Five cases of dizygotic triplet pregnancy following assisted reproductive techniques.

Of 816 patients who became clinically pregnant by assisted reproductive techniques between September 2000 and August 2004, we experienced 10 cases (1.2%) of monozygotic twinning, and in five of these 10 cases, implantation of another embryo resulted in dizygotic triplets. Here, we report these five cases of dizygotic triplets. Fresh embryo transfer was performed in all five cases. Intracytoplasmic sperm injection or assisted hatching was not carried out in these cases. Blastocyst transfer was performed in three cases. Three embryos were transferred in case 1 (40-year-old female). While only two embryos were transferred in the other four cases so as to avoid triplet pregnancy, triplet pregnancies were confirmed. Triplet pregnancy was maintained in three cases, but in the other two cases, monochorionic twinning resulted in miscarriage during the first trimester. For the three patients who delivered the triplets, while the postnatal growth has been normal for all nine babies, the mothers were hospitalized for a long period of time, and an emergency cesarean section was performed on two patients. Because triplet pregnancy could not be completely prevented even when only two embryos were transferred, physicians should be sure to obtain informed consent in similar cases. (Reprod Med Biol 2005; 4: 59-64).

Elective single embryo transfer: Comparison of blastocyst and cleavage-stage embryo transfer.

Objective: Blastocysts are reportedly suitable for preventing multiple pregnancies as a result of the high implantation rate per embryo. The present study compared clinical results for elective single embryo transfer (ET) between blastocysts and cleavage-stage embryos in order to ascertain the usefulness of blastocyst culturing in single ET. Methods: Between January 2002 and December 2004, conventional in vitro fertilization ET and/or intracytoplasmic sperm injection was carried out for single ET in 86 cycles, to prevent multiple pregnancies (for medical reasons or because of patient wishes). Results: Among the 80 cycles in which a fresh embryo was transferred, pregnancy/implantation rates per ET were 35.3% for day 2/3 ET and 50.0% for day 5 ET, and pregnancy/implantation rates per oocyte retrieval were 35.3% for day 2/3 ET and 44.2% for day 5 ET. Ongoing pregnancy/delivery rates per oocyte retrieval were 32.4% for day 2/3 ET and 38.5% for day 5 ET. Monozygotic twinning occurred in one case of day 5 ET. Conclusions: Pregnancy rates per single ET tended to be higher for day 5 ET than for day 2/3 ET. However, no marked differences were identified in ongoing pregnancy/delivery rates per oocyte retrieval between groups. (Reprod Med Biol 2005; 4: 197-201).

Effect of activation methods for bovine oocytes after intracytoplasmic injection.

A principal nuclear transfer procedure is to inject a donor cell into the perivitelline space in an enucleated oocyte and then electric fusion is performed (cell fusion method). The effects of activation methods in reconstructed oocytes for the serum-starved somatic cell cloning procedure were investigated in this study by means of intracytoplasmic injection (i.c.i.). Bovine oocytes were enucleated at 18-22 h for in vitro maturation, and subsequently the nucleus of cumulus cell collected from Japanese Black Bulls (JBCC) after 5-7 days of starved culture was injected into the recipient cytoplast with a piezo-micromanipulator. At 1 h after i.c.i., reconstructed oocytes were stimulated with ethanol (ET) or calcium ionophore (CaI) as the first activation treatment, followed by cycloheximide (CHX) or 6-dimethylaminopurin (DMAP) treatment as the second activation. In the experiment on the first activation method, the proportion of reconstructed oocytes developing to the blastocyst stage was significantly (p<0.01) higher in the ET activation method than that with CaI (10.5% and 4.7%, respectively). And the experiment on the second activation method after ET treatment showed similar proportions of blastocyst development in both CHX and DMAP treatments (5.9% and 2.8%, respectively). The present results indicated that combined activation treatment with ET and CHX was efficient for reconstructed bovine oocytes by i.c.i.

Effect of polyvinyl alcohol (PVA) concentration during vitrification of in vitro matured bovine oocytes.

Polyvinyl alcohol (PVA) was used as a substitute for serum in a vitrification solution for in vitro matured bovine oocytes. In vitro matured bovine oocytes were cryopreserved in various vitrification solutions (VS) supplemented with different concentrations (0.05, 0.1, 0.5, and 1%) of PVA, 20% fetal calf serum (FCS) or without macromolecule supplementation in a gel-loading tip (GL-tip). After warming, vitrified oocytes were examined for effects on survivability, fertilizability, and embryonic development in vitro. At 18 h in vitro fertilization after vitrifying and warming, the number of surviving mature oocytes vitrified in VS without macromolecule supplementation was significantly (P < 0.05) lower than those with macromolecule supplementation. For fertilizability after vitrification, there was no significant difference in the penetration rate of oocytes among fresh oocytes (98.7%); oocytes vitrified in VS supplemented with 0.1 (76.8%), 0.5 (70.2%), or 1% (80.3%) PVA; 20% (84.1%) FCS; or without supplementation (61.7%). Also, the normal fertilization rate was not significantly different in oocytes vitrified with 0.1 (56.5%), 0.5 (43.5%), or 1% (49.7%) PVA and 20% (60.6%) FCS, compared with fresh oocytes (84.0%). Subsequently, vitrified oocytes were examined for embryonic development effects in vitro. The highest proportion of cleaved oocytes after vitrification was obtained in VS supplemented with 0.1% (18.8%) PVA. Additionally, the proportion of development to morula stage (7.7%) in the oocytes vitrified in a VS supplemented with 0.1% PVA was significantly (P < 0.05) superior to that of the 0, 0.5, and 1% PVA-vitrified groups. However, the beneficial effect of PVA addition was not found in blastocyst development. Embryonic development of vitrified oocytes was significantly lower than that of fresh oocytes. In conclusion, the present results indicate that 0.1% PVA supplementation in VS results in a significantly higher rate of morula stage embryos than 0, 0.5, and 1% PVA supplementation, and could replace FCS in VS for vitrification of in vitro matured bovine oocytes.