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Cryopreservation of eleven whole human ovaries: histology, immunohistochemistry and technical details
were completely normal with good interval growth and fetal heartbeat of 170. A healthy pregnancy ensued. Follow-up bone scans one and a half years postop showed continuing resolution of her osteoporosis. CONCLUSIONS: To our knowledge, this is the first successful human intact whole ovary transplant leading to healthy pregnancy, now approaching term. The low serum FSH suggested minimal effects of the brief ischemia on ovarian reserve. The delay of 101 days (3 months) until the first menstruation precisely supports the theories of Gougeon on the dynamics of follicular growth in humans, i.e., that it requires 85 days for eggs in primordial follicles leaving their resting phase to reach sufficient maturity to enter the menstrual cycle and ovulate. Supported by: None.
Monday, November 10, 2008 3:30 pm O-103 CRYOPRESERVATION OF WHOLE HUMAN OVARIES TOGETHER WITH FALLOPIAN TUBES. P. Patrizio, A. Arav, J. Bromer, M. Martel, M. Azodi, S. Silber. Yale University Fertility Center, Yale University, New Haven, CT; Institute Animal Science, Volcani Center, Bet Dagan, Israel; Gynecology Oncology, Yale University, New Haven, CT; Infertility Center St. Louis, St. Luke’s Hospital, St. Louis, MO. OBJECTIVE: To report cryopreservation of whole human ovaries together with fallopian tubes and to assess the histological survival after thawing. DESIGN: Experimental research using cryopreservation/thawing of whole human ovaries and fallopian tubes. MATERIALS AND METHODS: Three patients (5 ovaries and 5 fallopian tubes) undergoing bilateral salpingo-oophorectomy were consented. Three ovaries with the accompanying fallopian tubes (transected at the isthmic level and with intact mesosalpinx) were allocated to the freeze/thaw experiment, while the contralateral served as fresh controls. For freezing, the ovarian artery was first perfused with PBS containing 200 iu/ml heparin, followed by cold (4 C) UW solution with 10% EG for 3 minutes. The same cryoprotectant was used to perfuse the lumen of the fallopian tube, after which both organs were loaded in a 26mm (OD) glass tube filled with UW 10%EG. Organ solidification using a unidirectional multi-gradient freezing device (MTGÒ, Core-Dynamics, Israel) reduced the temperature to –40 C (cooling rate of 0.3 C/min), after which the cryovials were plunged into LN. Thawing occurred 48 hrs later, placing the cryovials in 68 C water bath for 30 seconds and at 37 C for 2 minutes. The cryoprotectant solution was rinsed out with PBS containing 0.5M sucrose and 10 iu/ml heparin; the ovaries with the fallopian tubes were fixed in formalin and embedded in paraffin. Hematoxilin and eosin stained sections were analyzed by light microscopy. RESULTS: All three ovaries and fallopian tubes survived the freeze/thaw process without damage. The ovarian and tubal components showed normal architecture and the ovarian vascular pedicles and the mesosalpinx were intact and well preserved. Histological preparations of the tubal mucosa in all the three portions-isthmic, ampullary and fimbrial- as well the architecture of the fimbrial digitations, showed no significant differences from the fresh controls. The ovarian cortical components, including follicles and small vessels were morphologically similar in the frozen-thawed ovaries compared to the contralateral non-frozen. CONCLUSIONS: This is the first report of cryopreservation of whole human ovaries together with fallopian tubes. The excellent histological survival of both ovaries and fallopian tubes sets the stage for a new chapter in reproductive organ transplantation. In addition to whole ovary transplantation it is possible now to consider fallopian tube transplant for women with irreparable tubal disease. Supported by: None.
Monday, November 10, 2008 3:45 pm O-104 CRYOPRESERVATION OF ELEVEN WHOLE HUMAN OVARIES: HISTOLOGY, IMMUNOHISTOCHEMISTRY AND TECHNICAL DETAILS. P. Patrizio, J. Bromer, J. Johnson, M. Martel, S. Silber, A. Arav. Yale University Fertility Center, Yale University, New Haven, CT; Yale University, New Haven, CT; Pathology, Yale University, New Haven, CT; Infertility Center St. Louis, St. Luke’s Hospital, St. Louis, MO; Institute Animal Science, Volcani Center, Bet Dagan, Bet Dagan, Israel.
