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Fertility Following Microsurgical Removal of the Ampullary-Isthmic Junction in Rabbits*
Vol. 28, No. 10, October 1977 Printed in U.S.A.
FERTILITY AND STERILITY Copyright' 1977 The American Fertility Society
FERTILITY FOLLOWING MICROSURGICAL REMOVAL OF THE AMPULLARY-ISTHMIC JUNCTION IN RABBITS*
CARLTON A. EDDY, PH,D.t RENZO ANTONINI, JR., M.D.:!: CARL J. PAUERSTEIN, M.D.
Departments of Obstetrics and Gynecology and Physiology, Center for Research and Training in Reproductive Biology and Voluntary Regulation of Fertility, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
The role of the ampullary-isthmic junction (AIJ) in fertility was investigated in the rabbit by using microsurgical tuboplastic techniques. Microsurgical transection or resection of the AIJ failed to alter fertility significantly. Nine of ten rabbits which underwent unilateral transection of the AIJ became pregnant on the operated side and eight of the ten became pregnant on the unoperated control side. Of the ten rabbits which underwent unilateral AIJ resection, all became pregnant bilaterally. The results of this study indicate that the AIJ is not necessary for normal fertility in the rabbit.
lates neuroendocrine modulation of constriction at the AIJ.3'5 The recent development of microsurgical tuboplastic techniques 6• s provides the means with which to examine tubal function directly. The present study was undertaken to define the role of the AIJ in fertility of the rabbit.
Ovum transport through the mammalian oviduct is a complex, discontinuous process characterized by a pause of varying, species-specific duration at the ampullary-isthmic junction (AIJ) before final entrance of the embryo into the uterus. 1 The functional significance of this pause is unknown but may be important in synchronizing endometrial maturation and embryo development necessary for successful implantation. 2 The mechanism responsible for the pause remains undefined. Several theories have been advanced in explanation of this mechanism, such as transient edema, valve-like structures, and other anatomical and junctional obstructions to the free passage of the ovum or embryo. In the rabbit no anatomical structure has been defined, so that various functional constrictions have been favored. The most prevalent hypothesis po stu-
MATERIALS AND METHODS
Received May 16, 1977; revised June 13, 1977; accepted June 13, 1977. *Presented at the Ninth World Congress on Fertility and Sterility and the Thirty-Third Annual Meeting of The American Fertility Society, April 12 to 16, 1977, Miami Beach, Fla. tReprint requests: Carlton A. Eddy, Ph.D., University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Tex. 78229. :!:Rockefeller Foundation Fellow in Reproductive Biology.
Animals. Twenty adult virgin female New Zealand White rabbits were divided into two groups of ten animals each. In one group the AIJ was unilaterally transected and the oviduct rejoined by using microsurgical end-to-end anastomosis (surgical control group). In the second group a l-cm segment of oviduct containing the AIJ was unilaterally resected and the oviduct similarly anastomosed. Contralateral unoperated oviducts served as controls in both groups. Surgical Technique. Animals were anesthetized by the intravenous administration of sodium pentobarbital (20 to 32 mg/kg). A midventral laparotomy was performed and the reproductive tract exteriorized. A polyethylene catheter (external diameter 0.60 mm) attached to a syringe containing methylene blue diluted in physiologic saline was introduced through the tubal ostium
1090
Vol. 28, No. 10
1091
FERTILITY FOLLOWING AMPULLARY-ISTHMIC JUNCTION REMOVAL
FIG. 1. Appearance of the rabbit oviduct at the level of the AlJ (arrow) following intraluminal perfusion as seen through the operating microscope. The ampulla is to left of the arrow, the isthmus to the right. Note the ampullary distention and the abrupt transition in the mucosal fold pattern from ampulla to isthmus, delineating the position of the AIJ.
