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Role of stroma in regeneration of endometrial epithelium
volume 99
number 4
October 15, 1967
American Journal of
Obstetrics and Gynecology
GYNECOLOGY
Role of stroma in regeneration of endometrial epithelium MICHAELS. BAGGISH, M.D. CARL). PAUERSTEIN, M.D.*
J. DONALD WOODRUFF, M.D. Baltimore, Maryland Sections of menstruating endometria were studied by various techniques to determine the process by which the surface was re-epithelialized. Although the stumps of remaining glands projected over the surface, this residual epithelium seemed a metabolically inactive and unlikely source of a young growing cell. In many instances, the hyperchromatic stromal cells appeared to project between gland stumps and take part in the re-epithelization by a process simulating metaplasia. Metabolically this stroma-type cell compar~:d favorably with the small, "dark" indifferent cell which is noted at the stromoepithelial border and from which the gland ePithelium may reproduce. . -
S P E c u L A T r o N has suggested that regeneration of the endometrial lining takes place by either ( 1) a direct resurfacing from the stumps of the remaining glands; or (2) a metaplasia of the stromal cell into an epithelial type. Since mitoses 6 • 11 are rare at this stage, it is necessary to postulate some process of reproduction other than that envisioned in the usual "generative cycle." \A/hi!e
knowledge of the true process is possibly of only academic interest, nevertheless, certain changes and specific cells are characteristically noted at this time and deserve explanation. As noted above, it has been suggested that the upper iayers of the endometrium are sloughed and that the surface regenerates from the residual cpit..~elium of the gland stumps. 1 • 6 • 10 • 11 • 13 - 16 Herrell and Broders 6 stated that the glandular tissue seemed the most likely source of the reconstituted epithelium, and that there was no evidence to support the metaplasia theory. Sturgis and Meigs14 noted that the surface epithelium is
From the Departments of Gynecology and Obstetrics and Gynecologic Pathology, The Johns Hopkins University School of Medicine. *American Cancer Society Advanced Clinical Fellow.
459
460
Baggish, PauersteinJ and \A/oodruff
reformed within 48 hours, probably by a migration of epithelial cells from the exposed ends of glands. Bartelmez' studies' suggested that the surface was covered by cells which spread out from the gland mouths. Novak and TeLinde11 also felt that the source of
Figs. 1-6. For legends see opposite page.
October h. 196/ Arll . .f. Obst. & ( ;yw·r·
the new epithelium vvas chiefly the epidwliurn of the basal stumps of the uterine glands. McLennan and Rydell'1 noted that. "In the third day endometria, one can find gland remnants seemingly just beginning to pour out new epithelium to cowr quickly
Volume
Stroma and epithelial regene;ation 461
·~g
Number 4
the residual thin layer of spongiosa. On day 4, 5, or 6, definite areas of new epithelial surface are present." Others have proposed a stromal ongm for the regenerating epithelial layer. 3 • 5 • 7 • 8 • ~ Johnstone 8 believed the endometrium to be "lymphatic tissue and not mucous membrane." He stated that "feather and hair do not grow from epithelial tissue but directly from a lymphoid bed." Papanicolaou 12 recognized that the rapidity of regeneration and lack of mitotic activity could not be explained vvell by the generally accepted the= ory that the uterine epithelium regenerates from the epithelium of the deep glands which escaped destruction or from remnants of the old epithelium. He recognized that there was first desquamation of the epithelium and then "differentiation of the superficial cells of the tunica propria." Hitschmann and Adler' described this process as differentiation of a "false decidua." Heape 5 concurred in this feeling that the stroma was a likely source of the new surface epithelium. It seems basically illogical to suppose that the mature and well-differentiated epithelial cells of glands in the lower spongiosa and basalis can produce a new and immature cell. In a comparable situation, the regenerative process in the stratified epithelium is initiated by the immature basal or 1
"spindle" cell. This is evidenced by the loss of stratification, and the presence of only the less mature cells m the earliest stage of the resurfacing of a denuded area. It would seem necessary, if possible, to envision a process of regeneration that more nearly recapitulates that recognized in other tissues, namely, a process of maturation originating with an "immature or indifferent" cell. If such exists in the endometrium, it should have the potentiality, undoubtedly, of producing a mature stromal or epithelial ce!l since both of these elements are presumed to arise from the paramesonephric (Mullerian) system. Furthermore, this "undifferentiated mesodermal cell," technically speaking, may produce a variety of mesodermal tissues, e.g., those noted in the "mixed mesodermal tumor." Finally, as noted in the latter tumor, as well as in other anaplastic ( adenoacanthoma) and benign (acanthosis) conditions, this cell may produce a squamous type cell. It was on this thesis that the following study was developed. From the files of the Gynecologic Pathology Laboratory of The Johns Hopkins Hospital, random sections from 100 examples of menstruating endometrium have been reviewed. The following studies were performed: ( 1) routine hematoxylin and eosin stains; ( 2) periodic acid-Schiff stains, with
