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Chromosome constitution of human endometrium
Chromosome constitution of human endometrium MARGARET A. STANLEY, PH.D.* Adelaide, South Australia
Chromosome counts have been obtained, with a hypotonic squash technique, from 32 samples of nonmalignant endometrium. Seventy-nine per cent of the total population consisted of diploid cells exhibiting a normal female karyotype. A hypodiploid population of 16.3 per cent and a smaller hyperdiploid population of 2.4 per cent were present. Karyotype analysis indicated that the hypodiploid population consisted of metaphases with simple chromosome loss. No differences were observed in the distribution of chromosome count between proliferative and secretory endotnetriurn or between norrnal and hyperplastic endotnetriurn..
R E c E N T L Y it has been pointed out1 • 3 that the cytogenetic analysis of normal tissues has received relatively little attention, as compared to malignant material. As an example of the normal, endometrium has been chosen as an accessible solid tissue dividing in vivo. 3 The cytogenetic constitution of endometrium has nonetheless been the subject of somewhat controversial discussion/• 6 • 7 and argument continues as to the degree of aneuploidy existing in this tissue and to the extent to which this aneuploidy may depend upon technical factors. This report describes the cytogenetic data obtained from 32 cases of nonmalignant human endometrium. This chromosome study has been part of a larger investigation into the cytogenetic constitution of gynecologic malignant tumors, and endometrium has been one of the tissues selected as a normal control.
made using the technique previously described.8 These were examined immediately by phase-contrast microscopy. Permanent preparations were made with the method of Conger and Fairchild4 and stained \Vith Giemsa stain. All chromosome counts were made directly under the microscope. Karyotype analysis was carried out either visually or photographically, and all karyotypes were prepared according to the Patau classification and further subdivided using the Denver classification. Results
A total of 122 cases of nonmalignant endometria have been examined, of which 32 resulted in five or more accurate chromosome counts per case. The complete chromosome counts are given in Table I, together with a brief histologic description of the biopsy. The counts are arranged in two groups, according to histologic appearance, e.g., normal and hyperplastic endometria. Over-all, diploid cells constitute 79 per cent of all the cells counted, but there is a scatter of values on both sides of the modal number, 46. This scatter is more pronounced in the hypodiploid region and the total hypodiploid population is 16.3 per cent. A small percentage of the total count, 2.4 per cent, is in excess of 46 and, in addition, a
Material and methods
Squash preparations, with small tissue fragments obtained after curettage, were From the Department of Obstetrics and Gynaecology, The University of Adelaide. *Research Fellow, Anti-Cancer Foundation, The University of Adelaide, South Australia.
99
May I 1969
100 Stanley
Am.
J. Obst. & Gyncc.
Table I. Chromosome counts in nonmalignant endometrium -·-~--~-~--~-----~--~ -~-----~----~·~~·-·-···········---
Case
2 3 4 5 6
I Age
(yr.)
29 40 40
28
11
56 50 40 35 39 34 44
1~
28
7 8 9
10
13
14 15 16 17
42 50
18
50
19
76
20
47
21
56
22
50
23
64
24
56
25
50
26
78
27
61
28
43
29
44
30
53
31
47
32
44
:
I
I
.i
: r
Histology
Decidua Secretory Proliferative Secretory Proliferative Secretory Proliferative Proliferative Secretory Proliferative Proliferative Proliferative Proliferative Proliferative Secretory Proliferative Benign cystic pi asia Benign cystic plasia Benign cystic pi asia Benign cystic pi asia Benign cystic plasia Benign cystic plasia Benign cystic plasia Benign cystic plasia Benign cystic plasia Benign cystic plasia Benign cystic plasia Benign cystie plasia Benign cystic plasia Benign cystic plasia Benign cystic plasia cystic
---·--··-·
<35
35
..··
-,--
...
