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See detailCryopreservation of embryos : a way to reduce the number of housed animals and the genetic drift.
Remy, Benoît ULg; Ectors, Fabien ULg; Drion, Pierre ULg

Poster (2014, January 27)

The GIGA Mouse facility platform has recently improved its mouse line cryopreservation technique. The method of embryo cryopreservation by rapid cooling also called aseptic vitrification has been selected ... [more ▼]

The GIGA Mouse facility platform has recently improved its mouse line cryopreservation technique. The method of embryo cryopreservation by rapid cooling also called aseptic vitrification has been selected. Vitrification media, key steps and timing have been optimized and validated. After a first partial exposition of the embryos to cryoprotective solutions, they are immersed in a vitrifying mixture of penetrating and non-penetrating cryoprotectants for a short time. The straw containing the embryos is immediately sealed before to be plunged in LN2, resulting in a brutal solidification in which crystallization does not have time to occur. [less ▲]

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See detailGIGA ANIMAL CARE : Mice & Zebrafish Animal Facility and Transgenesis
Remy, Benoît ULg; Ectors, Fabien ULg; Winandy, Marie ULg et al

Poster (2014, January 27)

In fundamental research, animal models allow to place molecular and cellular observations back into their physiological context. In applied research, these models still remain a mandatory step to evaluate ... [more ▼]

In fundamental research, animal models allow to place molecular and cellular observations back into their physiological context. In applied research, these models still remain a mandatory step to evaluate the efficiency and the toxicity of potential treatments, before going to clinical trials. Mouse and Zebrafish (Danio rerio) are two very interesting models because of a short live cycle and a high prolificacy. They require a limited space. Their genome is well known and shows a high homology with the human. Many tools are available to produce transgenic mice or zebrafishes. Many tests are validated using both these species. [less ▲]

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See detailVitrification of immature equine oocytes
Gatez, Carine ULg; Ectors, Fabien ULg; Farnir, Frédéric ULg et al

Poster (2013, October 11)

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See detailLong term culture and characterization of chicken primordial germ cells
Tonus, Céline ULg; Cloquette, Karine; Ectors, Fabien ULg et al

Poster (2013, October)

Avian primordial germ cells (PGCs) can keep their germ cells properties and are foreseen as promising tools for developing avian transgenesis and preservation of genetic resources of endangered species ... [more ▼]

Avian primordial germ cells (PGCs) can keep their germ cells properties and are foreseen as promising tools for developing avian transgenesis and preservation of genetic resources of endangered species. We have developed original methods that allow long term (20 month) expansion of primary cultures of undifferentiated PGCs and their efficient cryopreservation. Blood samples were collected from stage 13-18 embryos, pooled, deposited in cell culture inserts and co-cultivated in the presence of irradiated BRL cells. This physically separated co-culture system along with selective culture medium promoted emergence, selection and proliferation of undifferentiated PGCs lines. Overall, 35% of blood samples gave rise to PGCs cell lines originating from three commercial layer breeds and two Belgian endangered breeds. Moreover, we recently isolate and cultivate a new PGC line from turkey. All PGCs lines were first characterised for the expression of the stem cells and PGCs characteristic marker SSEA-1 by FACS. RT-PCR confirmed expression of germ-line specific markers (CVH, CDH, DAZL), pluripotency markers (cPouV, cSox2, cNanog), telomerase and CXCR4 receptor. In addition, by means of a quantitative PCR amplification of a chromosome W specific sequence, we demonstrated a progressive drift of all our lines towards the male sex (WL), while they were initially isolated from pooled blood samples with statistically equivalent numbers of male and female embryos (35 females: 29 males). PGCs were subsequently efficiently cryopreserved by slow freezing or by a newly developed vitrification method. Labelled PGCs from 10 lines were injected in recipient embryos. At day 6, colonization of the genital ridges confirmed that PGCs retain their gonadal migratory ability, both after long-term culture (min 3, max 20 month) and after cryopreservation. In order to evaluate the germinal differentiation of cultured PGCs during the gonadal development as well as the germline transmission rate, we established a stably expressing GFP line that was successfully injected in emrbyos. Results are in progress. [less ▲]

