- Email: [email protected]
Orthotopic transplantation of fresh ovarian cortex: a report of two cases
Orthotopic transplantation of fresh ovarian cortex: a report of two cases Jacques Donnez, M.D., Ph.D., Jean Squifflet, M.D., Marie-Madeleine Dolmans, M.D., Belen Martinez-Madrid, V.M.D., Ph.D., Pascale Jadoul, M.D., and Anne Van Langendonckt, B.S., Ph.D. Department of Gynecology, Université Catholique de Louvain, Cliniques Universitaires Saint-Luc, Brussels, Belgium
Objective: To report two cases of orthotopic transplantation of fresh ovarian tissue. Setting: Academic hospital. Patient(s): Two patients with severe endometriosis, who underwent left oophorectomy for recurrent endometriosis. Intervention(s): Ovarian cortex was reimplanted in the heterolateral orthotopic site. Result(s): Biopsies of the grafted tissue were taken 3 months after reimplantation. Viable primordial follicles were found. The presence of a neovascular capillary network was demonstrated. Conclusion(s): Reimplantation of fresh ovarian cortex allows the survival of primordial follicles and may represent an alternative method for the preservation of ovarian cortex when oophorectomy is mandatory. (Fertil Steril威 2005;84:1018.e1–3. ©2005 by American Society for Reproductive Medicine.) Key Words: Ovarian cortex, ovary transplantation, orthotopic
This is a report of the first two cases of orthotopic transplantation of fresh ovarian tissue. To our knowledge, this is the first histological evaluation of the survival of primordial follicles after orthotopic reimplantation of fresh human ovarian tissue. The study was approved by the Ethics Committee of the Catholic University of Louvain. CASE REPORT Patient A A 25-year-old regularly menstruating woman was diagnosed with recurrent left ovarian endometriomas of 9 cm in size. She had previously undergone two laparatomies for left ovarian endometrioma resection in another department. At laparoscopy, the left part of the pelvis was found to be frozen. Severe and dense adhesions with the sigmoid colon and periureteral fibrosis were observed. After lysis of the sigmoid colon, the ureter was freed of periureteral fibrotic tissue. After careful dissection of the ovary, left ovarian vascularization appeared to be compromised and a left oophorectomy was decided upon. Before removal of the left ovary, two strips of 3– 4 by 12 mm of ovarian cortex were taken from residual healthy ovarian tissue. A window was created beneath the right ovarian hilus close to the ovarian blood vessels, as we previously described (1). The two strips of fresh ovarian cortex were placed in the Received April 26, 2005; revised and accepted June 27, 2005. Supported by a grant from the Fonds National de la Recherche Scientifique de Belgique “FNRS-Télévie” 7.4520.02 and a grant from A. Frère. Reprint requests: Professor J. Donnez, Department of Gynecology, Université Catholique de Louvain, Cliniques Universitaires Saint-Luc, Av. Hippocrate, 10 B-1200 Brussels, Belgium (FAX: 32-2-764-95-07; E-mail: [email protected]).
0015-0282/05/$30.00 doi:10.1016/j.fertnstert.2005.06.011
window and covered with Interceed (Ethicon, Johnson and Johnson, Neuchâtel, Switzerland). The remaining ovarian tissue was cryopreserved after sending a small piece (2 by 3 mm) for histological analysis. The patient was treated by GnRH agonist for 3 months and subsequently underwent a second-look laparoscopy for evaluation of residual endometriotic disease. Institutional Review Board and patient consent were obtained to evaluate the grafts. In the grafted area, macroscopically viable-looking ovarian tissue of ⫾1 cm in size was visible with a small follicular structure of 2–3 mm on the surface. A large biopsy was taken. The patient failed to conceive spontaneously and underwent three IVF attempts. She became pregnant on the third attempt. Patient B A 27-year-old woman was diagnosed with recurrent large endometriomas and rectovaginal nodules. She had previously undergone a laparoscopy in another department, which involved ovarian cystectomy for an endometrioma of 4 cm in size and excision of deep rectovaginal nodules. Four months later, she consulted us for a problem of persistent pelvic pain and dyspareunia. Clinical examination and vaginal echography revealed a left endometrioma of 8 cm in size and a rectovaginal nodule of 3– 4 cm in size with left lateral extension. Additional examinations (intravenous pyelography and barium enema) confirmed ureteral substenosis with pyelic dilatation and rectosigmoid perivisceritis. After discussion with the patient, it was decided that the ovarian cortex would be reimplanted if conservative surgery on the left side proved impossible. Because of previous surgery and dense adhesions provoking a frozen pelvis on the left side, but with normal right
Fertility and Sterility姞 Vol. 84, No. 4, October 2005 1018.e1 Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc.
adnexa, we performed sigmoidolysis, ureterolysis, rectovaginal nodule excision, and left oophorectomy. Dissection of the remaining healthy ovarian cortex from endometriotic tissue was performed intraperitoneally. Four strips of 3– 4 by 10 –12 mm were placed in a peritoneal window, as in patient A. The remaining healthy tissue was cryopreserved. A small biopsy was sent for histological analysis. Institutional Review Board and patient consent were obtained to evaluate the grafts.
