SYNBIOS® SYNTHETIC PROTEIN-FREE MEDIA ELIMINATES DISEASE TRANSMISSION , SPECIALTY SYNTHETIC PROTEIN-FREE & CONVENTIONAL PROTEIN-SUPPLEMENTED MEDIA

EFFICACY OF SYNBIOS® GAMETE & EMBRYO CULTURE & CRYO MEDIA

The SYNBIOS® signature synthetic protein-free and protein-supplemented conventional  handling, culture and cryopreservation media products have demonstrated efficacy comparable to other contemporary commercial conventional protein-supplemented media products.

I. HUMAN

A. Comparison of human sibling embryos generated by conventional IVF and ICSI in synthetic protein-free SYNBIOS® media and a commercially available (HSA) protein-supplemented ART culture media.

DESCRIPTION MEDIA WITH PROTEIN SYNBIOS® SYNTHETIC1,2 SIGNIFICANCE
A. Conventional IVF
% Fertilization rate (nos.) 79.2 (118/149) 85.3 (116/136) p=0.235
% Zygotes arrest rate 8.1 2.2 p=0.052
Mean blastomere number (±1SD) 3.4±1.0 3.4±1.0 p=0.865
Mean embryo grade (±1SD) 2.7±0.8 3.1±0.9 p=0.001
% Embryos >4-cell stage 56.2 58.4 p=0.847
% Embryos >grade 3 58.1 74.3 p=0.017
B. ICSI
% Fertilization rate (nos.) 69.4 (175/252) 77.8 (196/252) p=0.043
% Zygotes arrest rate 6.3 2.8 p=0.043
Mean blastomere (±1SD) 3.3±1.1 3.8±1.2 p=0.001
Mean embryo grade (±1SD) 2.8±0.8 2.9±0.7 p=0.080
% Embryos >4-cell stage 54.8 71.4 p=0.002
% Embryos >grade 3 58.6 63.5 p=0.413
(Embryo grade: 4 = excellent; 3 = good; 2 = fair; 1 = poor)

(Source: 1Ali et al., Hum Reprod. 15:145, 2000; 2Ali, MEFS J. 9:118,2004)

1,2% Clinical pregnancy rate (CPR) per retrieval

CONTROL(Protein-supplemented) media: 33% (IVF and ICSI)
SYNBIOS® synthetic media:
IVF:   50.0% (14 of 28) in all age groups;
          53.8% (14 of 26) in women < 40 years of age
ICSI:  46.2% (12 of 26) in all age groups;
          54.5% (12 of 22) in women < 40 years of age

1% Clinical pregnancy rate (CPR) per retrieval

Control (Protein-containing) media: 33% (IVF and ICSI; p=0.0002)
SYNBIOS® synthetic media:
IVF:   50.0% (14 of 28) in all age groups;
          53.8% (14 of 26) in women < 40 years of age
ICSI:  46.2% (12 of 26) in all age groups;
          54.5% (12 of 22) in women < 40 years of age

2% Term deliveries for SYNBIOS® synthetic media per retrieval for all age groups

Number of babies delivered:

High order births:

% Term deliveries: 

% Embryo viability rate:

ICSI: 

IVF:

53

13 patients (2x triplets; 13x Twins)

35% (38/109; p=0.0005 vs Control); 5 clinical pregnancies lost to follow-up

34%

41 babies  delivered; 4 pregnancies lost to follow-up

12 babies  delivered; 1 pregnancy lost to follow-up

(Source: 1Ali et al., Hum Reprod. 15:145, 2000; 2Ali, MEFS J. 9:118,2004)

ESHRE Statistics (for similar time period):

EU Countries: 26.9% (IVF) 28.5% (ICSI) for 2005; (Hum Reprod. 24:1267, 2009)

