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Successful vitrification of human oocytes and embryos: the Cryotop method
Abstracts - WARM: Beyond infertility: breakthroughs in reproductive medicine and technology
technologies are the experience of the embryologist, his daily overload, and his environmental conditions – the more experienced the embryologist, the better is the outcome – and the implementation of findings that improve the conditions and environment of the manipulated oocytes/embryos (e.g. tissue culture dishes, micropipettes, media, HA replacement for PVP). Nevertheless in ICSI there are tools for improving the outcome, for instance the use of high-magnification interference phase microscopes in real time of the sperm injection and a scoring system for optimizing fertilization rates and developmental potential. Efforts should always be focused on developing improvements in micromanipulation technologies. Those improvements would ultimately not only improve the potential of the embryo for successful implantation, but would also lead to a safer long-term outcome for the patients.
Innovations to optimize results Treating patients with polycystic ovarian syndrome with in-vitro maturation Abir R Infertility and IVF Unit, Helen Schneider Hospital for Women, Rabin Medical Center-Beilinson Hospital, Petach Tikvah, Israel Introduction: Polycystic ovarian syndrome (PCOS) patients undergoing ovulation induction show variability in ovarian response from resistance and poor response to increase in severe ovarian hyperstimulation syndrome (OHSS). In-vitro maturation (IVM) of human oocytes is an emerging technology that has promising potential for PCOS patients. PCOS patients whose ovaries contain large numbers of antral follicles might benefit from recovery of immature oocytes followed by IVM. The first pregnancies using IVM for these patients were already reported in the mid-1990s, although the embryo implantation rate was very low. Since then improvements in ultrasound imaging and IVM techniques have resulted in better maturation, fertilization and embryo implantation rates with consequent improved outcome. Materials and methods: The initial step in IVM protocols for patients with PCOS is induction of withdrawal bleeding by progestational preparations. Maturation and pregnancy rates can be improved by mild ovarian stimulation before oocyte retrieval with either recombinant follicle stimulating hormone (FSH) or human chorionic gonadotrophin (HCG, 36 h before retrieval). HCG also increases the number of retrieved oocytes. Patients with antral follicular counts above 20 are best suited for IVM, as the number of antral follicles and their size is directly correlated with successful IVM and high pregnancy rates. Oocytes from follicles larger than 6 mm have a greater maturation potential. Ideally oocyte retrieval is conducted when the largest follicles are 10–12 mm, and endometrium thickness is at least 6 mm. Good follicular visualization by transvaginal sonography (TVS) is the key for successful immature oocyte retrieval, and colour-flow Doppler may assist as well. Follicles are aspirated under TVS guidance using a 17-gauge single-lumen aspiration needle. Culture media as medium 199, Chang’s medium or Ham’s F-10 have been used successfully, and IVM culture media is available commercially.
S-20 Reproductive BioMedicine Online, Vol. 17, Suppl. 2, September 2008
Moreover, recombinant FSH and luteinizing hormone or human menopausal gonadotrophins and occasionally also oestrogen are added to the culture medium. The culture medium is also supplemented with a protein/growth factors source of either the patient’s own serum or human serum albumin. Oocyte maturation is evaluated within 48 h of culture with the identification of the first polar body, followed in most cases by intracytoplasmic sperm injection to avoid risk of zona hardening. Endometrial preparation and support should be conducted until week 12 of gestation, with administration of oestrogen combined with progesterone. Results: About 100 healthy babies are known to have been born after IVM in women with PCOS. The endometrial thickness is directly correlated with successful pregnancy rates. IVM in these patients results in pregnancy rates of 30–37% and implantation rates of 10–15%. A high rate of miscarriage (36%) was reported previously after IVM for patients with PCOS. There are no reports of increase in congenital malformations, although abnormalities in oocyte spindle organization have been noted. Conclusions: With improvements in IVM techniques, the method seems a promising alternative for women with PCOS. Further studies should be conducted to improve the culture conditions, endometrium thickness and to investigate possible chromosomal aberrations as a result of spindle abnormalities. Successful vitrification of human oocytes and embryos: the Cryotop method Okimura T, Kuwayama M Kato Ladies Clinic, Tokyo, Japan Recent drastic advances in cryobiology have made it possible to preserve various types of reproductive cells with relatively little loss of viability. Vitrification, the alternative cryopreservation method, seems to be a powerful tool for any biological specimens which cannot be preserved by the conventional slow-freezing method so far. Especially, ultrarapid cooling realizes the successful clinical use of vitrification not only for human pronuclei zygotes and blastocysts but also for oocytes. The purpose of this lecture is to introduce the history of vitrification of human IVF and the mechanism of vitrification of the cells, and how to maintain high viability of the oocytes and embryos under liquid nitrogen temperature, for basic knowledge. To provide evidence for the potential significance of vitrification, achievements with the Cryotop technology, an advanced version of the minimal volume approaches, is analysed. This technology alone has resulted in more healthy babies after cryopreservation of blastocysts than any other vitrification technique, and more successful human oocyte vitrification resulting in normal births than any other cryopreservation method. The value of this method is also demonstrated by achievements in the field of domestic animal embryology. The Cryotop method is now used for human oocytes and embryo cryopreservation in more than 350 IVF facilities in 12 countries and is producing excellent clinical results. Human oocyte banks for unmarried young cancer patients and egg donation programmes have already been established and have been enabling women to dream and have the courage to improve their quality of lives.
technologies are the experience of the embryologist, his daily overload, and his environmental conditions – the more experienced the embryologist, the better is the outcome – and the implementation of findings that improve the conditions and environment of the manipulated oocytes/embryos (e.g. tissue culture dishes, micropipettes, media, HA replacement for PVP). Nevertheless in ICSI there are tools for improving the outcome, for instance the use of high-magnification interference phase microscopes in real time of the sperm injection and a scoring system for optimizing fertilization rates and developmental potential. Efforts should always be focused on developing improvements in micromanipulation technologies. Those improvements would ultimately not only improve the potential of the embryo for successful implantation, but would also lead to a safer long-term outcome for the patients.