Fig. Jl. Gynecologic Pathology No. 201781. Residual endometrium showing secretory gland at arrow (1); decidua-like cells at arrow (2); stromal-type cells at surface arrow ( 3) beginning repair; and unidentified cells possibly fragmented gland epithelium or stroma at arrow (4). Generalized edema and cellular infiltrate. (x550.) Fig. 2. Gynecologic Pathology No. 197984. Disorganized surface layer at top. Cells have foamy cytoplasm and nuclei of various sizes with rare nucleolus. Compact underlying stroma shows more active appearing nuclei. Gland lining to left with occasional irregular cell with hyperchromatic nucleus between epithelium and stroma (? indifferent cell). (x1,000.) Fig. !1. Gynecologic Pathology No. 200655. Resurfacing process appearing "pseudostratified" and blending with the underlying stromal cell and with the gland epithelium to far right (at arrow) ( 48 to 72 hours after onset of bleeding). (x550.) Fig. 4. Gynecologic Pathology No. 200868. Surface cells appear inactive and degenerating. Underlying stromal cells contain prominent nucleoli. Hyperchromatic nuclei of ? indifferent cells at arrows between surface and stromal layers. (x1,400.) Fig. !i. Gynecologic Pathology No. 197984. Multilayered syncytium-like tissue at surface at arrows blending with the glandlike structure in center. Appearance of surface change simulates 11quamous metaplasia. (x550.) Fig. H. Gynecologic Pathology No. 200696. Periodic acid-Schiff-positive (after diastase) material in gland lumina in lower spongiosa-residue after slough of superficial layers. (x250.)
462 Baggish, Pauerstein, and Woodruff
and without diastase; (3) Wilder's reticulum stain, and ( 4) acridine orange fluorescence.
Results Hematoxylin and eosin. The routine sections demonstrated the usually reported findings. The compacta with portions of the
Figs. 7-12. For legends see opposite page.
( )( hlht• r !.1, It~(\ I :\111
I.
Oh~t .
& C vof"f·
spongiosa was dislodged from the basaJi,. The desquamated fragments were t~derna tous and infiltrated with polymorphonuclear cells and there was extensive intracellular hemorrhage. The degenerating decidual cells could be discerned by the presence of typical stromal cells containing abundant acido-
Volume 99 Numbe,. 4
philic cytoplasm and nuclei with coarse basophilic granules. In addition, epithelial cells from fragmented glands were scattered throughout and were difficult to differentiate from the changed stromal elements (Fig. 1). The latter contain large pale nuclei with irregular chromatin; no demonstrable nucleoli; and poorly defined cell borders. Other cells with similar nuclei but absent cytoplasm could not be specifically classified and probably represented the degenerating stages of the gland cell. The surface epithelial cells varied in appearance depending on stage of degeneration. The remaining endometrium revealed a compact stroma in which the nuclei uniformly were hyperchromatic and the cells without cytoplasm. Stumps of the glands usually were lined at the orifice by secretory epithelium, and the surface was devoid of definiti\·e layer. Early in the regenerative period, the superficial layer was composed of cells with pale nuclei, essentially no chromatin, and minimal light acidophilic foamy cytoplasm (Fig. 2). Initially, no line of demarcation was noted between the superficial cells and the underlying stroma; in fact, cells of the former seemed to be intermingled with the latter (Fig. 3). The surface layer actually appeared degenerative while, conversely, the
Stroma and epithelial regeneration
463
underlying "stromal-type" cells contained well-defined nuclei and nucleoli. A rare mitosis was seen and these were found only between stroma and lining cells. TI1e superficial zone seemed disorganized initially and presented a "multilayered" syncytium-like appearance (Fig. 4), simulating "squamous metaplasia"; although the uniformity of the latter was lacking. The surface cells linked up rapidly with the residual epithelium of the gland and became organized into a single layer (Fig. 5). Many of the hyperchromatic cells resembled the regularly seen, but illdefined, indifferent cell of the stromoepithelial junction in the mature endometrium. It seemed feasible that this cell, with its potential to develop into a stromal or epithelial type, might be the key to the regeneration of the endometrium. Periodic acid-Schiff ( P .A.S.) stains. Studies of additional sections from the same tissues as reported above demonstrated the presence of P.A.S.-positive material not dissolved with diastase in the lumina of the glands (Fig. 6). Although it was not uniform, there was a "basement" membrane beneath the well-formed residual epithelium of the basalis and distal spongiosa. Such was not present beneath the regenerating surface layers until organization took place. This
