Relationship Between Structure in the Frozen State and Viability of Thawed Ovarian Tissue After Cryopreservation

(62.3% more likely than Caucasians) after Asians. As hospital size increased, hysterectomy rates significantly decreased while myomectomy rates increased. Medicaid patients had significantly higher rates of hysterectomies (20.7%) and approximately three times fewer myomectomies than private paying patients. Overall, the Midwest, South, and Western regions of the country had significantly higher rates of hysterectomies and lower rates of myomectomies than the Northeast. CONCLUSIONS: The number of patients treated for uterine leiomyomas in the inpatient setting steadily increased from 1998-2002. Analyzing UL treatment patterns by patient and socioeconomic characteristics demonstrate that significant treatment variation exists across inpatients and suggests that unexplained disparities may exist. In light of the growing prevalence of inpatient treatment of uterine leiomyomas and the invasive nature of surgical treatments, more research is needed to understand which surgeries are most appropriate, and when women can benefit from more conservative, non-surgical treatment. Supported by: Dr. Becker is the Principal Investigator on a research grant from TAP Pharmaceutical Products Inc., and Dr. Horowitz has received consultative compensation for clinical comments and feedback.

CRYOPRESERVATION AND FROZEN EMBRYO TRANSFER Monday, October 17, 2005 3:45 p.m. O-81 Alteration of Protein Expression in Bovine Ovarian Tissue After Cryopreservation: Slow Freezing vs. Vitrification. S. Kim, H. Kim, H. C. Lee, H. Yang, H. H. Lee, Y. Yoon. Cedars-Sinai Medical Center, Los Angeles, CA; Hanyang University, College of Natural Sciences, Seoul, Republic of Korea; Eulji Life Science Institute, Seoul, Republic of Korea. OBJECTIVE: Ovarian tissue cryopreservation has been recognized as a promising strategy to preserve fertility in women with cancer. Ovarian tissue can be frozen successfully by slow freezing or vitrification. Nevertheless, the full scope of cryoinjury has not been discovered. The aims of this study are 1) to explore cryoinjury of ovarian tissue in the molecular level using proteomics technology, 2) to compare the changes in protein expression between the fresh, slow freezing and vitrification groups, 3) to identify altered proteins and their functions with significance. DESIGN: Controlled laboratory animal study MATERIALS AND METHODS: Fresh bovine ovaries were obtained from a local abattoir and processed to thin cortical sections. Prepared cortical sections were divided into three groups: fresh (A), slow freezing (B), and vitrification (C). Samples in the (B) group were frozen using a programmable freezer after equilibrating in 1.5 M DMSO. Samples in the (C) group were vitrified using 5.5M ethylene glycol as a cryoptotectant. Thawed ovarian tissue was homogenated and run on 2D gel electrophoresis. Quantitative analysis of digitalized images was carried out using the PDQuest software. Subsequently, protein spots in 2D gels were enzymatically digested and analyzed by MALDI-TOF. Proteins were identified by peptide mass finger printing. RESULTS: The total number of countable protein spots was 705 in fresh tissue, 646 in slow frozen tissue, and 708 in vitrified tissue. Thirty four spots showed a significant difference in density between the groups. From 34 spots, 31 proteins were identified using the protein database. All 31 identified proteins were expressed in fresh tissue, however, only 24 proteins in the slow freezing group and 27 in the vitrification group were expressed. The proteins suppressed after slow freezing and rapid thawing were related to actin and matrix proteins. However, laminin and gamma-actin were expressed normally in the vitrification group. The expression of an antioxidant related protein, Glutathione S-transferase, was missing in the slow freezing as well as vitrification group. CONCLUSIONS: We identified 31 proteins with significance in their expression after freezing and thawing. Identified proteins were mainly related to cytoskeletal structures and metabolism. Therefore, alteration of these proteins as a result of freezing and thawing could affect tissue survival. The present study suggested that integrity of proteins in ovarian tissue could be damaged by slow freezing, but in lesser degree by vitrification. Further investigation is required to confirm it. Supported by: Korea Research Foundation

