Patent Application: US-16228407-A

Abstract:
the invention relates to a method of assessing the viability of a thawed cell wherein the cell is a gamete , an embryo , a karyoplast , a putative stem cell population , a stem cell precursor population or a stem cell population . the method includes incubating the thawed cell in a culture medium including a plurality of amino acids and determining the change in concentration in the medium of at least one amino acid .

Description:
the invention is described by way of example only and with reference to the accompanying drawings : fig1 amino acid depletion and appearance by frozen thawed human embryos from day 2 to day 3 of development . n = 21 for embryos which developed to the blastocyst stage and n = 25 for embryos which arrested prior to the blastocyst stage . * p & lt ; 0 . 05 ; ** p & lt ; 0 . 01 ; *** p & lt ; 0 . 001 significance from zero . bars with the same superscript are significantly different ; a and b , p = 0 . 001 ; c , p = 0 . 0025 ; d p = 0 . 016 ; e , p = 0 . 032 ; f , p = 0 . 0448 . fig2 total amino acid production , depletion , turnover and balance by frozen thawed human embryos from day 2 to day 3 of development . * p & lt ; 0 . 05 ; ** p & lt ; 0 . 01 ; *** p & lt ; 0 . 001 significantly different from embryos which arrest . fig3 sum of glutamine , glycine and alanine utilisation by frozen thawed human embryos from day 2 - day 3 of development . *** p & lt ; 0 . 001 significantly different to embryos that develop . fig4 sum of glutamine , glycine and alanine for individual frozen thawed embryos which subsequently arrested or developed to blastocyst stage . fig5 amino acid depletion and appearance by grade i thawed human embryos from day 2 to day 3 of development . n = 6 for embryos which developed to the blastocyst stage and n = 13 for embryos which arrested prior to the blastocyst stage . * p & lt ; 0 . 05 ; ** p & lt ; 0 . 01 ; *** p & lt ; 0 . 001 significance from zero . bars with the same superscript are significantly different ; a , p = 0 . 014 ; b , p = 0 . 024 ; c , p = 0 . 029 ; p = 0 . 035 ; e , p = 0 . 0493 ; f , p = 0 . 050 . fig6 total amino acid production , depletion , turnover and balance by grade i thawed human embryos from day 2 to day 3 of development . * p & lt ; 0 . 05 significantly different from embryos which arrest . spare , frozen human embryos donated to research with informed patient consent were obtained from patients undergoing in vitro fertilisation ( ivf ) at the assisted conception unit at st james &# 39 ; s hospital , leeds . full ethical approval for this work was granted by the human fertilisation and embryology authority ( hfea ) as well as the ethics committees of the collaborating institutions . ovarian stimulation and oocyte collection were performed as previously described ( balen , 2001 ). in general , a long pituitary desensitisation protocol was used , with intranasal nafarelin followed by gonadotrophin stimulation with either human menopausal gonadotrophins ( hmg , menogon , ferring pharmaceuticals ltd ) or recombinant fsh ( puregon , organon laboratories ltd ). briefly , oocytes were collected by follicular aspiration 36 h after hcg administration and cultured at 37 ° c . in 5 % co 2 in medi - cult ivf medium under oil ( medi - cult ). the oocyte - cumulus complexes were inseminated with a final concentration of 70 , 000 motile sperm per ml at approximately 40 h post hcg and incubated overnight until fertilisation was confirmed by the presence of two pronuclei ( day 1 post - insemination ). prior to being used for research , zygotes were cultured in 70 μl drops of medi - cult ivf medium under oil as above . a maximum of 3 embryos were transferred on day 2 post - insemination and any remaining embryos frozen and stored in liquid nitrogen for potential future use . according to hfea guidelines , embryos may only be stored for 5 years and hence patients were contacted on a yearly basis to assess whether they wished their embryos to remain in storage , be discarded or donated to research . those embryos donated to research were transported to york in a dry shipper for use in this study . embryos were thawed using the sydney ivf thawing kit ( cook , queensland , australia ) and placed into 10 μl drops of ebss culture medium supplemented with 0 . 5 % hsa , 1 mm glucose , 0 . 