Kenji Hinokio

A combination of calcium ionophore and puromycin effectively produces human parthenogenones with one haploid pronucleus.

Parthenogenetic activation with various combinations of the calcium ionophore A23187 and protein synthesis or phosphorylation inhibitors was investigated as a means of producing human parthenogenones with one haploid pronucleus. Unfertilised human aged oocytes exposed to 5 microM A23187 for 5 min were treated with 10 microg/ml puromycin (puromycin group, 46 oocytes) or 2 mM 6-dimethylaminopurine (DMAP group, 42 oocytes) for 5 h. Oocytes treated only with A23187 served as a control (control group, 40 oocytes). After washing the oocytes, they were incubated for up to 37 h. Evidence of activation (pronuclear formation) and cleavage was observed 18 h and 42 h after A23187 treatment, respectively. Activation rates in the puromycin and DMAP groups were significantly higher than in the control group (91% (42/46) and 77% (34/44) vs 20% (8/40), p < 0.05, respectively). In the puromycin group, 81% (34/42) of the activated oocytes showed one pronucleus with the second polar body (2ndPB), whereas none (0/34) of the activated oocytes in the DMAP group extruded the 2ndPB. The cleavage rate in the puromycin group was significantly lower than in the DMAP group (38% vs 68%, p < 0.05). The activated oocytes which had one pronucleus with the 2ndPB in the puromycin group showed a haploid set of chromosomes (10/13). In conclusion, the combination of A23187 and puromycin is effective for producing human parthenogenones with one haploid pronucleus.

Lysophosphatidic acid stimulates nuclear and cytoplasmic maturation of golden hamster immature oocytes in vitro via cumulus cells.

Lysophosphatidic acid (LPA), a member of the phospholipid autacoid family, is induced in incubated human follicular fluid by lysophospholipase D. It is well known that LPA functions as a growth factor and the hypothesis that LPA in human follicular fluid takes a part in meiosis of oocytes is quite plausible. We studied the effects of LPA on the maturation of golden hamster immature oocytes in vitro. Hamster oocytes with a germinal vesicle were cultured in Tyrodes albumin lactate pyruvate (TALP) medium with 10(-5) M LPA, 10 ng/ml epidermal growth factor (EGF), 30 ng/ml insulin-like growth factor-1, 1 ng/ml tumor growth factor-alpha or 1 ng/ml basic fibroblast growth factor. The nuclear maturation rates in the LPA and EGF groups were significantly higher than in the control group and the other growth factors did not show any stimulatory effect (LPA group; 74.3% [75/101], EGF group; 82.4% [89/108] vs. control group; 60.2% [59/98], p < 0.05, p < 0.01, respectively). When the cells of cumulus were removed, EGF and LPA did not increase the nuclear maturation rates. Cotreatment EGF and LPA did not significantly enhance the stimulatory effect observed with LPA alone on maturation in vitro. The penetration rate determined by the zona-free hamster oocyte test was significantly higher in the LPA group than in the control group (26.7% vs. 13.2%, p < 0.05) and was comparable with that of oocytes matured in vivo. In conclusion, LPA stimulates the nuclear and cytoplasmic maturation of hamster immature oocytes via cumulus cells.

Effective activation method with A23187 and puromycin to produce haploid parthenogenones from freshly ovulated mouse oocytes.

Freshly ovulated mouse oocytes exposed to 5 mM calcium ionophore A23187 for 5 min and controls (not exposed) were cultured in TYH medium with 10 microg/ml puromycin (the puromycin group) or 2 mM 6-dimethylaminopurine (DMAP; the DMAP group) for 4 h. Among the controls, few oocytes were activated even if they were treated with DMAP or puromycin. In the oocytes exposed to A23187, in contrast, the activation rate, i.e. the rate of oocytes showing at least one pronucleus (PN) after the treatment, was 46.2% (48/104) in the DMAP group and 90.0% (118/131) in the puromycin group. Activation rate in the puromycin group was significantly higher than in the DMAP and control groups (p < 0.0001, respectively). Furthermore, 82.4% (108/131) of the activated oocytes in the puromycin group showed one PN with extrusion of the second polar body (PB). In the puromycin group, the DNA content of the PN of parthenogenones with 1PN2PB was half that of a set of metaphase II chromosomes. Chromosomal analysis was possible in 14 parthenogenones with 1PN2PB in the puromycin group. The parthenogenones possessed a normal set (n = 20) of haploid chromosomes. The combination of A23187 and puromycin proved to be an effective method of producing haploid parthenogenones.

Quality of embryo does not affect the implantation rate of IVF-ET in infertile women with antisperm antibody

OBJECTIVE To determine whether low quality score of embryos and advanced maternal age affect the implantation rate in infertile women with sperm-immobilizing antibody. DESIGN A retrospective study. SETTING The IVF Unit of the Department of Obstetrics and Gynecology at Tokushima University Hospital. PATIENT(S) Four infertile groups were studied: 20 women with sperm-immobilizing antibodies; 169 with tubal; 129 with male factor; and 72 with unexplained etiology. INTERVENTION(S) All women were hyperstimulated with GnRH analogue and scheduled ovarian stimulation with FSH and hMG for oocyte retrieval. MAIN OUTCOME MEASURE(S) Relationship of quality of transferred embryos, implantation rate and maternal age among four groups of infertile couples. RESULT(S) In the antisperm group, the fertilization rate (57.6%) and mean (+/- SD) score of transferred embryos (5.4+/-1.9) were significantly lower than those in the tubal group (72.4% and 6.2+/-1.9, respectively). However, the implantation rate in the antisperm group (23.6%) was significantly higher than those in other three groups (tubal, 8.6%; male factor, 9.5%; unexplained, 7.6%). With advancing maternal age, the implantation rate decreased in the three comparative groups. In contrast, the implantation rate in the antisperm group did not decrease with advancing maternal age. CONCLUSION(S) Women with antisperm antibodies have several disadvantages to overcome in order to achieve successful IVF-ET, such as a low fertilization rate and poor quality of transferred embryos. However, a high implantation rate was observed in this group, even in women at advanced age. The occurrence of a cellular or humoral immune reaction against sperm may augment the uterine receptivity for the implantation of fertilized ova or blastocyst.

