the potential differences in these cell types is manditory. Gerbie and colleagues1 have shown through enzyme studies that fibroblast-like cells have high concentrations of cystathionine synthetase while the epithelium-like cells showed high concentrations of histidase. The ratio between these two cell types is unpredictable in any one amniotic fluid cell culture. Researchers have long noted the presence From the Life Science Otterbein College. Received
for publication
Accepted
April
of polyploid (tetraploid) cells in cultured amniotic fluid cells. A large percentage of tetraploidy in amniotic fluid cell cultures appears to be compatible with the birth of a normal child.4l 5 Milunsky and colleagues4 hypothesized that the origin of tetraploid cells may well be the amnion itself. However, the reason for the wide variations in percentages of tetraploid cells observed in various samples remains unclear. In view of an ever-increasing number of prenatal diagnostic facilities, this phenomenon of tetraploidy needs intensive research.4 Some researchers suggest that culture technique may be responsible for the presence of these tetraploid cells in amniotic fluid cell cultures.4-G Colchicine and demecolcine” are known to interfere with cell division through their disruptive action on the mitotic spindle.7-g It is known that the percentage of tetraploidy varies among amniotic fluid specimens from case to case.4* 5* lo This investigation combined several of the aforementioned facts and personal hypotheses concerning tetraploidy in amniotic
Department, March
5, 1974.
26, 1974.
Reprint requests: Dr. T. R. Tegenkamp, Life Science Department, Otterbein College, Westerville, Ohio 43081. *Colcemid.
'Cokemid. 1066
Volume Number
120 8
Tetraploidy
1
2
3
4 CASES
5
and
amniotic
6
cell
types
1067
7
Fig. 1. Percentages of tetraploidy found in cultured human amniotic fluid cells following treatment with two concentrations of demecolcine. 0 = demecolcine concentration No. 1 ( 1.5 mcg. per milliliter of media). Culture exposure time was 2 hours. X = demecolcine concentration No. 2 (3.0 mcg. per milliliter of media). Culture exposure time was 2 hours. Cases 1 to 4 = fibroblast-like cells. Cases 5 to 7 = epithelium-like cells.
fluid cell cultures. The primary objectives were to isolate at least two morphologically distinct cell types, subject each culture to two different concentrations of demecolcine during harvesting, and observe any variances in the percentage of tetraploidy between the two cell type cultures. The null hypothesis is that no difference exists between the occurrence of tetraploidy in fibroblast-like and epithelium-like cells in amniotic fluid cells, in vitro. Material and methods This investigation made use of amniotic fluid from 7 women undergoing amniocentesis for either prenatal diagnosis or therapeutic abortion. Two 15 ml. tubes of amniotic fluid were received for each case. These specimens were
received through the mail in special packing containers. The age of each specimen ranged from 3 to 7 days; however, cell viability was not hindered as shown by cell growth. Four of the specimens were used for isolation of the fibroblast-like cells. Epitheliumlike cells were then isolated from the remaining 3 specimens. Cell type isolation was based on observations of initial cell growth for each specimen. The type of cell which predominated was used for isolation, while the rninority cell type was mechanically removed from the flask by observation with an inverted scope and the aid of a bent-end Pasteur pipet. Subcultures were made upon the formation of a monolayer to ensure the maintenance of the cell type. Epithelium-like cells were more difficult to isolate and subculture than
1068
Tegenkamp
and
Hux
the fibroblast-like cells. Once isolated, each type of cell grew well in vitro. However, it must be assumed that since these cultures did not originate from single cell clones a small percentage of a divergent cell type was probably present in each culture. Cases 1 to 4 were fibroblast-like cell cultures while Cases 5 to 7 represented epithelium-like cell cultures (Fig. 1 and Table 1). A standard harvesting procedure for amniotic fluid cell cultures was utilized except for the variance in concentration of demecolcine. Demecolcine concentration No. 1 was 1.5 mcg. per milliliter of media in the harvesting procedure. The concentration was doubled to 3.0 mcg. per milliliter of media for demecolcine concentration No. 2. All cultures were incubated in 5 per cent carbon dioxide for 2 hours following the addition of demecolcine. Three cultures were used as replications for each demecolcine concentration per case. Approximately 7 to 8 slides were obtained for each culture at the time of harvesting. A minimum of 200 cells were counted for each demecolcine concentration per case. A multivariate analysis of variance and Student’s t-distribution test were utilized to evaluate accumulated data after cell counts were completed. Results Table I contains the means and standard deviations of the percentage of tetraploid cells found in each demecolcine concentration per case. Table I is divided into those cases representing the fibroblast-like cell cultures and the cases of epithelium-like cell origin. Results of Student’s t-distribution test (Table I) give a highly significant value of p < 0.008, for the probable occurrence of such a wide variance in the per cent of tetraploid cells found between the two cell types. Fig. 1 is a graph of the results found in Table I with the percentage of tetraploidy found in demecolcine concentrations Nos. 1 and 2 plotted for each case.
