Survival of primordial follicles in vitrified-thawed whole ovary of New Zealand white rabbit

briefly and transferred. Morphological survival of each embryo and pregnancy rates resulting from each group of blastocysts were compared. MATERIALS AND METHODS: One thousand and ninety-six patients who gave informed consent to participate in this study were stimulated by treatment with clomiphene citrate, hMG and GnRH. Their oocytes were fertilized and allowed to develop into blastocysts. As embryos developed into blastocysts on a given day (days 4, 5 or 6), they were assigned a morphological grade (A, B, C) and were vitrified by the MVC method. Blastocysts were first incubated in a mixture of 1.3 M ethylene glycol (EG) ⫹ 1.1 M dimethylsulfoxide (DMSO) prepared in TCM199 medium for 10 –15 min, and then in a vitrification solution (VS) consisting of 2.7 M EG ⫹ 2.1 DMSO ⫹ 0.5 M sucrose for 30 sec. Individual blastocysts contained in ⬃0.1 ␮l of VS were placed onto the vitrification device (Cryotop, Kitazato Supply Co., Japan) and immediately submerged in LN2. After storage, vitrified embryos were liquefied by plunging the Cryotop directly into 1 M sucrose prepared in TCM199 medium at 37°C for 1 min. Embryos were rinsed in 0.5 M sucrose for 3 min, twice in TCM199 medium for 5 min, and then cultured for ⬃4 hrs before being transferred. Of the 1,175 blastocysts that were vitrified, 1,164 (99%) appeared morphologically normal after liquefaction and were judged to have survived based on re-expansion of the blastocoele during culture. All of these surviving blastocysts were transferred into 1,093 patients (1.1 blastocysts/patient). Pregnancies were diagnosed by ultrasound on the basis of formation of a gestational sac. RESULTS: Respective survival rates of blastocysts vitrified on Day 4, 5 and 6 were 97.1% (133/137), 99.2% (914/921) and 100% (117/117), and corresponding pregnancy rates were 60.6% (80/132), 41.4% (354/856) and 28.6% (30/105), respectively. Survival rates of the blastocysts based on their original quality grades A, B and C were 99.1% (214/216), 98.9% (371/375) and 99.1% (579/584), respectively; the corresponding pregnancy rates were 63.6% (133/209), 51.7% (187/362) and 27.6% (144/522). CONCLUSION: Since very high survival (99.1%) of vitrified blastocysts was obtained regardless of their quality, and since a high total pregnancy rate of 42.5% (464/1093) was achieved, these results strongly suggest that the MVC vitrification method is an effective way to cryopreserve human blastocysts. Supported by: Kato Ladies Clinic supported this research.

Wednesday, October 20, 2004 2:45 P.M. O-287 Artificial shrinkage (AS) of blastocoele prior to cooling step of vitrification improves the survival rate of vitrified human blastocysts. T. Mukaida. Hiroshima HART Clinic, Hiroshima, Japan. OBJECTIVE: For a good cumulative pregnancy rate per oocyte retrieval, it is essential to establish a reliable cryopreservation technique. We have reported clinical usefulness of a simple ultra-rapid vitrification procedure using a cryoloop with human blastocysts (BLs) (Hum. Reprod., 2003;18: 384 –391). According to the results of our vitrified BL program the survival rate of vitrified BLs was dependent on the developmental stage of BL and was negatively correlated with the expansion of the blastocoele. We postulated that a large blastocoele might lessen cryopreservative potential due to ice crystal formation in the short cooling duration of vitrification. Therefore we analyzed the effectiveness of reducing the volume of the blastocoele on expanded blastocysts by AS prior to cooling. DESIGN: Development of a new approach to improve the survival of vitrified BLs. MATERIALS AND METHODS: Patients who had spare embryos vitrified at the BL stage were enrolled in this study. The spare BLs were vitrified as described elsewhere. Briefly, the BLs were placed for 2 min in solution I, containing 7.5% dimethylsulfoxide (DMSO) and 7.5% ethylene glycol (EG) in human tubal fluid (HTF) supplemented with 0.5% human serum albumin (HSA), and then placed for 30 seconds in solution II, which contains 15% DMSO, 15% EG, 1% Ficoll 70 and 0.65 M sucrose in HTF/HSA at 37°C. The BLs in solution II were loaded on a small nylon loop (Hamilton Research, CA, USA) and submerged in liquid nitrogen. In the study group, AS of the blastocele on expanded(exp) BLs by puncturing with microneedle using a micromanipulator was performed prior to cooling. AS immediately induced collapsing of the blastocoelic cavity. Warming was carried out by placing the vitrified BLs in 0.33 M sucrose in HTF/HSA

