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Vanderzwalmen P.,Ivf Centers Prof Zech | Vanderzwalmen P.,University of Liege | Connan D.,University of Liege | Grobet L.,University of Liege | And 5 more authors.
Human Reproduction | Year: 2013

STUDY QUESTIONWhat is the intracellular concentration of cryoprotectant (ICCP) in mouse zygotes during vitrification (VIT) and slow-freezing (SLF) cryopreservation procedures?SUMMARY ANSWERContrary to common beliefs, it was observed that the ICCP in vitrified zygotes is lower than after SLF, although the solutions used in VIT contain higher concentrations of cryoprotectants (CPs).WHAT IS KNOWN ALREADYTo reduce the likelihood of intracellular ice crystal formation, which has detrimental effects on cell organelles and membranes, VIT was introduced as an alternative to SLF to cryopreserve embryos and gametes. Combined with high cooling and warming rates, the use of high concentrations of CPs favours an intracellular environment that supports and maintains the transition from a liquid to a solid glass-like state devoid of crystals. Although the up-to-date publications are reassuring in terms of obstetric and perinatal outcomes after VIT, a fear about exposing gametes and embryos to high amounts of CPs that exceed 3-4-fold those found in SLF was central to a debate initiated by advocates of SLF procedures.STUDY DESIGN, SIZE, DURATIONTwo experimental set-ups were applied. The objective of a first study was to determine the ICCP at the end of the exposure steps to the CP solutions with our VIT protocol (n = 31). The goal of the second investigation was to compare the ICCP between VIT (n = 30) and SLF (n = 30). All experiments were performed in triplicates using mouse zygotes. The study took place at the GIGA-Research Institute of the University of Liège.PARTICIPANTS/MATERIALS, SETTING, METHODSCell volume is modified by changes in extracellular osmolarity. Hence, we estimated the final ICCP after the incubation steps in the VIT solutions by exposing the cells to sucrose (SUC) solutions with defined molarities. The ICCP was calculated from the SUC concentration that produced no change in cell volume, i.e. when intra- and extracellular osmolarities were equivalent. Cell volume was monitored by microscopic cinematography. ICCP was compared between SLF and VIT based on the principle that a high ICCP lowers the probability of (re)crystallization during warming but increases the probability of over-swelling of the cell due to fast inflow of water. The survival rates of mouse zygotes after SLF or VIT were compared using either (i) various warming rates or (ii) various concentrations of SUC in the warming dilution medium.MAIN RESULTS AND THE ROLE OF CHANCEThe ICCP in mouse zygotes during the VIT procedure prior to plunging them in liquid nitrogen was ∼2.14 M, i.e. one-third of the concentration in the VIT solution. After SLF, the warming rate did not affect the zygote survival rate. In contrast, only 3/30 vitrified zygotes survived when warmed slowly but as many as 30/30 zygotes survived when warming was fast (>20 000°C/min). Vitrified zygotes showed significantly higher survival rates than slow-frozen zygotes when they were placed directly in the culture medium or in solutions containing low concentrations of SUC (P < 0.01). These two experiments demonstrate a lower ICCP after VIT than after SLF.LIMITATIONS, REASONS FOR CAUTIONThe results should not be directly extrapolated to other stages of development or to other species due to possible differences in membrane permeability to water and CPs.WIDER IMPLICATIONS OF THE FINDINGSThe low ICCP we observed after VIT removes the concern about high ICCP after VIT, at least in murine zygotes and helps to explain the observed efficiency and lack of toxicity of VIT.STUDY FUNDING / COMPETING INTEREST(S)The study was funded by the FNRS (National Funds for Scientific Research). The authors declare that they have no competing interests. © The Author 2013.

Papatheodorou A.,Fertility Center | Papatheodorou A.,University of Ioannina | Vanderzwalmen P.,Ivf Centers Prof Zech | Vanderzwalmen P.,Center Hospitalier Inter regional Cavell | And 7 more authors.
Reproductive BioMedicine Online | Year: 2013

Vitrification has been successfully applied in the cryopreservation of oocytes and embryos. It can be achieved either by direct (open system) or indirect (closed system) contact with liquid nitrogen. Unlike embryo vitrification, few reports have been published regarding oocyte vitrification in closed systems. In order to validate the effectiveness of a closed and aseptic vitrification approach for oocyte cryopreservation, a prospective, randomized study was performed. Sibling oocytes donated from the same donor were randomly and equally assigned into closed or open vitrification groups. A total of 75 vitrification-warming cycles were performed in each group. Apart from the survival rate (82.9% versus 91.0%, P < 0.05), no statistically significant differences were observed in pregnancy (β-human chorionic gonadotrophin positive) (42.7% versus 33.3%), clinical pregnancy (36.0% versus 28.0%), implantation (13.8% versus 10.1%), ongoing pregnancy (33.3% versus 24.0%) and live birth (36.0% versus 24.0%) rates between the closed and open groups, and 27 and 18 healthy babies were born, respectively. This study shows that the replacement of the open vitrification system by a closed system has no impact on clinical pregnancy and implantation rates. Therefore, the closed vitrification system provides an aseptic alternative to the open method for oocyte vitrification. © 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Ebner T.,IVF Unit | Vanderzwalmen P.,Center Hospitalier Inter regional Cavell | Vanderzwalmen P.,Ivf Centers Prof Zech | Shebl O.,IVF Unit | And 3 more authors.
Journal fur Reproduktionsmedizin und Endokrinologie | Year: 2011

