Skip to main content
Top
Published in: Reproductive Biology and Endocrinology 1/2019

Open Access 01-12-2019 | Progesterone | Research

A monocentric analysis of the efficacy of extracellular cryoprotectants in unfrozen solutions for cleavage stage embryos

Authors: Francesco Capodanno, Jessica Daolio, Gaetano De Feo, Angela Falbo, Daria Morini, Alessia Nicoli, Luca Braglia, MariaTeresa Villani, Giovanni B. La Sala, Lodovico Parmegiani, Lorenzo Aguzzoli

Published in: Reproductive Biology and Endocrinology | Issue 1/2019

Login to get access

Abstract

Background

In the absence of international guidelines indicating the usage of vitrification rather than slow-freezing, the study aim was to analyze a large cohort of slow-frozen/thawed embryos to produce a rationale supporting the standardization of IVF cryopreservation policy.

Methods

This retrospective analysis included 4779 cleavage stage embryos cryopreserved by slow-freezing/thawing from September 2009 to April 2017 at a single Center. Biological and clinical outcomes of three different commercial kits adopted sequentially, i.e. Vitrolife Cleave Kit® from Vitrolife (kit 1) vs. K-SICS-5000 Kit® and K-SITS-5000 Kit® from Cook Medical (kit 2) and Freeze/Thaw 1™ Kit® from Vitrolife (kit 3) were collected and compared in the light of cryoprotectants composition.

Results

Kit 3 compared to kit 1 and kit 2 showed significantly (P < 0.001) higher embryo survival (79.9% vs. 75.6 and 68.1%, respectively) and frozen embryo replacement (91.5% vs. 86.5 and 83.3%, respectively) rates, and significantly (P < 0.001) lower blastomere degeneration rate (41.5% vs. 43.6 and 52.4%, respectively). No significant difference for clinical outcomes was observed among kits. Only a slight positive trend was observed for kit 3 vs. kit 1 and kit 2 on delivery rate per thawing cycle (7.12% vs. 4.19 and 4.51%, respectively; P < 0.058) and live birth rate (3.07% vs. 2.59 and 1.93%, respectively, P < 0.069). Thawing solutions of kit 3 were similar to those of any warming protocol.