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See detailChapter 13: Adaptation of a Universal Procedure for Cryopreservation of Different Developmental Stages: Is it Conceivable?
Vanderzwalmen, Pierre ULg; Ectors, Fabien ULg; Grobet, Luc ULg et al

in Varghese, Alex C.; Sjöblom, Peter; Jayaprakasan, K (Eds.) A practical guide to setting up an IVF lab, embryo culture systems and running the unit (2013)

A RENEWED INTEREST IN CRYOPRESERVATION: WHY? It is now well recognized that the proportion of births following transfer of cryopreserved gametes or embryos will increase dramatically. Several reasons may ... [more ▼]

A RENEWED INTEREST IN CRYOPRESERVATION: WHY? It is now well recognized that the proportion of births following transfer of cryopreserved gametes or embryos will increase dramatically. Several reasons may explain this rising interest for embryo transfer (ET) after cryopreservation. The improvement of in vitro culture technique enables gaining better quality embryos associated with the policy of single embryo transfer (SET) results in the increasing proportion of supernumerary embryos cryopreserved at different stages of development. Moreover, with the increased efficiency of the cryopreservation technique such as vitrification, there is now a tendency to shift from fresh ET to cryopreserved ET in artificial or natural cycles. This attitude is relevant especially when the progesterone level before oocyte pick-up is above a physiological limiting value, a not optimal thickness of the endometrium or when embryos are originated from an in vitro maturation cycle. Cryopreserved ET is also an opportunity, when fresh ET cycles are cancelled because of hyperstimulation. With the introduction of vitrification, there is also an emergent change in the general attitude towards oocyte cryopreservation offering available solution, for example, in the field of preservation of fertility, ovarian hyperstimulation syndrome, poor responder, absence of sperm at the time of oocyte pick-up, cryo-banking for egg donation program or for social reason and finally to overcome ethical concerns and legal restrictions. [less ▲]

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See detailEvidence for cross-talk between the LH receptor and LH during implantation in mice
Gridelet, Virginie ULg; Tsampalas, Marie; Berndt, Sarah et al

in Reproduction, Fertility and Development (2013), 25

The present study investigated the first interaction that occurs between the blastocyst and endometrium during implantation. Given the ethical objections to studying implantation in humans, a mouse model ... [more ▼]

The present study investigated the first interaction that occurs between the blastocyst and endometrium during implantation. Given the ethical objections to studying implantation in humans, a mouse model was used to study the dialogue between luteinising hormone (LH) and luteinising hormone receptor (LHCGR). Several studies performed on LHCGR-knockout mice have generated controversy regarding the importance of the dialogue between LH and LHCGR during implantation. There has been no demonstration of a bioactive LH-like signal produced by the murine blastocyst. The first aim of the present study was to examine and quantify, using radioimmunoassay, the generation of a bioactive LH signal by the murine blastocyst. We went on to examine and quantify endometrial Lhcgr expression to validate the mouse model. Expression of LHCGR in mouse uteri was demonstrated using immunohistochemistry and western blot analysis. To quantify the expression of Lh in the mouse blastocyst and Lhcgr in the endometrium, reverse transcription–polymerase chain reaction (RT-PCR) and real-time quantitative (q) RT-PCR were performed. The results demonstrate that Lhcgr expression in BALB/c mouse endometrial epithelium is increased at the time of implantation and indicate that LHCGR may contribute to the implantation process. In support of this hypothesis, we identified a bioactive LH signal at the time of murine blastocyst implantation. [less ▲]

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See detailLower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentration of cryoprotectant solutions.
Vanderzwalmen, P.; Connan, Delphine ULg; Grobet, Luc ULg et al

in Human reproduction (Oxford, England) (2013)

STUDY QUESTION: What is the intracellular concentration of cryoprotectant (ICCP) in mouse zygotes during vitrification (VIT) and slow-freezing (SLF) cryopreservation procedures? SUMMARY ANSWER: Contrary ... [more ▼]