Am.
December J. Obstet.
15, 1974 Gynecol.
Table II is a summary of the computerized, multivariate analysis of variance program. The error factor in Table II represents all of the undefinable error not expressed in Factors A and B and Interaction AK described below. Factor A compares the means and standard deviations of the percentages of tetraploidy found between cases, with the assumption that each case should exhibit no difference in this per cent of tetraploid cells (Table II). Case 4, concentration No. 1, had 15 per cent tetraploid cells, while Case 6, concentration No. 1, exhibited only 1 per cent tetraploidy (Fig. 1) . Variances in tetraploidy among cases give a highly significant P value. Factor B compares differences between the percentage of tetraploidy found in demecoltine concentrations Nos. 1 and 2, with the percentages assumed to be equal for each concentration within an individual case (Table II). Therefore, Case 4 should show the same percentage of tetraploidy in concentrations Nos. 1 and 2 instead of deviating from 15 to 25 per cent (Fig. 1). Differences in the percentage of tetraploidy between cases, as shown in Factor A, are not involved with the Factor B consideration of significant variances between the two concentrations of demecolcine. Interaction AB notes any significant deviations between the increase in tetraploidy found between demecolcine concentrations Nos. 1 and 2 within each case. An increase from 15 to 25 per cent tetraploidy in Case 4 is greater than the 1 to 3 per cent increase in Case 6 (see Fig. 1). These deviations in percentage increase represent this interaction factor (AB) . Comment Results have shown varying increases in the percentage of tetraploidy from demecoltine concentrations No. 1 and No. 2. This would indicate a correlation between increases in demecolcine concentration and increases in observed tetraploids. A separate investigation on the relationship between the tetraploid cell and its possible origin, as
Volume Nwnber
120 8
Tetraploidy
and
amniotic
cell
types
1069
Table I. Mean percentages and standard deviations of tetraploidy found in 7 cases of cultured human fibroblast-like and epithelium-like amniotic fluid cells with 2 demecolcine concentrations with a culture exposure time of 2 hours Fibroblast-like
Case
/
1
zt$-
0 X 0 X 0 X 0 X
2 3 4
Epithelium-like
cells
1 tei:Edy
9.000 17.333 6.000 13.000 6.000 15.667 15.333 25.000
1 ZZfZLt
Case
1.000 2.082 1.732 2.646 2.646 4.163 5.508 3.000
1
Z2
5
7
Over-all mean per cent of tetraploidy: fibroblast-like cells, 13.417 Degrees of freedom = 12. Student’s distribution = 3.433. Probability
1 5.999 value
1 tezdy
0 X 0 X 0 X
6
cells
~iiT
1.500 4.667 1.000 3.667 3.000 8.667
0.707 2.082 0.000 1.518 1.ooo 1 5’8
(S.D.) ; epithelium-like cells, 3.750 0.008 (highly significant).
f. 2.523
(S.D.).
