Comparison of Quinn's Advantage fertilization medium and tissue culture medium 199 for in vitro maturation of oocytes

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  The purpose of the study was to compare the Quinn's Advantage fertilization medium (Q1) and the tissue culture medium 199 (TCM199) for in vitro maturation (IVM) of oocytes and ammonium production during IVM. The immature murine oocytes were
  Original Article Comparison of Quinn ’ s Advantage fertilization medium and tissueculture medium 199 for  in vitro  maturation of oocytes Yu-Hung Lin a , b , c , d , Jiann-Loung Hwang a , d , Lee-Wen Huang a , b , Kok-Min Seow a , e ,Bih-Chwen Hsieh a , b , * , Chii-Ruey Tzeng d a Department of Obstetrics and Gynecology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan b School of Medicine, Fu Jen Catholic University, Taipei, Taiwan c Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan d Department of Obstetrics and Gynecology, Taipei Medical University, Taipei, Taiwan e Department of Obstetrics and Gynecology, National Yang-Ming University, Taipei, Taiwan a r t i c l e i n f o  Article history: Accepted 30 December 2013 Keywords: ammoniumblastocyst in vitro  maturation a b s t r a c t Objective:  The purpose of the study was to compare the Quinn ’ s Advantage fertilization medium (Q1)and the tissue culture medium 199 (TCM199) for  in vitro  maturation (IVM) of oocytes and ammoniumproduction during IVM. Materials and methods:  The immature murine oocytes were randomly added into Q1 and TCM199.Ammonium concentrations were measured at the start and after 18 hours of IVM, and the mature oo-cytes were fertilized and cultured into blastocysts. The blastocysts were then stained for inner cell mass(ICM) and trophectoderm. Results:  The maturation rate was higher in Q1 than in TCM199 (85.7% vs. 76.6%,  p  ¼  0.024). The fertil-ization and blastocyst rates were slightly higher in Q1, but not signi fi cant. Differential staining of theblastocysts showed slightly higher ICM ratio in the blastocysts derived from Q1. Mean ammoniumconcentrations in Q1 and TCM199 at Time 0 were 184.9 and 339.2  m g/dL, respectively(  p ¼ 0.05), and after18 hours of IVM were 268.7 and 443.6  m g/dL, respectively (  p  ¼  0.045). Addition of ammonium chlorideinto Q1 adversely affects IVM. Conclusion:  Q1 is superior to TCM199 in terms of oocyte maturation, which may be due to lowerammonium concentration.Copyright  2014, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. Allrights reserved. Introduction Immatureoocyteretrievalfollowedby invitro maturation(IVM)isanattractivealternativeto invitrofertilization (IVF)[1,2].Becausefewor no injections of gonadotropins are needed, IVM reduces the pa-tient ’ s cost and suffering and avoids the side effects associated withovarianstimulation.Despiterecentprogresses,theimplantationrateof IVM is still lower than that of traditional IVF [3]. The inferioroutcome of human IVM was thought to be primarily due to abnor-malities of cytoplasmic maturation in  in vitro -matured oocytes.The maturation system is a vital component of IVM. ManystrategieshavebeendesignedtoimprovetheoutcomeofIVM,suchas supplementation of serum, gonadotropins, and growth factors[1], humanchorionic gonadotropinpriming[4], follicle-stimulating hormonepriming[3,5]beforeoocytesretrieval,andcoculture[6].It has been shown that the addition of glucose and amino acids insimple and complex media [e.g., tissue culture medium 199(TCM199)]duringbovineIVMcansupportembryonicdevelopment[7]. Zheng et al showed that the choice of amino acids in monkeyIVM system affected the competence of oocyte maturation [8].Amino acids are commonly added to the embryo culture media toenhance embryonic development, but their requirement for IVM isnot fully understood.Supplementationofembryoculturemediawithaminoacidshasbeen shown to bene fi t embryonic development in many mamma-lian species, including humans [9]. The effects of amino acids onembryonic development vary with regard to different amino acids Con fl icts of interest: The authors have no con fl icts of interest to declare. *  Corresponding author. Department of Obstetrics and Gynecology, Shin KongWu Ho-Su Memorial Hospital, Number 95, Wen Chang Road, Shih Lin District,Taipei, Taiwan. E-mail address: (B.