FIGURE 1 Biopsy of grafted ovarian cortex 3 months after orthotopic transplantation. After collagenase isolation, three partially isolated viable follicles were detected.
Three and a half months later, vaginal echography revealed a normal right ovary of 43 by 27 by 36 cm with a luteal cyst of 26 mm and, at a distance of 2 cm from the right ovary, a follicle of 12 mm in the grafted tissue. Two weeks later, a laparoscopy was carried out. The grafted area showed normal ovarian tissue (size ⫾1 cm) with a small cystic structure (follicle) not covered with peritoneum on the surface, which was biopsied. MATERIALS AND METHODS Biopsies of reimplanted tissue were studied by histology and vital fluorescent staining (calcein-AM and ethidium homodimer-1), according to the technique used by Donnez et al. (1) and Cortvrindt and Smitz (2). RESULTS In patient A, a large biopsy (5 by 5 by 1 mm) was taken and divided into two parts. In one part, three primordial follicles were found and their viability was proved by vital fluorescent staining. Active angiogenesis was demonstrated by the presence of numerous small vessels in the grafted tissue. Some areas of fibrosis were seen. In the other part, six viable follicles were detected after collagenase isolation (Fig. 1). In patient B, biopsy of the small follicular structure revealed granulosa cells but the oocyte was not recovered. Histology of another small biopsy (3 by 3 mm) revealed the presence of three primordial follicles and a primary follicle. Secondary follicles were never seen in either case. Vital fluorescent staining proved that the graft was completely revascularized 3 months after transplantation. The process of revascularization was demonstrated by the presence of numerous small vessels, whose lumen was covered with living endothelial cells. DISCUSSION The first live birth after orthotopic reimplantation of cryopreserved ovarian tissue was recently published in the Lancet but, to date, only five reports have described implantation of fresh human ovarian tissue in humans (autografts) (Table 1). All of them involved heterotopic autografts. Leporrier et al. (3) were the first to report heterotopic subcutaneous transplantation of an ovary to the arm, with 1018.e2 Donnez et al.
Transplantation of fresh ovarian tissue
Donnez. Transplantation of fresh ovarian tissue. Fertil Steril 2005.
vessel anastomosis. Ultrasound examination of the transplanted ovary showed normal follicular growth and ovulation. Accidental subcutaneous transplantation of ovarian tissue was reported by Marconi et al. (4) in 1997. During laparoscopic resection of an endometrioma, a piece of ovarian tissue was left in the subcutaneous area. Excision of a swelling in the umbilical area was performed and histology proved the presence of functional ovarian tissue with antral development and neovascularization. Heterotopic transplantation of fresh tissue to the forearm was described by Oktay et al. (5) in 2001. In one patient, follicle development was demonstrated by ultrasound 10 weeks after transplantation. The second patient experienced menstrual bleeding after 6 months. Long-term ovarian function evaluation after autografting of fresh human ovarian tissue to the abdominal wall was reported by Callejo et al. (6) and Kiran et al. (7), but all the women were premenopausal. Ovarian function was maintained for a short period only, probably due to a very low ovarian follicle reserve. Vol. 84, No. 4, October 2005
TABLE I Human autografts of fresh tissue. References Autografts Heterotopic Leporrier et al. (1987)
Type of graft
Graft site
Results
Left ovary
Forearm
Marconi et al. (1997) Oktay et al. (2001)
Strip Strip
Abdominal wall Forearm
Callejo et al. (2001)
Strip
Abdominal wall
Kiran et al. (2004)
Strip
Abdominal wall
Strip
Pelvic peritoneal window
Survival of primordial follicles, active angiogenesis
Large area of ovarian cortex
Decorticated ovary
Pregnancy
Orthotopic Donnez et al. (present study) Allograft Orthotopic Silber et al. (2005)
Follicular growth, ovulation, oocyte recovery Follicular development Ovulation, aspiration of mature oocytes Restoration of hormonal secretion Restoration of hormonal secretion
Donnez. Transplantation of fresh ovarian tissue. Fertil Steril 2005.
