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Comparison of Two Techniques of Suturing in Microsurgical Anastomosis of the Rabbit Oviduct*
Vol. 28, No. 11, November 1977 Printed in U.S.A.
FERTILITY AND STERILITY Copyright 1977 The American Fertility Society
COMPARISON OF TWO TECHNIQUES OF SUTURING IN MICROSURGICAL ANASTOMOSIS OF THE RABBIT OVIDUCT*
KENICHI SEKI, M.D.t CARLTON A. EDDY, PH.D. NANCY K. SMITH, PH.D. CARL J. PAUERSTEIN, M.D.:j:
Center for Research and Training in Reproductive Biology, Department of Obstetrics and Gynecology, and Department of Anatomy (Morphology Core Laboratory), The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
Microsurgical reconstruction of the rabbit oviduct was undertaken utilizing either through-and-through sutures including the mucosa, or sutures penetrating the serosa and myosalpinx but not the mucosa. Patency and pregnancy rates did not seem to vary with the suturing technique. However, scanning electron microscopic observation revealed abnormal mucosal fold patterns at 3 weeks after surgery. Fibrinous exudates over the surface of the intraluminal sutures increased with time until the entire surface was thickly veiled by 9 weeks. The observation also revealed regeneration of epithelial cells along the suture. These cells included many abnormal forms such as giant cells and misshapen cells. The majority of these were nonciliated. In theory, the intraluminal suture could form a nidus for epithelial hyperplasia which might cause future tubal obstruction. However, the patency and pregnancy rates obtained with through-and-through suturing were similar to those obtained when the endosalpinx was excluded, both in this study and in previous studies from these laboratories.
Recent years have witnessed a great increase in numbers of tubal ligations, particularly among younger women. Although most of these women are well pleased with the resultant sterility, a small percentage, especially among the younger women, subsequently seeks restoration offertility. Historically, gynecologists have not been optimistic about our ability to reverse tubal ligations successfully.lo5 The recent introduction of microsurgical techniques to gynecology has greatly raised our expectations and, indeed, in the brief experience to date, has yielded substantially Received June 13, 1977; accepted July 15, 1977. *Supported in part by Grant 1 P30 HD 10202-01Al from the National Institute of Child Health and Human Development. tRockefeller Foundation Postdoctoral Fellow in Reproductive Biology. :j:Reprint requests: Carl J. Pauerstein, M.D., University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Tex. 78229.
higher pregnancy rates. 6- 8 Yet, one may question whether this improvement is due to microsurgery per se or to emphasis on meticulous technique such as gentle handling of tissues and careful hemostasis. Certain critical questions remain to be answered, such as: How much tube must remain to assure normal function? Are some segments more important than others? Which details of the technique are critical? Is there a critical caliber of suture? This report details the results of a study undertaken to answer a simple, but quite specific, question. When the suture penetrates the endosalpinx, are the results of tubal anastomosis different from results obtained when the mucosa is carefully excluded? MATERIALS AND METHODS
Ten New Zealand White rabbits were subjected to uni- or bilateral microsurgical transection ofthe
1215
November 1977
SEKI ET AL.
1216
TABLE 2. Pregnancy after Microsurgical End-to-End Anastomosis of the Isthmus of the Rabbit Oviduct
TABLE 1. Patency after Microsurgical End-to-End Anastomosis of the Isthmus of the Rabbit Oviduct Suture placement
Animal no.
Oviduct
Through mucosa
1
Right Left Right Left Right Left Right Left Right Left
Time after surgery"
Animal no.
Patency
Oviduct and suture placement
Time after surgery"
wk
2
3
4 7
Excluding mucosa
5 6
Right Left Right Left
2 2
3 3 7
3 3
No. of implantation sites/no. of corpora Iutea
wk
Yes Yes Yes Partial Yes Yes Yes Yes Yes No
8 9
10
Right, not operated Left, through mucosa Right, not operated Left, through mucosa Right, not operated Left, through mucosa
9 9
14
5/6 4/6
617 6/14 0/6 3/8
"When the oviducts were evaluated.
