- Email: [email protected]
Autologous endometrial transplants within Millipore filters in monkeys
Autologous endometrial transplants within Millipore filters in monkeys ROGER B. SCOTT, M.D. RICHARD J, NOWAK, M.D. V. THOMAS MANNARELLI, M.D. Cleveland, Ohio
E N D o M E T R I A L tissue has a tremendous growth potential. Surgically excised fragments of endometrium grow readily when autologously transplanted to various locations in monkeys and other animals. Scott, TeLinde, and Wharton/• 5 • 7 in experiments with rhesus monkeys, have demonstrated the viability of even the endometrium cast off at the time of menstruation. Ridley and Edwards/ by injecting human menstrual material subcutaneously in human volunteers, have shown the viability of this shed tissue. The human female, as well as the rhesus monkey, has patent uterotubal junctions and cyclic menstruation. Endometrial fragments have been found free in the human tubal lumen and they can pass in a retrograde fashion out the tubes, implant on pelvic tissues, rapidly vascularize at this host site, and grow. This assumes that viability remains for the period of time necessary for tubal transport, without nourishment other than intraluminal and peritoneal fluid and juices. Millipore filters of cellulose ester are available in a pore size which will allow the passage of proteins, electrolytes, and other circulating chemical substances, but will exclude formed elements, even leukocytes. 1 • ~. •;
Endometrium transplanted within such a filter to various areas of the monkey's body would give us a method of testing the duration of viability before actual tissue vascularization occurs. Material and methods Millipore filter membranes of pore size 0.8p. ("AA'') and 0.45p. ("HA") were used. The former would allow the passage of formed elements, such as red cells and leukocytes, whereas the latter would exclude these. Leucite rings, as developed by Algire, Weaver, and Prehn/ were made and together with the filter wafers were sterilized in ethylene oxide gas chambers. By hysterotomy on adult female rhesus (Macaca mulatta) monkeys, fragments of endometrium were surgically excised and rapidly placed between two filters. The inner outside margins of the filters were glued to each side of the plastic ring, thus forming a sealing chamber in the center. These prepared transplants were then placed in various created spaces in the broad ligaments, pelvic retroperitoneal regions, and within the rectus muscles. Four rhesus monkeys were used for these experiments. The transplants were removed at intervals ranging from 2 to 7 days and histologically studied for degree of degeneration. When it was found that degenerative changes were marked after more than 2 days, "HA" filter chambers with endometrial fragments were again prepared, placed into similar sites in two monkeys, and left in place
From the Department of Obstetrics and Gynecology, Western Reserve University School of Medicine, and University Hospitals of Cleveland. Supported by United States Public Health Service Research Grant C-2892 (5).
1010
Volume 84 Number 8
for 2 days. After this 48 hour interval the animals were re-explored and the endometrial fragments removed from the filter chambers and placed directly into the tissue spaces. As a final test of viability the transplant sites were again investigated at intervals ranging from 7 to 27 months.
Autologous endomefrial transplants
1011
at the time of menstruation. These fragments can accept nourishment and a vascular supply when shed onto an unhealed area of the cervix, vagina, or vulva; the retrograde passage via the Fallopian tubes may take only a matter of a few hours and tubal and peritoneal fluids may well contain sufficient
Results
The histologic evidence of viability of endometrium within these filter chambers disappeared after more than 2 days and, with one exception, there was little difference between the two pore sizes. In one case the AA chamber (admitting cells) contained apparent viable endometrium after 3 weeks, but the filter had cracked (Fig. 1). The newer nylon-impregnated filters protect against cracking. Both the stroma and the epithelium maintained fair histologic evidence of viability (Fig. 2) after 48 hours in the HA filter chambers, but further evidence of this viability was obtained by removing the fragments and transferring them directly into the various tissue sites. Although the endometrium was exposed only to tissue proteins, electrolytes, and other chemical substances during this interim in the filter chambers, it remained capable of full generative power when placed directly into the tissues. Vascularization and growth occurred with nodules of endometriosis ranging in size from 0.3 to 0.5 em. when surgically investigated 7 to 27 months later (Fig. 3) .
Fig. 1. Endometrial transplant after 3 weeks in AA Millipore filter chamber. The tissue appears viable, but notice the crack in the filter. Other transplants in this type of filter chamber without cracking did not appear healthy for longer than 48 hours.
Comment
In these experiments surgically excised fragments of monkey endometrium were found to maintain viability for at least 48 hours without vascularization but in contact with the tissue juices of the autologous host. Endometrium cast off at the time of menstruation could be expected to have a somewhat lower survival time than the surgically removed material, but the difference is not answered in this study. Animal experiments 4 • 7 and clincial evidence5 have substantiated the implantation capability of fragments of endometrium shed
Fig. 2. Endometrial transplant after HA Millipore filter chamber placed peritoneal pelvic area of a monkey. elements penetrated the filter or chamber.