S34

Abstracts

Monday, October 17, 2005 4:00 p.m. O-82 Slow Freezing Alters the Proteome of Mouse Oocytes. M. G. Katz-Jaffe, C. Sheehan, W. B. Schoolcraft, D. K. Gardner. Colorado Center for Reproductive Medicine, Englewood, CO. OBJECTIVE: Oocyte cryopreservation holds enormous promise for assisted conception and fertility preservation in women. Methods used to date include both slow freezing and vitrification. However, there is limited basic research on the effects of either approach on the physiology of oocytes from animal models. It was therefore the aim of this work to determine the effects of slow freezing and vitrification on the protein composition (proteome) of mouse oocytes, facilitated by the development of a new system utilizing time-of-flight mass spectrometry. DESIGN: Experimental study MATERIALS AND METHODS: Metaphase II oocytes were collected from superovulated (C57BL/6 x CBA) mice at 12-14h post hCG. Denuded metaphase II oocytes were then cryopreserved using either a 1,2-propanediol slow freezing procedure or a two-step vitrification procedure using ethylene glycol and DMSO. Following cryopreservation groups of five oocytes were extracted, processed and analyzed by time-of-flight mass spectrometry. In-vivo metaphase II oocytes that were not cryopreserved served as the control. RESULTS: Slow freezing of oocytes resulted in the greatest differences between the two treatments and the control group. Both high and low molecular weight proteins/biomarkers were observed at significantly lower abundance following slow freezing (P⬍0.01). Interestingly of those proteins/biomarkers affected by slow freezing, one had a molecular weight of 44 kDa, which is consistent with the protein MAP kinase. In contrast oocytes that had undergone vitrification displayed a protein profile similar to that of the non-cryopreserved controls. CONCLUSIONS: Analysis of the oocyte following cryopreservation revealed that slow freezing induces significant perturbations of the proteome, indicating altered cell function. In contrast, vitrification had a minimal impact on the oocyte proteome. These data suggest that slow freezing induces greater trauma to the oocyte than vitrification. Possible mechanisms for such trauma include, damage to the plasma membrane resulting in protein efflux and/or degradation of the oocyte proteome. This approach to cellular function should help to facilitate improvements in cryopreservation regimens. Supported by: None

Monday, October 17, 2005 4:15 p.m. O-83 Relationship Between Structure in the Frozen State and Viability of Thawed Ovarian Tissue After Cryopreservation. R. G. Gosden, G. Morris, H. Yin, R. J. Bodine, K. Oktay. Weill Medical College of Cornell University, New York, NY; Asymptote Ltd, Cambridge, United Kingdom. OBJECTIVE: The relationship between cell morphology in the frozen state and viability on thawing is well-established for cell suspensions, including embryos and sperm. However, there are no equivalent data for tissues. This study compared the ultrastructure of cryopreserved rabbit ovarian tissue at -196°C after three different freezing protocols with tissue function in xenograft model. DESIGN: Laboratory analysis of the character and distribution of intracellular and extracellular ice in animal tissue cooled at different rates and analyzed using electron microscopy at liquid nitrogen temperatures (LN). MATERIALS AND METHODS: Adult rabbit ovaries were bisected longitudinally into 1.0-1.5 mm strips and randomized for: 1) equilibration in 1.5M 1,2-propanediol ⫹ 0.2M sucrose and cooling in a Planer automated freezer at -2°C min-1 followed by either (a) 0.3°C min-1 to -40°C, or (b) 3.0°C min-1 to -40°C, followed by cooling at 10°C min-1 to -140°C and plunging in LN; 2) direct plunging into LN (⬃300°C min-1) after equilibrating with the same cryoprotectants. Samples were stored in the same dewar. The cryovials were fractured under LN to dissect the tissue from the ice under LN using cooled forceps and scalpels. The tissue was cross-