Very recently, Silber et al. (8) reported successful reimplantation of fresh ovarian tissue using ovarian cortex donated by a monozygotous twin. Our findings, as well as those of Silber et al., prove that primordial follicles survive after orthotopic reimplantation of fresh ovarian tissue. Moreover, our study provides histological data after orthotopic transplantation of fresh ovarian cortex. Histological analysis proved primordial follicle survival and the presence of a neovascular network. In conclusion, our report describes orthotopic transplantation of fresh ovarian cortex (autograft), providing proof of the survival of primordial follicles, and describes a new technique to preserve ovarian tissue in case of severe and recurrent ovarian endometriosis when oophorectomy is mandatory. REFERENCES 1. Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;364:1405–10.
Fertility and Sterility姞
2. Cortvrindt RG, Smitz JE. Fluorescent probes allow rapid and precise recording of follicle density and staging in human ovarian cortical biopsy samples. Fertil Steril 2001;75:588 –93. 3. Leporrier M, von Theobald P, Roffe JL, Muller G. A new technique to protect ovarian function before pelvic irradiation. Cancer 1987;60: 2201– 4. 4. Marconi G, Quintana R, Rueda-Leverone NG, Vighi S. Accidental ovarian autograft after a laparoscopic surgery: case report. Fertil Steril 1997;68:364 – 6. 5. Oktay K, Economos K, Kan M, Rucinski J, Veeck L, Rosenwacks Z. Endocrine function and oocyte retrieval after autologous transplantation of ovarian cortical strips to the forearm. JAMA 2001;286: 1490 –3. 6. Callejo J, Salvador C, Miralles A, Vilaseca S, Lailla JM, Balasch J. Long-term ovarian function evaluation after autografting by implantation with fresh and frozen-thawed human ovarian tissue. J Clin Endocrinol Metab 2001;86:4489 –94. 7. Kiran G, Kiran H, Coban YK, Guven AM, Yuksel M. Fresh autologous transplantation of ovarian cortical strips to the anterior abdominal wall at the pfannenstiel incision site. Fertil Steril 2004;82:954 – 6. 8. Silber SJ, Lenahan KM, Levine DJ, Pineda JA, Gorman KS, Friez MJ, et al. Ovarian transplantation between monozygotic twins discordant for premature ovarian failure. N Engl J Med 2005;353:1– 6.
1018.e3
Objective: To report two cases of orthotopic transplantation of fresh ovarian tissue. Setting: Academic hospital. Patient(s): Two patients with severe endometriosis, who underwent left oophorectomy for recurrent endometriosis. Intervention(s): Ovarian cortex was reimplanted in the heterolateral orthotopic site. Result(s): Biopsies of the grafted tissue were taken 3 months after reimplantation. Viable primordial follicles were found. The presence of a neovascular capillary network was demonstrated. Conclusion(s): Reimplantation of fresh ovarian cortex allows the survival of primordial follicles and may represent an alternative method for the preservation of ovarian cortex when oophorectomy is mandatory. (Fertil Steril威 2005;84:1018.e1–3. ©2005 by American Society for Reproductive Medicine.) Key Words: Ovarian cortex, ovary transplantation, orthotopic
This is a report of the first two cases of orthotopic transplantation of fresh ovarian tissue. To our knowledge, this is the first histological evaluation of the survival of primordial follicles after orthotopic reimplantation of fresh human ovarian tissue. The study was approved by the Ethics Committee of the Catholic University of Louvain. CASE REPORT Patient A A 25-year-old regularly menstruating woman was diagnosed with recurrent left ovarian endometriomas of 9 cm in size. She had previously undergone two laparatomies for left ovarian endometrioma resection in another department. At laparoscopy, the left part of the pelvis was found to be frozen. Severe and dense adhesions with the sigmoid colon and periureteral fibrosis were observed. After lysis of the sigmoid colon, the ureter was freed of periureteral fibrotic tissue. After careful dissection of the ovary, left ovarian vascularization appeared to be compromised and a left oophorectomy was decided upon. Before removal of the left ovary, two strips of 3– 4 by 12 mm of ovarian cortex were taken from residual healthy ovarian tissue. A window was created beneath the right ovarian hilus close to the ovarian blood vessels, as we previously described (1). The two strips of fresh ovarian cortex were placed in the Received April 26, 2005; revised and accepted June 27, 2005. Supported by a grant from the Fonds National de la Recherche Scientifique de Belgique “FNRS-Télévie” 7.4520.02 and a grant from A. Frère. Reprint requests: Professor J. Donnez, Department of Gynecology, Université Catholique de Louvain, Cliniques Universitaires Saint-Luc, Av. Hippocrate, 10 B-1200 Brussels, Belgium (FAX: 32-2-764-95-07; E-mail: [email protected]).