Conclusions

A defined concentration of extracellular cryoprotectants in thawing/warming solutions had a beneficial effect on the embryo cryosurvival rate. Results could provide the rationale for the adoption of a single standardized warming protocol.
Literature
1.
go back to reference Shaw JM, Jones GM. Terminology associated with vitrification and other cryopreservation procedures for oocytes and embryos. Hum Reprod Update. 2003;9:583–605.CrossRef Shaw JM, Jones GM. Terminology associated with vitrification and other cryopreservation procedures for oocytes and embryos. Hum Reprod Update. 2003;9:583–605.CrossRef
2.
go back to reference Loutradi KE, Kolibianakis EM, Venetis CA, Papanikolaou EG, Pados G, Bontis I, Tarlatzis BC. Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertil Steril. 2008;90:186–93.CrossRef Loutradi KE, Kolibianakis EM, Venetis CA, Papanikolaou EG, Pados G, Bontis I, Tarlatzis BC. Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertil Steril. 2008;90:186–93.CrossRef
3.
go back to reference Veleva Z, Orava M, Nuojua-Huttunen S, Tapanainen JS, Martikainen H. Factors affecting the outcome of frozen thawed embryo transfer. Hum Reprod. 2013;28:2425–31.CrossRef Veleva Z, Orava M, Nuojua-Huttunen S, Tapanainen JS, Martikainen H. Factors affecting the outcome of frozen thawed embryo transfer. Hum Reprod. 2013;28:2425–31.CrossRef
4.
go back to reference Wong KM, Mastenbroek S, Repping S. Cryopreservation of human embryos and its contribution to in vitro fertilization success rates. Fertil Steril. 2014;102:19–26.CrossRef Wong KM, Mastenbroek S, Repping S. Cryopreservation of human embryos and its contribution to in vitro fertilization success rates. Fertil Steril. 2014;102:19–26.CrossRef
5.
go back to reference Sparks AE. Human embryo cryopreservation-methods, timing, and other considerations for optimizing an embryo cryopreservation program. Semin Reprod Med. 2015;33:128–44.CrossRef Sparks AE. Human embryo cryopreservation-methods, timing, and other considerations for optimizing an embryo cryopreservation program. Semin Reprod Med. 2015;33:128–44.CrossRef
6.
go back to reference Zegers-Hochschild F, David Adamson G, Dyer S, Racowsky C, de Mouzon J, Sokol R, Rienzi L, Sunde A, Schmidt L, Cooke ID, Simpson JL, van der Poel S. The international glossary on infertility and fertility care. Hum Reprod. 2017;32:1786–801.CrossRef Zegers-Hochschild F, David Adamson G, Dyer S, Racowsky C, de Mouzon J, Sokol R, Rienzi L, Sunde A, Schmidt L, Cooke ID, Simpson JL, van der Poel S. The international glossary on infertility and fertility care. Hum Reprod. 2017;32:1786–801.CrossRef
7.
go back to reference Vladimirov IK, Tacheva D, Diez A. Theory about the embryo Cryo-treatment. Reprod Med Biol. 2017;16:118–25.CrossRef Vladimirov IK, Tacheva D, Diez A. Theory about the embryo Cryo-treatment. Reprod Med Biol. 2017;16:118–25.CrossRef
9.
go back to reference European IVF-monitoring Consortium (EIM); European Society of Human Reproduction and Embryology (ESHRE). Assisted reproductive technology in Europe, 2013: results generated from European registers by ESHRE. Hum Reprod. 2017;32:1957–73.CrossRef European IVF-monitoring Consortium (EIM); European Society of Human Reproduction and Embryology (ESHRE). Assisted reproductive technology in Europe, 2013: results generated from European registers by ESHRE. Hum Reprod. 2017;32:1957–73.CrossRef
10.
go back to reference De Geyter C, Calhaz-Jorge C, Kupka MS, Wyns C, Mocanu E, Motrenko T, Scaravelli G, Smeenk J, Vidakovic S, Goossens V. European IVF-monitoring consortium (EIM) for the European Society of Human Reproduction and Embryology (ESHRE). ART in Europe, 2014: results generated from European registries by ESHRE: The European IVF-monitoring Consortium (EIM) for the European Society of Human Reproduction and Embryology (ESHRE). Hum Reprod. 2018;33:1586–601.CrossRef De Geyter C, Calhaz-Jorge C, Kupka MS, Wyns C, Mocanu E, Motrenko T, Scaravelli G, Smeenk J, Vidakovic S, Goossens V. European IVF-monitoring consortium (EIM) for the European Society of Human Reproduction and Embryology (ESHRE). ART in Europe, 2014: results generated from European registries by ESHRE: The European IVF-monitoring Consortium (EIM) for the European Society of Human Reproduction and Embryology (ESHRE). Hum Reprod. 2018;33:1586–601.CrossRef
11.
go back to reference Edgar DH, Gook DA. A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos. Hum Reprod Update. 2012;18:536–54.CrossRef Edgar DH, Gook DA. A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos. Hum Reprod Update. 2012;18:536–54.CrossRef