47 mm pyruvate , 5 mm lactate and a close to physiological mixture of amino acids ( tay et al ., 1997 ) under mineral oil for approximately 3 h . each patient &# 39 ; s embryos were randomised between treatment groups and the developmental grade and stage of the embryos assessed ( houghton et al ., 2002 ). the embryos were then cultured individually for 24 h , from day 2 to day 3 in 4 μl drops of the same medium in a humidified atmosphere containing 5 % co 2 and either 20 % or 5 % o 2 in air at 37 ° c . after incubation , embryo morphology was assessed , and the embryos placed individually into 10 μl drops of pre - equilibrated medium and incubated under either 5 % or 20 % o 2 until day 5 of development . the spent medium was stored at − 80 ° c . developmental stage and grade were again assessed before the embryos were placed individually into a 4 μl drop of pre - equilibrated medium under either 5 or 20 % o 2 for 24 h , until day 6 of development . embryo developmental stage and grade were assessed before removal from the drops . the spent medium was stored at − 80 ° c . embryo - free control drops were incubated in the same dish as those containing embryos to allow for any non specific changes in amino acid concentration throughout the culture period . the spent media were thawed and 2 μl aliquots diluted with 23 μl hplc grade water . amino acid analysis was performed by reverse - phase hplc using a kontron 500 attached to a jasco f920 fluorescence detector and a 4 . 5 × 250 mm hypersil ods - 16 column ( jones chromatography ) as previously described ( houghton et al ., 2002 ). briefly , derivatization was achieved by the automated reaction of 25 μl sample with an equal volume of reagent ( 10 μl 2mercaptoethanol and 5 ml o - phthaldialdehyde ( opa ) reagent ). the elution gradient operated at a flow rate of 1 . 3 ml per minute . solvent a consisted of 18 ml tetrahydrofuran ( fisher chemicals ), 200 ml methanol and 800 ml sodium acetate ( 83 mmol / l , ph 5 . 9 ). solvent b consisted of 800 ml methanol and 200 ml sodium acetate ( 83 mmol / l , ph 5 . 9 ). using this method it was not possible to detect proline and cysteine . all data were analysed to determine whether they were normally distributed , using the anderson - darling normality test . data for amino acid depletion / appearance were tested for significance from zero using either a 1 - sample t - test or 1 sample wilcoxon test , depending on whether the data were normally distributed . differences between amino acid profiles for arresting and developing embryos were analysed using either a student &# 39 ; s t - test or mann - whitney u test . differences between amino acid depletion , appearance , turnover and balance were analysed by student &# 39 ; s t - test . amino acid turnover by thawed embryos from day 2 to day 3 serine , glutamine , arginine , valine , isoleucine and leucine were significantly depleted from the medium ( fig1 ), while aspartate , glutamate , asparagine , alanine , tryptophan and phenylalanine appeared . thawed embryos which subsequently failed to develop to the blastocyst stage consumed glutamine , arginine , valine , isoleucine and leucine and produced aspartate , glutamate , glycine , alanine , tyrosine , tryptophan , phenylalanine and lysine . there was a significant difference in the utilisation of glutamine ( p = 0 . 001 ), alanine ( p = 0 . 001 ), glycine ( p = 0 . 0025 ), glutamate ( p = 0 . 016 ), arginine ( p = 0 . 032 ) and lysine ( p = 0 . 0448 ) between those thawed embryos which developed to the blastocyst stage and those which arrested prior to blastocyst formation ( fig1 ). surprisingly , the cohort of frozen / thawed day 2 to 3 embryos that subsequently failed to develop to the blastocyst stage contained more grade i embryos than those which developed ( table 1 ) but there was no difference in the average number of blastomeres per embryo between grade i embryos which subsequently developed to the blastocyst or that arrested prior to blastocyst formation . similarly , there was no difference between the total average blastomere number between the two groups ( table 1 ). overall , embryos that arrested prior to the blastocyst stage were metabolically more active in terms of amino acid turnover , depleting ( p = 0 . 011 ) and producing ( p = 0 . 009 ) significantly more amino acids than their developing counterparts ( fig2 ). interestingly it was found that developing embryos were balanced in terms of their amino acid turnover i . e ., the amount depleted equalled that produced , whereas arresting embryos had a negative balance i . e ., depleted more amino acids from the medium than they produced ( fig2 ). there was a significant difference ( p & lt ; 0 . 001 ) in the sum of glutamine , glycine and alanine from day 2 to day 3 between those embryos that developed to the blastocyst stage and those which arrested prior to blastocyst formation ( fig3 ). using the sum of glutamine , glycine and alanine for individual embryos , it was possible to predict with 91 % accuracy which embryos would develop to the blastocyst stage ( fig4 ). when the amino acid profile of grade i embryos was determined , there was a significant difference in amino acid depletion / appearance between those embryos which subsequently developed to the blastocyst stage compared with those grade i embryos which arrested prior to blastocyst formation ( fig5 ). specific differences between the two groups were in the utilisation of lysine ( p = 0 . 