Vol. 84, Suppl 1, September 2005

fractured and loaded onto the cryo-stage of a cryoSEM (Oxford Instruments XL30-FEG) without removing from LN. The stage was then warmed from -145°C to -90°C for etching the sample for 6 min. before recooling to -145°C. The sample was transferred to a stage for coating with 10-15 nm gold and returned to the cryoSEM stage for image recording. Specimens were also refractured into 1-2 mm thick segments and transferred under LN to a medium substitution chamber in a Reichert AFS containing 2% osmium tetroxide and 1% uranyl acetate in methyl alcohol. Samples were maintained at -90°C for 24h, warmed to -70°C at 3°C h-1 and then maintained at -70°C for 24h. They were warmed to room temperature at 3°C h-1, rinsed in methyl alcohol and embedded in Spurr’s epoxy resin. Sections were cut at 0.5␮m, stained with methylene blue and photographed using a Zeiss Axiophot. RESULTS: Protocol 1(a) was shown to be most compatible with tissue survival in a SCID mouse xenograft model. All ovarian tissue samples had an extensive internal network of ice whose structure was non-homogeneous, suggesting that ice had not propagated uniformly. After slow cooling [1(a)], ovarian tissue contained large ice crystals appearing to have propagated centripetally from the surface. Cells were compacted between crystals and extremely dehydrated. After faster cooling [1(b)], large ice crystals were still evident in tissue, but the cells were less compacted, and intracellular ice was evident in some of them. After cooling by plunging [2], the ice crystal dimensions were much smaller and cells contained intracellular and intraorganelle ice. CONCLUSIONS: Most evidence suggests that slow cooling produces the highest rates of follicular tissue survival and development after grafting. This protocol was found to create extreme cellular dehydration and disruption of tissue organization in the frozen state, but minimal intracellular damage. At faster rates of cooling, the organization was less perturbed but intracellular and intraorganelle ice was observed. The correspondence between findings from in vivo studies and the functional implications from ultrastructural evidence suggests that CryoSEM is a valuable technique for optimizing cryopreservation protocols for tissues. Supported by: None

Monday, October 17, 2005 4:30 p.m. O-84 Improving Oocyte Cryopreservation by Analyzing the Effects of Cryoprotectants on Intracellular Calcium. M. G. Larman, C. Sheehan, D. K. Gardner. Colorado Center for Reproductive Medicine, Englewood, CO. OBJECTIVE: Chen reported the first pregnancy following IVF of a cryopreserved human oocyte in 1986. Despite this only around 100 children have resulted from oocyte freezing techniques, to date. Together with the fact that the number of births per number of frozen oocytes is no greater than 5%, it is clear that oocyte freezing is an inefficient process at present. Cryopreservation of embryos is considered almost routine, which suggests that the oocyte is inherently sensitive to currently implemented freezing protocols. This is not surprising considering changes in an oocytes environment can lead to precocious activation events. One example of this is the induction of zona hardening during cryopreservation, which significantly inhibits IVF and may affect subsequent implantation. Zona hardening is brought about by fusion of cortical granules to the plasma membrane and the release of their contents into the zona pellucida layers. The membrane fusion event is calcium-dependent and is normally triggered by the increase in intracellular calcium initiated by sperm-egg fusion. Zona hardening can be overcome by ICSI, but the fact that the oocyte has undergone the cortical granule reaction means that the oocyte has been artificially activated prior to actual fertilization. This might explain the poor efficiency of oocyte freezing. Since the cortical granule reaction is calcium-dependent we investigated if commonly used cryoprotectants affect the intracellular calcium concentration ([Ca2⫹]i). We subsequently used this data to assess the affect of calcium-free vitrification and empirically determine the optimal cryoprotectant concentrations and combinations to reduce any calcium increase, therefore increasing IVF success. DESIGN: Experimental Study MATERIALS AND METHODS: Changes in [Ca2⫹]i of F1 (C57BL/6 x CBA) mouse eggs were recorded using whole cell fluorescence with Indo-1. Vitrification was carried out in a two-step cryoloop method with a nomi-