Fig. 7. Gynecologic Pathology No. 200655. Glycogen granules prominent in superficial layers (top) and present to a minimal degree in the immediately underlying stromal cells. Again differentiation between the various types is essentially impossible ( 48 to 72 hours alter onset of bleeding). (xl,250.) Fig. 8. Gynecologic Pathology No. 200842. Variable secretory activities in adjoining epithelial cells showing dark mucoid material at arrow characteristic of that seen in the endocervix while adjacent cells are devoid of such activity. Other areas demonstrating similar variations are obvious. ( x460.) Fig. 9. Gynecologic Pathology No. 200539. Residual surface epithelium at arrow with semblance of basal reticulum but this disappears at far left where syncytium-like surface cells blend into the stroma and adjacent gland. Fragmented reticulum is evident in center. (Wild·~r's reticulum stain. x480.) Fig. 1(}. Gynecologic Pathology No. 200539. Multilayered surface syncytium-like tissue with residua of basal reticulum layer at arrow (same field as Fig. 9, 24 hours after onset of bleed· ing). !"Wilder's reticulum stain. xl,250.) Fig. 11. Acridine orange fluorescence. Syncytium-like surface cells showing yellow rim at nuclear edge with dark center, occasional bright nucleoli, and light green cytoplasm. The underlying stromal cells vary in staining quality, many similar to those of the surface, but others, some in the position of the indifferent cell, have brilliant yellow nuclei of the metabolically active (DNA) cell. Fig. 12. Acridine orange fluorescence. The multilayered superficial cells are similar to those seen in Fig. I 1 with no line of demarcation from the underlying stroma except in the differential metabolic activity. Scattered brilliant yellow nuclei are easily recognized.
464 Baggish, Pauerstein, and Woodruff
might be interpreted as "growth of the epithelium over the surface," however, transitions between the already organizing surface layers and the "pseudostratified" (pseudometaplastic) regenerating layers were apparent. The newly formed epithelium demonstrated great variation in cytoplasmic contents. Of interest was the variability in staining of the cytoplasm of the surface cells. Glycogen granules were prominent. Since the same was true to a lesser degree in the underlying stromal cells, and since in the early stages of regeneration, no line of demarcation between epithelium and stroma could be discerned; it seemed quite feasible to suggest that the surface cell was the mature counterpart of the less well-differentiated "stromal" cell (Fig. 7). Of similar interest was the difference in staining reactions of the more well-defined surface cells. Some demonstrated brilliantly the presence of mucoid material (Fig. 8), while adjacent groups were devoid of any P.A.S.-positive substance. Similar reactions are noted in the mature epithelia of the internal genitals where heterotopia is well known, and mucus-secreting cells characteristic of the endocervix may be recognized in endometrium and tube. Such variations of the paramesonephric (Mullerian) epithelia probably arise from the "indifferent" cell, as may the early changes in the regenerating endometrial epithelia.
Ut. & (;~""'.
mg that at least part of the reconstitution of
the surface layer of the endometrium was derived by ditrerentiation from tile underlving stroma. Acridine orange fluorescence
The technique described in a previous publication 4 was used on the sections of men· struating endometria. The cells of the surface epithelium, where visualized, were characterized by rather brilliant orange cytoplasm and nuclei with minimal acridine uptake and darkened zone. suggestive of the mature inactive nuclei in the most superficial layers of the stratified squamous epithelium. It would seem unlikely, as noted previously, that such a mature cell, presumably at the end of its life-span, could produce the new surface epithelium. In contrast, the underlying stromal cells showed brilliant yellow nuclei (Fig. 111. As described previously by routine histologic study, cytoplasm was larg-ely absent in these cells. At times, these cells were seen adjacent to the definitive epithelium, so that the contrast was striking (Fig. 12). In addition to these types, the socalled indifferent ct>ll could be recognized in some of the deeper epithelia, and the nuclei of these cells were prominent due to the striking ye!low color. These resembled, metabolically, the stromal cell and suggested that both stroma and epithelium might be derived by differentiation from the same stem cell. Obviously, from an embryologic point of \'iew, this seems logical.