B. Independent Evaluation of the Synthetic SYNBIOS®IUI Medium: A Multi-Center Trial

DESCRIPTION %PREGNANCY (P/N) SINGLETONS TWINS AGE
Natural cycle 20% (1/5) 1 0 20-34
Stimulated cycle 29.5% (28/95) 24 4 20-40
Total 29% (29/100) 25 4 20-403
3Two (n=2) singleton pregnancies in 9 patients in the age group 35-40yrs
Source: Cellcura ASA, Norway

C. Excellent Sperm Motility Preservation After Cryopreservation Using Synthetic Protein-Free SYNBIOS®Sperm Freeze Medium

DESCRIPTION % MEAN MOTILITY±1SD
Fresh sperm 55.7±17.3
Frozen with synthetic cryopreservation medium4 54.9±13.3
Number 10
Significance P>0.05 (p=0.222)

4Viable pregnancy with live-birth in the human has been obtained following cryopreservation of testicular sperm using synthetic protein-free SYNBIOS® Sperm Freeze Medium. The testicular sperm was held in cold-storage for 14 months.

Source: International Conference for Innovation in Biomedical Engineering and Life Sciences. ICIBEL2015, 6–8 December 2015, Putrajaya, Malaysia Putrajaya, Malaysia. Springer Publishers,  pg. 172

D. Excellent Survival of Cryopreserved Day 2 Human Embryos After Vitrification With SYNBIOS®VS14/EG5.5 Solution5

DEVELOPMENTAL STAGE FRESH CONTROL %SURVIVAL AFTER VITRIFICATION SIGNIFICANCE
Survival (day 2) No intervention (n=39) 100 % survival (n=24) -
1st cleavage (day 3) 89.7 % (n=35) 83.3 % (n=20) p>0.05 (p=0.4580)
Morula/Comp morula (day 4) 60.0 %(n=23) 75.0 %(n=18) p>0.05 (p=0.1951)
Early cavitation 41.0 %(n=16) 37.5 %(n=9) p>0.05 (p=0.7812)
Expanded blastocyst cryostored 16.7 %(n=4) -
Hatched blastocyst - 4.2 %(n=1) -
5Source: Ali et al., Successful vitrification of human embryos. Proceedings of the 1994 Annual Scientific Meeting of the Fertility Society of Australia, Brisbane, 4-7 October 1994; Ali et al., 1995. Med. Sci. Res.(UK) 23:539-540, 1995. This is the first report of successful vitrification of the human embryo.

Many workers (Refs 1-21) from around the world successfully vitrified oocytes and embryos of various species including human, some with live-births, from the 1990s and onwards using the VS14 (also called EG5.5) formulation, in some instances with minor modifications (Refs 22-24) to the formulation. The successful application of VS14 in a number of species including humans proved its versatility has been reviewed (25).

II. ANIMALS

A. MOUSE

a. In Vitro Survival of Swiss Outbred Mouse Embryos Vitrified with SYNBIOS® VS14/EG5.5 Vitrification Solution

DESCRIPTION FRESH EMBRYOS % VITRIFIED EMBRYOS SIGNIFICANCE
1.In vitro survival
Blastocyst 100 (n=35) 97% (n=36) p>0.05
Early Blastocyst 100 (n=39) 97% (n=38) p>0.05
Morulae 98% (n=48) 98% (n=48) p>0.05
8-cell embryo 90% (n=69) 87% (n=55) p>0.05
4-cell embryo 81% (n=36) 70% (n=33) p>0.05
2-cell embryo 86% (n=51) 71% (n=51) p>0.05
1-cell embryo 46% (n=37) 19% (n=36) p<0.05 (p=0.0160)
F1 1-cell embryo6 91% (n=23) 92% (n=24) p>0.05
2.In vivo survival in surrogates
Day 4 embryos 19.1% (n=125) 26.1% (n=36) p>0.05 (p=0.2253)