Yes Yes Yes Yes
"When the oviducts were evaluated.
isthmus followed immediately by microsurgical end-to-end tubal anastomosis. The anastomosis was performed by using 10-0 monofilament nylon suture mounted on a 130-M-m needle. In eight of the rabbits four sutures were placed at 90 intervals around the perimeter ofthe tube through the serosa, myosalpinx, and mucosa. In two rabbits the sutures were placed through the myosalpinx and serosa, excluding the mucosa. Anastomotic sites were reperitonized and the defects of the mesosalpinx were repaired with interrupted 10-0 nylon sutures. At the end of surgery, tubal patency was ascertained by perfusing methylene blue solution from the fimbriated end. Three of the rabbits in which the anastomosis included 0
the endosalpinx were mated after recovery (two at 3 weeks and one at 8 weeks postoperation). They were examined 3 weeks after mating to diagnose pregnancy. The animals were subsequently killed, and the oviducts were removed for evaluation. The animals were killed at intervals of 2 to 14 weeks after surgery. After ascertaining tubal patency, the anastomotic sties were longitudinally incised to expose the endosalpinx and prepared for examination by scanning electron microscopy. The tissues were fixed in a 2.5% glutaraldehyde-2% paraformaldehyde mixture in 0.1 M cacodylate buffer (pH 7.4) for 4 hours and postfixed in buffered. 1% osmium tetroxide in 0.1 M cacodylate buffer for 2 hours. They were then dehydrated in graded concentrations of ethanol followed by acetone and dried by the critical-point drying method. They were then coated with gold and palladium by sputtering. Specimens were observed in a JSM-35 scanning electron microscope.
FIGs. 1 AND 2. Low-power view of the anastomotic site 2 weeks after surgery. Sutures were placed to include the mucosa in Figure 1 and the mucosa was excluded from the suture line in Figure 2. Mucosal folds show the same irregular pattern and gathering toward the center in both cases. Both figures show slight constriction in this area (x95).
Vol. 28, No. 11
COMPARISON OF TWO TECHNIQUES OF SUTURING IN MICROSURGERY
FIG. 3. Three weeks after surgery. Fibrinous exudates partially cover the suture (x4690).
RESULTS
All oviducts were patent when tested at surgery. As shown in Table 1, two oviducts did not demonstrate free passage of the dye at the remote examination after sacrifice. One oviduct, in an animal killed 2 weeks after surgery, demonstrated a partial obstruction at the anastomotic site. The other oviduct, obtained from an animal killed 7 weeks after surgery, showed no patency. Both tubes were palpably thickened and indurated at the anastomotic site. In the remaining 14 tubes, dye
FIG.4. Seven weeks after surgery. Fibrinous exudates have become thick and cover more of the surface of the suture (x2870).
1217
FIG. 5. Nine weeks after surgery. The suture is now completely veiled with a thick exudate (x3050).
passed freely over the entire length of the oviduct. The three mated rabbits demonstrated normally implanted embryos in both uterine horns 3 weeks after mating (Table 2). In oviducts examined 2 weeks after surgery we observed some fibrosis on the luminal surface of the oviduct, although patency was normal. By 3 weeks after surgery, no fibrosis or scarring was seen. We noted slight constriction of the lumen at the anastomotic site. The mucosal folds were disturbed, showing some discontinuity, and were bunched slightly to· the center (Fi_gs. 1 and 2). Similar changes were observed both in tubes that
FIG. 6. Seven weeks after surgery. Epithelial regeneration has started along the suture (x620).
SEKI ET AL.
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November 1977
FIG. 7. Nine weeks after surgery. The suture is now covered with giant and abnormally shaped epithelial cells, the majority of which are nonciliated (x 1270).
FIG. 8. Higher magnification of the right edge of Figure 7. Dense microvilli cover the regenerating cells. Ciliated cells are scattered among the nonciliated cells (x3130).
had been sutured to include the mucosa and in those in which the mucosa had been excluded. The surface of the intraluminal suture also demonstrated changes with the passage of time. At 3 weeks, part of the surface of the suture was covered by a fibrinous exudate (Fig. 3). The exudate was thicker and covered more of the surface of the suture at 7 weeks (Fig. 4). By 9 weeks after surgery the entire surface was covered (Fig. 5). We also noted proliferation of oviductal epithelial cells along the suture. The proliferation had started by 7 weeks (Fig. 6), and at 9 weeks most of the intraluminal sutures were completely epithelialized (Fig. 7). These cells regenerated and were covered with dense microvilli; they were very large and misshapen in comparison with normal epithelial cells (Fig. 8).
that the majority of the animals showed partial or complete absence of the mucosal fold pattern at the anastomotic site, maximal at 6 weeks, with a return to normal after 16 weeks. In our animals, at 3 weeks the mucosa also showed an irregular pattern with gathering of the mucosal folds. In addition, the lumina were slightly constricted. These observations were true in both types of suturing. The abnormal mucosal pattern, including the giant cell forms seen on the intraluminal sutures, did not interfere with patency nor with normal fertility. Thus, in spite of the demonstration of epithelial abnormalities in the presence of sutures that penetrate the mucosa and the theoretical possibility of subsequent tubal occlusion, there seems to be no practical advantage to placing the sutures to exclude the endosalpinx.