48 hours in in a retroNo formed intervening
1012 Scott, Nowak, and Mannarelli ·\J11.
t: Jc!• .dtt'l 15 . E.Hi..: & L ynr·t; ,
T. Oil:-I,
tum. the shed endometrial rnaterial is fairly necrotic and this together with tlw rapid healing potential of the squamous nmCOIIS membrane of the lower genital tract reduces the frequency of implantation in this particular region. However. adding delayed healing and subsequent menses with the discharge of relati,·ely healthy endometrium, a fertile soil has been prepared. Another example of fertile and highly acceptable soil is the liDhealed. recently cauterized cen·ix. Summary
Fig. 3. Endometriosis actively growing in the posterior cul-de-sac of a monkey. The autologous endometrial transplant was removed from the HA filter chamber after 48 hours and directly placed into a space in the posterior cul-de-sac. This biopsy was obtained 7 months following the direct transplant.
nutrients until a favorable site for more direct survival is located. Fragments of endometrium within a hematometra may be so impeded in their passage through the thick hemorrhagic medium that they lose their viability in transit. Post par-
Surgically removed fragments of monkPy endometrium were autologously transplanted within Millipore filter chambers into various tissue spaces. When the pore size admitted only tissue fluids and chemicals. but no formed elements, the endometrium maintained histologic evidence of viability for 48 hours. The true viability of this tissue was established by removing the fragments from the filter chamber after 48 hours, directly implanting them in the tissue spaces, and demonstrating active growth at subsequent surgical exploration. The evidence is strong that endometrial tissue fragments shed at the time of a menstrual period are viable and capable of implantation and growth. These experiments suggest an ability of endometrium to survive the time of retrograde tubal transport, a time without active \·ascularization for nourishment.
REFERENCES
1. :\lgire, E. W., Weaver, J. M ., and Prehn, R . T .: J. Nat. Cancer lnst. 15: 4-93, 1954. 2. Castellanos, H., and Sturgis, S. H.: Obst. & Gynec. 12: 603, 1958. 3. Ridley, J. H., and Edwards, I. K.: AM. J. 0BST. & GYNEC. 76: 783, 1958. 4. Scott, R. B., TeLinde, R. W., and Wharton, L. R., Jr.: AM. J. 0BsT. & GYNEC. 66: 1082, 1953.
5. Scott, R. B. , and TeLinde, R . W.: Obst. & Gynec. 4: 502, 1954. 6. Sturgis, S. H ., and Castellanos , H.: .\M. J. 0BST. & GYNEC. 76: 1132, 1958. 7. TeLinde, R. W., and Scott, R. B.: :hr. J. 0BST. & GYNEC. 60: 1147, 1950. University Circle Cleveland 6, Ohio
E N D o M E T R I A L tissue has a tremendous growth potential. Surgically excised fragments of endometrium grow readily when autologously transplanted to various locations in monkeys and other animals. Scott, TeLinde, and Wharton/• 5 • 7 in experiments with rhesus monkeys, have demonstrated the viability of even the endometrium cast off at the time of menstruation. Ridley and Edwards/ by injecting human menstrual material subcutaneously in human volunteers, have shown the viability of this shed tissue. The human female, as well as the rhesus monkey, has patent uterotubal junctions and cyclic menstruation. Endometrial fragments have been found free in the human tubal lumen and they can pass in a retrograde fashion out the tubes, implant on pelvic tissues, rapidly vascularize at this host site, and grow. This assumes that viability remains for the period of time necessary for tubal transport, without nourishment other than intraluminal and peritoneal fluid and juices. Millipore filters of cellulose ester are available in a pore size which will allow the passage of proteins, electrolytes, and other circulating chemical substances, but will exclude formed elements, even leukocytes. 1 • ~. •;
Endometrium transplanted within such a filter to various areas of the monkey's body would give us a method of testing the duration of viability before actual tissue vascularization occurs. Material and methods Millipore filter membranes of pore size 0.8p. ("AA'') and 0.45p. ("HA") were used. The former would allow the passage of formed elements, such as red cells and leukocytes, whereas the latter would exclude these. Leucite rings, as developed by Algire, Weaver, and Prehn/ were made and together with the filter wafers were sterilized in ethylene oxide gas chambers. By hysterotomy on adult female rhesus (Macaca mulatta) monkeys, fragments of endometrium were surgically excised and rapidly placed between two filters. The inner outside margins of the filters were glued to each side of the plastic ring, thus forming a sealing chamber in the center. These prepared transplants were then placed in various created spaces in the broad ligaments, pelvic retroperitoneal regions, and within the rectus muscles. Four rhesus monkeys were used for these experiments. The transplants were removed at intervals ranging from 2 to 7 days and histologically studied for degree of degeneration. When it was found that degenerative changes were marked after more than 2 days, "HA" filter chambers with endometrial fragments were again prepared, placed into similar sites in two monkeys, and left in place
From the Department of Obstetrics and Gynecology, Western Reserve University School of Medicine, and University Hospitals of Cleveland. Supported by United States Public Health Service Research Grant C-2892 (5).