-C. Hsieh). Contents lists available at ScienceDirect Taiwanese Journal of Obstetrics & Gynecology journal homepage:    2014, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved. Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 17 e 20  and the stages of the embryos [10]. However, spontaneous aminoacidbreakdown,mainlyglutamine,generatesammonium,whichinturn induces aberrant blastocyst development and alters fetaldevelopment [11]. In contrast to the commonly used TCM199,Quinn ’ s Advantage fertilization medium (Q1; SAGE, Trumbull, CT,USA) contains heat-stable alanyl-glutamine rather than glutamine.Therefore, we conducted a study to compare the outcomes of IVMwith the two media, and ammonium production during IVM. Materials and methods The mice used in the study were FVB/NJNarl mice (NationalLaboratory Animal Center, Taipei, Taiwan). The experiment wasapproved by the Institutional Animal Care and Use Committee. Thechemicals were obtained from Sigma Chemical Company (St. Louis,MO, USA) unless stated otherwise. The experiment was repeatedsix times. IVM of oocytes Immature oocytes were collected from 3- to 5-week-old femalemice, 48 hours after intraperitoneal injection with 5 IU pregnantmare serum gonadotropin. Immature oocytes with cumulus cellswere collected by teasing the ovarian follicles. Only cumulus e oocyte complexes that comprised oocytes with homogeneouscytoplasm and more than three layers of cumulus cells were usedfor the study.The maturation medium consisted of Q1 or TCM199, plus75 mIU/mL   human menopausal  gonadotropin (Merional; IBSA,Lamone, Switzerland), 0.2 mM pyruvate, and 10% fetal bovineserum (FBS). The immature oocytes were randomly added into Q1and TCM199, and were incubated in groups of 10 at 37  C in anatmosphere of 5% CO 2  in air. Two blank media, namely, Q1-basedand TCM199-based maturation media without oocytes, wereincubated at the same time. Ammonium concentrations in themediaweremeasuredatthestartandafter18hoursofIVMculture.After 18 hours of IVM, the oocytes were assessed for maturity.Only oocytes that displayed a distinct  fi rst polar body were classi- fi ed as in metaphase II (MII) and were considered further forfertilization and culture. IVF  Spermatozoa were retrieved from the cauda epididymis of 5- to7-week-old male mice. The spermatozoa were dispersed in thehuman tubal  fl uid (HTF) medium (Irvine Scienti fi c, Santa Ana, CA,USA)containing5mg/mLFBS,dilutedtoaconcentrationof1  10 6 /mL. The MII oocytes were incubated with the spermatozoa for4 hours. The oocytes were then washed to eliminate excess sper-matozoa and cultured overnight in a drop of mKSOM (SpecialityMediaInc.,Phillipsburg,NJ,USA)coveredwithmineraloil.Thenextmorning, the number of two-cell embryos was counted, and theembryos were transferred to fresh droplets of mKSOM undermineral oil and cultured to blastocysts. The maturation rates,fertilization rates, and embryo development were recorded. Differential staining of blastocysts Forthedifferentialstainingof trophectoderm(TE)andinnercellmass (ICM), blastocysts were incubated in 0.05% pronase solutionin mKSOM for 5 minutes at 37  C to remove the zona pellucida. Theblastocysts were washed in mKSOM and incubated in 0.5% 2,4,6-trinitrobenzene sulfonic acid solution for 10 minutes