Reticulum stains
The Wilder reticulum stains demonstrated the usual "basket-weave" pattern in the basal area and fragmentation in the upper spongiosa and compacta, as reported by Craig 2 and others. 10 In general, there was an incomplete, but discernible, reticulum surrounding the glands in the desquamated zone. This could be visualized beneath the surface epithelium (Fig. 9), but disappeared as the groups of "stromal-type cells" were approached and abutted the recognizable epithelial layer (Fig. 10). Although little of a specific nature could be determined from this study, it did provide another link in the chain of evidence suggest.
Comment
Sections of the menstruating endometria from both curettage and hysterectomy specimens were studied for evidence of re-epithelization of the surface. The residual stromal cell at this stage showed a deep basophilic nucleus and scant cytoplasm. Clumps of these cells were seen clinging to the remaining endometrial layers. The stumps of the glands projected on the surface and occasionally seemed to creep over the adjacent stromal surface. Mitoses were rare in the epithelium and stroma. The small, dark "indifferent" cell noted at the stromoepithelial border in
Volume 99 Numbe:· 4
the normal endometrium, was prominent in the regenerating tissue and in many respects, especially with acridine orange fluorescence, simulated the hyperchromatic "stromal cell." Characteristically, groups of stroma-type cells extended to the surface, abutted the epithelium of the gland stumps and assumed a multi.layered effect suggestive of "metaplasia." These cells contained pale-staining nuclei with scant slightly acidophilic cytoplasm. Differentiation of this layer from the underlying stroma by routine and special stain was generally impossible. This finding suggested that the stromal cell or the indifferent variety lying in the stroma played an active role in the regeneration of the surface epithelium. The epithelium at the tips of the residual glands was seen to project over the surface and collapse onto, but remain unattached to,
RI:FERENCES l. Bartelmez, W. G.: Contrib. Embryo!. 24: 143, 1S133. 2. Craig, J. M.: AM. J. OBsT. & GYNEC. 86: 421, 1963. 3. Duval, M. M.: Compt. rend. Soc. bioi. 2: 697, 1890. 4. Friedrich, E. G., Julian, C. G., and Woodruff, J. D.: A~L J. OnsT. & GYNEC. 90: 1281, 1964. 5. Heape, W.: Tr. Obst. Soc. London. 40: 161, 1898. 6. Herrell, W. E., and Broders, A. C.: Surg., Gynec. & Obst. 61: 751, 1935. 7. Hitschmann, F., and Adler, L.: Monatschr. Geburtsh. u. Gynak. 27: 1, 1908. 8 . .Johnstone, A. W.: AM. J. 0BsT. & GYNEC. 24: 681, 1891.
Stroma and epithelial regeneration
465
the stroma. In some instances, it seemed to form a framework upon which the new epithelium developed but there was no suggestion that this "metabolically inactive epithelium" was producing new cells. Thus, the reconstitution of the endometrium was found to follow a more or less particular sequence of regeneration. The keystone in this series of events appears to be a cell which lies at the stromoepithelial border and has the propensity for fom1ing first a metaplastic (squamoid) type of lining to be later replaced by the mature cylindrical variety. The authors wish to express their gratitude to Mr. Raymond Lund, for the photography, and Miss Grace Ann Loftus, for technical assistance in preparation of the paper.
9. McLennan, C. E., and Rydell, A. F.: Obst. & Gynec. 26: 605, 1965. 10. Novak, E.: Surg., Gynec. & Obst. 21: 336, 1915. 1L Novak, E., and TeLinde, R. W.: J. A.M. A. 83: 900, 1924. 12. Papanicolaou, G.: AM. J. 0BST. & GYNEC. 25: 30, 1933. 13. Schroder, R.: Arch. Gynak. 104: 27, 1915. 14. Sturgis, S. H., and Meigs, J. V.: Am. J. Surg. 33: 369, 1936. 15. Westphalen, F.: Arch. Gynak. 52: 37, 1896. 16. Williams, J. W.: J. Obst. & Gynaec. Brit. Emp. 2: 681, 1874.