=
Table II. Multivariate analysis of variance for 7 cases of human fibroblast-like epithelium-like amniotic fluid cells with 2 demecolcine concentrations with a culture exposure time of 2 hours
and
.--
Sum of Source
Error factor Cases (A) Concentrations AB interaction *Highly
squares
(B)
Degrees freedom
178.500 1277.542 460.227 66.506
26 6 1 6
of Mean squares
6.865 212.924 460.227 11.084
j
fa,For
31.014 67.036 1.615
1
P
0.001* o.rlol* 0.183
significant.
related to demecolcine concentration, will be published elsewhere. However, more important to this investigation than the effect of demecolcine on amniotic fluid cell cultures are the differences between the percentages of tetraploidy found in the two cell types isolated. Fibroblast-like cells represented in Cases 1 to 4 showed a highly significant increase in tetraploid expression over the epithelium-like cell lines represented in Cases 5 to 7 (Fig. 1 and Table I). Consequently, the null hypothesis is rejected, indicating there is indeed a difference between the occurrence of tetraploidy and cell types. If tetraploids are predominantly originating from the fibroblast-like cell line, then variances in tetraploidy among amniotic fluid specimens could be explained, inasmuch as cell type ratios vary from specimen to specimen.
The origin of tetraploidy appears to be related to culturing techniques based on the type of cells initially present. Further investigation into the origin of tetraploidy, as related to cell type, is necessary to further assure accurate cytogenetic analysis, as well as the metabolic aspects of these cell types (as indicated in previous literature) . Perhaps the mechanism resulting in this alteration of the chromosomal complement may partially account for alterations of any metabolic variances found in the different cell types.l
The authors wish to thank Dr. M. N. MacIntyre, Mrs. Irene Tegenkamp, Searle Diagnostic, Inc., and Otterbein College for their guidance and financial assistance during the investigation. A special thanks is extended to Dr. Alan Schenck and Battelle Memorial Institute for use of their statistical analysis program.
070
Tegenkamp
and
December Am. J. Obstet.
Hux
REFERENCES
1.
2. 3. 4. 5.
Gerbie, A. B., Melancon, S. B., Ryan, and Nadler, H. L.: AM. J. OBSTET. GYNECOL. 3: 314, 1972. Steele, M. W., and Breg, W. R.: Lancet 383, 1966. Melancon, S. B., Lee, S. Y., and Nadler, L.: Science 173: 627, 1971. Milunsky, A., Atkins, L., and Littlefield, W.: J. Pediatr. 79: 305, 1971. Walker, S., Lee, C. L. Y., and Gregson, M.: Lancet 2: 1937, 1970.
6. C., 7. 1:
8. 9.
J. 10. J. N.
15, 1974 Gynecol.
Sperling, K., and Saling, E.: Hum. Genet. 11: 139, 1971. Fitzgerald, P. H., and Brehaut, L. A.: Cell Tissue Kinetics 3: 252, 1970. Taylor, E. W.: J. Cell Biol. 25: 145, 1965. Cox, D. M., and Puck, T. T.: Cytogenetics 8: 158, 1969. Kohn, G., and Robinson, A.: Lancet 2: 778, 1970.
R.
TEGENKAMP,
CHARLES Westeruille,
H.
PH.D. HUX,
B.A.
Ohio
Human amniotic fluid cells obtained by amniocentesis performed on 7 women during the thirteenth to sixteenth weeks of pregnancy were cultured. Four of the specimens were used for the isolation of the fibroblast-like cells. Epithelial cells were isolated from the 3 remaining specimens. Higher frequencies of tetraploidy were noted in the fibroblast-like cell cultures when harvested, Two different concentrations of demecolcine+ were utilized prior to harvesting. If tetraploid cells originated predominantly from the fibroblast-like cells, then variances in tetragloidy among amniotic @id specimens could be explained, inasmuch as cell type ratios vary from specimen to specimen. Metabolic variances between the cell types have previously been reported. This investigation shows there is a diflerence in chromosomal analysis (tetraQloid percentage) as related to cell type.