DISCUSSION
In experiments of this type, the experience of the surgeon might greatly affect the results. In this study, all surgery was performed by the same surgeon. Only one tube was not patent, and normal intrauterine implantation was obtained in all of the animals tested. More significantly, the results with through-and-through sutures were also not different from those in another group of animals operated upon in our laboratory, in which the sutures were placed to avoid the mucosa. 9 , 10 After reconstructive surgery of the uterotubal junction of the rabbit, Khoo and Mackayll reported
REFERENCES 1. Siegler AM, Hellman LH: Fallopian tubes in sterility. Fertil Steril 7:170, 1956 2. Siegler AM: Tubal plastic surgery: the past, the present and the future. Obstet Gynecol Survey 15:680, 1960 3. Crane M, Woodruff JD: Factors influencing the success of tuboplastic procedures. Fertil Steril 19:810, 1968 4. Umezaki C, Katayama KP, Jones HW Jr: Pregnancy rates after reconstructive surgery on the fallopian tubes. Obstet Gynecol 43:418, 1974 5. Patton GW, Kistner RW: Surgical reconstruction of the oviduct. In Progress in Infertility, Second Edition, Edited by SJ Behrman, RW Kistner. Boston, Little, Brown and Co, 1975, p 223
T
I Vol. 28, No. 11
COMPARISON OF TWO TECHNIQUES OF SUTURING IN MICROSURGERY
6. Gomel V: Tubal reanastomosis by microsurgery. Fertil Steril 28:59, 1977 7. Gomel V: Reconstructive surgery of the oviduct. J Reprod Med 18:181, 1977 8. Winston RML: Microsurgical tubocornual anastomosis for reversal of sterilization. Lancet 1:284, 1977 9. Eddy CA, Antonini R Jr, Pauerstein CJ: Fertility followingmicrosurgical removal of the ampullary-isthmic junction in rabbits. Fertil Steril 28:1090, 1977
1219
10. Eddy CA, Balmaceda JP, Pauerstein CJ: Effect of resection of the ampullary-isthmic junction on estrogen induced tube locking of ova in the rabbit. BioI Reprod. In press 11. Khoo SK, Mackay EV: Reactions in rabbit fallopian tube after plastic reconstruction. I. Gross pathology, tubal patency, and pregnancy. Fertil Steril 23:201, 1972
FERTILITY AND STERILITY Copyright 1977 The American Fertility Society
COMPARISON OF TWO TECHNIQUES OF SUTURING IN MICROSURGICAL ANASTOMOSIS OF THE RABBIT OVIDUCT*
KENICHI SEKI, M.D.t CARLTON A. EDDY, PH.D. NANCY K. SMITH, PH.D. CARL J. PAUERSTEIN, M.D.:j:
Center for Research and Training in Reproductive Biology, Department of Obstetrics and Gynecology, and Department of Anatomy (Morphology Core Laboratory), The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
Microsurgical reconstruction of the rabbit oviduct was undertaken utilizing either through-and-through sutures including the mucosa, or sutures penetrating the serosa and myosalpinx but not the mucosa. Patency and pregnancy rates did not seem to vary with the suturing technique. However, scanning electron microscopic observation revealed abnormal mucosal fold patterns at 3 weeks after surgery. Fibrinous exudates over the surface of the intraluminal sutures increased with time until the entire surface was thickly veiled by 9 weeks. The observation also revealed regeneration of epithelial cells along the suture. These cells included many abnormal forms such as giant cells and misshapen cells. The majority of these were nonciliated. In theory, the intraluminal suture could form a nidus for epithelial hyperplasia which might cause future tubal obstruction. However, the patency and pregnancy rates obtained with through-and-through suturing were similar to those obtained when the endosalpinx was excluded, both in this study and in previous studies from these laboratories.
Recent years have witnessed a great increase in numbers of tubal ligations, particularly among younger women. Although most of these women are well pleased with the resultant sterility, a small percentage, especially among the younger women, subsequently seeks restoration offertility. Historically, gynecologists have not been optimistic about our ability to reverse tubal ligations successfully.lo5 The recent introduction of microsurgical techniques to gynecology has greatly raised our expectations and, indeed, in the brief experience to date, has yielded substantially Received June 13, 1977; accepted July 15, 1977. *Supported in part by Grant 1 P30 HD 10202-01Al from the National Institute of Child Health and Human Development. tRockefeller Foundation Postdoctoral Fellow in Reproductive Biology. :j:Reprint requests: Carl J. Pauerstein, M.D., University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Tex. 78229.