number 4
October 15, 1967
American Journal of
Obstetrics and Gynecology
GYNECOLOGY
Role of stroma in regeneration of endometrial epithelium MICHAELS. BAGGISH, M.D. CARL). PAUERSTEIN, M.D.*
J. DONALD WOODRUFF, M.D. Baltimore, Maryland Sections of menstruating endometria were studied by various techniques to determine the process by which the surface was re-epithelialized. Although the stumps of remaining glands projected over the surface, this residual epithelium seemed a metabolically inactive and unlikely source of a young growing cell. In many instances, the hyperchromatic stromal cells appeared to project between gland stumps and take part in the re-epithelization by a process simulating metaplasia. Metabolically this stroma-type cell compar~:d favorably with the small, "dark" indifferent cell which is noted at the stromoepithelial border and from which the gland ePithelium may reproduce. . -
S P E c u L A T r o N has suggested that regeneration of the endometrial lining takes place by either ( 1) a direct resurfacing from the stumps of the remaining glands; or (2) a metaplasia of the stromal cell into an epithelial type. Since mitoses 6 • 11 are rare at this stage, it is necessary to postulate some process of reproduction other than that envisioned in the usual "generative cycle." \A/hi!e
knowledge of the true process is possibly of only academic interest, nevertheless, certain changes and specific cells are characteristically noted at this time and deserve explanation. As noted above, it has been suggested that the upper iayers of the endometrium are sloughed and that the surface regenerates from the residual cpit..~elium of the gland stumps. 1 • 6 • 10 • 11 • 13 - 16 Herrell and Broders 6 stated that the glandular tissue seemed the most likely source of the reconstituted epithelium, and that there was no evidence to support the metaplasia theory. Sturgis and Meigs14 noted that the surface epithelium is
From the Departments of Gynecology and Obstetrics and Gynecologic Pathology, The Johns Hopkins University School of Medicine. *American Cancer Society Advanced Clinical Fellow.
459
460
Baggish, PauersteinJ and \A/oodruff
reformed within 48 hours, probably by a migration of epithelial cells from the exposed ends of glands. Bartelmez' studies' suggested that the surface was covered by cells which spread out from the gland mouths. Novak and TeLinde11 also felt that the source of
Figs. 1-6. For legends see opposite page.
October h. 196/ Arll . .f. Obst. & ( ;yw·r·
the new epithelium vvas chiefly the epidwliurn of the basal stumps of the uterine glands. McLennan and Rydell'1 noted that. "In the third day endometria, one can find gland remnants seemingly just beginning to pour out new epithelium to cowr quickly
Volume
Stroma and epithelial regene;ation 461
·~g
Number 4
the residual thin layer of spongiosa. On day 4, 5, or 6, definite areas of new epithelial surface are present." Others have proposed a stromal ongm for the regenerating epithelial layer. 3 • 5 • 7 • 8 • ~ Johnstone 8 believed the endometrium to be "lymphatic tissue and not mucous membrane." He stated that "feather and hair do not grow from epithelial tissue but directly from a lymphoid bed." Papanicolaou 12 recognized that the rapidity of regeneration and lack of mitotic activity could not be explained vvell by the generally accepted the= ory that the uterine epithelium regenerates from the epithelium of the deep glands which escaped destruction or from remnants of the old epithelium. He recognized that there was first desquamation of the epithelium and then "differentiation of the superficial cells of the tunica propria." Hitschmann and Adler' described this process as differentiation of a "false decidua." Heape 5 concurred in this feeling that the stroma was a likely source of the new surface epithelium. It seems basically illogical to suppose that the mature and well-differentiated epithelial cells of glands in the lower spongiosa and basalis can produce a new and immature cell. In a comparable situation, the regenerative process in the stratified epithelium is initiated by the immature basal or 1
"spindle" cell. This is evidenced by the loss of stratification, and the presence of only the less mature cells m the earliest stage of the resurfacing of a denuded area. It would seem necessary, if possible, to envision a process of regeneration that more nearly recapitulates that recognized in other tissues, namely, a process of maturation originating with an "immature or indifferent" cell. If such exists in the endometrium, it should have the potentiality, undoubtedly, of producing a mature stromal or epithelial ce!l since both of these elements are presumed to arise from the paramesonephric (Mullerian) system. Furthermore, this "undifferentiated mesodermal cell," technically speaking, may produce a variety of mesodermal tissues, e.g., those noted in the "mixed mesodermal tumor." Finally, as noted in the latter tumor, as well as in other anaplastic ( adenoacanthoma) and benign (acanthosis) conditions, this cell may produce a squamous type cell. It was on this thesis that the following study was developed. From the files of the Gynecologic Pathology Laboratory of The Johns Hopkins Hospital, random sections from 100 examples of menstruating endometrium have been reviewed. The following studies were performed: ( 1) routine hematoxylin and eosin stains; ( 2) periodic acid-Schiff stains, with