into the distal ampulla. Digital pressure was gently applied around the ostium to prevent leakage. A small volume of saline was slowly perfused into the oviduct. The resultant tubal distention was largely limited to the thin-walled ampulla. The limits of distention marked the transition to the thick-walled, nondistended isthmus and thus served to identify the AIJ. Observation of the tubal mucosal pattern through an operating microscope served to delineate further the position of the AIJ (Fig. 1). Lengths of elastic cord 0.5 mm in diameter were placed above and below the area of the AIJ by piercing the mesosalpinx and mesotubarium superius in avascular areas with curved no. 7
FIG. 3. Microsurgical technique used to repair the defect in the mesosalpinx and reperitonize the anastomosis site. Inset, appearance of the anastomosis site following completion of microsurgery.
jeweler's forceps. These cords were then drawn through the supporting structures and tightened sufficiently with small serrefines to occlude the tubal vascular arcade but not the tubal lumen. A Zeiss OPMI 6 operating microscope fitted with a 300-mm objective lens was positioned and the surgery conducted under magnification ranging from x10 to x50. Major collateral blood vessels not occluded by the ligatures in the area of oviduct to be transected or resected were underrun with 10-0 monofilament nylon suture and ligated. The oviduct was then incised perpendicular to its long axis at the AIJ, using Vannas scissors. In animals undergoing resection of the AIJ, the oviduct was divided above and below the AIJ, defining a segment 1 cm in length. The dissection was continued parallel to and flush with the segment, severing its attachments to the mesosalpinx. The excised segment was retained for histologic confirmation that the AIJ had been resected. Hemostasis was further assisted by constant irrigation with a 1% solution of 1:1000 adrenaline in normal saline. End-to-end tubal anastomosis was performed by using four or five interrupted sutures of 10-0 monofilament nylon placed through the myosalpinx, excluding the endosalpinx (Fig. 2). The defect in the mesoTABLE 1. Pregnancy following Unilateral Transection or Resection of the Ampullary-Isthmic Junction AIJ transection
FIG. 2. Microsurgical technique used to perform end-to-end tubal anastomosis following transection or resection of the AIJ. Sutures are placed through the myosalpinx only, specifically avoiding the endosalpinx.
No. of pregnant uteri Total no. of uteri % Pregnant
AIJ resection
Operated side
Control side
Operated side
9 10 90
8 10 80
10 10 100
Control side
10
1'0 100
EDDY ET AL.
1092
October 1977
TABLE 2. Fertility following Unilateral Microsurgical Transection or Resection of the Ampullary-Isthmic Junction" AIJ transection
No. of corpora lutea No. of implants Nidation index "Values are means
±
AIJ resection
Operated side
Control side
Operated side
Control side
5.1 ± 0.85 2.2 ± 0.53 55.9 ± 12.0
5.4 ± 0.45 2.6 ± 0.63 54.2 ± 13.4
4.7 ± 0.67 2.5 ± 0.34 60.6 ± 8.98
5.0 ± 0.67 3.5 ± 0.40 77.1 ± 7.88
standard error; N
=
10. Differences between row means were not statistically significant (P > 0.05).
salpinx was repaired with interrupted sutures and the anastomosis site reperitonized with additional interrupted sutures (Fig. 3). The abdomen was closed in two layers and the animals were allowed to recover. Fertility Tests. Three to five weeks following microsurgery the animals were mated to a male of proven fertility and given an intravenous injection of 100 IV of human chorionic gonadotropin to ensure ovulation. Two weeks later, laparotomy was performed under sodium pentobarbital anesthesia and the reproductive tracts were examined. The number of corpora lute a on each ovary and the number of implantations in each uterine horn were recorded. The abdomen was again closed in two layers and the pregnancies were allowed to go to term prior to use of the animals in subsequent studies.