STUDY QUESTION: What is the intracellular concentration of cryoprotectant (ICCP) in mouse zygotes during vitrification (VIT) and slow-freezing (SLF) cryopreservation procedures? SUMMARY ANSWER: Contrary 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 ALREADY: To 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, DURATION: Two 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 Liege. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cell 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 CHANCE: The ICCP in mouse zygotes during the VIT procedure prior to plunging them in liquid nitrogen was approximately 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 degrees 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 CAUTION: The 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 FINDINGS: The 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. [less ▲]

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See detailBlastocyst transfer after aseptic vitrification of zygotes: an approach to overcome an impaired uterine environment
Vanderzwalmen, Pierre ULg; Zech, Nicolas; Ectors, Fabien ULg et al

in Reproductive Biomedicine Online (2012), 25

In some IVF cycles, no fresh embryo transfer in the stimulated cycle is advisable. The cryopreservation of zygotes and the transfer of blastocysts in a cryo-embryo transfer is an option to circumvent an ... [more ▼]

In some IVF cycles, no fresh embryo transfer in the stimulated cycle is advisable. The cryopreservation of zygotes and the transfer of blastocysts in a cryo-embryo transfer is an option to circumvent an inadequate uterine environment due to risk of ovarian hyperstimulation syndrome, inappropriate endometrium build up, endometrial polyps or uterine myomas. For this strategy, highly secure and safe cryopreservation protocols are advisable. This study describes a protocol for aseptic vitrification of zygotes that results in high survival rates and minimizes the potential risk of contamination in liquid nitrogen during cooling and long-term storage. In mouse zygotes, there was no difference in efficiency as compared with a conventional open vitrification system. In IVF patients, aseptically vitrified zygotes showed no difference in blastocyst formation rate as compared with sibling zygotes kept in fresh culture. A clinical study comprising 173 cryo-cycles with a transfer of blastocysts originating from vitrified zygotes showed an ongoing pregnancy rate of 40.9%. The live birth rate per patient was 36.8%. A combination of good clinical results and increased safety conditions due to aseptic vitrification encourages the use of cryo-embryo transfer for patients with a suboptimal uterine environment in a fresh cycle. [less ▲]

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See detailLong term culture and characterization of chicken primordial germ cells
Tonus, Céline ULg; Waroux, Olivier ULg; Cloquette, Karine et al

Poster (2012, November)

Avian primordial germ cells (PGCs), can keep their germ cells properties and are foreseen as promising tools for developing avian transgenesis and preservation of genetic resources of endangered species ... [more ▼]

Avian primordial germ cells (PGCs), can keep their germ cells properties and are foreseen as promising tools for developing avian transgenesis and preservation of genetic resources of endangered species. We have developed original methods that allow long term (20 month) expansion of primary cultures of undifferentiated PGCs and their efficient cryopreservation. Blood samples were collected from stage 13-18 embryos, pooled, deposited in cell culture inserts and co-cultivated in the presence of irradiated BRL cells. This physically separated co-culture system along with selective culture medium promoted emergence, selection and proliferation of undifferentiated PGCs lines. Overall, 35% of blood samples gave rise to PGCs cell lines originating from three commercial layer breeds and two Belgian endangered breeds. PGCs lines were first characterised for the expression of the stem cells and PGCs characteristic marker SSEA-1 by FACS (expression rate: 90-99%). RT-PCR confirmed expression of germ-line specific markers (CVH, CDH, DAZL), pluripotency markers (cPouV, cSox2, cNanog), telomerase and CXCR4 receptor. In addition, by means of a quantitative PCR amplification of a chromosome W specific sequence, we demonstrated a drift of all our lines towards the male sex (WL), while they were initially isolated from pooled blood samples with statistically equivalent numbers of male and female embryos (35 females: 29 males). PGCs were subsequently efficiently cryopreserved by slow freezing or by a newly developed vitrification method. Labelled PGCs from 10 lines were injected in recipient embryos. Colonization of the genital ridges confirmed that PGCs retain their gonadal migratory ability, both after long-term culture (min 3, max 20 month) and after cryopreservation. [less ▲]

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See detailOPTIMIZATION OF HUMAN ES CELLS (hESCs) CRYOPRESERVATION
Connan, Delphine ULg; Ectors, Fabien ULg; Grobet, Luc ULg et al