Fig. Jl. Gynecologic Pathology No. 201781. Residual endometrium showing secretory gland at arrow (1); decidua-like cells at arrow (2); stromal-type cells at surface arrow ( 3) beginning repair; and unidentified cells possibly fragmented gland epithelium or stroma at arrow (4). Generalized edema and cellular infiltrate. (x550.) Fig. 2. Gynecologic Pathology No. 197984. Disorganized surface layer at top. Cells have foamy cytoplasm and nuclei of various sizes with rare nucleolus. Compact underlying stroma shows more active appearing nuclei. Gland lining to left with occasional irregular cell with hyperchromatic nucleus between epithelium and stroma (? indifferent cell). (x1,000.) Fig. !1. Gynecologic Pathology No. 200655. Resurfacing process appearing "pseudostratified" and blending with the underlying stromal cell and with the gland epithelium to far right (at arrow) ( 48 to 72 hours after onset of bleeding). (x550.) Fig. 4. Gynecologic Pathology No. 200868. Surface cells appear inactive and degenerating. Underlying stromal cells contain prominent nucleoli. Hyperchromatic nuclei of ? indifferent cells at arrows between surface and stromal layers. (x1,400.) Fig. !i. Gynecologic Pathology No. 197984. Multilayered syncytium-like tissue at surface at arrows blending with the glandlike structure in center. Appearance of surface change simulates 11quamous metaplasia. (x550.) Fig. H. Gynecologic Pathology No. 200696. Periodic acid-Schiff-positive (after diastase) material in gland lumina in lower spongiosa-residue after slough of superficial layers. (x250.)
462 Baggish, Pauerstein, and Woodruff
and without diastase; (3) Wilder's reticulum stain, and ( 4) acridine orange fluorescence.
Results Hematoxylin and eosin. The routine sections demonstrated the usually reported findings. The compacta with portions of the
Figs. 7-12. For legends see opposite page.
( )( hlht• r !.1, It~(\ I :\111
I.
Oh~t .
& C vof"f·
spongiosa was dislodged from the basaJi,. The desquamated fragments were t~derna tous and infiltrated with polymorphonuclear cells and there was extensive intracellular hemorrhage. The degenerating decidual cells could be discerned by the presence of typical stromal cells containing abundant acido-
Volume 99 Numbe,. 4
philic cytoplasm and nuclei with coarse basophilic granules. In addition, epithelial cells from fragmented glands were scattered throughout and were difficult to differentiate from the changed stromal elements (Fig. 1). The latter contain large pale nuclei with irregular chromatin; no demonstrable nucleoli; and poorly defined cell borders. Other cells with similar nuclei but absent cytoplasm could not be specifically classified and probably represented the degenerating stages of the gland cell. The surface epithelial cells varied in appearance depending on stage of degeneration. The remaining endometrium revealed a compact stroma in which the nuclei uniformly were hyperchromatic and the cells without cytoplasm. Stumps of the glands usually were lined at the orifice by secretory epithelium, and the surface was devoid of definiti\·e layer. Early in the regenerative period, the superficial layer was composed of cells with pale nuclei, essentially no chromatin, and minimal light acidophilic foamy cytoplasm (Fig. 2). Initially, no line of demarcation was noted between the superficial cells and the underlying stroma; in fact, cells of the former seemed to be intermingled with the latter (Fig. 3). The surface layer actually appeared degenerative while, conversely, the
Stroma and epithelial regeneration
463
underlying "stromal-type" cells contained well-defined nuclei and nucleoli. A rare mitosis was seen and these were found only between stroma and lining cells. TI1e superficial zone seemed disorganized initially and presented a "multilayered" syncytium-like appearance (Fig. 4), simulating "squamous metaplasia"; although the uniformity of the latter was lacking. The surface cells linked up rapidly with the residual epithelium of the gland and became organized into a single layer (Fig. 5). Many of the hyperchromatic cells resembled the regularly seen, but illdefined, indifferent cell of the stromoepithelial junction in the mature endometrium. It seemed feasible that this cell, with its potential to develop into a stromal or epithelial type, might be the key to the regeneration of the endometrium. Periodic acid-Schiff ( P .A.S.) stains. Studies of additional sections from the same tissues as reported above demonstrated the presence of P.A.S.-positive material not dissolved with diastase in the lumina of the glands (Fig. 6). Although it was not uniform, there was a "basement" membrane beneath the well-formed residual epithelium of the basalis and distal spongiosa. Such was not present beneath the regenerating surface layers until organization took place. This