at 4  C in thedark, followed by a wash in mKSOM. The blastocysts were incu-bated at room temperature for 40 minutes in the dark in a stainsolution, which contained 1  m g/mL bisbenzimide (Hoechst 33258)and 1  m g/mL propidium iodide in mKSOM. The stained blastocystswere washed again in mKSOM before  fi xation in 4% formalin so-lution.Theywerethenstoredinthedarkat4  Cuntilobservation.Asingle-layered image was obtained by applying gentle pressure ontheblastocysts.Theblastocystswereobservedundera fl uorescencemicroscope (Olympus AH3; Tokyo, Japan) equipped with an ultra-violet  fi lter. The nuclei of ICM appeared blue and the nuclei of TEappeared pink.  Addition of ammonium chloride To assess the effects of ammonium on IVM, ammonium wasadded into Q1. In the  fi rst part of the experiment, the meanammonium concentration in Q1 at Time 0 was 184.9    5.1  m g/dL;180  m g/dL (Group 2) and 540  m g/dL (Group 3) ammonium chloride(Sigma) were added into Q1 to make ammonium concentrationsapproximatelytwoandfourtimesthatofQ1only(Group1;i.e.,360and 720  m g/dL). The immature oocytes were now added into thethree media. After 18 hours of IVM, the mature oocytes werefertilized,andculturedtoblastocysts.Theexperimentwasrepeatedsix times.Ammonium concentrations in the maturation media weredeterminedinduplicateswiththeenzymaticmethodandreadonaspectrophotometer (model COBAS INTEGRA 800; Roche Di-agnostics, Berlin, Germany) set at 340 nm. The intra-assay coef  fi -cient of variation from  fi ve samples was 4.8%. Statistical analysis Values were expressed as mean    standard deviation. Ammo-nium concentrations were compared using the Mann e Whitney  U  test. Comparison of percentages between groups was carried outusingthe c 2 testorone-wayanalysisofvariance,asappropriate.A  p value < 0.05 was taken to be signi fi cant. Analyses were carried outusing the SPSS statistical package, version 14.0 (SPSS Inc., Chicago,IL, USA). Results At Time 0, ammonium concentrations in Q1 and the TCM199were 184.9    5.1 and 339.2    7.6  m g/dL, respectively (  p  ¼  0.05).After 18 hours of IVM, ammonium concentrations rose slightly inQ1andTCM199(both  p ¼ 0.05)to268.7  13.4and443.6  15.5 m g/dL, respectively. The ammonium concentration after 18 hours waslower in Q1 than in TCM199 (  p  ¼  0.045). At the same time,ammonium concentrations in the blank Q1 and in the blankTCM199 without oocytes were 247.1  22.0 and 457.0  12.0  m g/dL,respectively. Ammonium concentrations in the same mediumwithand without oocytes were not signi fi cantly different.The outcomes of IVM are shown in Table 1. The maturation ratewas higher in Q1 than in TCM199. The fertilization rate anddevelopment to blastocysts were slightly higher in Q1, but notsigni fi cant.  Table 1 Outcomes of IVM in TCM199 and Q1.TCM199 Q1  p No. of oocytes 210 210MII oocytes 161 (76.6%) 180 (85.7%) 0.024Fertilization 105 (65.2%) 128 (71.1%) 0.24Morula 32 (30.5%) 45 (35.2%) 0.48Blastocyst 16 (15.2%) 23 (18.0%) 0.6IVM ¼ in vitro  maturation; MII ¼ metaphase II. Y.-H. Lin et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 17  e  20 18  The results of differential staining of blastocysts are shown inTable 2. There were no signi fi cant differences in total cell numbersand cell numbers of ICM and TE of the blastocysts derived from thetwo media. However, the ICM ratio was slightly higher in theblastocysts derived from Q1.In the second part of the experiment in which ammoniumchloride was added into Q1, mean ammonium concentrations atthe start and after 18 hours of IVM in the three groups were142.1    1.9, 324.7    12.6, 685.4    11.6  m g/dL and 199.7    3.7,373.3  5.2,728.5  1.6 m g/dL,respectively.TheresultsofIVMinthethreegroupsareshowninTable3.Thematurationrate,fertilizationrate, and development to morulae and