RESULTS
Table 1 shows the over-all effect of microsurgery on the establishment of pregnancy. In the group that underwent unilateral AIJ transection only, one animal failed to become pregnant on the operated side whereas two animals. failed to become pregnant on the unoperated control side. In no case, however, did any animal in the group fail to become pregnant on at least one side. All of the animals that underwent unilateral AIJ resection exhibited bilateral pregnancy. The effects of AIJ transection and resection on number of ova ovulated, number of implantations, and the resultant nidation index (number of implantations/number of ovulations x 100) are summarized in Table 2. There was no statistically significant variation in any of the three parameters between operated and control sides of both groups r.p > 0.05). DISCUSSION
Although histologically the transition between ampulla and isthmus is abrupt,9 prospective identification of the AIJ on the basis of gross tubal morphology at laparotomy is difficult. The use of
tubal insumation with methylene blue diluted in saline in conjunction with microscopic examination of the tubal mucosa proved to be a highly effective means of localizing the AIJ, as was subsequently confirmed histologically. The technical adequacy of the microsurgery involved is amply confirmed by the statistically unaltered fertility associated with microsurgical AIJ transection or resection. Previous studies have shown that under normal mating conditions only 4.5% of rabbit ova fail to become fertilized,10 while the over-all prenatal mortality rate is 29.7%.11 The results obtained in the present study compare favorably with the latter figure. Failure of AIJ resection to alter fertility demonstrates that in the rabbit no anatomical sphincter exists at the AIJ. Furthermore, any functional obstruction to ovum transport is not dependent upon the specific presence of the AIJ. In light of recent data 12 in which AIJ resection failed to abolish estrogen-induced tubal blockade of ova at the entrance to the isthmus, it appears likely that it is the isthmus itself, or more simply the transition from ampulla to isthmus, which is responsible for the transient retardation of the ovum's journey through the oviduct. In addition to allowing these biologic inferences, the results ofthese experiments provide the opportunity to gain new insights into the mechanisms controlling tubal gamete transport and emphasize the evolving feasibility and importance of tuboplastic microsurgery as both a research tool and a clinical procedure. Acknowledgment. Appreciation is expressed to Mr. Mark Weakley for the medical illustrations. REFERENCES 1. Blandau RJ: The Mammalian Oviduct. Chicago, Univer-
sity of Chicago Press, 1968, p 129 2. Chang MC: Development and fate oftransferred rabbit ova or blastocyst in relation to the ovulation time of recippients. J Exp Zool 114:197, 1950 3. Brundin J: An occlusive mechanism in the fallopian tube of the rabbit. Acta Physiol Scand 61:219, 1964 4. Brundin J: Distribution and function of adrenergic nerves in the rabbit fallopian tube. Acta Physiol Scand 66:5, 1965
Vol. 28, No. 10
FERTILITY FOLLOWING AMPULLARY -ISTHMIC JUNCTION REMOVAL
5. Hodgson BJ, Eddy CA: The autonomic nervous system and its relationship to tubal ovum transport-a reappraisal. Gynecol Invest 6:162, 1975 6. Winston RML, McClure-Browne JC: Pregnancy following autograft transplantation of fallopian tube and ovary in the rabbit. Lancet 2:494, 1974 7. Paterson P, Wood C: The use of microsurgery in the reanastomosis of the rabbit fallopian tube. Fertil Steril 25: 757, 1974 8. Eddy CA, Hoffman JJ, Pauerstein CJ: Pregnancy following segmental isthmic reversal of the rabbit oviduct. Experientia 32:1194, 1976
1093
9. Pauerstein CJ, Anderson V, Chatkoff ML, Hodgson BJ: Effect of estrogen and progesterone on the time course of tubal ovum transport in rabbits. Am J Obstet Gynecol 120:299, 1974 10. Adams CE: A study offertilization in the rabbit: the effect of post-coital ligation ofthe fallopian tube or uterine horn. J Endocrinol 13:296, 1956 11. Adams CE: Studies on prenatal mortality in the rabbit Oryctolagus cuniculus: the amount and distribution ofloss before and after implantation. J Endocrinol 19:325, 1960 12. Eddy CA, Balmaceda JP, Pauerstein CJ: Effect of resection ofthe ampullary-isthmicjunction on estrogen induced tube locking of ova in the rabbit. BioI Reprod. In press
FERTILITY AND STERILITY Copyright' 1977 The American Fertility Society
FERTILITY FOLLOWING MICROSURGICAL REMOVAL OF THE AMPULLARY-ISTHMIC JUNCTION IN RABBITS*
CARLTON A. EDDY, PH,D.t RENZO ANTONINI, JR., M.D.:!: CARL J. PAUERSTEIN, M.D.
