Is early embryo development as observed by time-lapse microscopy dependent on whether fresh or frozen sperm was used for ICSI? A cohort study

  • Jessica EastickEmail author
  • Christos Venetis
  • Simon Cooke
  • Ashleigh Storr
  • Daisy Susetio
  • Michael Chapman
Embryo Biology



The aim of this study was to compare timings of key events of embryo development from those originating from either fresh or cryopreserved ejaculate sperm using time-lapse technology.


In this retrospective observational cohort study, time-lapse technology was used to monitor 1927 embryos from 234 women undergoing intracytoplasmic sperm injection (ICSI) and utilizing either fresh (n = 172 cycles) or cryopreserved ejaculate sperm (n = 62 cycles) for insemination were included in the study. Key developmental events as described in time-lapse were compared with the use of generalized estimating equations (GEE) to adjust for any auto-correlation between the observations. In addition, multivariable logit regression models were used to account for any known baseline differences between the two groups.


There were no differences in conventional embryo development such as number of 8-cell embryos by 72 h (p = 0.359), the number of blastocysts by 120 h (p = 0.417), and the number of top quality blastocysts (p = 0.956) between the two groups compared. There were no statistical differences in the timings of any of the key embryo developmental events (PN_t1, NEBD, cytokinesis, t2, t3, t4, t5, t6, t7, t8, tM, tSB, tEB, tHB, s1, s2, s3, cc2, and cc3) when either fresh or cryopreserved ejaculate sperm was used for ICSI. This was also confirmed with conventional morphological assessment.


This observational cohort study has shown that there are no differences in the morphokinetic parameters of early embryo development when either fresh or frozen ejaculate sperm are used for ICSI insemination.


Time-lapse Morphokinetic Semen cryopreservation Embryo development ICSI 


Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflicts of interest.


