Human ovarian tissues can be cultured for 15 days under high oxygen concentration with the organ culture system used here. This technique could make it possible to utilize ovarian tissue for preservation of reproductive competence in cancer patients.

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.

Effects of several Cl(-) channel blockers on ionic currents in mouse embryos were studied using whole-cell patch-clamp and microelectrode methods. Microelectrode measurements showed that the resting membrane potential of early embryonic cells (1-cell stage) was -23 mV and that reduction of extracellular Cl(-) concentration depolarized the membrane, suggesting that Cl(-) conductance is a major contributor for establishing the resting membrane potential. Membrane currents recorded by whole-cell voltage clamp showed outward rectification and confirmed that a major component of these embryonic currents are carried by Cl(-) ions. A Cl(-) channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), suppressed the outward rectifier current in a voltage- and concentration-dependent manner. Other Cl(-) channel blockers (5-nitro-2-[3-phenylpropyl-amino] benzoic acid and 2-[3-(trifluoromethyl)-anilino] nicotinic acid [niflumic acid]) similarly inhibited this current. Simultaneous application of niflumic acid with DIDS further suppressed the outward rectifier current. Under high osmotic condition, niflumic acid, but not DIDS, inhibited the Cl(-)current, suggesting the presence of two types of Cl(-) channels: a DIDS-sensitive (swelling-activated) channel, and a DIDS-insensitive (niflumic acid-sensitive) Cl(-) channel. Anion permeability of the DIDS-insensitive Cl(-) current differed from that of the compound Cl(-) current: Rank order of anion permeability of the DIDS-sensitive Cl(-) channels was I(-) = Br(-) > Cl(-) > gluconate(-), whereas that of the DIDS-insensitive Cl(-) channel was I(-) = Br(-) > Cl(-) >> gluconate(-). These results indicate that early mouse embryos have a Cl(-) channel that is highly permeable to amino acids, which may regulate intracellular amino acid concentration.

In previous studies, patients with severe peri-ovarian adhesions have been found to show low pregnancy rates and a poor response to gonadotrophin stimulation during in-vitro fertilization (IVF) treatment. The purpose of this retrospective pharmacokinetic study was to assess the diffusion of exogenous human chorionic gonadotrophin (HCG) in patients with peri-ovarian adhesions by examining the concentration of exogenous HCG in the follicular fluid in patients undergoing down-regulation and IVF due to infertility. The patients underwent laparoscopic examination for the scoring of peri-ovarian adhesions (using the classification of adnexal adhesions adopted by the American Fertility Society, a score of 0 means no adhesions, and a score of 32 represents bilateral expanded dense adhesions). Oocytes were recovered after human menopausal gonadotrophin-human chorionic gonadotrophin (HMG-HCG) stimulation with gonadotrophin-releasing hormone agonist. Serum and follicular fluid were collected at the time of oocyte recovery for measuring the HCG ratio (the follicular HCG concentration to the serum HCG concentration; a reflection of the diffusion of exogenous gonadotrophin) by time-resolved fluoroimmunoassay. A negative correlation was found between the number of oocytes recovered and the peri-ovarian adhesion score (r = -0.62, P < 0.01). In a given patient, the follicular HCG concentration was always lower than the serum HCG at the time of oocyte recovery. The HCG ratio in all samples was 0.9 or less (0.51 +/- 0.20; range, 0.09-0.90). Significant negative correlations were found between the peri-ovarian adhesion score and both the follicular HCG concentration (r = -0.80, P < 0.01) and the HCG ratio (r = -0.75, P < 0.01). In conclusion, severe peri-ovarian adhesions interfered with the diffusion of exogenous gonadotrophin into the follicular fluid during IVF treatment. Thus, the diffusion of exogenous gonadotrophin into the follicular fluid may represent a new parameter in the assessment of ovarian blood flow and IVF outcome.

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