Poster (2012, October 22)

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See detailAseptic and automatable vitrification of human embryonic stem cells using defined media
Ectors, Fabien ULg

Conference (2012, October 22)

Aseptic and automatable vitrification of human embryonic stem cells using defined media Efficient recovery of biologically safe and undifferentiated hESCs is a major challenge after cryobanking ... [more ▼]

Aseptic and automatable vitrification of human embryonic stem cells using defined media Efficient recovery of biologically safe and undifferentiated hESCs is a major challenge after cryobanking, especially when their use for human therapy is foreseen. One drawback of actual cryopreservation protocols is the direct contact of the cells with liquid nitrogen, therefore cells can be in contact with heavy metals, bacteria, viruses or fungi during cooling and storage. We describe a novel cryopreservation method which uses aseptic vitrification1 (no direct contact with LN2) and only chemically defined materials and media, and is automatable. 1: Vitrification is a cryopreservation method based on the conversion of a liquid into a glass-like state by an infinite enhancement of its viscosity and without formation of ice crystals. [less ▲]

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See detailCryopréservation d’ovocytes et d’embryons par congélation ou vitrification dans le cadre de l’assistance médicale à la procréation
Vanderzwalmen, Pierre ULg; Ectors, Fabien ULg; Papras, Y et al

in Poncelet, Christophe; Sifer, Christophe (Eds.) Physiologie, pathologie et thérapie de la reproduction chez l’humain (2011)

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See detailSupport fermé: une réalité clinique pour vitrifier en conditions aseptiques les ovocytes et embryons
Vanderzwalmen, Pierre ULg; Zech, Nicolas; Prapas, Y. et al

in Gynécologie Obstétrique & Fertilité (2010), 38(9), 541-546

Vitrification with the use of ‘‘Open’’ carrier devices (Cryoloop, cryotop, cryoleaf, Vitriplug) which allowed the contact with liquid nitrogen has become a more popular way to achieve cooling rate ... [more ▼]

Vitrification with the use of ‘‘Open’’ carrier devices (Cryoloop, cryotop, cryoleaf, Vitriplug) which allowed the contact with liquid nitrogen has become a more popular way to achieve cooling rate superior to 20.000 °C/min. Even though the question of contamination with liquid nitrogen during ultra-rapid cooling and storage remain debatable with the use of ‘‘open’’ devices, it is important to revise the carrier system in a way, which minimizes the risk of contamination. According to the EU tissues and cells directive, it is advisable that the cooling and storage should be carried out in embryo carrier devices ensuring complete separation of the embryos from liquid nitrogen in a way, which minimizes the risk of contamination. The consequence of a reduction in the cooling rate resulting from the heat-insulating barrier of aseptic devices has to be counteracted by gradually increasing intracellular concentrations of cryoprotectants without inducing a toxic effect. We developed an aseptic vitrification method of vitrification for MII oocytes and embryos at different stage of development using the ‘‘VitriSafe’’ as ‘‘closed’’ carrier device. [less ▲]

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See detailLa vitrification et l’utilisation de concentrations élevées en cryoprotecteurs : ceci justifie-t-il la préférence pour la congélation lente ?
Vanderzwalmen, Pierre ULg; Zech, Nicolas; Lejeune, Bernard et al

in Gynécologie Obstétrique & Fertilité (2010), 38(9), 536-40

The use of high levels of cryoprotectants (CPs) in solutions applied to vitrify oocytes or embryos is an argument to still prefer slow freezing procedure. Is it a justified argument? Out of three studies ... [more ▼]

The use of high levels of cryoprotectants (CPs) in solutions applied to vitrify oocytes or embryos is an argument to still prefer slow freezing procedure. Is it a justified argument? Out of three studies using mice zygotes we may assume that (i) the intracellular concentration of CPs is far lower than the one in the vitrification solutions, (ii) the intracellular concentration of CPs in the vitrified zygote is in contrary to the common beliefs even lower than the one observed after a slow freezing procedure, (iii) survival after slow freezing reflects the presence of an intracellular vitrified state in these cells. [less ▲]

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