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Oocyte cryopreservation and in vitro culture affect calcium signalling during human fertilization

Dimitra Nikiforaki (UGent) , Frauke Vanden Meerschaut (UGent) , Chen Qian (UGent) , Ilse De Croo (UGent) , Yuechao Lu (UGent) , Tom Deroo (UGent) , Etienne Van den Abbeel (UGent) , Björn Heindryckx (UGent) and Petra De Sutter (UGent)
(2014) HUMAN REPRODUCTION. 29(1). p.29-40
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Abstract
What are the precise patterns of calcium oscillations during the fertilization of human oocytes matured either in vivo or in vitro or aged in vitro and what is the effect of cryopreservation? Human oocytes matured in vivo exhibit a specific pattern of calcium oscillations, which is affected by in vitro maturation, in vitro ageing and cryopreservation. Oscillations in cytoplasmic calcium concentration are crucial for oocyte activation and further embryonic development. While several studies have described in detail the calcium oscillation pattern during fertilization in animal models, studies with human oocytes are scarce. This was a laboratory-based study using human MII oocytes matured in vivo or in vitro either fresh or after cryopreservation with slow freezing or vitrification. Altogether, 205 human oocytes were included in the analysis. In vivo and in vitro matured human oocytes were used for this research either fresh or following vitrification/warming (V/W) and slow freezing/thawing (F/T). Human oocytes were obtained following written informed consent from patients undergoing ovarian hyperstimulation. For the calcium pattern analysis, oocytes were loaded with the ratiometric calcium indicator fluorescent dye Fura-2. Following ICSI using sperm from a single donor, intracellular calcium was measured for 16 h at 37C under 6 CO2. The calcium oscillation parameters were calculated for all intact oocytes that showed calcium oscillations and were analyzed using the MannWhitney U-test. Human in vivo MII oocytes display a specific pattern of calcium oscillations following ICSI. This pattern is significantly affected by in vitro ageing, with the calcium oscillations occurring over a longer period of time and with a lower frequency, shorter duration and higher amplitude (P 0.05). In vitro matured oocytes from the GV and MI stage exhibit a different pattern of calcium oscillations with calcium transients being of lower frequency and shorter duration compared with in vivo matured MII. In MI oocytes that reached the MII stage within 3 h the calcium oscillations additionally appear over a longer period of time (P 0.05). In vivo MII oocytes show a different calcium oscillation pattern following V/W with calcium oscillations occurring over a longer period of time, with a higher amplitude and a lower frequency (P 0.05). In vitro matured oocytes, either from the GV or the MI stage, also display an altered pattern of calcium oscillations after V/W and the parameters that were similarly affected in all these oocyte groups are the frequency and the amplitude of the calcium transients. Slow freezing/thawing differentially affects the calcium oscillation pattern of in vitro matured and in vitro aged oocytes. The relationship between a specific pattern of calcium oscillations and subsequent human embryonic development could not be evaluated since the calcium indicator used and the high-intensity excitation light impair development. Furthermore, all oocytes were derived from stimulated cycles and immature oocytes were denuded prior to in vitro maturation. Our data show for the first time how calcium signalling during human fertilization is affected by oocyte in vitro maturation, in vitro ageing as well as V/W and slow freezing/thawing. The analysis of calcium oscillations could be used as an oocyte quality indicator to evaluate in vitro culture and cryopreservation techniques of human oocytes. This work was supported by a clinical research mandate from the Flemish Foundation of Scientific Research (FWO-Vlaanderen, FWO09/ASP/063) to F.V.M, a fundamental clinical research mandate from the FWO-Vlaanderen (FWO05/FKM/001) to P.D.S and a Ghent University grant (KAN-BOF E/01321/01) to B.H. The authors have no conflict of interest to declare.
Keywords
fertilization, calcium oscillations, oocyte maturation, oocyte vitrification, PHOSPHOLIPASE-C-ZETA, INTRACYTOPLASMIC SPERM INJECTION, MOUSE EGG ACTIVATION, HUMAN MATURE OOCYTES, II HUMAN OOCYTES, CA2+ OSCILLATIONS, EMBRYO DEVELOPMENT, PLC-ZETA, CHROMOSOME CONFIGURATIONS, INTRACELLULAR CALCIUM