Fig. 7. Gynecologic Pathology No. 200655. Glycogen granules prominent in superficial layers (top) and present to a minimal degree in the immediately underlying stromal cells. Again differentiation between the various types is essentially impossible ( 48 to 72 hours alter onset of bleeding). (xl,250.) Fig. 8. Gynecologic Pathology No. 200842. Variable secretory activities in adjoining epithelial cells showing dark mucoid material at arrow characteristic of that seen in the endocervix while adjacent cells are devoid of such activity. Other areas demonstrating similar variations are obvious. ( x460.) Fig. 9. Gynecologic Pathology No. 200539. Residual surface epithelium at arrow with semblance of basal reticulum but this disappears at far left where syncytium-like surface cells blend into the stroma and adjacent gland. Fragmented reticulum is evident in center. (Wild·~r's reticulum stain. x480.) Fig. 1(}. Gynecologic Pathology No. 200539. Multilayered surface syncytium-like tissue with residua of basal reticulum layer at arrow (same field as Fig. 9, 24 hours after onset of bleed· ing). !"Wilder's reticulum stain. xl,250.) Fig. 11. Acridine orange fluorescence. Syncytium-like surface cells showing yellow rim at nuclear edge with dark center, occasional bright nucleoli, and light green cytoplasm. The underlying stromal cells vary in staining quality, many similar to those of the surface, but others, some in the position of the indifferent cell, have brilliant yellow nuclei of the metabolically active (DNA) cell. Fig. 12. Acridine orange fluorescence. The multilayered superficial cells are similar to those seen in Fig. I 1 with no line of demarcation from the underlying stroma except in the differential metabolic activity. Scattered brilliant yellow nuclei are easily recognized.
464 Baggish, Pauerstein, and Woodruff
might be interpreted as "growth of the epithelium over the surface," however, transitions between the already organizing surface layers and the "pseudostratified" (pseudometaplastic) regenerating layers were apparent. The newly formed epithelium demonstrated great variation in cytoplasmic contents. Of interest was the variability in staining of the cytoplasm of the surface cells. Glycogen granules were prominent. Since the same was true to a lesser degree in the underlying stromal cells, and since in the early stages of regeneration, no line of demarcation between epithelium and stroma could be discerned; it seemed quite feasible to suggest that the surface cell was the mature counterpart of the less well-differentiated "stromal" cell (Fig. 7). Of similar interest was the difference in staining reactions of the more well-defined surface cells. Some demonstrated brilliantly the presence of mucoid material (Fig. 8), while adjacent groups were devoid of any P.A.S.-positive substance. Similar reactions are noted in the mature epithelia of the internal genitals where heterotopia is well known, and mucus-secreting cells characteristic of the endocervix may be recognized in endometrium and tube. Such variations of the paramesonephric (Mullerian) epithelia probably arise from the "indifferent" cell, as may the early changes in the regenerating endometrial epithelia.
U
mg that at least part of the reconstitution of
the surface layer of the endometrium was derived by ditrerentiation from tile underlving stroma. Acridine orange fluorescence
The technique described in a previous publication 4 was used on the sections of men· struating endometria. The cells of the surface epithelium, where visualized, were characterized by rather brilliant orange cytoplasm and nuclei with minimal acridine uptake and darkened zone. suggestive of the mature inactive nuclei in the most superficial layers of the stratified squamous epithelium. It would seem unlikely, as noted previously, that such a mature cell, presumably at the end of its life-span, could produce the new surface epithelium. In contrast, the underlying stromal cells showed brilliant yellow nuclei (Fig. 111. As described previously by routine histologic study, cytoplasm was larg-ely absent in these cells. At times, these cells were seen adjacent to the definitive epithelium, so that the contrast was striking (Fig. 12). In addition to these types, the socalled indifferent ct>ll could be recognized in some of the deeper epithelia, and the nuclei of these cells were prominent due to the striking ye!low color. These resembled, metabolically, the stromal cell and suggested that both stroma and epithelium might be derived by differentiation from the same stem cell. Obviously, from an embryologic point of \'iew, this seems logical.
