Noriko Kagawa

Successful vitrification of bovine and human ovarian tissue

Ovariectomy and ovarian tissue cryopreservation has the potential to preserve the natural fertility of cancer patients prior to sterilizing chemo- and radiotherapies. Ovarian tissue cryopreservation with the conventional slow-freezing method has yielded limited success, partly because of oocyte loss during freeze-thaw and subsequent transplant. Based on the high-efficiency vitrification Cryotop method, a practical vitrification procedure for murine, bovine and human ovarian tissue was developed. A Cryotissue method was designed for cryopreservation of ovarian tissue, and vitrification experiments were performed in a bovine animal model with ovarian size and structure similar to the human. There was no difference in oocyte viability (>89%) between fresh and vitrified ovarian cortical tissue in either bovine or human samples. Ovarian tissue was successfully autotransplanted to six cattle. Autotransplantation of vitrified-warmed tissue back to the cattle resulted in no loss of oocyte viability. In addition, human ovarian tissue from cancer patients, and from ovary transplant donors was also vitrified by the Cryotissue method. After warming, high oocyte survival in human tissue (similar to bovine tissue) was obtained. These results indicate that an ultra-rapid cooling vitrification method has the potential for clinical use in human ovarian tissue cryopreservation.

Production of the first offspring from oocytes derived from fresh and cryopreserved pre-antral follicles of adult mice

Although mammalian ovaries contain hundreds of thousands of pre-antral follicles, fewer than 1% of these reach maturity and ovulation. Obtaining immature eggs from the pre-antral follicles of ovarian tissue could increase the possibility of preserving fertility in women undergoing anti-cancer treatment, and in women who wish to delay pregnancy and child raising until they are older. This study reports the birth of 10 healthy mouse pups derived from oocytes obtained from pre-antral follicles after adult ovary tissue cryopreservation and allotransplantation. High in-vitro maturation (55.1%), fertilization (76.3%) and cleavage (98.3%) rates were achieved using these oocytes, and there was no significant difference between the vitrified and control samples except in maturation rate (55.1 versus 72.8%, P < 0.05). After an ultra-rapid vitrification procedure, the warmed tissue fragments were transplanted beneath the kidney capsule of severe combined immunodeficient mice for onward in-vivo culture. Within 10 days of culture, 138 full size oocytes developed from the 456 transplanted pre-antral follicles. In-vivo growth of follicles was followed by in-vitro oocyte maturation, in-vitro fertilization and subsequent embryo transfer, leading to the birth of 10 healthy pups. These results may lead to increasing the possibility of preserving fertility by cryopreservation of ovarian tissue.

Production of fertile zebrafish (Danio rerio) possessing germ cells (gametes) originated from primordial germ cells recovered from vitrified embryos

This study aimed to produce fertile zebrafish (Danio rerio) possessing germ cells (gametes) that originated from cryopreserved primordial germ cells (PGCs). First, to improve the vitrification procedure of PGCs in segmentation stage embryos, dechorionated yolk-intact and yolk-removed embryos, the PGCs of which were labeled with green fluorescent protein, were cooled rapidly after serial exposures to equilibration solution (ES) and vitrification solution (VS), which contained ethylene glycol, DMSO, and sucrose. Yolk removal well prevented ice formation in the embryos during cooling and improved the viability of cryopreserved PGCs. The maximum recovery rate of live PGCs in the yolk-removed embryos vitrified after optimum exposure to ES and VS was estimated to be about 90%, and about 50% of the live PGCs showed pseudopodial movement. Next, to elucidate the ability of cryopreserved PGCs to differentiate into functional gametes, PGCs recovered from the yolk-removed embryos (striped-type) that were vitrified under the optimum exposure to ES and VS were transplanted individually into 218 sterilized recipient blastulae (golden-type). Two days after the transplantation, 7.5% (14/187) of morphologically normal embryos had PGC(s) in the genital ridges. Six (5 males and 1 female) of the 14 recipient embryos developed into mature fish and generated progeny with characteristics inherited from PGC donors. In conclusion, we demonstrated the successful cryopreservation of PGCs by vitrification of yolk-removed embryos and the production of fertile zebrafish possessing germ cells that originated from the PGCs in vitrified embryos.