blastocysts in Group 1 werehigher than those in Groups 2 and 3, and ammonium had a dose-dependent adverse effect on oocyte maturation. Discussion Although srcinally designed for bovine IVM, TCM199 is stillusedinsomecentersforhumanIVM[3,12 e 14].Nopreviousstudieshave compared the outcomes of IVM in TCM199 and Q1. Our studyshows that the ammonium concentration was lower in Q1, and theoocyte maturation rate was slightly higher in Q1 than in TCM199.The fertilization rates and embryo development were slightlyhigher in Q1, but not signi fi cant. TCM199 contains all essential andnonessential amino acids, and is supplemented with vitamins. Q1was modi fi ed from the HTF medium, which was  fi rst described byQuinn et al [15]. Modi fi cations include low phosphate and additionof citrate, lactate, ethylenediaminetetraacetic acid, selectednonessential amino acids, taurine, and alanyl-glutamine (productinsert, Table 4).Certain amino acids are bene fi cial for embryo development[9,16] and IVM [7,17]. Supplementation of nonessential and essen- tial amino acids in the bovine oocyte maturation media was asso-ciated with enhanced developmental frequencies, increasedblastocyst cell number, and elevated oocyte maternal messengerRNA levels [17]. However, spontaneous breakdown of amino acids,especially glutamine, produced ammonium [16,18]. Instead of glutamine in TCM199, Q1 contains a heat-stable derivative of glutamine, alanyl-glutamine. Replacing glutamine with alanyl-glutamine reduced the accumulation of ammonium and resultedin signi fi cantly lower ammonium levels [11]. The present studyshowed that ammonium had a dose-dependent adverse effect onoocyte maturation. Addition of ammonium into Q1 to make itsconcentration similar to that in TCM199 also impaired IVM.Therefore, we speculate that the inferiority of TCM199 is due to ahigher ammonium concentration.This is the  fi rst study to show that ammonium was produced athigh concentrations in TCM199. The ammonium concentrationsincreased slightly after 18 hours of IVM in both media. Ammoniumconcentrationsinthesamemediumwithandwithoutoocyteswerenot signi fi cantly different, indicating that ammonium resultedmostly from the medium  per se , rather than from the oocytes.AdditionofammoniumintoQ1tomakeitsconcentrationsimilartothat in TCM199 signi fi cantly reduced maturation and fertilizationrate,andblastocystdevelopment,butTCM199onlyproducedlowermaturation rate than Q1, implying that TCM199 partially overcamethe adverse effects of ammonium. Because the embryos weremoved to mKSOM on the next day after fertilization, they were nolonger affected by the ammonium in the maturation media. Theadverse effects of ammonium on IVM are probably time and dosedependent. Yuan and Krisher showed that ammonium at concen-trations  2 mM decreased porcine oocyte maturation, but embryodevelopmentwasnotaffecteduntilanammoniumconcentrationof   20 mM [19]. Gardner and Lane showed that ammonium pro-ductionduringembryo culture increased linearlywith time,anditslevels increased to potentially inhibitory levels after 18 hours [16].In human IVM, the oocytes take 24 e 48 hours to mature. Therefore,the inhibitory effect of ammonium may be more pronounced inhuman IVM.Ammonium is present in follicular  fl uid and its concentrationsare highest in the smallest follicles and decrease as follicles grow[20]. Themeanammoniumconcentration inpreovulatoryfollicular fl uid is 38.87  m M (67.0  m g/dL) [21], which is much lower than the concentrations in freshly prepared maturation media and after18 hours of IVM in our study. Few studies have investigated theeffects of ammonium on IVM. Hammon et al showed that exposingbovine oocytes to 29 e 356  m M (50 e 613.8  m g/dL) of ammoniumduring IVM did not adversely in fl uence embryonic