36
37
38
39
40
41
42
q •)
2
2
hyperhyperhyperhyperhyperhyperhyperhyperhyperhyperhyperhyperhyperhyperhyperhyper-
Total No. of cells
% Cell number
Chromosome --~~· ·------~--·~·-
0.17
0.17
2
6
6
4
0.34
1.0
1.0
0.70
5
0.86
Volume 104 Number i
Chromosome constitution of endometrium
1OJ
No. per cell
43
I I
44
45
47
1
46 5
4
3
18
2
2 2
2
48
49
92
50
7 nn
L.:J
6 4 8 1
6 5 12
9
17 22 24
5
21 24 2
5
9
1
23 54 11 21
26 60
1
2 1
3
5
2
13
29
..,._
35
'FI
2 2
3
4
2
14 47
17 64
2
8
9
3
6
81
24
82
28 5
4 2
!Total No. of cells! I case
2
2
17
3
30
2
74
14
2
8
8
13
14
6
6
11
12
2
15
2
18
18
21
"" ""
17
19
4
9
3
10
14
30
28
478
11
2
2.4
5.0
4.7
79.1
1.9
0.34
74 81 82 1 1 1 0.17 0.17 0.17
-
0.17
3
17
10
602
1.8
l\ L; 'r 1• 1~~~ i~ 1
102 Stanley
\111
( JIN!. & Cyu~·c
Table II. Distribution of chromosome count between normal and hyperplastic endmetrium Chromosome No. per ,;ell
I35T37___ JB
39
40
4J-142T4.J. -4-4r-4s-' 46 :47
48
i
49
74
J
81
82
l 92
1
Total
Normal No. of cells % cell number
2
4 1.'\
fl.:l I
u
0.31 0.6:1
Ul
1.9 :i. 7
IS
5. 7
'\ l i
257
81.1
n.::1
Hyperplastic No. of cells '/rJ cell number
I)
2
"
.)
0.36 0. 72 0. 72 0. 7:Z l.l
l :2
•)
.I
l.l
2 9 4 .~i
tetraploid population of 1.8 per cent is apparent. This distribution of counts is very similar to that obtained in marrow using the direct squash techniqueY No significant differences \vere apparent in the distribution of counts between normal and hyperplastic endometrium (Table II). Diploid cells constitute 81 per cent of the total count in normal endometrium and 77 per cent in hyperplastic endometrium. The percentage of hypodiploid cells is similar in both groups ( 17.1 per cent for normal endometrium and 15.2 per cent in hyperplastic tissues) . The hyperdiploid population is a little larger in the hyperplastic tissue, 3 per cent as compared to 0.3 per cent. Karyotypes of 30 cells with 46 chromosomes were prepared. No morphologic abnormalities were encountered, and a normal female diploid karyotype was obtained in each case. Only limited analyses of cells with other than 46 chromosomes vvere performed, but in these instances no rearrangement of chromosomes within the groups was encountered, and simple loss of homologues had occurred. One karyotype of a cell with more than 46 chromosomes was prepared; this had 4 7 chromosomes, the addi tiona! chromosome being a member of Group F. Comment 1 ne cytogenetic status of endometrium has been the subject of a number of investi-
1(! 21 ~ 3.6
76.il 2.:') 7.'.!.
() o.36
:276
o.36 o.:J6 :u
gations, many of which occurred prior to the introduction of the hypotonic pretreatment in the analysis of the human chromosome complement. Such studies are technically suspect and of historical interest only. Hov.rever, even \vith the use of acceptable techniques, a wide range in the extent of the normal diploid population in nonmalignant endometria is apparent. Studies involving direct examiualion of biopsy material describe total diploid populations of 49 per cent/ 57 per cent/ and 91 per cent,'~ and, in the present study, 79 per cent. A similar range is apparent in the results of in vitro studies, with diploid populations of 32 per cent6 ' ' and 73 per cent1 having been described, as compared to a report 11 in \vhich only normal diploid cells with a small proportion of real tetraploids could be demonstrated. This discrepancy in observations suggests that the problems are essentially technicaL In this series, apparently broken cells were not included in the analysis, but the real extent of breakage is not known. Certainly the negative skew distribution of counts would suggest some factor other than chance in the production of the hypodiploid counts, and cell breakage is an obvious choice. Evidence that this assumption is valid in relation to this pattern of chromosome distribution has been presented by Ford" in the analysis of the somatic tissues of a large
Volume 104 Number·l
number of mammals, other than man, and similar conclusions have been drawn in the analysis of human tissues. 5 • 9 In all cases direct analysis was employed. Furthermore, if aneuploidy is a real feature of endometrium, then a hyperdiploid population of comparable size to the hypodiploid should be apparent. In no report involving the direct squash technique has this been shown, although a substantial hyperdiploid population was reported in in vitro cultures by Hughes and Csermely. 6 • 7 However, further information on the influence of the in vitro environment on endometrium is required before the significance of this report can be assessed. Changes in the histologic appearance of endometrium were not accompanied by changes in cytogenetic constitution. No dif-
Chromosome constitution of endometrium
103
ference in the distribution of chromosome count was apparent between proliferative and secretory endometrium, and, surprisingly, no difference except for a small increase in the tetraploid population vvas observed between normal and hyperplastic endometria. In view of these results it would appear that hyperplastic endometrium exhibiting a cytogenetic constitution similar to norIna! endon1etriurn is not related to invasive carcinoma. The findings in atypical endometrial hyperplasia are being reported elsewhere. My thanks are due Professor L. W. Cox and the clinical members of the Department of Obstetrics and Gynaecology for the collection of material, Dr. J. A. Kirkland for histologic assessment, and Mrs, M. L, Tregloan and Miss S. Sims for expert technical assistance.