Roscovitine in combination with calcium ionophore induces oocyte activation through reduction of M-phase promoting factor activity in mice

The aim of the present study was to determine oocyte activation and change in M-phase promoting factor (MPF) activity induced by treatment with calcium ionophore and roscovitine in comparison with those induced by treatment with roscovitine alone and treatment with calcium ionophore and puromycin in mice. Freshly ovulated oocytes obtained from 6-8-week-old mice were divided into five groups (no activation treatment; 5 μM calcium ionophore A23187; 50 μM roscovitine; 5 μM calcium ionophore and 10 μg/ml puromycin; and 5 μM calcium ionophore and 50 μM roscovitine) and were incubated for 6 h. Oocyte activation, assessed by morphological changes, and changes in MPF activity in the five groups at 0, 2, 4 and 6 h of incubation were examined. Activated oocytes were defined as oocytes with at least one pronucleus. Oocytes treated with roscovitine alone were not activated during the 6-h incubation period. All of the oocytes in the calcium ionophore with puromycin group and in the calcium ionophore with roscovitine group were activated. The percentage activity of MPF in oocytes treated with roscovitine alone was decreased after 2 h and increased after 4 h of incubation. The percentage activity of MPF in oocytes treated with calcium ionophore and roscovitine was significantly decreased with suppression of MPF activity being maintained for 6 h, and this change was similar to that in oocytes treated with calcium ionophore and puromycin. Roscovitine with calcium ionophore is effective for induction of oocyte activation through suppression of MPF activity in mice.

Intra-follicular kisspeptin levels are related to oocyte maturation and gonadal hormones in patients who are undergoing assisted reproductive technology

To assess the kisspeptin concentrations in follicular fluid and their relationship with clinical outcomes during assisted reproductive technology.

Effects of preservation of mouse spermatozoa in electrolyte- free solution at 4°C on the outcome of mouse in vitro fertilization.

Purpose: The aim was to assess the fertilizing capacity ofspermatozoa cool-preserved in electrolyte-free (EF)solution. Methods: Mouse spermatozoa were cool-preserved in EFsolution and the acrosomal status of the spermatozoa wascompared before and after preservation using chlortetracyclinestain. Mouse oocytes were inseminated by spermatozoacool-preserved in EF solution for 2, 4, or 7 days and fertilizationand blastocyst rates were evaluated. Results: Acrosomal status of spermatozoa cool-preservedin EF solution was not different from spermatozoa beforepreservation, but the capacitated and acrosome-reactedspermatozoa significantly increased after reinitiation. Cool-preservationin EF solution for up to 4 days did not affectfertilization rate. Blastocyst rate of embryos derived fromspermatozoa cool-preserved for 4 or 7 days in EF solutionwas significantly lower than that of embryos derived fromfresh spermatozoa. Conclusions: Mouse spermatozoa cool-preserved in EFsolution possesses as much fertilizing capacity as fresh spermatozoa.However, prolonged preservation affects theembryonic development.

Successful Advanced Maternal Age Pregnancy with Mosaic Turner Syndrome Conceived after Ovulation Induction with Clomiphene Citrate: A Case Report

Turner women typically experience gonadal dysfunction that results in amenorrhea and sterility. We encountered a case of mosaic Turner syndrome where conception was possible after ovulation induction with clomiphene citrate (CC). The patients ovaries were overresponsive to induction with CC. The challenges and successful outcome are reported.

Blastocyst transfer, its risk of Ectopic Pregnancy in Assisted Reproduction

ABSTRACT The clinical benefit of Blasotcyst transfer (BT) in IVF-ET is still controversial. Recent study elucidated the benefit of BT to reduce ectopic pregnancy in assisted reproduction. Retrospective analysis of pregnancies with BT showed lower ectopic pregnancy rate compared to the cleaved stage transfer, especially among patients with tubal factor. There could be few chance of embryo migration into the tube after BT because of a limited period from transfer to implantation, and a reduced frequency of uterine contraction at the time of transfer.

The Scheduled Ovarian Hyperstimulation Method Makes it Easy to Perform ICSI with Fresh Testicular Sperm (ICSI/TESE)

The authors evaluated whether scheduled ovarian stimulation makes it easy to perform ICSI with fresh testicular sperm. Scheduled ovarian hyperstimulation was applied for testicular sperm extraction and ICSI with fresh testicular spermatozoa. Fifteen cycles in 10 couples were included in the present study; all couples were azoospermic, 5 were obstructive, and the remaining 5 were nonobstructive. No cycles were canceled, and all oocyte retrievals were performed on the scheduled day. Testicular sperm were obtained in 14 treatment cycles (93%). The mean numbers of retrieved and injected oocytes were 9.4 and 6.4, respectively. The fertilization and cleavage rates were 47 and 91%, respectively. Embryo transfers were performed in 12 cycles except 2 cycles that had no embryos. The number of transferred embryos was 2.3. Two clinical pregnancies were obtained. This scheduled ovarian hyperstimulation was applicable for ICSI with fresh testicular sperm.