Departments of Obstetrics and Gynecology and Physiology, Center for Research and Training in Reproductive Biology and Voluntary Regulation of Fertility, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
The role of the ampullary-isthmic junction (AIJ) in fertility was investigated in the rabbit by using microsurgical tuboplastic techniques. Microsurgical transection or resection of the AIJ failed to alter fertility significantly. Nine of ten rabbits which underwent unilateral transection of the AIJ became pregnant on the operated side and eight of the ten became pregnant on the unoperated control side. Of the ten rabbits which underwent unilateral AIJ resection, all became pregnant bilaterally. The results of this study indicate that the AIJ is not necessary for normal fertility in the rabbit.
lates neuroendocrine modulation of constriction at the AIJ.3'5 The recent development of microsurgical tuboplastic techniques 6• s provides the means with which to examine tubal function directly. The present study was undertaken to define the role of the AIJ in fertility of the rabbit.
Ovum transport through the mammalian oviduct is a complex, discontinuous process characterized by a pause of varying, species-specific duration at the ampullary-isthmic junction (AIJ) before final entrance of the embryo into the uterus. 1 The functional significance of this pause is unknown but may be important in synchronizing endometrial maturation and embryo development necessary for successful implantation. 2 The mechanism responsible for the pause remains undefined. Several theories have been advanced in explanation of this mechanism, such as transient edema, valve-like structures, and other anatomical and junctional obstructions to the free passage of the ovum or embryo. In the rabbit no anatomical structure has been defined, so that various functional constrictions have been favored. The most prevalent hypothesis po stu-
MATERIALS AND METHODS
Received May 16, 1977; revised June 13, 1977; accepted June 13, 1977. *Presented at the Ninth World Congress on Fertility and Sterility and the Thirty-Third Annual Meeting of The American Fertility Society, April 12 to 16, 1977, Miami Beach, Fla. tReprint requests: Carlton A. Eddy, Ph.D., University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Tex. 78229. :!:Rockefeller Foundation Fellow in Reproductive Biology.
Animals. Twenty adult virgin female New Zealand White rabbits were divided into two groups of ten animals each. In one group the AIJ was unilaterally transected and the oviduct rejoined by using microsurgical end-to-end anastomosis (surgical control group). In the second group a l-cm segment of oviduct containing the AIJ was unilaterally resected and the oviduct similarly anastomosed. Contralateral unoperated oviducts served as controls in both groups. Surgical Technique. Animals were anesthetized by the intravenous administration of sodium pentobarbital (20 to 32 mg/kg). A midventral laparotomy was performed and the reproductive tract exteriorized. A polyethylene catheter (external diameter 0.60 mm) attached to a syringe containing methylene blue diluted in physiologic saline was introduced through the tubal ostium
1090
Vol. 28, No. 10
1091
FERTILITY FOLLOWING AMPULLARY-ISTHMIC JUNCTION REMOVAL
FIG. 1. Appearance of the rabbit oviduct at the level of the AlJ (arrow) following intraluminal perfusion as seen through the operating microscope. The ampulla is to left of the arrow, the isthmus to the right. Note the ampullary distention and the abrupt transition in the mucosal fold pattern from ampulla to isthmus, delineating the position of the AIJ.