S38
Abstracts
OBJECTIVE: To report cryopreservation of whole human ovaries, their survival after thawing and technical details for successful ovarian perfusion. DESIGN: Experimental research. MATERIALS AND METHODS: A total of 11 pre-menopausal patients (22 ovaries; age range 35 to 48) undergoing bilateral oophorectomy, were consented. One ovary was allocated to the freeze/thaw, while the contralateral served as fresh control. Before freezing, the ovarian artery was first perfused with PBS containing 200 iu/ml heparin followed by cryo-solution of cold (4 C) UW with 10% EG for 3 minutes. Unidirectional solidification using a multi-gradient freezing device (CoreDynamics, Israel) reduced the temperature to –40 C (cooling rate of 0.3 C/min), after which the cryovial glass tubes were plunged into LN. Thawing occurred between 48 to 96 hrs. by immersing the tubes in 68 C water bath for 30 seconds and then at 37 C for 2 minutes. The cryo-solution was rinsed out using PBS with 0.5M sucrose and 10 iu/ml heparin. The thawed ovaries were fixed in formalin and embedded for hematoxylin and eosin sections analyzed by light microscopy. Pre-freeze and post-thaw cortical biopsies were biochemically assessed for tissue viability (CellTiterGLO-assay); immuno-histochemistry (IHC) for phospho-p53 Serine 15 (P-p53 Ser15) and active caspase 3; and Western blotting (WB). RESULTS: All the ovaries survived the freeze/thaw and showed intact ovarian and vascular components. In 3 of the 11 ovaries (27%) the perfusion was suboptimal for an extremely short (<1 cm.) pedicle. Histological preparations revealed similar morphology (and number) of follicles, cortical stroma, and hilar and other small blood vessels in the frozen-thawed ovaries (including those sub-optimally perfused) compared to fresh controls. In all specimens, biochemical tissue viability was not different between fresh and frozen/thawed tissue. The IHC preparations revealed some increase in P-p53 Ser 15 (also confirmed by WB) and active caspase 3 in frozen/thawed tissue vs. controls. CONCLUSIONS: This report documents the largest series of whole human ovary cryopreservation experiments. The effectiveness of unidirectional freezing is confirmed by maintenance of tissue ultrastructure and excellent biochemical viability. The increase in P-p53 Ser 15 supports the presence of some cryodamage, but the clinical significance for re-transplantation success is unclear. It is very important to leave a long infundibulopelvic pedicle (R 2 cm.) for proper organ perfusion and future re-anastomosis. Supported by: None.
Monday, November 10, 2008 4:00 pm O-105 SALINE INFUSION SONOGRAPHY VS. FLEXIBLE OFFICE HYSTEROSCOPY, COMPARING PATIENT SATISFACTION AND PAIN PERCEPTION. S. Senapati, K. C. Wang, D. I. Lebovic, A. H. Song, S. As-Sanie. Obstetrics and Gynecology, Evanston Northwestern Healthcare, Evanston, IL; Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI; University of Michigan, Ann Arbor, MI. OBJECTIVE: Abnormal uterine bleeding is a common complaint presenting to gynecology clinics. Work-up of this is varied given the multiple diagnostic tools that can be utilized. Studies have demonstrated that saline infusion sonography (SIS) and office hysteroscopy have similar diagnostic ability. Few studies have examined which procedure is better tolerated. The purpose of this study is to determine if there is a difference in pain perception and satisfaction in patients undergoing SIS compared to flexible office hysteroscopy (fOHS). DESIGN: Prospective, randomized, cross-over study. MATERIALS AND METHODS: Women who were scheduled to have fOHS from 6/2006-4/2008 were eligible. Following enrollment, participants were randomized to either having the fOHS first or the SIS. A fifteen minute washout period was allowed prior to the subsequent procedure. After each procedure, participants completed a questionnaire regarding their experience. Pain and tolerance were analyzed with a Visual Analogue Scale (VAS). RESULTS: 42 women enrolled. Average age was 40.5 years. No significant difference was noted in demographics between the two groups.76% Caucasian, 79% parous, and 12% menopausal. Average time taken for fOHS was 255 seconds vs. 296 seconds for SIS. There was no significant difference between identification of pathology between with the two imaging