AT L E AS T TWO morphologically distinct cell types, fibroblast-like and epithelium-like cells, have been found and described in cultured amniotic fluid cells.*, ’ Melancon and associates3 have described distinct differences in enzyme activity in these two cell types. They concluded that if amniotic fluid cells are to be utilized for intrauterine detection of genetic disorders an understanding of the potential differences in these cell types is manditory. Gerbie and colleagues1 have shown through enzyme studies that fibroblast-like cells have high concentrations of cystathionine synthetase while the epithelium-like cells showed high concentrations of histidase. The ratio between these two cell types is unpredictable in any one amniotic fluid cell culture. Researchers have long noted the presence From the Life Science Otterbein College. Received
for publication
Accepted
April
of polyploid (tetraploid) cells in cultured amniotic fluid cells. A large percentage of tetraploidy in amniotic fluid cell cultures appears to be compatible with the birth of a normal child.4l 5 Milunsky and colleagues4 hypothesized that the origin of tetraploid cells may well be the amnion itself. However, the reason for the wide variations in percentages of tetraploid cells observed in various samples remains unclear. In view of an ever-increasing number of prenatal diagnostic facilities, this phenomenon of tetraploidy needs intensive research.4 Some researchers suggest that culture technique may be responsible for the presence of these tetraploid cells in amniotic fluid cell cultures.4-G Colchicine and demecolcine” are known to interfere with cell division through their disruptive action on the mitotic spindle.7-g It is known that the percentage of tetraploidy varies among amniotic fluid specimens from case to case.4* 5* lo This investigation combined several of the aforementioned facts and personal hypotheses concerning tetraploidy in amniotic
Department, March
5, 1974.
26, 1974.
Reprint requests: Dr. T. R. Tegenkamp, Life Science Department, Otterbein College, Westerville, Ohio 43081. *Colcemid.
'Cokemid. 1066
Volume Number
120 8
Tetraploidy
1
2
3
4 CASES
5
and
amniotic
6
cell
types
1067
7
Fig. 1. Percentages of tetraploidy found in cultured human amniotic fluid cells following treatment with two concentrations of demecolcine. 0 = demecolcine concentration No. 1 ( 1.5 mcg. per milliliter of media). Culture exposure time was 2 hours. X = demecolcine concentration No. 2 (3.0 mcg. per milliliter of media). Culture exposure time was 2 hours. Cases 1 to 4 = fibroblast-like cells. Cases 5 to 7 = epithelium-like cells.
