Time-lapse monitoring of zona pellucida-free embryos obtained through in vitro fertilization: a retrospective case series.

OBJECTIVE: To report time-lapse monitoring of human oocytes in which the damaged zona pellucida was removed, producing zona-free (ZF) oocytes that were cultured until the blastocyst stage in time-lapse incubators. DESIGN: Retrospective case series. SETTING: Private infertility clinic. PATIENT(S): Infertile patients (n = 32) undergoing minimal ovarian stimulation or natural cycle IVF treatment between October 2012 and June 2014. INTERVENTION(S): Intracytoplasmic sperm injection (ICSI) fertilization of ZF oocytes, prolonged embryo culture in time-lapse incubators, elective vitrification, and subsequent single vitrified-thawed blastocyst transfer (SVBT). MAIN OUTCOME MEASURE(S): Rate of fertilization, cleavage and blastocyst development, live-birth rate per SVBT cycle. RESULT(S): In spite of advanced maternal age (39 ± 4.2; range, 30-46 years), good fertilization (94%), cleavage (94%), and blastocyst development rates (38%) were reached after fertilization and culturing of ZF oocytes/embryos. All thawed ZF blastocysts survived, and up to this date seven SVBT transfers were performed, yielding three (43%) term live births with healthy newborns. CONCLUSION(S): Time-lapse imagery gives a unique insight into the dynamics of embryo development in ZF embryos. Moreover, our case series demonstrate that an oocyte with a damaged zona pellucida that has been removed could be successfully fertilized with ICSI, cultured until blastocyst stage in a time-lapse incubator and vitrified electively for subsequent use.

[Clinical result of testicular sperm extraction (TESE) to male-factor infertility in Toyota Memorial Hospital].

Owing to progress of assisted reproduction technology in recent years, it has become possible for couples with infertility problems to have children. Between March 1998 and May 2003 testicular sperm extraction (TESE) was performed on 30 men with male-factor infertility in our hospital. Consequently, we succeeded in recovering 20 spermatozoa. Intracytoplasmic sperm injection was subsequently performed in 15 couples and resulted in 8 pregnancies. There was a statistically significant difference in follicle-stimulating hormone, luteirizing hormone and Johnsen's score between the non-obstructive groups with successful TESE and those with unsuccessful TESE.

Embryo evaluation by analysing blastomere nuclei.

BACKGROUND: To create a more effective selection standard for early embryos, we developed a new grading system consisting of conventional morphological evaluation in combination with analysis of blastomere nuclei. METHODS: A total of 744 embryos used during 459 cycles of embryo transfer on day 2 and blastocyst transfer were subjected to retrospective analysis. The overall implantation rate was 15.5% (115/744). Morphological evaluation of the embryos was performed on day 2 by referring to both the size of blastomere and fragmentation (conventional method) and the nucleic features of the blastomeres--either multinucleated or anucleic (nuclei counting method). The implantation rate for every transferred embryo and blastocyst was examined. RESULTS: Although a high implantation rate was observed with the highest quality embryos as judged by either the conventional method (24.1%; 57/237) or the nuclei counting method (26.1%; 104/399), the nuclei counting method predicted implantation rate better than the conventional method. The embryos that were considered to be high quality according to the conventional method, but low quality according to the nuclei counting method, had a limited implantation success rate of 6.3% (4/66). Also, after blastocyst transfer, implantation occurred most often when high quality embryos evaluated by the nuclei counting method were used (25.5%; 25/98), while the blastocysts from low quality embryos seldom implanted (3.2%; 2/63). CONCLUSIONS: When choosing which embryo to transfer, the normality of blastomere nuclei may be a more important index of quality than standard fragmentation features and/or blastomere uniformity analysis. When choosing among embryos, if nucleic status is identical, then embryos with the least fragmentation should be chosen. Moreover, in blastocyst transfer, a blastocyst whose nuclei were judged normal on day 2 should be selected on day 5 over any other blastocysts.

Interleukin-1beta stimulates placental leucine aminopeptidase/oxytocinase expression in BeWo choriocarcinoma cells.

In addition to prostaglandins, inflammatory cytokines induce uterine contraction via oxytocin (OT). Placental leucine aminopeptidase (P-LAP), an oxytocinase that is identical to cystine aminopeptidase, destroys OT activity. Patients with spontaneous preterm delivery have higher concentrations of inflammatory cytokines and lower P-LAP activities than those with normal delivery. In addition, the P-LAP promoter region contains putative binding sites for cytokine-induced transcription factors. We therefore postulated that inflammatory cytokines suppress P-LAP expression and examined this notion using BeWo choriocarcinoma cells cultured in the presence of cytokines. However, interleukin-1beta (IL-1beta) increased P-LAP activity in a time- and dose-dependent manner. Furthermore, Western blot analysis showed a dose-dependent increase of P-LAP proteins. We also detected IL-1 type I receptor mRNA in BeWo cells by RT-PCR. Semi-quantitative RT-PCR and Southern blot analysis showed that IL-1beta also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assays did not reveal any regulatory regions that could explain IL-1beta-induced P-LAP mRNA accumulation within 1.1 kb upstream of the P-LAP gene. Immunohistochemical analysis of human placenta with chorioamnionitis demonstrated prominent P-LAP staining at sites of abundant inflammatory cell infiltration. These findings indicated that prolonged exposure to IL-1beta induces P-LAP in the trophoblasts, possibly via other de-novo protein synthesis, which contradicted our initial hypothesis.

Ap-2 and Ikaros regulate transcription of human placental leucine aminopeptidase/oxytocinase gene.

P-LAP is identical with cystine aminopeptidase as oxytocinase. We previously located on the placental leucine aminopeptidase (P-LAP) gene the footprint site with the high promoter activity (FP3: -214 to -183) and found a possible interaction between it and AP-2 in choriocarcinoma cells. Here, we investigated FP3 in detail and identified the elements responsible for the high basal rate of transcription. Electrophoretic mobility shift assays demonstrated that FP3 would interact with Ikaros as well as AP-2. Further analysis using antibody against Ikaros confirmed that Ikaros was indeed present and bound to FP3. In addition, AP-2alpha and AP-2gamma antibodies supershifted the second complex at FP3. Functionally, mutations that eliminate AP-2 binding reduced promoter activity significantly, while those that eliminate Ikaros binding reduced promoter activity insignificantly. Double mutations of AP-2 and Ikaros decreased promoter activity progressively. We conclude that AP-2 is the main activator and Ikaros functions cooperatively with it for maximal expression of the human P-LAP gene.