We report here the first case of successful pregnancy and delivery after the blastocyst transfer of twice-vitrified embryos produced following in vitro maturation (IVM) and ICSI. The patient received 5000 IU hCG on day 12 of the treatment cycle, and oocyte retrieval was carried out 36 h after hCG injection. A total of 22 immature oocytes were obtained. Following incubation for 26 h in IVM medium, 15 oocytes (68.2%) reached metaphase II stage. In total, 13 oocytes (86.7%) were fertilized after ICSI with the husband's sperm, and 11 embryos at the pronuclear stage and two cleaved embryos on day 2 were vitrified because of thin endometrial thickness. Eight cryopreserved embryos at the pronuclear stage were warmed and cultured until the day 3 stage. Three embryos were transferred, and three embryos were twice vitrified. Unfortunately, these transferred embryos did not implant. Three twice-vitrified embryos were rewarmed and cultured until the day 5 stage, and two embryos were transferred. The second transfer attempt of twice-vitrified embryos resulted in the full-term delivery of a healthy infant. This case report demonstrates that twice-vitrified embryos, developed using an IVM protocol, retain the developmental competence for full-term, healthy infants.
In-vitro maturation (IVM) of immature oocytes has been proposed as a potential alternative to conventional IVF treatment following ovarian stimulation. However, the effects of the oocyte retrieval conditions on subsequent development have not been well understood. This study assessed the effects of different aspiration vacuums during oocyte retrieval on the developmental competence of immature oocytes following IVM, IVF and embryo transfer, retrospectively. Immature oocytes were aspirated with 20-gauge needles with a vacuum of 180 or 300 mmHg. Immature oocytes were cultured in IVM medium for 26 h. All mature oocytes were inseminated by intracytoplasmic sperm injection (ICSI). Embryo transfer was carried out 2 or 3 days after ICSI. The percentage of cumulus-cell enclosed oocytes and of transferable embryos per retrieved oocytes in 180 mmHg (69.7% and 23.8%, respectively) were significantly higher (P < 0.01) than those in 300 mmHg (46.2% and 12.8%, respectively). The ongoing pregnancy rate per retrieval cycle in 180 mmHg (30%) was higher (P < 0.01) than that in 300 mmHg (4.3%). The data indicate that lower pressure of vacuum aspiration with a 20-gauge needle improves the developmental competence of immature oocytes following IVM, IVF and embryo transfer.
The incidence of monozygotic twinning (MZT) appears to be increasing within the field of assisted reproductive technology (ART), although the factors contributing to the phenomenon are still far from being identified. On the contrary, in vitro maturation (IVM) of oocytes is becoming more accepted and more and more babies have been born worldwide using this procedure. Assessing its safety and impact on monozygotic twinning (MZT), and following up the health of these babies, is essential. We report here a first case of successful monozygotic (MZ) twin delivery following IVM. The patient was a 28-year-old Japanese female, referred to the IVF clinic for primary infertility. Several previous cycles of ovarian stimulation had resulted in ovarian hyperstimulation syndrome (OHSS). The patient received norethisterone-mestranol to initiate the menstruation, and oocyte retrieval was performed 36 h after hCG. A total of 22 immature oocytes were obtained. Following incubation for 24 h in IVM medium, 50% of the oocytes were matured to the metaphase II (MII) stage. Nine oocytes were fertilized after ICSI with the husband's sperm. Three day 3 embryos were transferred into the uterus on the fourth day following oocyte retrieval. Three weeks after embryo transfer, a single gestational sac was visualized in the uterus. At 7 weeks of gestation, two fetal poles with cardiac activity were seen in the single gestational sac. Serial ultrasound examinations revealed a MZ, monochorionic diamniotic pregnancy. After intensive perinatal monitoring, two healthy male infants were delivered by Caesarean section at 35 weeks of gestation.
Intergeneric crosses were made between Brassica oleracea and Moricandia arvensis utilizing embryo rescue. Six F 1 hybrid plants were generated in the cross-combination of B. oleracea · M. arvensis from 64 pods by the placenta-embryo culture technique, whereas three plants were produced in the reciprocal cross from 40 pods by the ovary culture technique. The hybrid plants were ascertained to be amphihaploid with 2n ¼ 23 chromosomes in mitosis and a meiotic chromosome association of (0-3)II + (17-23)I at metaphase I (M I). In the backcross with B. oleracea, some of these hybrids developed sesquidiploid BC 1 plants with 2n ¼ 32 chromosomes that predominantly exhibited a meiotic configuration of (9II + 14I) in pollen mother cells. The following backcross of BC 2 plants to B. oleracea generated 48 BC 3 progeny with somatic chromosomes from 2n ¼ 19 to 2n ¼ 41. The 2n ¼ 19 plants showed a chromosomal association type of (9II + 1I) and a chromosomal distribution type of (9 1 / 2 + 9 1 / 2 ) or (9 + 10) at M I and M II, respectively. These facts might suggest that they were monosomic addition lines (MALs) of B. oleracea carrying a single chromosome of M. arvensis that could offer potential for future genetic and breeding research, together with other novel hybrid progeny developed in this intergeneric hybridization.Key words: Brassica oleracea -Moricandia arvensisintergeneric hybrid -embryo rescue -monosomic addition line Genetic diversification of crop species through wide hybridization can be a first step in plant breeding programme. In Brassica oleracea that comprises many staple vegetables throughout the world, the genetic and breeding efforts have been focused on the tolerance or resistance to both biotic and abiotic stresses. A closely related species, Moricandia arvensis of the subtribe Moricandiinae, has been reported as a C 3 -C 4 intermediate possessing the genetic potential for improvement of photosynthetic ability (Apel et al. 1978(Apel et al. , 1984. Moreover, this wild relative exhibits greater resistance to clubroot (Kuginuki et al. 1991), and tolerance to drought and insects (Warwick 1993). It is also worth noting that M. arvensis has been used for the development of cytoplasmic male sterility (CMS) and genic restorer systems for CMS that might offer sources for hybrid seed production in Brassica and Raphanus crops (Prakash et al. 1998, Bang et al. 2002, Bhat et al. 2005. In pioneering work on somatic hybridization between Brassica and Moricandia species, amphidiploid hybrids were successfully developed through protoplast fusion by Toriyama et al. (1987). Takahata (1990) also produced amphihaploid hybrids from cultured ovaries from intergeneric hybridization when M. arvensis was used as a female parent. Recently, Ishikawa et al. (2003) generated somatic hybrid plants between these two species and evaluated them from the viewpoint of chloroplast and mitocondrial DNA, cytogenetics, leaf anatomy and fertility. In the present study, the novel production and characterization of intergeneric hybrids of B. oler...
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