FERTILITY & STERILITY威

for 2 min and then 0.2 M sucrose in HTF/HSA for 3 min. Embryo transfer was performed 2–3 hours after warming and the BLs with morphologically normal inner cell mass and trophectoderm and re-expanding blastocoel were considered to have survived and transferred. The uterine endometrium was prepared with oral estrogen and intramuscular injection of progesterone (50 mg/d). Pregnancy was defined as the presence of gestational sac(s). RESULTS: A total of 124 exp. BLs from 66 patients underwent AS and were transferred from June 2003 to March 2004. This was compared with 131 exp BLs from 68 patients transferred without AS from November 2002 to May 2003, as a control. Patients’ characteristics were not different between the two groups. The survival rate and re-expansion rate that reached full BLs 3 hours after warming was significantly higher in the study group (99.2%, 123/124 and 76.3%, 99/124) than in the control group (21.2%, 55/260 and 59/131) (p⬍0.01).The pregnancy rate was also increased but not significantly different (53.0%, 35/66 vs. 45.6%, 31/68). CONCLUSION: AS on exp BLs prior to cooling was a safe procedure and significantly improved the survival and re-expansion rate. This suggests that the large blastocele could be an obstacle to the dehydration and permeating step of cryoprotectants, and that the problem is avoided by collapsing of blastocele using AS procedure. Supported by: None

Wednesday, October 20, 2004 3:00 P.M. O-288 Survival of primordial follicles in vitrified-thawed whole ovary of New Zealand white rabbit. K. Y. Cha, D. R. Lee, J. J. Lim, H. J. Kim, H. Yoon, T. K. Yoon. Infertility Medical Center of CHA General Hospital, CHA Research Institute, Pochon CHA University, Seoul, Republic of Korea; Department of Anatomy, Pochon CHA University, Sungnam, Republic of Korea. OBJECTIVE: One of the greatest challenges in cryobiology is the cryopreservation of entire organs. In spite of its difficulty, whole ovary freezing will have been one of developed tools of reproductive medicine for the preservation of the reproductive function in young women who are faced with premature ovarian failure through cancer therapy. The purpose of present study was to establish the cryopreservation method for whole ovary by using vitrification following perfusion with cryoprotective.agent (CPA). DESIGN: Effect of the CPA concentration and its penetration time during whole ovary perfusion on the survival of ovarian tissue after vitrificationthawing. MATERIALS AND METHODS: For whole ovary vitrification, New Zealand white rabbits (2month, 2.5kg) were anaesthetized using sodium pentobarbital. Ovaries and the related blood vessels were removed by cut of aorta and vena cava. We perfused for each 10 or 20 min with CPA (VS40 (40%); PBS containing 3.1 M of diemethyl sulfoxide and 2.2 M 1.2propanediol) increased concentrations gradient (10〫40%; 10〫 20〫40%). And whole ovaries and reproductive organs in container after perfusion were cooled ultra-rapidly by placing on liquid nitrogen. After storing for several days at -80°C, the container was quickly thawed in 37°C water bath and the CPA was removed from whole ovaries and reproductive organs by re-perfusion with a reversed concentration gradient. Vitrified-thawed ovaries were sliced and cultured in vitro for 0 or 6 hours. In order to check their viability, cultured ovarian tissues were fixed and examined by H/E staining and immunohistochemical analysis using PCNA (cellular proliferation marker). RESULTS: Even though vitrified-thawed ovary showed poor morphology compared to fresh one, most of stromal tissues were survived by H/E staining. At thawing and rehydration, survival rate of primordial follicles was not different in all groups. However, in 6hours after in vitro culture, survival of follicles in perfusion group for 20 min interval of 3step (10 – 20-40%) was higher than others group. Also, the localization of PCNA was observed in a same pattern. CONCLUSION: These results demonstrated that rabbit’s whole ovary can be, in part, successfully cryopreserved by vitrification conjunction with perfusion. Therefore, this method of whole ovary freezing could be a fertility option for women and children who will have been undergoing sterilizing chemotherapy in the future. However, further research for improving the survival rate of follicles will be needed in order to apply for clinical trial.

