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 Human Embryos After Vitrification With SYNBIOS®VS14/EG5.5 Solution5

DEVELOPMENTAL STAGE FRESH CONTROL VITRIFIED SIGNIFICANCE
Survival (day 2) No intervention (n=39) 100 % (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

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  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.
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  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
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