0015-0282/05/$30.00 doi:10.1016/j.fertnstert.2005.06.011
window and covered with Interceed (Ethicon, Johnson and Johnson, Neuchâtel, Switzerland). The remaining ovarian tissue was cryopreserved after sending a small piece (2 by 3 mm) for histological analysis. The patient was treated by GnRH agonist for 3 months and subsequently underwent a second-look laparoscopy for evaluation of residual endometriotic disease. Institutional Review Board and patient consent were obtained to evaluate the grafts. In the grafted area, macroscopically viable-looking ovarian tissue of ⫾1 cm in size was visible with a small follicular structure of 2–3 mm on the surface. A large biopsy was taken. The patient failed to conceive spontaneously and underwent three IVF attempts. She became pregnant on the third attempt. Patient B A 27-year-old woman was diagnosed with recurrent large endometriomas and rectovaginal nodules. She had previously undergone a laparoscopy in another department, which involved ovarian cystectomy for an endometrioma of 4 cm in size and excision of deep rectovaginal nodules. Four months later, she consulted us for a problem of persistent pelvic pain and dyspareunia. Clinical examination and vaginal echography revealed a left endometrioma of 8 cm in size and a rectovaginal nodule of 3– 4 cm in size with left lateral extension. Additional examinations (intravenous pyelography and barium enema) confirmed ureteral substenosis with pyelic dilatation and rectosigmoid perivisceritis. After discussion with the patient, it was decided that the ovarian cortex would be reimplanted if conservative surgery on the left side proved impossible. Because of previous surgery and dense adhesions provoking a frozen pelvis on the left side, but with normal right
Fertility and Sterility姞 Vol. 84, No. 4, October 2005 1018.e1 Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc.
adnexa, we performed sigmoidolysis, ureterolysis, rectovaginal nodule excision, and left oophorectomy. Dissection of the remaining healthy ovarian cortex from endometriotic tissue was performed intraperitoneally. Four strips of 3– 4 by 10 –12 mm were placed in a peritoneal window, as in patient A. The remaining healthy tissue was cryopreserved. A small biopsy was sent for histological analysis. Institutional Review Board and patient consent were obtained to evaluate the grafts.
FIGURE 1 Biopsy of grafted ovarian cortex 3 months after orthotopic transplantation. After collagenase isolation, three partially isolated viable follicles were detected.
Three and a half months later, vaginal echography revealed a normal right ovary of 43 by 27 by 36 cm with a luteal cyst of 26 mm and, at a distance of 2 cm from the right ovary, a follicle of 12 mm in the grafted tissue. Two weeks later, a laparoscopy was carried out. The grafted area showed normal ovarian tissue (size ⫾1 cm) with a small cystic structure (follicle) not covered with peritoneum on the surface, which was biopsied. MATERIALS AND METHODS Biopsies of reimplanted tissue were studied by histology and vital fluorescent staining (calcein-AM and ethidium homodimer-1), according to the technique used by Donnez et al. (1) and Cortvrindt and Smitz (2). RESULTS In patient A, a large biopsy (5 by 5 by 1 mm) was taken and divided into two parts. In one part, three primordial follicles were found and their viability was proved by vital fluorescent staining. Active angiogenesis was demonstrated by the presence of numerous small vessels in the grafted tissue. Some areas of fibrosis were seen. In the other part, six viable follicles were detected after collagenase isolation (Fig. 1). In patient B, biopsy of the small follicular structure revealed granulosa cells but the oocyte was not recovered. Histology of another small biopsy (3 by 3 mm) revealed the presence of three primordial follicles and a primary follicle. Secondary follicles were never seen in either case. Vital fluorescent staining proved that the graft was completely revascularized 3 months after transplantation. The process of revascularization was demonstrated by the presence of numerous small vessels, whose lumen was covered with living endothelial cells. DISCUSSION The first live birth after orthotopic reimplantation of cryopreserved ovarian tissue was recently published in the Lancet but, to date, only five reports have described implantation of fresh human ovarian tissue in humans (autografts) (Table 1). All of them involved heterotopic autografts. Leporrier et al. (3) were the first to report heterotopic subcutaneous transplantation of an ovary to the arm, with 1018.e2 Donnez et al.
