Lower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentration of cryoprotectant solutions.
; Connan, Delphine ; Grobet, Luc 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 ▲]Detailed reference viewed: 14 (2 ULg)
Relationship of human follicular diameter with oocyte fertilization and development after in-vitro fertilization or intracytoplasmic sperm injection.
Ectors, Fabien ; ; et al
in Human Reproduction (1997), 12(9), 2002-2005
The aim of this work was to evaluate the relationship between follicular size at the time of oocyte retrieval, and the subsequent oocyte competence to be fertilized and to develop in vitro. All the ... [more ▼]
The aim of this work was to evaluate the relationship between follicular size at the time of oocyte retrieval, and the subsequent oocyte competence to be fertilized and to develop in vitro. All the obtained oocytes were classified according to the corresponding volume of aspirated follicular fluid. Aspirated volume of follicular fluid <2 ml corresponded to a follicular diameter <16 mm and constituted the small size group. Volume of follicular fluid from 2 to 6 ml corresponded to a diameter from 16 to 23 mm and constituted the medium size group. The large size group contained follicles with diameter >23 mm and corresponded to an aspirated volume of follicular fluid of >6 ml. A progressive and significant increase in the rates of oocytes with a first polar body was observed from the small size group to the other groups and from the medium to the large size group: 75.3, 85.9 and 95.3% respectively. After classical in-vitro fertilization (IVF), significantly better rates of fertilization and development were obtained in the medium size group compared to the two other groups. Moreover, a positive relationship was observed between follicular diameter and rates of embryos scored as 'good' when oocytes were fertilized by intracytoplasmic sperm injection (ICSI). These results demonstrated that follicular size is positively related to the oocyte ability to be fertilized and to develop. Although oocytes from small follicles gave lower percentages of development probably due to partial oocyte incompetence, they allowed an increase in the total number of embryos scored as 'good'. [less ▲]Detailed reference viewed: 11 (5 ULg)
The behaviour of cow blastocyst in vitro: cinematographic and morphometric analysis.
; ; et al
in Journal of Anatomy (1982), 134(Pt 2), 399-405
The behaviour of the cow blastocyst in vitro was studied by time-lapse cinematography and analysed by morphometry. Three types of behaviour were observed: continuous expansion followed by hatching ... [more ▼]
The behaviour of the cow blastocyst in vitro was studied by time-lapse cinematography and analysed by morphometry. Three types of behaviour were observed: continuous expansion followed by hatching; discontinuous expansion interrupted by few contractions and followed by hatching; discontinuous expansion interrupted by several rapid contractions without hatching. This demonstrated that the pulsatile activity of the blastocyst is not a necessary condition of hatching but also that only a moderate pulsatile activity is compatible with normal hatching. The time of hatching in vitro corresponded approximately with the time of zona loss in vivo (9-10 days). Rupture of the zona occurred at any point of the trophoblast layer. Hatching by herniation through a reduced opening of the zona was occasionally observed. The behavior of the embryos from a particular animal was very similar but differences were noted between embryos from different animals. [less ▲]Detailed reference viewed: 19 (2 ULg)