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Supported by: This work was supported by a grant from the INTERDISCIPLINARY RESEARCH PROGRAM of the KOSEF (1999 –2-205– 002-5).

Wednesday, October 20, 2004 4:45 P.M. O-289 Detection of microscopic metastasis of solid tumors and hematological malignancies in cryopreserved ovaries. S. E. Elizur, D. Ben-Yehuda, I. Hardan, J. Dor, D. Meirow. IVF unit, Sheba Medical Center, Ramat Gan, Israel; Hematology department, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; Hematology department, Sheba Medical Center, Ramat Gan, Israel. OBJECTIVE: Cryopreservation of ovarian tissue has been used to preserve fertility in female patients subjected to cancer treatments that can cause sterilization. In some cases it might be dangerous to graft the tissue back as microscopic undetected malignant cells could remain within the cryopreserved-thawed grafts. We describe here sensitive techniques used for the detection of microscopic metastases in ovarian tissue in order to increase the safety of transplantation. DESIGN: Ovarian tissue was obtained by laparoscopic procedure. Both ovaries were screened by sonography and during laparoscopy and then ovarian tissue was taken from one side. The tissue was prepared and divided into slices of 5mm X 10mm. From each slice small fragment was taken for detection of possible malignant cells. For each disease different methods and tumor markers were used. MATERIALS AND METHODS: In cases of leukemia and lymphoma the presence of specific chromosomal translocations and mutations in malignant cells served as tumor markers. Performing targeting reverse transcriptase polymerase chain reaction (RT-PCR) assays in order to detect those markers in ovarian tissue increased tremendously the detection rate of ovarian microscopic metastases. In patients with breast cancer, immunohistochemical staining of ovarian tissue was performed using specific tumor antigens, while for Hodgkin’s lymphoma patients a pathological evaluation of the ovarian tissue was performed in order to detect Reed-Sternberg cells. RESULTS: In a 20-year-old female, with chronic myeloid leukemia (CML), RT-PCR was performed in order to detect the presence of Philadelphia chromosome. PCR product was detected only in bone marrow but not in ovarian tissue. In two patients with non-Hodgkin’s lymphoma, RT-PCR was performed in ovarian tissue for specific mutations rearrangement in the T cell receptor. No PCR product was detected in ovarian samples of both women; however lymphoma cells were detected in the bone marrow. In all patients with Hodgkin’s lymphoma no Reed-Sternberg cells were detected in ovarian tissue. In a 38-year-old woman with breast cancer, immunohistochemical staining for HER2 showed microscopic metastasis in ovarian tissue. Thus cryopreservation was not performed. CONCLUSION: In order to reduce the risk of transferring malignant cells back to cured cancer patient during ovarian tissue cryopreservation-transplantation procedure, it is highly recommended to use the best available methods in order to detect microscopic ovarian metastases. These methods can also be applied in the future in cases of testicular tissue cryopreservation from prepubertal boys facing chemotherapy or bone marrow transplantation due to cancer. Supported by: None

Wednesday, October 20, 2004 5:00 P.M. O-290 Cryopreservation of unstimulated immature oocytes. J. B. Russell, S. Pritchard, M. Gibbs, R. Church, C. Lott, K. Staab. Delaware Institute for Reproductive Medicine, P.A., Newark, DE. OBJECTIVE: The ability to cryopreserve oocytes provides tremendous reproductive opportunities for preserving fertility. The ability to cryopreserve unstimulated immature oocytes alleviates numerous factors such as the administration of gonadotropins, ovarian hyperstimulation, follicular monitoring, and the associated costs. The purpose of this study was to