fluid cell cultures. The primary objectives were to isolate at least two morphologically distinct cell types, subject each culture to two different concentrations of demecolcine during harvesting, and observe any variances in the percentage of tetraploidy between the two cell type cultures. The null hypothesis is that no difference exists between the occurrence of tetraploidy in fibroblast-like and epithelium-like cells in amniotic fluid cells, in vitro. Material and methods This investigation made use of amniotic fluid from 7 women undergoing amniocentesis for either prenatal diagnosis or therapeutic abortion. Two 15 ml. tubes of amniotic fluid were received for each case. These specimens were
received through the mail in special packing containers. The age of each specimen ranged from 3 to 7 days; however, cell viability was not hindered as shown by cell growth. Four of the specimens were used for isolation of the fibroblast-like cells. Epitheliumlike cells were then isolated from the remaining 3 specimens. Cell type isolation was based on observations of initial cell growth for each specimen. The type of cell which predominated was used for isolation, while the rninority cell type was mechanically removed from the flask by observation with an inverted scope and the aid of a bent-end Pasteur pipet. Subcultures were made upon the formation of a monolayer to ensure the maintenance of the cell type. Epithelium-like cells were more difficult to isolate and subculture than
1068
Tegenkamp
and
Hux
the fibroblast-like cells. Once isolated, each type of cell grew well in vitro. However, it must be assumed that since these cultures did not originate from single cell clones a small percentage of a divergent cell type was probably present in each culture. Cases 1 to 4 were fibroblast-like cell cultures while Cases 5 to 7 represented epithelium-like cell cultures (Fig. 1 and Table 1). A standard harvesting procedure for amniotic fluid cell cultures was utilized except for the variance in concentration of demecolcine. Demecolcine concentration No. 1 was 1.5 mcg. per milliliter of media in the harvesting procedure. The concentration was doubled to 3.0 mcg. per milliliter of media for demecolcine concentration No. 2. All cultures were incubated in 5 per cent carbon dioxide for 2 hours following the addition of demecolcine. Three cultures were used as replications for each demecolcine concentration per case. Approximately 7 to 8 slides were obtained for each culture at the time of harvesting. A minimum of 200 cells were counted for each demecolcine concentration per case. A multivariate analysis of variance and Student’s t-distribution test were utilized to evaluate accumulated data after cell counts were completed. Results Table I contains the means and standard deviations of the percentage of tetraploid cells found in each demecolcine concentration per case. Table I is divided into those cases representing the fibroblast-like cell cultures and the cases of epithelium-like cell origin. Results of Student’s t-distribution test (Table I) give a highly significant value of p < 0.008, for the probable occurrence of such a wide variance in the per cent of tetraploid cells found between the two cell types. Fig. 1 is a graph of the results found in Table I with the percentage of tetraploidy found in demecolcine concentrations Nos. 1 and 2 plotted for each case.
Am.
December J. Obstet.
15, 1974 Gynecol.
Table II is a summary of the computerized, multivariate analysis of variance program. The error factor in Table II represents all of the undefinable error not expressed in Factors A and B and Interaction AK described below. Factor A compares the means and standard deviations of the percentages of tetraploidy found between cases, with the assumption that each case should exhibit no difference in this per cent of tetraploid cells (Table II). Case 4, concentration No. 1, had 15 per cent tetraploid cells, while Case 6, concentration No. 1, exhibited only 1 per cent tetraploidy (Fig. 1) . Variances in tetraploidy among cases give a highly significant P value. Factor B compares differences between the percentage of tetraploidy found in demecoltine concentrations Nos. 1 and 2, with the percentages assumed to be equal for each concentration within an individual case (Table II). Therefore, Case 4 should show the same percentage of tetraploidy in concentrations Nos. 1 and 2 instead of deviating from 15 to 25 per cent (Fig. 1). Differences in the percentage of tetraploidy between cases, as shown in Factor A, are not involved with the Factor B consideration of significant variances between the two concentrations of demecolcine. Interaction AB notes any significant deviations between the increase in tetraploidy found between demecolcine concentrations Nos. 1 and 2 within each case. An increase from 15 to 25 per cent tetraploidy in Case 4 is greater than the 1 to 3 per cent increase in Case 6 (see Fig. 1). These deviations in percentage increase represent this interaction factor (AB) . Comment Results have shown varying increases in the percentage of tetraploidy from demecoltine concentrations No. 1 and No. 2. This would indicate a correlation between increases in demecolcine concentration and increases in observed tetraploids. A separate investigation on the relationship between the tetraploid cell and its possible origin, as
Volume Nwnber
120 8
Tetraploidy
and
amniotic
cell
types
1069
Table I. Mean percentages and standard deviations of tetraploidy found in 7 cases of cultured human fibroblast-like and epithelium-like amniotic fluid cells with 2 demecolcine concentrations with a culture exposure time of 2 hours Fibroblast-like
Case
/
1
zt$-
0 X 0 X 0 X 0 X
2 3 4
Epithelium-like
cells
1 tei:Edy
9.000 17.333 6.000 13.000 6.000 15.667 15.333 25.000
1 ZZfZLt
Case
1.000 2.082 1.732 2.646 2.646 4.163 5.508 3.000
1
Z2
5
7
Over-all mean per cent of tetraploidy: fibroblast-like cells, 13.417 Degrees of freedom = 12. Student’s distribution = 3.433. Probability
1 5.999 value
1 tezdy
0 X 0 X 0 X
6
cells
~iiT
1.500 4.667 1.000 3.667 3.000 8.667
0.707 2.082 0.000 1.518 1.ooo 1 5’8
(S.D.) ; epithelium-like cells, 3.750 0.008 (highly significant).