into the distal ampulla. Digital pressure was gently applied around the ostium to prevent leakage. A small volume of saline was slowly perfused into the oviduct. The resultant tubal distention was largely limited to the thin-walled ampulla. The limits of distention marked the transition to the thick-walled, nondistended isthmus and thus served to identify the AIJ. Observation of the tubal mucosal pattern through an operating microscope served to delineate further the position of the AIJ (Fig. 1). Lengths of elastic cord 0.5 mm in diameter were placed above and below the area of the AIJ by piercing the mesosalpinx and mesotubarium superius in avascular areas with curved no. 7
FIG. 3. Microsurgical technique used to repair the defect in the mesosalpinx and reperitonize the anastomosis site. Inset, appearance of the anastomosis site following completion of microsurgery.
jeweler's forceps. These cords were then drawn through the supporting structures and tightened sufficiently with small serrefines to occlude the tubal vascular arcade but not the tubal lumen. A Zeiss OPMI 6 operating microscope fitted with a 300-mm objective lens was positioned and the surgery conducted under magnification ranging from x10 to x50. Major collateral blood vessels not occluded by the ligatures in the area of oviduct to be transected or resected were underrun with 10-0 monofilament nylon suture and ligated. The oviduct was then incised perpendicular to its long axis at the AIJ, using Vannas scissors. In animals undergoing resection of the AIJ, the oviduct was divided above and below the AIJ, defining a segment 1 cm in length. The dissection was continued parallel to and flush with the segment, severing its attachments to the mesosalpinx. The excised segment was retained for histologic confirmation that the AIJ had been resected. Hemostasis was further assisted by constant irrigation with a 1% solution of 1:1000 adrenaline in normal saline. End-to-end tubal anastomosis was performed by using four or five interrupted sutures of 10-0 monofilament nylon placed through the myosalpinx, excluding the endosalpinx (Fig. 2). The defect in the mesoTABLE 1. Pregnancy following Unilateral Transection or Resection of the Ampullary-Isthmic Junction AIJ transection
FIG. 2. Microsurgical technique used to perform end-to-end tubal anastomosis following transection or resection of the AIJ. Sutures are placed through the myosalpinx only, specifically avoiding the endosalpinx.
No. of pregnant uteri Total no. of uteri % Pregnant
AIJ resection
Operated side
Control side
Operated side
9 10 90
8 10 80
10 10 100
Control side
10
1'0 100
EDDY ET AL.
1092
October 1977
TABLE 2. Fertility following Unilateral Microsurgical Transection or Resection of the Ampullary-Isthmic Junction" AIJ transection
No. of corpora lutea No. of implants Nidation index "Values are means
±
AIJ resection
Operated side
Control side
Operated side
Control side
5.1 ± 0.85 2.2 ± 0.53 55.9 ± 12.0
5.4 ± 0.45 2.6 ± 0.63 54.2 ± 13.4
4.7 ± 0.67 2.5 ± 0.34 60.6 ± 8.98
5.0 ± 0.67 3.5 ± 0.40 77.1 ± 7.88
standard error; N
=
10. Differences between row means were not statistically significant (P > 0.05).
salpinx was repaired with interrupted sutures and the anastomosis site reperitonized with additional interrupted sutures (Fig. 3). The abdomen was closed in two layers and the animals were allowed to recover. Fertility Tests. Three to five weeks following microsurgery the animals were mated to a male of proven fertility and given an intravenous injection of 100 IV of human chorionic gonadotropin to ensure ovulation. Two weeks later, laparotomy was performed under sodium pentobarbital anesthesia and the reproductive tracts were examined. The number of corpora lute a on each ovary and the number of implantations in each uterine horn were recorded. The abdomen was again closed in two layers and the pregnancies were allowed to go to term prior to use of the animals in subsequent studies.