higher pregnancy rates. 6- 8 Yet, one may question whether this improvement is due to microsurgery per se or to emphasis on meticulous technique such as gentle handling of tissues and careful hemostasis. Certain critical questions remain to be answered, such as: How much tube must remain to assure normal function? Are some segments more important than others? Which details of the technique are critical? Is there a critical caliber of suture? This report details the results of a study undertaken to answer a simple, but quite specific, question. When the suture penetrates the endosalpinx, are the results of tubal anastomosis different from results obtained when the mucosa is carefully excluded? MATERIALS AND METHODS
Ten New Zealand White rabbits were subjected to uni- or bilateral microsurgical transection ofthe
1215
November 1977
SEKI ET AL.
1216
TABLE 2. Pregnancy after Microsurgical End-to-End Anastomosis of the Isthmus of the Rabbit Oviduct
TABLE 1. Patency after Microsurgical End-to-End Anastomosis of the Isthmus of the Rabbit Oviduct Suture placement
Animal no.
Oviduct
Through mucosa
1
Right Left Right Left Right Left Right Left Right Left
Time after surgery"
Animal no.
Patency
Oviduct and suture placement
Time after surgery"
wk
2
3
4 7
Excluding mucosa
5 6
Right Left Right Left
2 2
3 3 7
3 3
No. of implantation sites/no. of corpora Iutea
wk
Yes Yes Yes Partial Yes Yes Yes Yes Yes No
8 9
10
Right, not operated Left, through mucosa Right, not operated Left, through mucosa Right, not operated Left, through mucosa
9 9
14
5/6 4/6
617 6/14 0/6 3/8
"When the oviducts were evaluated.
Yes Yes Yes Yes
"When the oviducts were evaluated.
isthmus followed immediately by microsurgical end-to-end tubal anastomosis. The anastomosis was performed by using 10-0 monofilament nylon suture mounted on a 130-M-m needle. In eight of the rabbits four sutures were placed at 90 intervals around the perimeter ofthe tube through the serosa, myosalpinx, and mucosa. In two rabbits the sutures were placed through the myosalpinx and serosa, excluding the mucosa. Anastomotic sites were reperitonized and the defects of the mesosalpinx were repaired with interrupted 10-0 nylon sutures. At the end of surgery, tubal patency was ascertained by perfusing methylene blue solution from the fimbriated end. Three of the rabbits in which the anastomosis included 0
the endosalpinx were mated after recovery (two at 3 weeks and one at 8 weeks postoperation). They were examined 3 weeks after mating to diagnose pregnancy. The animals were subsequently killed, and the oviducts were removed for evaluation. The animals were killed at intervals of 2 to 14 weeks after surgery. After ascertaining tubal patency, the anastomotic sties were longitudinally incised to expose the endosalpinx and prepared for examination by scanning electron microscopy. The tissues were fixed in a 2.5% glutaraldehyde-2% paraformaldehyde mixture in 0.1 M cacodylate buffer (pH 7.4) for 4 hours and postfixed in buffered. 1% osmium tetroxide in 0.1 M cacodylate buffer for 2 hours. They were then dehydrated in graded concentrations of ethanol followed by acetone and dried by the critical-point drying method. They were then coated with gold and palladium by sputtering. Specimens were observed in a JSM-35 scanning electron microscope.
FIGs. 1 AND 2. Low-power view of the anastomotic site 2 weeks after surgery. Sutures were placed to include the mucosa in Figure 1 and the mucosa was excluded from the suture line in Figure 2. Mucosal folds show the same irregular pattern and gathering toward the center in both cases. Both figures show slight constriction in this area (x95).
Vol. 28, No. 11
COMPARISON OF TWO TECHNIQUES OF SUTURING IN MICROSURGERY
FIG. 3. Three weeks after surgery. Fibrinous exudates partially cover the suture (x4690).
RESULTS
All oviducts were patent when tested at surgery. As shown in Table 1, two oviducts did not demonstrate free passage of the dye at the remote examination after sacrifice. One oviduct, in an animal killed 2 weeks after surgery, demonstrated a partial obstruction at the anastomotic site. The other oviduct, obtained from an animal killed 7 weeks after surgery, showed no patency. Both tubes were palpably thickened and indurated at the anastomotic site. In the remaining 14 tubes, dye
FIG.4. Seven weeks after surgery. Fibrinous exudates have become thick and cover more of the surface of the suture (x2870).