Prevention of mitochondrial disease inheritance by assisted reproductive technologies: Prospects and challenges

BACKGROUND Mitochondrial diseases are caused by the mutations in both nuclear and mitochondrial DNA (mtDNA) and the treatment options for patients who have mitochondrial disease are rather limited. Mitochondrial DNA is transmitted maternally and does not follow a Mendelian pattern of inheritance. Since reliable and predictable detection of mitochondrial disorders in embryos and oocytes is unattainable at present, an alternative approach to this problem has emerged as partial or complete replacement of mutated mtDNA with the wild-type mtDNA through embryo manipulations. Currently available methods to achieve this goal are germinal vesicle transfer (GVT), metaphase chromosome transfer (CT), pronuclear transfer (PNT) and ooplasmic transfer (OT). SCOPE OF REVIEW We summarize the state of the art regarding these technologies and discuss the implications of recent advances in the field for clinical practice. MAJOR CONCLUSIONS CT, PNT and GVT techniques hold promise to prevent transmission of mutant mtDNA through ARTs. However, it is clear that mtDNA heteroplasmy in oocytes, embryos and offspring produced by these methods remains as a legitimate concern. GENERAL SIGNIFICANCE New approaches to eliminate transmission of mutant mtDNA certainly need to be explored in order to bring the promise of clinical application for the treatment of mitochondrial disorders. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010.

Oophorectomy for Fertility Preservation via Reduced-Port Laparoscopic Surgery

Background. For fertility preservation of women patients scheduled to undergo chemotherapy or radiotherapy, unilateral oophorectomy was performed, and the ovary was cryopreserved. Methods. Two-port surgery was conducted in 3 patients, and single-port surgery using a single-incision laparoscopic surgery port in 3. An 18-G Cathelin needle equipped with a syringe was directly inserted transabdominally to reach the small follicle on the ovarian surface; then, follicular fluid was recovered by aspiration through the syringe as with in vitro fertilization procedures, and immature oocytes were collected from the resulting culture medium under microscopy and cryopreserved. Vitrification of the ovarian tissue was performed using the cryotissue method. Results. The operative time and estimated blood loss were 39.7 minutes (17-57) and 8.6 mL (2-20), and the numbers of ovarian cortical tissues and immature oocytes collected were 10.1 (5.5-15) and 16.3 (0-36), respectively. Conclusions. It is suggested that fertility preservation operations before chemotherapy or radiotherapy can be safely done using reduced-port surgery.

Successful Ovarian Vitrification and Back-Transplantation to Preserve Fertility in a Patient Requiring Chemotherapy for Malignant Lymphoma

For women with hematologic malignancy anticipating chemotherapy or radio therapy but wishing to preserve fertility, it is preferable that recovery from oophorectomy be as rapid as possible. Considering Reduced-Port Surgery (RPS) to be potentially suitable for such patients, we used RPS for a patient with malignant lymphoma scheduled for pre-bone marrow transplantation chemotherapy. The patient was a 28-year-old woman, gravida 0, who had requested fertility preservation. Thus, with ethics committee approval, the left ovary was removed by RPS, and ovarian cortex with primordial ovarian follicles was cryopreserved by vitrification. The cortex was divided into 12 pieces, 1 cm×1 cm×1 mm each, placed in a container, and stored in liquid nitrogen. The patient’s post-bone marrow transplantation course was good. Because she was judged to have lost ovarian function, ovarian back-transplantation was performed by RPS. The cortex of the remaining right ovary was removed with a laparoscopic cold knife and scissors while heparin saline was instilled to maintain blood flow and a 2 cm×1 cm base was created. Two thawed ovarian cortex pieces were laparoscopically sewed to the base with 5-0 absorbable sutures. By postoperative day 173, the ovarian follicle had increased to 10 mm in diameter, and the patient’s estradiol level had risen to 101 pg/ml, suggesting recovery of ovarian function. Our experience in this lymphoma case suggests that ovarian cryopreservation and back-transplantation for fertility preservation can be performed safely by RPS and is a viable option for selected patients anticipating chemotherapy and bone marrow transplantation for hematologic malignancy.

Ovarian Tissue Vitrification for Fertility Preservation

In a notable 2012 case, a Japanese woman treated for leukemia delivered a healthy baby after implantation of an egg collected before bone marrow transplant. It has since become possible for women facing cancer treatment to avoid iatrogenic infertility and anticipate pregnancy through ovarian tissue preservation and back-transplantation. It is essential to obtain and preserve the patient’s tissue safely and efficiently through advanced surgical techniques and cryopreservation methods. For widespread benefit, cooperation between institutions and between departments is necessary. Herein, vitrification of ovarian tissue for cryopreservation is described in detail, and recent clinical achievements along with future directions are introduced.