RESULTS
Table 1 shows the over-all effect of microsurgery on the establishment of pregnancy. In the group that underwent unilateral AIJ transection only, one animal failed to become pregnant on the operated side whereas two animals. failed to become pregnant on the unoperated control side. In no case, however, did any animal in the group fail to become pregnant on at least one side. All of the animals that underwent unilateral AIJ resection exhibited bilateral pregnancy. The effects of AIJ transection and resection on number of ova ovulated, number of implantations, and the resultant nidation index (number of implantations/number of ovulations x 100) are summarized in Table 2. There was no statistically significant variation in any of the three parameters between operated and control sides of both groups r.p > 0.05). DISCUSSION
Although histologically the transition between ampulla and isthmus is abrupt,9 prospective identification of the AIJ on the basis of gross tubal morphology at laparotomy is difficult. The use of
tubal insumation with methylene blue diluted in saline in conjunction with microscopic examination of the tubal mucosa proved to be a highly effective means of localizing the AIJ, as was subsequently confirmed histologically. The technical adequacy of the microsurgery involved is amply confirmed by the statistically unaltered fertility associated with microsurgical AIJ transection or resection. Previous studies have shown that under normal mating conditions only 4.5% of rabbit ova fail to become fertilized,10 while the over-all prenatal mortality rate is 29.7%.11 The results obtained in the present study compare favorably with the latter figure. Failure of AIJ resection to alter fertility demonstrates that in the rabbit no anatomical sphincter exists at the AIJ. Furthermore, any functional obstruction to ovum transport is not dependent upon the specific presence of the AIJ. In light of recent data 12 in which AIJ resection failed to abolish estrogen-induced tubal blockade of ova at the entrance to the isthmus, it appears likely that it is the isthmus itself, or more simply the transition from ampulla to isthmus, which is responsible for the transient retardation of the ovum's journey through the oviduct. In addition to allowing these biologic inferences, the results ofthese experiments provide the opportunity to gain new insights into the mechanisms controlling tubal gamete transport and emphasize the evolving feasibility and importance of tuboplastic microsurgery as both a research tool and a clinical procedure. Acknowledgment. Appreciation is expressed to Mr. Mark Weakley for the medical illustrations. REFERENCES 1. Blandau RJ: The Mammalian Oviduct. Chicago, Univer-
sity of Chicago Press, 1968, p 129 2. Chang MC: Development and fate oftransferred rabbit ova or blastocyst in relation to the ovulation time of recippients. J Exp Zool 114:197, 1950 3. Brundin J: An occlusive mechanism in the fallopian tube of the rabbit. Acta Physiol Scand 61:219, 1964 4. Brundin J: Distribution and function of adrenergic nerves in the rabbit fallopian tube. Acta Physiol Scand 66:5, 1965
Vol. 28, No. 10
FERTILITY FOLLOWING AMPULLARY -ISTHMIC JUNCTION REMOVAL
5. Hodgson BJ, Eddy CA: The autonomic nervous system and its relationship to tubal ovum transport-a reappraisal. Gynecol Invest 6:162, 1975 6. Winston RML, McClure-Browne JC: Pregnancy following autograft transplantation of fallopian tube and ovary in the rabbit. Lancet 2:494, 1974 7. Paterson P, Wood C: The use of microsurgery in the reanastomosis of the rabbit fallopian tube. Fertil Steril 25: 757, 1974 8. Eddy CA, Hoffman JJ, Pauerstein CJ: Pregnancy following segmental isthmic reversal of the rabbit oviduct. Experientia 32:1194, 1976
1093
9. Pauerstein CJ, Anderson V, Chatkoff ML, Hodgson BJ: Effect of estrogen and progesterone on the time course of tubal ovum transport in rabbits. Am J Obstet Gynecol 120:299, 1974 10. Adams CE: A study offertilization in the rabbit: the effect of post-coital ligation ofthe fallopian tube or uterine horn. J Endocrinol 13:296, 1956 11. Adams CE: Studies on prenatal mortality in the rabbit Oryctolagus cuniculus: the amount and distribution ofloss before and after implantation. J Endocrinol 19:325, 1960 12. Eddy CA, Balmaceda JP, Pauerstein CJ: Effect of resection ofthe ampullary-isthmicjunction on estrogen induced tube locking of ova in the rabbit. BioI Reprod. In press