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Abstracts

investigate three different crypreservation protocols on unstimulated immature oocytes. DESIGN: Prospective cohort study of immature oocyte survival after freezing and thawing, presence of Germinal Vesicle (GV), Metaphase I (M1) at 24 hours. MATERIALS AND METHODS: Immature oocytes were retrieved transvaginally with ultrasound guidance from follicles ranging from 4 to 10 mm in size from unstimulated ovaries. Normal appearing immature oocytes were cryopreserved by one of three different cryopreservation protocols. Group 1) Slow freeze/fast thaw using Na-depleted Phosphate Buffered Saline (PBS) with 1.5 Molar (M) Propanediol (PPD) and 0.2 M sucrose. The slow freeze-fast thaw was performed in cryovials using a KRYO 10 Biological Freezer (Planer Biomed, UK). Group 2) Fast freeze/fast thaw using 40% Ethylene Glycol (EG) and 1.0 M sucrose Group 3) Fast freeze/fast thaw using 15% DiMethylSulfoxide (DMSO)/ 15% EG and 1.0 M sucrose. All freezing by the vitrification methods was performed using 1/4⬙ diameter straws (TS Scientific, Perkasie, PA). Thawing the oocytes from Group 1 was performed in 30oC water bath followed by serial dilutions of lower conentrations of sucrose. Group 2 and Group 3 were thawed with exposure of the straw to room temperature for 20 seconds and then the straw was plunged into a 30o water bath. The contents were placed in a 1.0 M sucrose solution and diluted in lower concentrations every 2.5 minutes in a stepwise fashion. Survival was assessed by morphological apperance post thaw, 12 and 24 hours later. The apperance of the GV and M1 development was recorded at 12 and 24 hours later. RESULTS:

CONCLUSION: Immature oocytes can be retrieved transvaginally during the follicular phase of the menstrual cycle and cryopreserved successfully utilizing either a slow/fast protocol using PPD or a fast/fast protocol using 40% EG. The vitrification protocol without DMSO with 40% EG appears to be superior for survival immediately post thaw and at 24 hours. Supported by: None

Wednesday, October 20, 2004 5:15 P.M.

O-291 Trypan blue staining: A quick test to attest the quality of ovarian cryopreservation. C. Roux, P. Fauque, C. Joanne, C. Tripogney, G. Agnani, J. L. Bresson. Service de Ge´ ne´ tique, Histologie, Biologie du De´ veloppement et de la Reproduction, CECOS (EA 3185 : ge´ ne´ tique et reproduction & IFR 133 : IBCT) ⫺ CHU Saint Jacques, Besanc¸ on, France; Service de Gyne´ cologie Obste´ trique ⫺ CHU Saint Jacques, Besanc¸ on, France. OBJECTIVE: Ovarian tissue cryopreservation is a technique proposed to young females to prevent the effects of gonadotoxic treatments. This practice is yet experimental, and so far no children have been born after utilisation of thawed ovarian tissue. The aim of the study was to check the efficiency of the cryopreservation procedure by using ovarian test fragments to attest the quality of frozen-thawed ovarian tissue. DESIGN: Prospective study on cryopreserved ovarian tissue MATERIALS AND METHODS: Ovarian cortical biopsies from 12 patients with PCOS were obtained at the time of ovarian resection by laparoscopy, prior to electrocoagulation of the puncture sites. The study was approved by the local ethical committee. For each patient, one part of the tissue was cryopreserved using a slow freezing ⫺ rapid thawing protocol (with: seeding during freezing, 1.5M DMSO and 0.1M sucrose as cryoprotectants), and the other part was used as control. Both fresh and frozen/ thawed tissue samples were embedded in paraffin for histological evaluation

Vol. 82, Suppl. 2, September 2004