Innovations to optimize results Treating patients with polycystic ovarian syndrome with in-vitro maturation Abir R Infertility and IVF Unit, Helen Schneider Hospital for Women, Rabin Medical Center-Beilinson Hospital, Petach Tikvah, Israel Introduction: Polycystic ovarian syndrome (PCOS) patients undergoing ovulation induction show variability in ovarian response from resistance and poor response to increase in severe ovarian hyperstimulation syndrome (OHSS). In-vitro maturation (IVM) of human oocytes is an emerging technology that has promising potential for PCOS patients. PCOS patients whose ovaries contain large numbers of antral follicles might benefit from recovery of immature oocytes followed by IVM. The first pregnancies using IVM for these patients were already reported in the mid-1990s, although the embryo implantation rate was very low. Since then improvements in ultrasound imaging and IVM techniques have resulted in better maturation, fertilization and embryo implantation rates with consequent improved outcome. Materials and methods: The initial step in IVM protocols for patients with PCOS is induction of withdrawal bleeding by progestational preparations. Maturation and pregnancy rates can be improved by mild ovarian stimulation before oocyte retrieval with either recombinant follicle stimulating hormone (FSH) or human chorionic gonadotrophin (HCG, 36 h before retrieval). HCG also increases the number of retrieved oocytes. Patients with antral follicular counts above 20 are best suited for IVM, as the number of antral follicles and their size is directly correlated with successful IVM and high pregnancy rates. Oocytes from follicles larger than 6 mm have a greater maturation potential. Ideally oocyte retrieval is conducted when the largest follicles are 10–12 mm, and endometrium thickness is at least 6 mm. Good follicular visualization by transvaginal sonography (TVS) is the key for successful immature oocyte retrieval, and colour-flow Doppler may assist as well. Follicles are aspirated under TVS guidance using a 17-gauge single-lumen aspiration needle. Culture media as medium 199, Chang’s medium or Ham’s F-10 have been used successfully, and IVM culture media is available commercially.
S-20 Reproductive BioMedicine Online, Vol. 17, Suppl. 2, September 2008
Moreover, recombinant FSH and luteinizing hormone or human menopausal gonadotrophins and occasionally also oestrogen are added to the culture medium. The culture medium is also supplemented with a protein/growth factors source of either the patient’s own serum or human serum albumin. Oocyte maturation is evaluated within 48 h of culture with the identification of the first polar body, followed in most cases by intracytoplasmic sperm injection to avoid risk of zona hardening. Endometrial preparation and support should be conducted until week 12 of gestation, with administration of oestrogen combined with progesterone. Results: About 100 healthy babies are known to have been born after IVM in women with PCOS. The endometrial thickness is directly correlated with successful pregnancy rates. IVM in these patients results in pregnancy rates of 30–37% and implantation rates of 10–15%. A high rate of miscarriage (36%) was reported previously after IVM for patients with PCOS. There are no reports of increase in congenital malformations, although abnormalities in oocyte spindle organization have been noted. Conclusions: With improvements in IVM techniques, the method seems a promising alternative for women with PCOS. Further studies should be conducted to improve the culture conditions, endometrium thickness and to investigate possible chromosomal aberrations as a result of spindle abnormalities. Successful vitrification of human oocytes and embryos: the Cryotop method Okimura T, Kuwayama M Kato Ladies Clinic, Tokyo, Japan Recent drastic advances in cryobiology have made it possible to preserve various types of reproductive cells with relatively little loss of viability. Vitrification, the alternative cryopreservation method, seems to be a powerful tool for any biological specimens which cannot be preserved by the conventional slow-freezing method so far. Especially, ultrarapid cooling realizes the successful clinical use of vitrification not only for human pronuclei zygotes and blastocysts but also for oocytes. The purpose of this lecture is to introduce the history of vitrification of human IVF and the mechanism of vitrification of the cells, and how to maintain high viability of the oocytes and embryos under liquid nitrogen temperature, for basic knowledge. To provide evidence for the potential significance of vitrification, achievements with the Cryotop technology, an advanced version of the minimal volume approaches, is analysed. This technology alone has resulted in more healthy babies after cryopreservation of blastocysts than any other vitrification technique, and more successful human oocyte vitrification resulting in normal births than any other cryopreservation method. The value of this method is also demonstrated by achievements in the field of domestic animal embryology. The Cryotop method is now used for human oocytes and embryo cryopreservation in more than 350 IVF facilities in 12 countries and is producing excellent clinical results. Human oocyte banks for unmarried young cancer patients and egg donation programmes have already been established and have been enabling women to dream and have the courage to improve their quality of lives.