  1. 1.
    Bunge RG, Sherman JK. Fertilizing capacity of frozen human spermatozoa. Nature. 1953;172(4382):767–8.CrossRefPubMedGoogle Scholar
  2. 2.
    O'Connell M, McClure N, Lewis SEM. The effects of cryopreservation on sperm morphology, motility and mitochondrial function. Hum Reprod. 2002;17(3):704–9.CrossRefPubMedGoogle Scholar
  3. 3.
    Zribi N, et al. Effects of cryopreservation on human sperm deoxyribonucleic acid integrity. Fertil Steril. 2010;93(1):159–66.CrossRefPubMedGoogle Scholar
  4. 4.
    Donnelly ET, McClure N, Lewis SEM. Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity. Fertil Steril. 2001;76(5):892–900.CrossRefPubMedGoogle Scholar
  5. 5.
    Nallella KP, et al. Cryopreservation of human spermatozoa: comparison of two cryopreservation methods and three cryoprotectants. Fertil Steril. 2004;82(4):913–8.CrossRefPubMedGoogle Scholar
  6. 6.
    Di Santo M, et al. Human sperm cryopreservation: update on techniques, effect on DNA integrity, and implications for ART. Advances in Urology. 2012;2012:854837.CrossRefPubMedGoogle Scholar
  7. 7.
    Thomson LK, et al. Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Hum Reprod. 2009;24(9):2061–70.Google Scholar
  8. 8.
    Boitrelle F, Albert M, Theillac C, Ferfouri F, Bergere M, Ois Vialard F, Wainer R, Bailly M, Selva J. Cryopreservation of human spermatozoa decreases the number of motile normal spermatozoa, induces nuclear vacuolization and chromatin decondensation. J Androl. 2012;33(6):1371–8.CrossRefPubMedGoogle Scholar
  9. 9.
    Loutradi KE, et al. The effects of sperm quality on embryo development after intracytoplasmic sperm injection. J Assist Reprod Genet. 2006;23(2):69–74.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Van Steirteghem A, et al. Results of intracytoplasmic sperm injection with ejaculated, fresh and frozen–thawed epididymal and testicular spermatozoa. Hum Reprod. 1998;13(suppl 1):134–42.CrossRefPubMedGoogle Scholar
  11. 11.
    Lu Y-h, et al. Different sperm sources and parameters can influence intracytoplasmic sperm injection outcomes before embryo implantation. Journal of Zhejiang University Science B. 2012;13(1):1–10.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Holden CA, et al. Frozen-thawed epididymal spermatozoa for intracytoplasmic sperm injection. Fertil Steril. 1997;67(1):81–7.CrossRefPubMedGoogle Scholar
  13. 13.
    Tournaye H, et al. No differences in outcome after intracytoplasmic sperm injection with fresh or with frozen–thawed epididymal spermatozoa. Hum Reprod. 1999;14(1):90–5.CrossRefPubMedGoogle Scholar
  14. 14.
    Cayan S, et al. A comparison of ICSI outcomes with fresh and cryopreserved epididymal spermatozoa from the same couples. Hum Reprod. 2001;16(3):495–9.CrossRefPubMedGoogle Scholar
  15. 15.
    Ohlander S, et al. Impact of fresh versus cryopreserved testicular sperm upon intracytoplasmic sperm injection pregnancy outcomes in men with azoospermia due to spermatogenic dysfunction: a meta-analysis. Fertil Steril. 2014;101(2):344–9.CrossRefPubMedGoogle Scholar
  16. 16.
    Karacan M, et al. Outcome of intracytoplasmic sperm injection cycles with fresh testicular spermatozoa obtained on the day of or the day before oocyte collection and with cryopreserved testicular sperm in patients with azoospermia. Fertil Steril. 2013;100(4):975–80.CrossRefPubMedGoogle Scholar
  17. 17.
    Wood S, et al. Reproductive potential of fresh and cryopreserved epididymal and testicular spermatozoa in consecutive intracytoplasmic sperm injection cycles in the same patients. Fertil Steril. 2002;77(6):1162–6.CrossRefPubMedGoogle Scholar
  18. 18.
    Park Y-S, et al. Influence of motility on the outcome of in vitro fertilization/intracytoplasmic sperm injection with fresh vs. frozen testicular sperm from men with obstructive azoospermia. Fertil Steril. 2003;80(3):526–30.CrossRefPubMedGoogle Scholar
  19. 19.
    Friedler S, et al. The outcome of intracytoplasmic injection of fresh and cryopreserved epididymal spermatozoa from patients with obstructive azoospermia—a comparative study. Hum Reprod. 1998;13(7):1872–7.CrossRefPubMedGoogle Scholar
  20. 20.
    Borges E Jr, et al. Fertilization and pregnancy outcome after intracytoplasmic injection with fresh or cryopreserved ejaculated spermatozoa. Fertil Steril. 2007;87(2):316–20.CrossRefPubMedGoogle Scholar
  21. 21.
    Braga DPAF, et al. The negative influence of sperm cryopreservation on the quality and development of the embryo depends on the morphology of the oocyte. Andrology. 2015;3(4):723–8.CrossRefPubMedGoogle Scholar
  22. 22.
    Kuczyński W, et al. The outcome of intracytoplasmic injection of fresh and cryopreserved ejaculated spermatozoa—a prospective randomized study. Hum Reprod. 2001;16(10):2109–13.CrossRefPubMedGoogle Scholar
  23. 23.
    Ciray HN, et al. Proposed guidelines on the nomenclature and annotation of dynamic human embryo monitoring by a time-lapse user group. Hum Reprod. 2014;29(12):2650–60.CrossRefPubMedGoogle Scholar
  24. 24.
    Lammers J, et al. Does sperm origin affect embryo morphokinetic parameters? J Assist Reprod Genet. 2015;32(9):1325–32.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Sundvall L, et al. Inter- and intra-observer variability of time-lapse annotations. Hum Reprod. 2013;28(12):3215–21.CrossRefPubMedGoogle Scholar
  26. 26.
    Meseguer M, et al. The use of morphokinetics as a predictor of embryo implantation. Hum Reprod. 2011;26(10):2658–71.CrossRefPubMedGoogle Scholar
  27. 27.
    Kirkegaard K, et al. Time-lapse parameters as predictors of blastocyst development and pregnancy outcome in embryos from good prognosis patients: a prospective cohort study. Hum Reprod. 2013;28(10):2643–51.CrossRefPubMedGoogle Scholar
  28. 28.
    Kirkegaard K, et al. Timing of human preimplantation embryonic development is confounded by embryo origin. Human Reproduction (Oxford, England). 2016;31(2):324–31.Google Scholar
  29. 29.
    Basile N, et al. The use of morphokinetics as a predictor of implantation: a multicentric study to define and validate an algorithm for embryo selection. Hum Reprod. 2015;30(2):276–83.CrossRefPubMedGoogle Scholar
  30. 30.
    Storr A, et al. Morphokinetic parameters using time-lapse technology and day 5 embryo quality: a prospective cohort study. J Assist Reprod Genet. 2015;32(7):1151–60.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Gardner DKS, William B. Culture and transfer of human blastocysts. Current Opinion in Obstetrics and Gynaecology. 1999;11(3):307–11.CrossRefGoogle Scholar
  32. 32.
    Muela García L, et al. Does semen cryopreservation have influence on embryo kinetics? Fertil Steril. 2012;98(3, Supplement):S164.CrossRefGoogle Scholar
  33. 33.
    Liu Y, et al. Time-lapse videography of human embryos: using pronuclear fading rather than insemination in IVF and ICSI cycles removes inconsistencies in time to reach early cleavage milestones. Reprod Biol. 2015;15(2):122–5.CrossRefPubMedGoogle Scholar
  34. 34.
    Hardin JW, Hilbe JM. Generalized Estimating Equations. Chapman & Hall/CRC; 2003Google Scholar
  35. 35.
    Payne D, Flaherty SP, Barry MF, Matthews CD. Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography. Hum Reprod. 1997;12(3):532–41.CrossRefPubMedGoogle Scholar
  36. 36.
    Lemmen JG, Agerholm I, Ziebe S. Kinetic markers of human embryo quality using time-lapse recordings of IVF/ICSI-fertilized oocytes. Reprod BioMed Online. 2008;17(3):385–91.CrossRefPubMedGoogle Scholar
  37. 37.
    Cruz M, et al. Embryo quality, blastocyst and ongoing pregnancy rates in oocyte donation patients whose embryos were monitored by time-lapse imaging. J Assist Reprod Genet. 2011;28(7):569–73.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Petersen BM, et al. Development of a generally applicable morphokinetic algorithm capable of predicting the implantation potential of embryos transferred on day 3. Hum Reprod. 2016;31(10):2231–44.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Liu Y, et al. Clinical significance of intercellular contact at the four-cell stage of human embryos, and the use of abnormal cleavage patterns to identify embryos with low implantation potential: a time-lapse study. Fertil Steril. 2015;103(6):1485–1491.e1.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Hunter IVF, IVF AustraliaNew LambtonAustralia
  2. 2.University of New South WalesSydneyAustralia

Personalised recommendations