The topic whether blastocyst culture and transfer is a promising tool in IVF laboratories has been discussed controversially. Discrepancies in outcome may be explained by the fact that formation of a blastocyst on day 5 does not automatically correspond to its viability. Adequate morphological scoring at blastocyst stage (quality of inner cell mass and trophectoderm, expansion, screening for anomalies) would definitely help to significantly reduce the number of blastocysts being replaced, which in turn would limit the number of multiple gestations. Such a strategy could automatically increase the number of blastocysts to be frozen for later replacement. Currently, vitrification challenges slow freezing as the cryopreservation method of choice, since it is faster, more cost-effective and yields at least comparable thawing results. With respect to this, four morphological parameters of vitrified/warmed blastocyts are reported (re-expansion, hatching, necrotic foci and cytoplasmic defects) which can successfully be used for selection purposes in frozen blastocyst transfer. To conclude, blastocyst culture, transfer and cryopreservation is certainly a valuable method in the hands of IVF practitioners and has gained acceptance by many programs throughout the world.

Wogatzky J.,Centers Prof Zech Bregenz | Wirleitner B.,Centers Prof Zech Bregenz | Stecher A.,Centers Prof Zech Bregenz | Vanderzwalmen P.,Centers Prof Zech Bregenz | And 6 more authors.
Reproductive Biology and Endocrinology | Year: 2012

Background: Poor sperm quality can negatively affect embryonic development and IVF outcome. This study is aimed at investigating the influence of various lifestyle factors on semen quality according to MSOME (motile sperm organelle morphology examination) criteria.Methods: 1683 male patients undergoing assisted reproductive technologies (ART) in our clinic were surveyed about their age, BMI (body mass index), ejaculation frequency, nutrition, sports, sleeping habits and social behavior. Semen samples were collected and evaluation of semen parameters according to MSOME and WHO criteria was performed. Results were grouped and statistically analyzed.Results: Although single parameters had minor effects on sperm parameter, the combination of age, BMI, coffee intake, ejaculatory frequency and duration of sexual abstinence were identified as factors having a negative effect on sperm motility. Additionally, we could demonstrate that MSOME quality was reduced. The negative impact of age, BMI and coffee intake on sperm quality could be compensated if patients had a high ejaculation frequency and shorter periods of sexual abstinence.Conclusions: Combinations of adverse lifestyle factors could have a detrimental impact on sperm, not only in terms of motility and sperm count but also in terms of sperm head vacuolization. This negative impact was shown to be compensated by higher ejaculation frequency and a shorter period of sexual abstinence. The compensation is most likely due to a shorter storage time in the male gonads, thus reducing the duration of sperms' exposure to reactive oxygen species (ROS). © 2012 Wogatzky et al.; licensee BioMed Central Ltd.

Panagiotidis Y.,Iakentro Advanced Medical Center | Panagiotidis Y.,Democritus University of Thrace | Vanderzwalmen P.,Center Hospitalier Inter regional Cavell | Vanderzwalmen P.,Ivf Centers Prof Zech | And 10 more authors.
Reproductive BioMedicine Online | Year: 2013

The use of open carriers for embryo vitrification has raised safety concerns and therefore vitrification in closed systems has been proposed. However, the drop in the cooling rate emerges as a major drawback. The objective of the present study was to compare the efficiency of vitrification in open versus closed conditions. Blastocysts were randomly allocated either to open ultra-rapid vitrification (group I) or closed aseptic vitrification (group II). In group I, blastocysts were exposed to two solutions of ethylene glycol/dimethylsulphoxide (10%/10% and 20%/20%), while in group II, blastocysts were pretreated with a solution of lower concentration (5%/5%). A total of 208 and 224 vitrification-warming cycles were performed for groups I and II, respectively. Both groups were equal in terms of maternal age, sperm parameters and number and quality of blastocysts vitrified, warmed and transferred per cycle. Importantly, there was no significant difference between the groups in the analysed outcomes; embryo survival rate (84.1% versus 82.1%), clinical pregnancy rate (45.9% versus 42.4%), implantation rate (25.6% versus 24.5%), cycle cancellation rate (6.7% versus 8.5%) and live birth rate (41.2% versus 41.0%). These data suggest that ultra-rapid vitrification may be replaced by aseptic vitrification without affecting clinical efficiency. © 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

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