REFERENCES
1. Arakaki, D. T., Waxman, S. H., and Smith, J. B.: AM. J. On sT. & GvNEc, 97: 868, 1967. 2. Auersperg, N., Corey, M. ]., and Worth, A.: Cancer Res. 27: 1394, 1967. 3. Bowey, C. E., and Spriggs, A. 1.: J. M. Genet. 4: 91, 1967. 4. Conger, A. D., and Fairchild, L. N.: Stain Techno!. 28: 281, 1954. 5. Ford, C. E.: Cytogenetics of Ceiis in Cuiture, in Harris, R. J. C., editor: Symposia of the International Society in Cell Biology, New York and London, 1964, .A~..cademic Press, Inc., vol. III. 6. Hughes, E. C., and Csermely, T. V.: AM. J. 0BST. & GYNEC. 93: 777, 1965.
7. Hughes, E. C., and Csermely, T. V.: Nature 209: 326, 1966, 8. Kirkland, J. A., Stanley, M. A., and Cellier, K.: Cancer 20: 1934, 1967. 9. Sandberg, A. A., Ishihara, T., Miwa, T., and Hauschka, T. S.: Cancer Res. 21: 678, 1961. 10. Takemura, T.: J. Jap. Obst. & Gynec. Soc. 7: 300, 1960. 11. Tijio, J. H., and Puck, T. T.: J. Exper. Med. 108: 259, 1958. 12. Wakonig-Vaartaja, R.: Australia-New Zealand J. Obst. & Gynaec. 3: 170, 1963.
Chromosome counts have been obtained, with a hypotonic squash technique, from 32 samples of nonmalignant endometrium. Seventy-nine per cent of the total population consisted of diploid cells exhibiting a normal female karyotype. A hypodiploid population of 16.3 per cent and a smaller hyperdiploid population of 2.4 per cent were present. Karyotype analysis indicated that the hypodiploid population consisted of metaphases with simple chromosome loss. No differences were observed in the distribution of chromosome count between proliferative and secretory endotnetriurn or between norrnal and hyperplastic endotnetriurn..
R E c E N T L Y it has been pointed out1 • 3 that the cytogenetic analysis of normal tissues has received relatively little attention, as compared to malignant material. As an example of the normal, endometrium has been chosen as an accessible solid tissue dividing in vivo. 3 The cytogenetic constitution of endometrium has nonetheless been the subject of somewhat controversial discussion/• 6 • 7 and argument continues as to the degree of aneuploidy existing in this tissue and to the extent to which this aneuploidy may depend upon technical factors. This report describes the cytogenetic data obtained from 32 cases of nonmalignant human endometrium. This chromosome study has been part of a larger investigation into the cytogenetic constitution of gynecologic malignant tumors, and endometrium has been one of the tissues selected as a normal control.
made using the technique previously described.8 These were examined immediately by phase-contrast microscopy. Permanent preparations were made with the method of Conger and Fairchild4 and stained \Vith Giemsa stain. All chromosome counts were made directly under the microscope. Karyotype analysis was carried out either visually or photographically, and all karyotypes were prepared according to the Patau classification and further subdivided using the Denver classification. Results
A total of 122 cases of nonmalignant endometria have been examined, of which 32 resulted in five or more accurate chromosome counts per case. The complete chromosome counts are given in Table I, together with a brief histologic description of the biopsy. The counts are arranged in two groups, according to histologic appearance, e.g., normal and hyperplastic endometria. Over-all, diploid cells constitute 79 per cent of all the cells counted, but there is a scatter of values on both sides of the modal number, 46. This scatter is more pronounced in the hypodiploid region and the total hypodiploid population is 16.3 per cent. A small percentage of the total count, 2.4 per cent, is in excess of 46 and, in addition, a
Material and methods
Squash preparations, with small tissue fragments obtained after curettage, were From the Department of Obstetrics and Gynaecology, The University of Adelaide. *Research Fellow, Anti-Cancer Foundation, The University of Adelaide, South Australia.