1010
Volume 84 Number 8
for 2 days. After this 48 hour interval the animals were re-explored and the endometrial fragments removed from the filter chambers and placed directly into the tissue spaces. As a final test of viability the transplant sites were again investigated at intervals ranging from 7 to 27 months.
Autologous endomefrial transplants
1011
at the time of menstruation. These fragments can accept nourishment and a vascular supply when shed onto an unhealed area of the cervix, vagina, or vulva; the retrograde passage via the Fallopian tubes may take only a matter of a few hours and tubal and peritoneal fluids may well contain sufficient
Results
The histologic evidence of viability of endometrium within these filter chambers disappeared after more than 2 days and, with one exception, there was little difference between the two pore sizes. In one case the AA chamber (admitting cells) contained apparent viable endometrium after 3 weeks, but the filter had cracked (Fig. 1). The newer nylon-impregnated filters protect against cracking. Both the stroma and the epithelium maintained fair histologic evidence of viability (Fig. 2) after 48 hours in the HA filter chambers, but further evidence of this viability was obtained by removing the fragments and transferring them directly into the various tissue sites. Although the endometrium was exposed only to tissue proteins, electrolytes, and other chemical substances during this interim in the filter chambers, it remained capable of full generative power when placed directly into the tissues. Vascularization and growth occurred with nodules of endometriosis ranging in size from 0.3 to 0.5 em. when surgically investigated 7 to 27 months later (Fig. 3) .
Fig. 1. Endometrial transplant after 3 weeks in AA Millipore filter chamber. The tissue appears viable, but notice the crack in the filter. Other transplants in this type of filter chamber without cracking did not appear healthy for longer than 48 hours.
Comment
In these experiments surgically excised fragments of monkey endometrium were found to maintain viability for at least 48 hours without vascularization but in contact with the tissue juices of the autologous host. Endometrium cast off at the time of menstruation could be expected to have a somewhat lower survival time than the surgically removed material, but the difference is not answered in this study. Animal experiments 4 • 7 and clincial evidence5 have substantiated the implantation capability of fragments of endometrium shed
Fig. 2. Endometrial transplant after HA Millipore filter chamber placed peritoneal pelvic area of a monkey. elements penetrated the filter or chamber.
48 hours in in a retroNo formed intervening
1012 Scott, Nowak, and Mannarelli ·\J11.
t: Jc!• .dtt'l 15 . E.Hi..: & L ynr·t; ,
T. Oil:-I,
tum. the shed endometrial rnaterial is fairly necrotic and this together with tlw rapid healing potential of the squamous nmCOIIS membrane of the lower genital tract reduces the frequency of implantation in this particular region. However. adding delayed healing and subsequent menses with the discharge of relati,·ely healthy endometrium, a fertile soil has been prepared. Another example of fertile and highly acceptable soil is the liDhealed. recently cauterized cen·ix. Summary
Fig. 3. Endometriosis actively growing in the posterior cul-de-sac of a monkey. The autologous endometrial transplant was removed from the HA filter chamber after 48 hours and directly placed into a space in the posterior cul-de-sac. This biopsy was obtained 7 months following the direct transplant.
nutrients until a favorable site for more direct survival is located. Fragments of endometrium within a hematometra may be so impeded in their passage through the thick hemorrhagic medium that they lose their viability in transit. Post par-
Surgically removed fragments of monkPy endometrium were autologously transplanted within Millipore filter chambers into various tissue spaces. When the pore size admitted only tissue fluids and chemicals. but no formed elements, the endometrium maintained histologic evidence of viability for 48 hours. The true viability of this tissue was established by removing the fragments from the filter chamber after 48 hours, directly implanting them in the tissue spaces, and demonstrating active growth at subsequent surgical exploration. The evidence is strong that endometrial tissue fragments shed at the time of a menstrual period are viable and capable of implantation and growth. These experiments suggest an ability of endometrium to survive the time of retrograde tubal transport, a time without active \·ascularization for nourishment.
REFERENCES
1. :\lgire, E. W., Weaver, J. M ., and Prehn, R . T .: J. Nat. Cancer lnst. 15: 4-93, 1954. 2. Castellanos, H., and Sturgis, S. H.: Obst. & Gynec. 12: 603, 1958. 3. Ridley, J. H., and Edwards, I. K.: AM. J. 0BST. & GYNEC. 76: 783, 1958. 4. Scott, R. B., TeLinde, R. W., and Wharton, L. R., Jr.: AM. J. 0BsT. & GYNEC. 66: 1082, 1953.
5. Scott, R. B. , and TeLinde, R . W.: Obst. & Gynec. 4: 502, 1954. 6. Sturgis, S. H ., and Castellanos , H.: .\M. J. 0BST. & GYNEC. 76: 1132, 1958. 7. TeLinde, R. W., and Scott, R. B.: :hr. J. 0BST. & GYNEC. 60: 1147, 1950. University Circle Cleveland 6, Ohio