FERTILITY & STERILITY威

nally-free or calcium containing base medium and appropriate concentrations of cyroprotectant(s). RESULTS: It was determined that DMSO, ethylene glycol and 1,2propanediol all caused increases in oocyte [Ca2⫹]i. However, each of the calcium release profiles differed as did their response to calcium-free incubation. DMSO and ethylene glycol both caused transient increases in calcium whereas 1,2-propanediol maintained elevated calcium during the course of the exposure. Furthermore, the calcium response to both ethylene glycol and 1,2-propanediol were significantly reduced in calcium-free media, whereas the DMSO response was unaffected. CONCLUSIONS: Because DMSO was unaffected by calcium-free treatment it suggests that the calcium increase is solely derived from internal stores. In contrast, both ethylene glycol and 1,2-propanediol appear to cause an influx of calcium from the external medium that can be prevented by calcium-free treatment. Based on these results we have designed a protocol that reduces zona hardening, thus allowing significantly more oocytes to be fertilized in vitro following vitrification. Finally, these data may help explain the poor embryo development reported following oocyte cryopreservation when 1,2-propanediol is used, as the sustained calcium increase upon exposure could result in premature oocyte activation, or at least compromised cell function. Supported by: Grant support of Vitrolife AB.

Monday, October 17, 2005 4:45 p.m. O-85 Is Ectopic Pregnancy More Common After Frozen Versus Fresh IVFET? A. A. Al Shaikh, P. Claman, M. Leveille. Division of Reproductive Medicine, Department of Obstetrics and Gynecology, University of Ottawa, Ottawa Hospital, Ottawa, ON, Canada. OBJECTIVE: It has been shown that ectopic pregnancy (EP) is more common after IVF (3-5%) compared to naturally conceived pregnancies (1%). At the 59th annual meeting of the ASRM in October 2003 (P168), Keefe et al suggested that EP was higher after frozen embryo transfer 31.6% (6/19) versus 1.8% (9/490) after fresh embryo transfer (RR 17.2, 95% CI; 6.8-43, p⬍0.0001). This reported higher rate of EP after frozen versus fresh embryo transfer was widely reported in the international media resulting in wide spread concern by patients and public policy makers. The aim of this paper is to study a large number of pregnant IVF cycles in order to: Calculate and compare the incidence of ectopic pregnancy after fresh and frozen embryo transfer cycles. Evaluate and compare risk factors for ectopic pregnancy in patients who achieved a clinical pregnancy after fresh versus frozen embryo transfer cycles. DESIGN: A retrospective cohort study. MATERIALS AND METHODS: Ectopic pregnancy rates were calculated and compared for all fresh and frozen IVF pregnancies that occurred between July 1, 1992 and December 31, 2002 at the University of Ottawa IVF program. The two groups were analyzed and compared for EP risk factors including: maternal age, number of embryos transferred, tubal disease, pelvic surgery, previous ectopic pregnancy, and smoking. Chi-squared and Fisher exact tests were used where appropriated for statistical analysis. RESULTS: A total of 109 frozen and 1098 fresh ET cycles resulting in clinical pregnancy were compared. There was no significant difference in the rate of ectopic pregnancy following frozen [2.75% (3/109)] versus fresh [3.46% (38/1098)] embryo transfer pregnancies (P⫽1.0).There were no differences between the frozen versus fresh pregnant IVF patients in respect to: maternal age (33.5⫾2.7 vs.33.5⫾3.1), average number of embryos transferred (2.3⫾0.9 vs.2.5⫾0.7), diagnosis of tubal disease (46.7% vs.37.2%; p⫽0.065), history of pelvic surgery (41.1% vs. 36.2%; p⫽0.37), previous ectopic pregnancy (12.2% vs. 12.7%; p⫽0.88), or smoking (15.9% vs. 12.4% ;p⫽0.38). CONCLUSIONS: There is no difference in the rate of EP after fresh versus frozen embryo transfer. Patients should be reassured about the safety of frozen embryo transfer. The option of embryo cryopreservation and subsequent transfer is an important part of a strategy to reduce the incidence of multiple pregnancies after IVF by limiting the number of fresh embryos transferred to patients after IVF treatment. Supported by: None

S35