12.
go back to reference Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature. 1983;305:707–9.CrossRef Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature. 1983;305:707–9.CrossRef
13.
go back to reference Kupka MS, D’Hooghe T, Ferraretti AP, de Mouzon J, Erb K, Castilla JA, Calhaz-Jorge C, De Geyter C, Goossens V. Assisted reproductive technology in Europe, 2011: results generated from European registers by ESHRE. Hum Reprod. 2016;31:233–48.PubMed Kupka MS, D’Hooghe T, Ferraretti AP, de Mouzon J, Erb K, Castilla JA, Calhaz-Jorge C, De Geyter C, Goossens V. Assisted reproductive technology in Europe, 2011: results generated from European registers by ESHRE. Hum Reprod. 2016;31:233–48.PubMed
14.
go back to reference Van Landuyt L, Van de Velde H, De Vos A, Haentjens P, Blockeel C, Tournaye H, Verheyen G. Influence of cell loss after vitrification or slow-freezing on further in vitro development and implantation of human day 3 embryos. Hum Reprod. 2013;28:2943–9.CrossRef Van Landuyt L, Van de Velde H, De Vos A, Haentjens P, Blockeel C, Tournaye H, Verheyen G. Influence of cell loss after vitrification or slow-freezing on further in vitro development and implantation of human day 3 embryos. Hum Reprod. 2013;28:2943–9.CrossRef
15.
16.
go back to reference Guan Y, Fan H, Styer AK, Xiao Z, Li Z, Zhang J, Sun L, Wang X, Zhang Z. A modified natural cycle results in higher live birth rate in vitrified-thawed embryo transfer for women with regular menstruation. Syst Biol Reprod Med. 2016;62:335–42.CrossRef Guan Y, Fan H, Styer AK, Xiao Z, Li Z, Zhang J, Sun L, Wang X, Zhang Z. A modified natural cycle results in higher live birth rate in vitrified-thawed embryo transfer for women with regular menstruation. Syst Biol Reprod Med. 2016;62:335–42.CrossRef
17.
go back to reference Balaban B, Urman B, Ata B, Isiklar A, Larman MG, Hamilton R, Gardner DK. A randomized controlled study of human day 3 embryo cryopreservation by slow-freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Hum Reprod. 2008;23:1976–82.CrossRef Balaban B, Urman B, Ata B, Isiklar A, Larman MG, Hamilton R, Gardner DK. A randomized controlled study of human day 3 embryo cryopreservation by slow-freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Hum Reprod. 2008;23:1976–82.CrossRef
18.
go back to reference Wilding MG, Capobianco C, Montanaro N, Kabili G, Di Matteo L, Fusco E, Dale B. Human cleavage-stage embryo vitrification is comparable to slow-rate cryopreservation in cycles of assisted reproduction. J Assist Reprod Genet. 2010;27:549–54.CrossRef Wilding MG, Capobianco C, Montanaro N, Kabili G, Di Matteo L, Fusco E, Dale B. Human cleavage-stage embryo vitrification is comparable to slow-rate cryopreservation in cycles of assisted reproduction. J Assist Reprod Genet. 2010;27:549–54.CrossRef
19.
go back to reference Fasano G, Fontenelle N, Vannin AS, Biramane J, Devreker F, Englert Y, Delbaere A. A randomized controlled trial comparing two vitrification methods versus slow-freezing for cryopreservation of human cleavage stage embryos. J Assist Reprod Genet. 2014;31:241–7.CrossRef Fasano G, Fontenelle N, Vannin AS, Biramane J, Devreker F, Englert Y, Delbaere A. A randomized controlled trial comparing two vitrification methods versus slow-freezing for cryopreservation of human cleavage stage embryos. J Assist Reprod Genet. 2014;31:241–7.CrossRef
20.
go back to reference Debrock S, Peeraer K, Fernandez Gallardo E, De Neubourg D, Spiessens C, D’Hooghe TM. Vitrification of cleavage stage day 3 embryos results in higher live birth rates than conventional slow-freezing: a RCT. Hum Reprod. 2015;30:1820–30.CrossRef Debrock S, Peeraer K, Fernandez Gallardo E, De Neubourg D, Spiessens C, D’Hooghe TM. Vitrification of cleavage stage day 3 embryos results in higher live birth rates than conventional slow-freezing: a RCT. Hum Reprod. 2015;30:1820–30.CrossRef
21.
go back to reference Pouget O, Scalici E, Hoa-Ferrieres A, Deutsch-Bringer S, Gala A, Dechaud H, Hamamah S. Comparison of frozen embryo transfer outcomes at blastocyst stage according to freezing method and type of endometrial preparation. Gynecol Obstet Fertil. 2015;43:219–24.CrossRef Pouget O, Scalici E, Hoa-Ferrieres A, Deutsch-Bringer S, Gala A, Dechaud H, Hamamah S. Comparison of frozen embryo transfer outcomes at blastocyst stage according to freezing method and type of endometrial preparation. Gynecol Obstet Fertil. 2015;43:219–24.CrossRef
22.
