The time aspect in storing vitrified blastocysts: its impact on survival rate, implantation potential and babies born.

STUDY QUESTION: Does the storage time of vitrified human blastocysts negatively impact their survival, the implantation potential of embryos or the malformation rate of babies born? SUMMARY ANSWER: There was no evidence that storage times of up to 6 years after vitrification (VIT) had a negative impact on blastocyst survival, the implantation potential of embryos or the malformation rate of babies born. WHAT IS KNOWN ALREADY: Although several thousand children have been born after blastocyst VIT, many aspects of this technique remain to be elucidated. New applications, such as fertility preservation, lead to long storage times of vitrified gametes or embryos but it remains to be determined if these vitrified embryos are stable over time. STUDY DESIGN, SIZE, DURATION: A retrospective study including 603 transfers was conducted between January 2009 and April 2012. Blastocysts were vitrified using a closed system. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients underwent the transfer of aseptically vitrified/warmed blastocysts in a cryo-cycle. A total of 1077 blastocysts were transferred. Survival rates (SRs), implantation potential, birth rates and characteristics of the children born were evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: We found that the storage of vitrified blastocysts in aseptic conditions neither impaired blastocyst viability (SR after warming during the first year of storage was 83.0% compared with 83.1% after 5-6 years of storage: NS) nor decreased pregnancy rates (clinical pregnancy rate after 1 year of storage was 40.0 versus 38.5% after 6 years: NS). In addition, no increase in the malformation rate over time was observed. LIMITATIONS, REASONS FOR CAUTION: Our study only included the transfer of blastocysts which had been vitrified aseptically (i.e. using a closed system). Therefore, our results might not be applicable to 'open' VIT systems. The long-term follow-up of children born will be necessary to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS: The results suggest that vitrified human blastocysts can be stored for long periods of time without significant negative consequences for the offspring. Therefore, the method should be of benefit to those patients who need to consider taking measures for fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): No external funding was sought for this study and the authors have no conflict of interest to declare.

Individual demands of human embryos on IVF culture medium: influence on blastocyst development and pregnancy outcome.

The elucidation of the metabolic requirements of human embryos in vivo or in vitro remains, despite being intensively investigated, a work in progress. The adoption of extended embryo culture to the blastocyst stage during the last decade has entailed new challenges. With the increased attention to culture media formulations, more evidence on the sensitivity of embryos to their early environmental conditions is accumulating which might affect phenotype and developmental potential. A retrospective study was conducted that comprised 286 IVF cycles to evaluate the effect of two different culture media on blastocyst development and pregnancy outcome. Embryos were either cultured in a one step or a sequential medium. Higher fertilization rates and augmented blastocyst rates as well as higher implantation rates were observed when embryos were cultured in one step medium (P<0.05). Interestingly, the transfer of two embryos where one embryo was cultured in either medium resulted in a significantly higher rate of twin pregnancies. Although multiple pregnancies should be avoided in assisted reproduction treatment to reduce risks for offspring and mother, this higher frequency of twin pregnancies resulting from the transfer of embryos derived from different culture media suggests that each embryo makes individual demands on its early environment.

Characterization and functional analysis of the rat endothelin-1 promoter.

To define the molecular mechanisms of endothelin-1 (ET-1) gene regulation, we cloned, sequenced, and characterized the rat ET-1 promoter. A sequence consisting of the first 1329 bp of the rat ET-1 promoter was investigated in greater detail. Sequence analysis identified putative binding sites for a number of transcriptional factors that may be involved in ET-1 gene regulation. Several of these factors have been proposed earlier to be involved in cell-specific gene regulation and may be responsible for directing ET-1 expression in vivo. For functional analysis of the ET-1 promoter, we generated a reporter gene construct using luciferase as reporter gene under control of the promoter fragment isolated. The construct was transfected transiently into bovine aortic endothelial cells, and luciferase expression was evaluated. The results indicated that the promoter segment used showed high expression in endothelial cells comparable to that induced by viral promoters. Since ET-1 is regulated by a number of vasoactive substances, we studied the effect of angiotensin II on endothelin transcription. We could demonstrate a dose-dependent transcriptional activation of ET-1 transcription by angiotensin.

Regulation of the endothelin system in transgenic rats expressing the human endothelin-2 gene.

We have established a transgenic rat model for the expression of the human endothelin-2 (ET-2). These animals exhibit overexpression of the transgene in tissues as well as in plasma. Despite these changes, blood pressure remains normal. To understand the regulatory mechanisms for normotension in the presence of increased ET-2 levels, we have investigated the ET system in more detail. We used competitive reverse transcription-polymerase chain reaction (RT-PCR) to evaluate possible overexpression or downregulation of endothelin A and B receptors at the mRNA level. PCR analyses revealed no significant differences of ETA and ETB receptor expression. In conclusion, the expression of human ET-2 in transgenic rats does not result in hypertension. Normotension in the transgenic animals is independent of ET receptor regulation. The reason for this may be counterregulation by other vasoactive systems, such as the NO system. Future studies will take this into account and will also concentrate on possible histomorphologic alterations caused by mitogenic properties of the endothelins.