014 ), glycine ( p = 0 . 024 ), tryptophan ( p = 0 . 029 ), arginine ( p = 0 . 035 ), glutamate ( p = 0 . 0493 ) and glutamine ( p = 0 . 050 ). it should be noted that there was no significant difference in the average number of blastomeres between grade i arresting and developing embryos ( table 1 ). grade i embryos displayed no significant difference in total amino acid depletion , appearance or in the balance of amino acids ( fig6 ). however , arresting grade i embryos were metabolically more active in terms of amino acid turnover than those that developed to the blastocyst stage . the metabolism of frozen thawed human embryos from day 2 to day 3 has been determined in terms of amino acid depletion from , and appearance in the culture medium and used to predict their capacity to develop to the blastocyst stage . this is the first study to investigate the energy metabolism of cryopreserved human embryos . the amino acid profiles obtained can be used to predict which thawed embryo will develop to the blastocyst stage ; a phenomenon which is independent of embryo grade and blastomere number on day 2 . those embryos with the ability to develop to the blastocyst stage are metabolically more quiet , having a lower rate of amino acid production , depletion and turnover than arresting embryos . amino acid profiling of cryopreserved embryos will also be able to predict implantation and live offspring similar to the study by brison et al . ( 2004 ). these investigators used retrospective analysis of amino acid profiles of icsi embryos measured from day 1 to day 2 of development . the embryos were selected for transfer on day 2 , based on morphology alone and it was found that the turnover of asparagine , glycine and leucine were correlated with a clinical pregnancy . specifically they were also independent of known predictors such as female age , basal fsh levels , embryo cell number and grade . the technique provides a sensitive method which can readily be translated into ivf clinics and used by embryologists to select developmentally competent thawed embryos for transfer . the use of amino acid profiling has proved efficient not only in the retrospective selection of which frozen thawed embryo will develop to the blastocyst stage but also by providing the embryologist with a tool with which to distinguish between a population of the best grade i embryos in terms of developmental potential . balen a h ( 2001 ). gnrh agonists and superovulation for assisted conception . infertility and reproductive medicine clinics of north america 12 , 89 - 104 . bourgain c and devroey , p ( 2003 ). the endometrium in stimulated cycles for ivf . human reproduction update 9 , 515 - 22 . brison d r , houghton f d , falconer d et al ( 2004 ). identification of viable embryos in ivf by non - invasive measurement of amino acid turnover . human reproduction 19 , 2319 - 2324 . devroey p , bourgain c , macklon n s and fauser b c ( 2004 ). reproductive biology and ivf : ovarian stimulation and endometrial receptivity . trends in endocrinology and metabolism . 15 , 84 - 90 . dulioust e , toyama k , busnel m c et al ( 1995 ). long - term effects of embryo freezing in mice . proceedings of the national academy of sciences usa ., 92 , 589 - 93 . edgar d h , bourne h , speirs a l and mcbain jc ( 2000 ). a quantitative analysis of the impact of cyropreservation on the implantation potential of human early cleavage stage embryos . human reproduction 15 , 175 - 179 . edgar , dh , archer j and bourne h ( 2005 ). the application and impact of cryopreservation of early cleavage stage embryos in assisted reproduction . human fertility 8 , 225 - 230 . el - toukhy t , khalaf y , al - darazi k et al ( 2003 ). effect of blastomere loss on the outcome of frozen embryo replacement cycles . fertility and sterility 79 , 1106 - 1111 . emiliani , s , van den bergh m , vannin a - s , biramane j and englert y ( 2000 ). comparison of ethylene glycol , 1 , 2 - propanediol and glycerol for cryopreservation of slow - cooled mouse zygotes , 4 - cell embryos and blastocysts . human reproduction , 15 , 905 - 910 . ho , y , wrigglesworth k , eppig j j and schultz r m ( 1995 ). preimplantation development of mouse embryos in ksom : augmentation by amino acids and analysis of gene expression . molecular reproduction and development 41 , 232 - 238 . houghton f d , hawkhead j a , humpherson p g et al ( 2002 ). non - invasive amino acid turnover predicts human embryo developmental capacity . human reproduction 17 , 999 - 1005 . 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