99
May I 1969
100 Stanley
Am.
J. Obst. & Gyncc.
Table I. Chromosome counts in nonmalignant endometrium -·-~--~-~--~-----~--~ -~-----~----~·~~·-·-···········---
Case
2 3 4 5 6
I Age
(yr.)
29 40 40
28
11
56 50 40 35 39 34 44
1~
28
7 8 9
10
13
14 15 16 17
42 50
18
50
19
76
20
47
21
56
22
50
23
64
24
56
25
50
26
78
27
61
28
43
29
44
30
53
31
47
32
44
:
I
I
.i
: r
Histology
Decidua Secretory Proliferative Secretory Proliferative Secretory Proliferative Proliferative Secretory Proliferative Proliferative Proliferative Proliferative Proliferative Secretory Proliferative Benign cystic pi asia Benign cystic plasia Benign cystic pi asia Benign cystic pi asia Benign cystic plasia Benign cystic plasia Benign cystic plasia Benign cystic plasia Benign cystic plasia Benign cystic plasia Benign cystic plasia Benign cystie plasia Benign cystic plasia Benign cystic plasia Benign cystic plasia cystic
---·--··-·
<35
35
..··
-,--
...
36
37
38
39
40
41
42
q •)
2
2
hyperhyperhyperhyperhyperhyperhyperhyperhyperhyperhyperhyperhyperhyperhyperhyper-
Total No. of cells
% Cell number
Chromosome --~~· ·------~--·~·-
0.17
0.17
2
6
6
4
0.34
1.0
1.0
0.70
5
0.86
Volume 104 Number i
Chromosome constitution of endometrium
1OJ
No. per cell
43
I I
44
45
47
1
46 5
4
3
18
2
2 2
2
48
49
92
50
7 nn
L.:J
6 4 8 1
6 5 12
9
17 22 24
5
21 24 2
5
9
1
23 54 11 21
26 60
1
2 1
3
5
2
13
29
..,._
35
'FI
2 2
3
4
2
14 47
17 64
2
8
9
3
6
81
24
82
28 5
4 2
!Total No. of cells! I case
2
2
17
3
30
2
74
14
2
8
8
13
14
6
6
11
12
2
15
2
18
18
21
"" ""
17
19
4
9
3
10
14
30
28
478
11
2
2.4
5.0
4.7
79.1
1.9
0.34
74 81 82 1 1 1 0.17 0.17 0.17
-
0.17
3
17
10
602
1.8
l\ L; 'r 1• 1~~~ i~ 1
102 Stanley
\111
( JIN!. & Cyu~·c
Table II. Distribution of chromosome count between normal and hyperplastic endmetrium Chromosome No. per ,;ell
I35T37___ JB
39
40
4J-142T4.J. -4-4r-4s-' 46 :47
48
i
49
74
J
81
82
l 92
1
Total
Normal No. of cells % cell number
2
4 1.'\
fl.:l I
u
0.31 0.6:1
Ul
1.9 :i. 7
IS
5. 7
'\ l i
257
81.1
n.::1
Hyperplastic No. of cells '/rJ cell number
I)
2
"
.)
0.36 0. 72 0. 72 0. 7:Z l.l
l :2
•)
.I
l.l
2 9 4 .~i
tetraploid population of 1.8 per cent is apparent. This distribution of counts is very similar to that obtained in marrow using the direct squash techniqueY No significant differences \vere apparent in the distribution of counts between normal and hyperplastic endometrium (Table II). Diploid cells constitute 81 per cent of the total count in normal endometrium and 77 per cent in hyperplastic endometrium. The percentage of hypodiploid cells is similar in both groups ( 17.1 per cent for normal endometrium and 15.2 per cent in hyperplastic tissues) . The hyperdiploid population is a little larger in the hyperplastic tissue, 3 per cent as compared to 0.3 per cent. Karyotypes of 30 cells with 46 chromosomes were prepared. No morphologic abnormalities were encountered, and a normal female diploid karyotype was obtained in each case. Only limited analyses of cells with other than 46 chromosomes vvere performed, but in these instances no rearrangement of chromosomes within the groups was encountered, and simple loss of homologues had occurred. One karyotype of a cell with more than 46 chromosomes was prepared; this had 4 7 chromosomes, the addi tiona! chromosome being a member of Group F. Comment 1 ne cytogenetic status of endometrium has been the subject of a number of investi-
1(! 21 ~ 3.6
76.il 2.:') 7.'.!.