1217
FIG. 5. Nine weeks after surgery. The suture is now completely veiled with a thick exudate (x3050).
passed freely over the entire length of the oviduct. The three mated rabbits demonstrated normally implanted embryos in both uterine horns 3 weeks after mating (Table 2). In oviducts examined 2 weeks after surgery we observed some fibrosis on the luminal surface of the oviduct, although patency was normal. By 3 weeks after surgery, no fibrosis or scarring was seen. We noted slight constriction of the lumen at the anastomotic site. The mucosal folds were disturbed, showing some discontinuity, and were bunched slightly to· the center (Fi_gs. 1 and 2). Similar changes were observed both in tubes that
FIG. 6. Seven weeks after surgery. Epithelial regeneration has started along the suture (x620).
SEKI ET AL.
1218
November 1977
FIG. 7. Nine weeks after surgery. The suture is now covered with giant and abnormally shaped epithelial cells, the majority of which are nonciliated (x 1270).
FIG. 8. Higher magnification of the right edge of Figure 7. Dense microvilli cover the regenerating cells. Ciliated cells are scattered among the nonciliated cells (x3130).
had been sutured to include the mucosa and in those in which the mucosa had been excluded. The surface of the intraluminal suture also demonstrated changes with the passage of time. At 3 weeks, part of the surface of the suture was covered by a fibrinous exudate (Fig. 3). The exudate was thicker and covered more of the surface of the suture at 7 weeks (Fig. 4). By 9 weeks after surgery the entire surface was covered (Fig. 5). We also noted proliferation of oviductal epithelial cells along the suture. The proliferation had started by 7 weeks (Fig. 6), and at 9 weeks most of the intraluminal sutures were completely epithelialized (Fig. 7). These cells regenerated and were covered with dense microvilli; they were very large and misshapen in comparison with normal epithelial cells (Fig. 8).
that the majority of the animals showed partial or complete absence of the mucosal fold pattern at the anastomotic site, maximal at 6 weeks, with a return to normal after 16 weeks. In our animals, at 3 weeks the mucosa also showed an irregular pattern with gathering of the mucosal folds. In addition, the lumina were slightly constricted. These observations were true in both types of suturing. The abnormal mucosal pattern, including the giant cell forms seen on the intraluminal sutures, did not interfere with patency nor with normal fertility. Thus, in spite of the demonstration of epithelial abnormalities in the presence of sutures that penetrate the mucosa and the theoretical possibility of subsequent tubal occlusion, there seems to be no practical advantage to placing the sutures to exclude the endosalpinx.
DISCUSSION
In experiments of this type, the experience of the surgeon might greatly affect the results. In this study, all surgery was performed by the same surgeon. Only one tube was not patent, and normal intrauterine implantation was obtained in all of the animals tested. More significantly, the results with through-and-through sutures were also not different from those in another group of animals operated upon in our laboratory, in which the sutures were placed to avoid the mucosa. 9 , 10 After reconstructive surgery of the uterotubal junction of the rabbit, Khoo and Mackayll reported
REFERENCES 1. Siegler AM, Hellman LH: Fallopian tubes in sterility. Fertil Steril 7:170, 1956 2. Siegler AM: Tubal plastic surgery: the past, the present and the future. Obstet Gynecol Survey 15:680, 1960 3. Crane M, Woodruff JD: Factors influencing the success of tuboplastic procedures. Fertil Steril 19:810, 1968 4. Umezaki C, Katayama KP, Jones HW Jr: Pregnancy rates after reconstructive surgery on the fallopian tubes. Obstet Gynecol 43:418, 1974 5. Patton GW, Kistner RW: Surgical reconstruction of the oviduct. In Progress in Infertility, Second Edition, Edited by SJ Behrman, RW Kistner. Boston, Little, Brown and Co, 1975, p 223
T
I Vol. 28, No. 11
COMPARISON OF TWO TECHNIQUES OF SUTURING IN MICROSURGERY
6. Gomel V: Tubal reanastomosis by microsurgery. Fertil Steril 28:59, 1977 7. Gomel V: Reconstructive surgery of the oviduct. J Reprod Med 18:181, 1977 8. Winston RML: Microsurgical tubocornual anastomosis for reversal of sterilization. Lancet 1:284, 1977 9. Eddy CA, Antonini R Jr, Pauerstein CJ: Fertility followingmicrosurgical removal of the ampullary-isthmic junction in rabbits. Fertil Steril 28:1090, 1977
1219
10. Eddy CA, Balmaceda JP, Pauerstein CJ: Effect of resection of the ampullary-isthmic junction on estrogen induced tube locking of ova in the rabbit. BioI Reprod. In press 11. Khoo SK, Mackay EV: Reactions in rabbit fallopian tube after plastic reconstruction. I. Gross pathology, tubal patency, and pregnancy. Fertil Steril 23:201, 1972