f. 2.523
(S.D.).
=
Table II. Multivariate analysis of variance for 7 cases of human fibroblast-like epithelium-like amniotic fluid cells with 2 demecolcine concentrations with a culture exposure time of 2 hours
and
.--
Sum of Source
Error factor Cases (A) Concentrations AB interaction *Highly
squares
(B)
Degrees freedom
178.500 1277.542 460.227 66.506
26 6 1 6
of Mean squares
6.865 212.924 460.227 11.084
j
fa,For
31.014 67.036 1.615
1
P
0.001* o.rlol* 0.183
significant.
related to demecolcine concentration, will be published elsewhere. However, more important to this investigation than the effect of demecolcine on amniotic fluid cell cultures are the differences between the percentages of tetraploidy found in the two cell types isolated. Fibroblast-like cells represented in Cases 1 to 4 showed a highly significant increase in tetraploid expression over the epithelium-like cell lines represented in Cases 5 to 7 (Fig. 1 and Table I). Consequently, the null hypothesis is rejected, indicating there is indeed a difference between the occurrence of tetraploidy and cell types. If tetraploids are predominantly originating from the fibroblast-like cell line, then variances in tetraploidy among amniotic fluid specimens could be explained, inasmuch as cell type ratios vary from specimen to specimen.
The origin of tetraploidy appears to be related to culturing techniques based on the type of cells initially present. Further investigation into the origin of tetraploidy, as related to cell type, is necessary to further assure accurate cytogenetic analysis, as well as the metabolic aspects of these cell types (as indicated in previous literature) . Perhaps the mechanism resulting in this alteration of the chromosomal complement may partially account for alterations of any metabolic variances found in the different cell types.l
The authors wish to thank Dr. M. N. MacIntyre, Mrs. Irene Tegenkamp, Searle Diagnostic, Inc., and Otterbein College for their guidance and financial assistance during the investigation. A special thanks is extended to Dr. Alan Schenck and Battelle Memorial Institute for use of their statistical analysis program.
070
Tegenkamp
and
December Am. J. Obstet.
Hux
REFERENCES
1.
2. 3. 4. 5.
Gerbie, A. B., Melancon, S. B., Ryan, and Nadler, H. L.: AM. J. OBSTET. GYNECOL. 3: 314, 1972. Steele, M. W., and Breg, W. R.: Lancet 383, 1966. Melancon, S. B., Lee, S. Y., and Nadler, L.: Science 173: 627, 1971. Milunsky, A., Atkins, L., and Littlefield, W.: J. Pediatr. 79: 305, 1971. Walker, S., Lee, C. L. Y., and Gregson, M.: Lancet 2: 1937, 1970.
6. C., 7. 1:
8. 9.
J. 10. J. N.
15, 1974 Gynecol.
Sperling, K., and Saling, E.: Hum. Genet. 11: 139, 1971. Fitzgerald, P. H., and Brehaut, L. A.: Cell Tissue Kinetics 3: 252, 1970. Taylor, E. W.: J. Cell Biol. 25: 145, 1965. Cox, D. M., and Puck, T. T.: Cytogenetics 8: 158, 1969. Kohn, G., and Robinson, A.: Lancet 2: 778, 1970.