Vol. 90, Suppl 1, September 2008
Monday, November 10, 2008 3:30 pm O-103 CRYOPRESERVATION OF WHOLE HUMAN OVARIES TOGETHER WITH FALLOPIAN TUBES. P. Patrizio, A. Arav, J. Bromer, M. Martel, M. Azodi, S. Silber. Yale University Fertility Center, Yale University, New Haven, CT; Institute Animal Science, Volcani Center, Bet Dagan, Israel; Gynecology Oncology, Yale University, New Haven, CT; Infertility Center St. Louis, St. Luke’s Hospital, St. Louis, MO. OBJECTIVE: To report cryopreservation of whole human ovaries together with fallopian tubes and to assess the histological survival after thawing. DESIGN: Experimental research using cryopreservation/thawing of whole human ovaries and fallopian tubes. MATERIALS AND METHODS: Three patients (5 ovaries and 5 fallopian tubes) undergoing bilateral salpingo-oophorectomy were consented. Three ovaries with the accompanying fallopian tubes (transected at the isthmic level and with intact mesosalpinx) were allocated to the freeze/thaw experiment, while the contralateral served as fresh controls. For freezing, the ovarian artery was first perfused with PBS containing 200 iu/ml heparin, followed by cold (4 C) UW solution with 10% EG for 3 minutes. The same cryoprotectant was used to perfuse the lumen of the fallopian tube, after which both organs were loaded in a 26mm (OD) glass tube filled with UW 10%EG. Organ solidification using a unidirectional multi-gradient freezing device (MTGÒ, Core-Dynamics, Israel) reduced the temperature to –40 C (cooling rate of 0.3 C/min), after which the cryovials were plunged into LN. Thawing occurred 48 hrs later, placing the cryovials in 68 C water bath for 30 seconds and at 37 C for 2 minutes. The cryoprotectant solution was rinsed out with PBS containing 0.5M sucrose and 10 iu/ml heparin; the ovaries with the fallopian tubes were fixed in formalin and embedded in paraffin. Hematoxilin and eosin stained sections were analyzed by light microscopy. RESULTS: All three ovaries and fallopian tubes survived the freeze/thaw process without damage. The ovarian and tubal components showed normal architecture and the ovarian vascular pedicles and the mesosalpinx were intact and well preserved. Histological preparations of the tubal mucosa in all the three portions-isthmic, ampullary and fimbrial- as well the architecture of the fimbrial digitations, showed no significant differences from the fresh controls. The ovarian cortical components, including follicles and small vessels were morphologically similar in the frozen-thawed ovaries compared to the contralateral non-frozen. CONCLUSIONS: This is the first report of cryopreservation of whole human ovaries together with fallopian tubes. The excellent histological survival of both ovaries and fallopian tubes sets the stage for a new chapter in reproductive organ transplantation. In addition to whole ovary transplantation it is possible now to consider fallopian tube transplant for women with irreparable tubal disease. Supported by: None.
Monday, November 10, 2008 3:45 pm O-104 CRYOPRESERVATION OF ELEVEN WHOLE HUMAN OVARIES: HISTOLOGY, IMMUNOHISTOCHEMISTRY AND TECHNICAL DETAILS. P. Patrizio, J. Bromer, J. Johnson, M. Martel, S. Silber, A. Arav. Yale University Fertility Center, Yale University, New Haven, CT; Yale University, New Haven, CT; Pathology, Yale University, New Haven, CT; Infertility Center St. Louis, St. Luke’s Hospital, St. Louis, MO; Institute Animal Science, Volcani Center, Bet Dagan, Bet Dagan, Israel.