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MLA
Nikiforaki, Dimitra, Frauke Vanden Meerschaut, Chen Qian, et al. “Oocyte Cryopreservation and in Vitro Culture Affect Calcium Signalling During Human Fertilization.” HUMAN REPRODUCTION 29.1 (2014): 29–40. Print.
APA
Nikiforaki, D., Vanden Meerschaut, F., Qian, C., De Croo, I., Lu, Y., Deroo, T., Van den Abbeel, E., et al. (2014). Oocyte cryopreservation and in vitro culture affect calcium signalling during human fertilization. HUMAN REPRODUCTION, 29(1), 29–40.
Chicago author-date
Nikiforaki, Dimitra, Frauke Vanden Meerschaut, Chen Qian, Ilse De Croo, Yuechao Lu, Tom Deroo, Etienne Van den Abbeel, Björn Heindryckx, and Petra De Sutter. 2014. “Oocyte Cryopreservation and in Vitro Culture Affect Calcium Signalling During Human Fertilization.” Human Reproduction 29 (1): 29–40.
Chicago author-date (all authors)
Nikiforaki, Dimitra, Frauke Vanden Meerschaut, Chen Qian, Ilse De Croo, Yuechao Lu, Tom Deroo, Etienne Van den Abbeel, Björn Heindryckx, and Petra De Sutter. 2014. “Oocyte Cryopreservation and in Vitro Culture Affect Calcium Signalling During Human Fertilization.” Human Reproduction 29 (1): 29–40.
Vancouver
1.
Nikiforaki D, Vanden Meerschaut F, Qian C, De Croo I, Lu Y, Deroo T, et al. Oocyte cryopreservation and in vitro culture affect calcium signalling during human fertilization. HUMAN REPRODUCTION. 2014;29(1):29–40.
IEEE
[1]
D. Nikiforaki et al., “Oocyte cryopreservation and in vitro culture affect calcium signalling during human fertilization,” HUMAN REPRODUCTION, vol. 29, no. 1, pp. 29–40, 2014.
@article{4382454,
  abstract     = {What are the precise patterns of calcium oscillations during the fertilization of human oocytes matured either in vivo or in vitro or aged in vitro and what is the effect of cryopreservation?
Human oocytes matured in vivo exhibit a specific pattern of calcium oscillations, which is affected by in vitro maturation, in vitro ageing and cryopreservation.
Oscillations in cytoplasmic calcium concentration are crucial for oocyte activation and further embryonic development. While several studies have described in detail the calcium oscillation pattern during fertilization in animal models, studies with human oocytes are scarce.
This was a laboratory-based study using human MII oocytes matured in vivo or in vitro either fresh or after cryopreservation with slow freezing or vitrification. Altogether, 205 human oocytes were included in the analysis.
In vivo and in vitro matured human oocytes were used for this research either fresh or following vitrification/warming (V/W) and slow freezing/thawing (F/T). Human oocytes were obtained following written informed consent from patients undergoing ovarian hyperstimulation. For the calcium pattern analysis, oocytes were loaded with the ratiometric calcium indicator fluorescent dye Fura-2. Following ICSI using sperm from a single donor, intracellular calcium was measured for 16 h at 37C under 6 CO2. The calcium oscillation parameters were calculated for all intact oocytes that showed calcium oscillations and were analyzed using the MannWhitney U-test.
Human in vivo MII oocytes display a specific pattern of calcium oscillations following ICSI. This pattern is significantly affected by in vitro ageing, with the calcium oscillations occurring over a longer period of time and with a lower frequency, shorter duration and higher amplitude (P 0.05). In vitro matured oocytes from the GV and MI stage exhibit a different pattern of calcium oscillations with calcium transients being of lower frequency and shorter duration compared with in vivo matured MII. In MI oocytes that reached the MII stage within 3 h the calcium oscillations additionally appear over a longer period of time (P 0.05). In vivo MII oocytes show a different calcium oscillation pattern following V/W with calcium oscillations occurring over a longer period of time, with a higher amplitude and a lower frequency (P 0.05). In vitro matured oocytes, either from the GV or the MI stage, also display an altered pattern of calcium oscillations after V/W and the parameters that were similarly affected in all these oocyte groups are the frequency and the amplitude of the calcium transients. Slow freezing/thawing differentially affects the calcium oscillation pattern of in vitro matured and in vitro aged oocytes.
The relationship between a specific pattern of calcium oscillations and subsequent human embryonic development could not be evaluated since the calcium indicator used and the high-intensity excitation light impair development. Furthermore, all oocytes were derived from stimulated cycles and immature oocytes were denuded prior to in vitro maturation.
Our data show for the first time how calcium signalling during human fertilization is affected by oocyte in vitro maturation, in vitro ageing as well as V/W and slow freezing/thawing. The analysis of calcium oscillations could be used as an oocyte quality indicator to evaluate in vitro culture and cryopreservation techniques of human oocytes.
This work was supported by a clinical research mandate from the Flemish Foundation of Scientific Research (FWO-Vlaanderen, FWO09/ASP/063) to F.V.M, a fundamental clinical research mandate from the FWO-Vlaanderen (FWO05/FKM/001) to P.D.S and a Ghent University grant (KAN-BOF E/01321/01) to B.H. The authors have no conflict of interest to declare.},
  author       = {Nikiforaki, Dimitra and Vanden Meerschaut, Frauke and Qian, Chen and De Croo, Ilse and Lu, Yuechao and Deroo, Tom and Van den Abbeel, Etienne and Heindryckx, Björn and De Sutter, Petra},
  issn         = {0268-1161},
  journal      = {HUMAN REPRODUCTION},
  keywords     = {fertilization,calcium oscillations,oocyte maturation,oocyte vitrification,PHOSPHOLIPASE-C-ZETA,INTRACYTOPLASMIC SPERM INJECTION,MOUSE EGG ACTIVATION,HUMAN MATURE OOCYTES,II HUMAN OOCYTES,CA2+ OSCILLATIONS,EMBRYO DEVELOPMENT,PLC-ZETA,CHROMOSOME CONFIGURATIONS,INTRACELLULAR CALCIUM},
  language     = {eng},
  number       = {1},
  pages        = {29--40},
  title        = {Oocyte cryopreservation and in vitro culture affect calcium signalling during human fertilization},
  url          = {http://dx.doi.org/10.1093/humrep/det404},
  volume       = {29},
  year         = {2014},
}

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