() o.36
:276
o.36 o.:J6 :u
gations, many of which occurred prior to the introduction of the hypotonic pretreatment in the analysis of the human chromosome complement. Such studies are technically suspect and of historical interest only. Hov.rever, even \vith the use of acceptable techniques, a wide range in the extent of the normal diploid population in nonmalignant endometria is apparent. Studies involving direct examiualion of biopsy material describe total diploid populations of 49 per cent/ 57 per cent/ and 91 per cent,'~ and, in the present study, 79 per cent. A similar range is apparent in the results of in vitro studies, with diploid populations of 32 per cent6 ' ' and 73 per cent1 having been described, as compared to a report 11 in \vhich only normal diploid cells with a small proportion of real tetraploids could be demonstrated. This discrepancy in observations suggests that the problems are essentially technicaL In this series, apparently broken cells were not included in the analysis, but the real extent of breakage is not known. Certainly the negative skew distribution of counts would suggest some factor other than chance in the production of the hypodiploid counts, and cell breakage is an obvious choice. Evidence that this assumption is valid in relation to this pattern of chromosome distribution has been presented by Ford" in the analysis of the somatic tissues of a large
Volume 104 Number·l
number of mammals, other than man, and similar conclusions have been drawn in the analysis of human tissues. 5 • 9 In all cases direct analysis was employed. Furthermore, if aneuploidy is a real feature of endometrium, then a hyperdiploid population of comparable size to the hypodiploid should be apparent. In no report involving the direct squash technique has this been shown, although a substantial hyperdiploid population was reported in in vitro cultures by Hughes and Csermely. 6 • 7 However, further information on the influence of the in vitro environment on endometrium is required before the significance of this report can be assessed. Changes in the histologic appearance of endometrium were not accompanied by changes in cytogenetic constitution. No dif-
Chromosome constitution of endometrium
103
ference in the distribution of chromosome count was apparent between proliferative and secretory endometrium, and, surprisingly, no difference except for a small increase in the tetraploid population vvas observed between normal and hyperplastic endometria. In view of these results it would appear that hyperplastic endometrium exhibiting a cytogenetic constitution similar to norIna! endon1etriurn is not related to invasive carcinoma. The findings in atypical endometrial hyperplasia are being reported elsewhere. My thanks are due Professor L. W. Cox and the clinical members of the Department of Obstetrics and Gynaecology for the collection of material, Dr. J. A. Kirkland for histologic assessment, and Mrs, M. L, Tregloan and Miss S. Sims for expert technical assistance.
REFERENCES
1. Arakaki, D. T., Waxman, S. H., and Smith, J. B.: AM. J. On sT. & GvNEc, 97: 868, 1967. 2. Auersperg, N., Corey, M. ]., and Worth, A.: Cancer Res. 27: 1394, 1967. 3. Bowey, C. E., and Spriggs, A. 1.: J. M. Genet. 4: 91, 1967. 4. Conger, A. D., and Fairchild, L. N.: Stain Techno!. 28: 281, 1954. 5. Ford, C. E.: Cytogenetics of Ceiis in Cuiture, in Harris, R. J. C., editor: Symposia of the International Society in Cell Biology, New York and London, 1964, .A~..cademic Press, Inc., vol. III. 6. Hughes, E. C., and Csermely, T. V.: AM. J. 0BST. & GYNEC. 93: 777, 1965.
7. Hughes, E. C., and Csermely, T. V.: Nature 209: 326, 1966, 8. Kirkland, J. A., Stanley, M. A., and Cellier, K.: Cancer 20: 1934, 1967. 9. Sandberg, A. A., Ishihara, T., Miwa, T., and Hauschka, T. S.: Cancer Res. 21: 678, 1961. 10. Takemura, T.: J. Jap. Obst. & Gynec. Soc. 7: 300, 1960. 11. Tijio, J. H., and Puck, T. T.: J. Exper. Med. 108: 259, 1958. 12. Wakonig-Vaartaja, R.: Australia-New Zealand J. Obst. & Gynaec. 3: 170, 1963.