go back to reference Fernandez Gallardo E, Spiessens C, D'Hooghe T, Debrock S. Effect of day 3 embryo morphometrics and morphokinetics on survival and implantation after slow freezing-thawing and after vitrification-warming: a retrospective cohort study. Reprod Biol Endocrinol. 2017. https://doi.org/10.1186/s12958-017-0299-5. Fernandez Gallardo E, Spiessens C, D'Hooghe T, Debrock S. Effect of day 3 embryo morphometrics and morphokinetics on survival and implantation after slow freezing-thawing and after vitrification-warming: a retrospective cohort study. Reprod Biol Endocrinol. 2017. https://​doi.​org/​10.​1186/​s12958-017-0299-5.
23.
go back to reference Rienzi L, Gracia C, Maggiulli R, LaBarbera AR, Kaser DJ, Ubaldi FM, Vanderpoel S, Racowsky C. Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. Hum Reprod Update. 2017;23:139–55.PubMed Rienzi L, Gracia C, Maggiulli R, LaBarbera AR, Kaser DJ, Ubaldi FM, Vanderpoel S, Racowsky C. Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. Hum Reprod Update. 2017;23:139–55.PubMed
24.
go back to reference Zhu HY, Xue YM, Yang LY, Jiang LY, Ling C, Tong XM, Zhang SY. Slow freezing should not be totally substituted by vitrification when applied to day 3 embryo cryopreservation: an analysis of 5613 frozen cycles. J Assist Reprod Genet. 2015;32:1371–7.CrossRef Zhu HY, Xue YM, Yang LY, Jiang LY, Ling C, Tong XM, Zhang SY. Slow freezing should not be totally substituted by vitrification when applied to day 3 embryo cryopreservation: an analysis of 5613 frozen cycles. J Assist Reprod Genet. 2015;32:1371–7.CrossRef
25.
go back to reference Fang L, Jin L, Li E, Cui L, Ye Y. Clinical evaluation of two formulations of slow-freezing solutions for cleavage stage embryos. J Assist Reprod Genet. 2016;33:1389–93.CrossRef Fang L, Jin L, Li E, Cui L, Ye Y. Clinical evaluation of two formulations of slow-freezing solutions for cleavage stage embryos. J Assist Reprod Genet. 2016;33:1389–93.CrossRef
26.
27.
go back to reference Alpha Scientists in Reproductive Medicine. The alpha consensus meeting on cryopreservation key performance indicators and benchmarks: proceedings of an expert meeting. Reprod BioMed Online. 2012;25:146–67.CrossRef Alpha Scientists in Reproductive Medicine. The alpha consensus meeting on cryopreservation key performance indicators and benchmarks: proceedings of an expert meeting. Reprod BioMed Online. 2012;25:146–67.CrossRef
28.
go back to reference Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil Steril. 1985;44:645–51.CrossRef Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil Steril. 1985;44:645–51.CrossRef
29.
go back to reference R Core Team. R: a language and environment for statistical computing. Vienna: R Foundation for statistical computing; 2018. R Core Team. R: a language and environment for statistical computing. Vienna: R Foundation for statistical computing; 2018.
30.
go back to reference Zeilmaker GH, Alberda AT, van Gent I, Rijkmans CM, Drogendijk AC. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril. 1984;42:293–6.CrossRef Zeilmaker GH, Alberda AT, van Gent I, Rijkmans CM, Drogendijk AC. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril. 1984;42:293–6.CrossRef
31.
go back to reference Cobo A, de los Santos MJ, Castellò D, Gámiz P, Campos P, Remohí J. Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: evaluation of 3,150 warming cycles. Fertil Steril. 2012;98:1138–46.CrossRef Cobo A, de los Santos MJ, Castellò D, Gámiz P, Campos P, Remohí J. Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: evaluation of 3,150 warming cycles. Fertil Steril. 2012;98:1138–46.CrossRef
32.
go back to reference Rodriguez-Wallberg KA. Clinical aspects and perinatal outcomes after cryopreservation of embryos and gametes. Minerva Ginecol. 2015;67:207–15.PubMed Rodriguez-Wallberg KA. Clinical aspects and perinatal outcomes after cryopreservation of embryos and gametes. Minerva Ginecol. 2015;67:207–15.PubMed
33.
go back to reference Capalbo A, Rienzi L, Buccheri M, Maggiulli R, Sapienza F, Romano S, Colamaria S, Iussig B, Giuliani M, Palagiano A, Ubaldi F. The worldwide frozen embryo reservoir: methodologies to achieve optimal results. Ann N Y Acad Sci. 2011;1221:32–9.CrossRef Capalbo A, Rienzi L, Buccheri M, Maggiulli R, Sapienza F, Romano S, Colamaria S, Iussig B, Giuliani M, Palagiano A, Ubaldi F. The worldwide frozen embryo reservoir: methodologies to achieve optimal results. Ann N Y Acad Sci. 2011;1221:32–9.CrossRef
34.