S38
Abstracts
OBJECTIVE: To report cryopreservation of whole human ovaries, their survival after thawing and technical details for successful ovarian perfusion. DESIGN: Experimental research. MATERIALS AND METHODS: A total of 11 pre-menopausal patients (22 ovaries; age range 35 to 48) undergoing bilateral oophorectomy, were consented. One ovary was allocated to the freeze/thaw, while the contralateral served as fresh control. Before freezing, the ovarian artery was first perfused with PBS containing 200 iu/ml heparin followed by cryo-solution of cold (4 C) UW with 10% EG for 3 minutes. Unidirectional solidification using a multi-gradient freezing device (CoreDynamics, Israel) reduced the temperature to –40 C (cooling rate of 0.3 C/min), after which the cryovial glass tubes were plunged into LN. Thawing occurred between 48 to 96 hrs. by immersing the tubes in 68 C water bath for 30 seconds and then at 37 C for 2 minutes. The cryo-solution was rinsed out using PBS with 0.5M sucrose and 10 iu/ml heparin. The thawed ovaries were fixed in formalin and embedded for hematoxylin and eosin sections analyzed by light microscopy. Pre-freeze and post-thaw cortical biopsies were biochemically assessed for tissue viability (CellTiterGLO-assay); immuno-histochemistry (IHC) for phospho-p53 Serine 15 (P-p53 Ser15) and active caspase 3; and Western blotting (WB). RESULTS: All the ovaries survived the freeze/thaw and showed intact ovarian and vascular components. In 3 of the 11 ovaries (27%) the perfusion was suboptimal for an extremely short (<1 cm.) pedicle. Histological preparations revealed similar morphology (and number) of follicles, cortical stroma, and hilar and other small blood vessels in the frozen-thawed ovaries (including those sub-optimally perfused) compared to fresh controls. In all specimens, biochemical tissue viability was not different between fresh and frozen/thawed tissue. The IHC preparations revealed some increase in P-p53 Ser 15 (also confirmed by WB) and active caspase 3 in frozen/thawed tissue vs. controls. CONCLUSIONS: This report documents the largest series of whole human ovary cryopreservation experiments. The effectiveness of unidirectional freezing is confirmed by maintenance of tissue ultrastructure and excellent biochemical viability. The increase in P-p53 Ser 15 supports the presence of some cryodamage, but the clinical significance for re-transplantation success is unclear. It is very important to leave a long infundibulopelvic pedicle (R 2 cm.) for proper organ perfusion and future re-anastomosis. Supported by: None.
Monday, November 10, 2008 4:00 pm O-105 SALINE INFUSION SONOGRAPHY VS. FLEXIBLE OFFICE HYSTEROSCOPY, COMPARING PATIENT SATISFACTION AND PAIN PERCEPTION. S. Senapati, K. C. Wang, D. I. Lebovic, A. H. Song, S. As-Sanie. Obstetrics and Gynecology, Evanston Northwestern Healthcare, Evanston, IL; Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI; University of Michigan, Ann Arbor, MI. OBJECTIVE: Abnormal uterine bleeding is a common complaint presenting to gynecology clinics. Work-up of this is varied given the multiple diagnostic tools that can be utilized. Studies have demonstrated that saline infusion sonography (SIS) and office hysteroscopy have similar diagnostic ability. Few studies have examined which procedure is better tolerated. The purpose of this study is to determine if there is a difference in pain perception and satisfaction in patients undergoing SIS compared to flexible office hysteroscopy (fOHS). DESIGN: Prospective, randomized, cross-over study. MATERIALS AND METHODS: Women who were scheduled to have fOHS from 6/2006-4/2008 were eligible. Following enrollment, participants were randomized to either having the fOHS first or the SIS. A fifteen minute washout period was allowed prior to the subsequent procedure. After each procedure, participants completed a questionnaire regarding their experience. Pain and tolerance were analyzed with a Visual Analogue Scale (VAS). RESULTS: 42 women enrolled. Average age was 40.5 years. No significant difference was noted in demographics between the two groups.76% Caucasian, 79% parous, and 12% menopausal. Average time taken for fOHS was 255 seconds vs. 296 seconds for SIS. There was no significant difference between identification of pathology between with the two imaging
Vol. 90, Suppl 1, September 2008