Reticulum stains
The Wilder reticulum stains demonstrated the usual "basket-weave" pattern in the basal area and fragmentation in the upper spongiosa and compacta, as reported by Craig 2 and others. 10 In general, there was an incomplete, but discernible, reticulum surrounding the glands in the desquamated zone. This could be visualized beneath the surface epithelium (Fig. 9), but disappeared as the groups of "stromal-type cells" were approached and abutted the recognizable epithelial layer (Fig. 10). Although little of a specific nature could be determined from this study, it did provide another link in the chain of evidence suggest.
Comment
Sections of the menstruating endometria from both curettage and hysterectomy specimens were studied for evidence of re-epithelization of the surface. The residual stromal cell at this stage showed a deep basophilic nucleus and scant cytoplasm. Clumps of these cells were seen clinging to the remaining endometrial layers. The stumps of the glands projected on the surface and occasionally seemed to creep over the adjacent stromal surface. Mitoses were rare in the epithelium and stroma. The small, dark "indifferent" cell noted at the stromoepithelial border in
Volume 99 Numbe:· 4
the normal endometrium, was prominent in the regenerating tissue and in many respects, especially with acridine orange fluorescence, simulated the hyperchromatic "stromal cell." Characteristically, groups of stroma-type cells extended to the surface, abutted the epithelium of the gland stumps and assumed a multi.layered effect suggestive of "metaplasia." These cells contained pale-staining nuclei with scant slightly acidophilic cytoplasm. Differentiation of this layer from the underlying stroma by routine and special stain was generally impossible. This finding suggested that the stromal cell or the indifferent variety lying in the stroma played an active role in the regeneration of the surface epithelium. The epithelium at the tips of the residual glands was seen to project over the surface and collapse onto, but remain unattached to,
RI:FERENCES l. Bartelmez, W. G.: Contrib. Embryo!. 24: 143, 1S133. 2. Craig, J. M.: AM. J. OBsT. & GYNEC. 86: 421, 1963. 3. Duval, M. M.: Compt. rend. Soc. bioi. 2: 697, 1890. 4. Friedrich, E. G., Julian, C. G., and Woodruff, J. D.: A~L J. OnsT. & GYNEC. 90: 1281, 1964. 5. Heape, W.: Tr. Obst. Soc. London. 40: 161, 1898. 6. Herrell, W. E., and Broders, A. C.: Surg., Gynec. & Obst. 61: 751, 1935. 7. Hitschmann, F., and Adler, L.: Monatschr. Geburtsh. u. Gynak. 27: 1, 1908. 8 . .Johnstone, A. W.: AM. J. 0BsT. & GYNEC. 24: 681, 1891.
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the stroma. In some instances, it seemed to form a framework upon which the new epithelium developed but there was no suggestion that this "metabolically inactive epithelium" was producing new cells. Thus, the reconstitution of the endometrium was found to follow a more or less particular sequence of regeneration. The keystone in this series of events appears to be a cell which lies at the stromoepithelial border and has the propensity for fom1ing first a metaplastic (squamoid) type of lining to be later replaced by the mature cylindrical variety. The authors wish to express their gratitude to Mr. Raymond Lund, for the photography, and Miss Grace Ann Loftus, for technical assistance in preparation of the paper.
9. McLennan, C. E., and Rydell, A. F.: Obst. & Gynec. 26: 605, 1965. 10. Novak, E.: Surg., Gynec. & Obst. 21: 336, 1915. 1L Novak, E., and TeLinde, R. W.: J. A.M. A. 83: 900, 1924. 12. Papanicolaou, G.: AM. J. 0BST. & GYNEC. 25: 30, 1933. 13. Schroder, R.: Arch. Gynak. 104: 27, 1915. 14. Sturgis, S. H., and Meigs, J. V.: Am. J. Surg. 33: 369, 1936. 15. Westphalen, F.: Arch. Gynak. 52: 37, 1896. 16. Williams, J. W.: J. Obst. & Gynaec. Brit. Emp. 2: 681, 1874.