go back to reference Parmegiani L, Tatone C, Cognigni GE, Bernardi S, Troilo E, Arnone A. Rapid warming increases survival of slow-frozen sibling oocytes: a step towards a single warming procedure irrespective of the freezing protocol? Reprod BioMed Online. 2014;28:614–23.CrossRef Parmegiani L, Tatone C, Cognigni GE, Bernardi S, Troilo E, Arnone A. Rapid warming increases survival of slow-frozen sibling oocytes: a step towards a single warming procedure irrespective of the freezing protocol? Reprod BioMed Online. 2014;28:614–23.CrossRef
35.
go back to reference Parmegiani L, Arnone A, Cognigni G, Quintero L, Vinoles R, Filicori M. Universal warming protocol for a transnational egg donation program with vitrified oocytes. Fertil Steril. 2018;110:e230. Parmegiani L, Arnone A, Cognigni G, Quintero L, Vinoles R, Filicori M. Universal warming protocol for a transnational egg donation program with vitrified oocytes. Fertil Steril. 2018;110:e230.
36.
go back to reference Parmegiani L, Arnone A, Bernardi S, Cognigni G, Filicori M. Universal warming protocol for vitrified human embryos: a retrospective study on combination of 3 different vitrification/warming kits. Fertil Steril. 2018;110:e230. Parmegiani L, Arnone A, Bernardi S, Cognigni G, Filicori M. Universal warming protocol for vitrified human embryos: a retrospective study on combination of 3 different vitrification/warming kits. Fertil Steril. 2018;110:e230.
37.
go back to reference Serdarogullari M, Coban O, Boynukalin FK, Bilgin EM, Findikli N, Bahceci M. Successful application of a single warming protocol for embryos cryopreserved by either slow freezing or vitrification techniques. Syst Biol Reprod Med. 2019;65:12–9.CrossRef Serdarogullari M, Coban O, Boynukalin FK, Bilgin EM, Findikli N, Bahceci M. Successful application of a single warming protocol for embryos cryopreserved by either slow freezing or vitrification techniques. Syst Biol Reprod Med. 2019;65:12–9.CrossRef
38.
go back to reference Vanderzwalmen P, Zech NH, Ectors F, Stecher A, Lejeune B, Vanderzwalmen S, Wirleitner B. Blastocyst transfer after aseptic vitrification of zygotes: an approach to overcome an impaired uterine environment. Reprod BioMed Online. 2012;25:591–9.CrossRef Vanderzwalmen P, Zech NH, Ectors F, Stecher A, Lejeune B, Vanderzwalmen S, Wirleitner B. Blastocyst transfer after aseptic vitrification of zygotes: an approach to overcome an impaired uterine environment. Reprod BioMed Online. 2012;25:591–9.CrossRef
39.
go back to reference Vanderzwalmen P, Connan D, Grobet L, Wirleitner B, Remy B, Vanderzwalmen S, Zech N, Ectors FJ. Lower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentration of cryoprotectant solutions. Hum Reprod. 2013;28:2101–10.CrossRef Vanderzwalmen P, Connan D, Grobet L, Wirleitner B, Remy B, Vanderzwalmen S, Zech N, Ectors FJ. Lower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentration of cryoprotectant solutions. Hum Reprod. 2013;28:2101–10.CrossRef
40.
go back to reference Parmegiani L, Arnone A, Bernardi S, Cognigni G, Filicori M. A single “universal warming protocol” for vitrified human embryos: a randomized controlled study. Fertil Steril. 2017;108:e173.CrossRef Parmegiani L, Arnone A, Bernardi S, Cognigni G, Filicori M. A single “universal warming protocol” for vitrified human embryos: a randomized controlled study. Fertil Steril. 2017;108:e173.CrossRef
41.
go back to reference Parmegiani L, Beilby KH, Arnone A, Bernardi S, Maccarini AM, Nardi E, Cognigni GE, Filicori M. Testing the efficacy and efficiency of a single “universal warming protocol” for vitrified human embryos: prospective randomized controlled trial and retrospective longitudinal cohort study. J Assist Reprod Genetics. 2018;35:1887–95.CrossRef Parmegiani L, Beilby KH, Arnone A, Bernardi S, Maccarini AM, Nardi E, Cognigni GE, Filicori M. Testing the efficacy and efficiency of a single “universal warming protocol” for vitrified human embryos: prospective randomized controlled trial and retrospective longitudinal cohort study. J Assist Reprod Genetics. 2018;35:1887–95.CrossRef
Metadata
Title
A monocentric analysis of the efficacy of extracellular cryoprotectants in unfrozen solutions for cleavage stage embryos
Authors
Francesco Capodanno
Jessica Daolio
Gaetano De Feo
Angela Falbo
Daria Morini
Alessia Nicoli
Luca Braglia
MariaTeresa Villani
Giovanni B. La Sala
Lodovico Parmegiani
Lorenzo Aguzzoli
Publication date
01-12-2019
Publisher
BioMed Central
Keyword
Progesterone
Published in
Reproductive Biology and Endocrinology / Issue 1/2019
Electronic ISSN: 1477-7827
DOI
https://doi.org/10.1186/s12958-019-0519-2

Other articles of this Issue 1/2019

Reproductive Biology and Endocrinology 1/2019 Go to the issue