Transplantation of fresh ovarian tissue
Donnez. Transplantation of fresh ovarian tissue. Fertil Steril 2005.
vessel anastomosis. Ultrasound examination of the transplanted ovary showed normal follicular growth and ovulation. Accidental subcutaneous transplantation of ovarian tissue was reported by Marconi et al. (4) in 1997. During laparoscopic resection of an endometrioma, a piece of ovarian tissue was left in the subcutaneous area. Excision of a swelling in the umbilical area was performed and histology proved the presence of functional ovarian tissue with antral development and neovascularization. Heterotopic transplantation of fresh tissue to the forearm was described by Oktay et al. (5) in 2001. In one patient, follicle development was demonstrated by ultrasound 10 weeks after transplantation. The second patient experienced menstrual bleeding after 6 months. Long-term ovarian function evaluation after autografting of fresh human ovarian tissue to the abdominal wall was reported by Callejo et al. (6) and Kiran et al. (7), but all the women were premenopausal. Ovarian function was maintained for a short period only, probably due to a very low ovarian follicle reserve. Vol. 84, No. 4, October 2005
TABLE I Human autografts of fresh tissue. References Autografts Heterotopic Leporrier et al. (1987)
Type of graft
Graft site
Results
Left ovary
Forearm
Marconi et al. (1997) Oktay et al. (2001)
Strip Strip
Abdominal wall Forearm
Callejo et al. (2001)
Strip
Abdominal wall
Kiran et al. (2004)
Strip
Abdominal wall
Strip
Pelvic peritoneal window
Survival of primordial follicles, active angiogenesis
Large area of ovarian cortex
Decorticated ovary
Pregnancy
Orthotopic Donnez et al. (present study) Allograft Orthotopic Silber et al. (2005)
Follicular growth, ovulation, oocyte recovery Follicular development Ovulation, aspiration of mature oocytes Restoration of hormonal secretion Restoration of hormonal secretion
Donnez. Transplantation of fresh ovarian tissue. Fertil Steril 2005.
Very recently, Silber et al. (8) reported successful reimplantation of fresh ovarian tissue using ovarian cortex donated by a monozygotous twin. Our findings, as well as those of Silber et al., prove that primordial follicles survive after orthotopic reimplantation of fresh ovarian tissue. Moreover, our study provides histological data after orthotopic transplantation of fresh ovarian cortex. Histological analysis proved primordial follicle survival and the presence of a neovascular network. In conclusion, our report describes orthotopic transplantation of fresh ovarian cortex (autograft), providing proof of the survival of primordial follicles, and describes a new technique to preserve ovarian tissue in case of severe and recurrent ovarian endometriosis when oophorectomy is mandatory. REFERENCES 1. Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;364:1405–10.
Fertility and Sterility姞
2. Cortvrindt RG, Smitz JE. Fluorescent probes allow rapid and precise recording of follicle density and staging in human ovarian cortical biopsy samples. Fertil Steril 2001;75:588 –93. 3. Leporrier M, von Theobald P, Roffe JL, Muller G. A new technique to protect ovarian function before pelvic irradiation. Cancer 1987;60: 2201– 4. 4. Marconi G, Quintana R, Rueda-Leverone NG, Vighi S. Accidental ovarian autograft after a laparoscopic surgery: case report. Fertil Steril 1997;68:364 – 6. 5. Oktay K, Economos K, Kan M, Rucinski J, Veeck L, Rosenwacks Z. Endocrine function and oocyte retrieval after autologous transplantation of ovarian cortical strips to the forearm. JAMA 2001;286: 1490 –3. 6. Callejo J, Salvador C, Miralles A, Vilaseca S, Lailla JM, Balasch J. Long-term ovarian function evaluation after autografting by implantation with fresh and frozen-thawed human ovarian tissue. J Clin Endocrinol Metab 2001;86:4489 –94. 7. Kiran G, Kiran H, Coban YK, Guven AM, Yuksel M. Fresh autologous transplantation of ovarian cortical strips to the anterior abdominal wall at the pfannenstiel incision site. Fertil Steril 2004;82:954 – 6. 8. Silber SJ, Lenahan KM, Levine DJ, Pineda JA, Gorman KS, Friez MJ, et al. Ovarian transplantation between monozygotic twins discordant for premature ovarian failure. N Engl J Med 2005;353:1– 6.
1018.e3