6 Day 1 embryos with F1 cytoplasm (C57BL/6xSJL) were more resistant to cooling and warming stress.

Source: Ali and Shelton, J Reprod Fertil. 1993; 98:459-465

B. SHEEP

 b. Vitrification of Day 6 Sheep Embryos withSYNBIOS® VS14/EG5.5 Vitrification Solution

DESCRIPTION F% IN VITRO SURVIVAL % IN VIVO VIABILITY
Morulae 50% 100%
Blastocyst 100% 100%

C. CATTLE

Please see reports (1,2,26) in refs below

III. QUALITY CONTROL

Quality Control Tests for SYNBIOS® synthetic PROTEIN-FREE Culture Medium 

Results Routinely Obtained

Sterility (Ph.Eur, USP) tested for the absence of bacteria, yeast and fungi

pH (Ph.Eur, USP) 7.2-7.4 at 37°C in carbon dioxide atmosphere

Osmolality (Ph.Eur, USP)

Endotoxins (Ph.Eur, USP) <0.05EU/ml

Mouse embryo assays (MEA) > 90% expanded blastocyst formation after 96 hours 100%

Certificate of release/analysis and MSDS available on request.

Sterile

pH 7.3

285mOsm/kg

References

  1. Martino A, Songsasen N, Leibo SP.Biol Reprod 1996;54:1059-69.
  2. Papis K, Avery H, Holm P, et al. Theriogenology 1995; 43, 293 (abstr)
  3. Ali, J., Bongso, A., Ratnam, S.S. (1995). Med. Sci. Res.(UK) 23:539-540
  4. Ali J.  VS14. Med Sci Res 1996a;24:377–8.
  5. Ali J.  Med Sci Res 1996b;24:837–8.
  6. Hong SW, Hyung MS, Chung HM, et al. (1999) Fertil Steril 1999;72:142 –6.
  7. Chen SU, Lien YR, Chen HF, et al. Hum Reprod 2000a;15:2598–603.
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  13. Yin H, Kim SS, Fisher J, et al. Fertil Steril  2001;76: Suppl.1, pp. S101(abstr)
  14. Kim, TJ, Hong SW,Park, SE, et al. Fertil Steril 2003;80:Suppl. 3, pp.143 (abstr)
  15. Kim T,Hong S, Cha K. Fertil Steril 2005;84:Suppl.1, pp.S179 (abstr)
  16. .Kim SH, Ku SY, Sung KC, et al. Yonsei Med J 2006;  30;47(3):399-404.
  17. Kim, TJ, Hong SW,Chung HM, et al. Fertil Steril 2005;83:Suppl.5, pp.S13 (abstr)
  18. .Park SE, Chung HM, Cha KY, et al. Fertil Steril 2001;75(6):1177-84.
  19. Park SE, Kim TJ, Hong SW, et al. Fertil Steril 80:Suppl. 3, pp.64-5 (abstr)
  20. 19.Hong S, Kim T, Lee S, et al. Fertil Steril 2005:84:Suppl.1, pp.S178-S179 (abstr)
  21. Martins RD, Costa EP, Chagas JSC, et al. AnimReprod 2005;2:128-34
  22. Cha et al., 2006: Proceedings of FIGO Conference, Kuala Lumpur, 2006.
  23. El-Danasouri I,  Selman HA.Fertil Steril 2001;76:400-2.
  24. Selman HA, El-Danasouri I.Fertil Steril 2002;77:422-3.
  25. Rama Raju GA, Haranath GB, Krishna KM, et al. Reprod Biomed Online 2005;11:434-7
  26. Ali J, Shelton JN. Development of vitrification solutions. In: Vitrification in Assisted Reproduction. A User’s Manual and Troubleshooting Guide. M.Tucker& J. Liebermann, Eds. Informa Healthcare Medical Books, UK,p45-63, 2006
  27. Papis K, Stachowiak E, Kruszewski M, Iwanenko T and Bartlomiejczyk T
  28. Reprod Fertil Dev. 2005; 18(2) 162 – 162
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