development[20].However,theydidnotinvestigatetheeffectsofammoniumonoocyte maturation. The present study is compatible with theirstudy in that development to blastocysts was unaffected, but wefound that the maturation rate was affected by ammonium.ThemechanismofthedetrimentaleffectsofammoniumonIVMmay be twofold. Ammonium reduced  in vitro  growth and meta-bolism of granulosa cells, and impaired their ability to support IVMof oocytes [22]. Second, exposure to ammonium may decrease  Table 2 Blastocyst cell numbers in different media.TCM199 Q1  p Total cell number 83.1  2.9 83.0  3.2 0.749ICM 17.0  0.9 20.0  1.1 0.969TE cells 66.1  2.1 63.0  2.2 0.651ICM ratios 0.20 0.23 0.026Values are mean  standard deviation.ICM ¼ inner cell mass; TE ¼ trophectoderm.  Table 3 Outcomes of IVM in Q1 and addition of ammonium.Group 1  p  (Group 1vs.Group 2)Group 2  p  (Group 2vs.Group 3)Group 3No. of oocytes 180 180 180MII oocytes 156 (86.7%) 0.001 116 (64.4%) 0.037 98 (54.4%)Fertilization 106 (67.9%) 0.006 58 (50%) 0.958 50 (51.0%)Morula 52 (49.1%) 0.009 12 (20.7%) 0.087 2 (4.0%)Blastocyst 28 (26.4%) 0.003 4 (6.9%) 0.211 0Group1,Q1;Group2,Q1 þ 180 m g/dLammoniumchloride;Group3,Q1 þ 540 m g/dL ammonium chloride.IVM ¼ in vitro  maturation; MII ¼ metaphase II.  Table 4 Comparison of components of the TCM199 and Q1.Components TCM199 Q1Essential amino acids  þ  e Nonessential amino acids  þ  L  -Asparagine, L  -aspartic acid,glycine,  L  -proline, L  -serine, taurineAlanyl-glutamine  e  þ Inorganic salts(e.g., NaCl, KCl, MgSO 4 ,NaHCO 3 , potassium phosphate) þ þ Glucose  þ þ Pyruvate  þ þ Lactate  e  þ Sodium citrate  e  þ EDTA  e  þ Vitamins  þ  e EDTA ¼ ethylenediaminetetraacetic acid. Y.-H. Lin et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 17  e  20  19  intracellular pH [11], and intracellular pH regulatory mechanisms are inactive in immature oocytes [23].In conclusion, the study showed that Q1 was superior toTCM199 for IVM. Ammonium was produced in the maturationmedia and had adverse effects on IVM. Utilizing media or matu-ration systems that produce less ammonium may improve theoutcome of IVM.  Acknowledgments The authors are grateful to Chyi-Hue Bai, PhD, for the assistanceof statistical analysis. This study was supported by the Shin KongWu Ho-Su Memorial Hospital (SKH-8302-97-DR-18). References [1] Chian RC, Buckett WM, Tan SL.  In-vitro  maturation of human oocytes. ReprodBiomed Online 2004;8:148 e 66.[2] Lin YH, Hwang JL.  In vitro  maturation of human oocytes. Taiwan J ObstetGynecol 2006;45:95 e 9.[3] Lin YH, Hwang JL, Huang LW, Mu SC, Seow KM, Chung J, et al. Combination of FSH priming and hCG priming for  in-vitro  maturation of human oocytes. HumReprod 2003;18:1632 e 6.[4] Chian RC, Gülekli B, Buckett WM, Tan SL. Priming with human chorionicgonadotropin before retrieval of immature oocytes in women with infertilitydue to the polycystic ovary syndrome. N Engl J Med 1999;341:1624 e 6.[5] Mikkelsen AL, Lindenberg S. Bene fi t of FSH priming of women with PCOS tothe  in vitro  maturation procedure and the outcome: a randomized prospectivestudy. Reproduction 2001;122:587 e 92.[6] Ge L, Sui HS, Lan GC, Liu N, Wang JZ, Tan JH. Coculture with cumulus cells im-proves maturation of mouse oocytes denuded of the cumulus oophorus: ob-servations of nuclear and cytoplasmic events. Fertil Steril 2008;90:2376 e 88.[7] Rose-Hellekant TA, Libersky-Williamson EA, Bavister BD. Energy substratesand amino acids provided during  in vitro  maturation of bovine oocytes alteracquisition of developmental competence. Zygote 1998;6:285 e 94.[8] Zheng P, Bavister BD, Ji WZ. Amino acid requirements for maturation of rhesusmonkey( Macaccamulatta )oocytesinculture.Reproduction2002;124:515 e 22.[9] Devreker F, Hardy K, Van den Bergh M, Vannin AS, Emiliani S, Englert Y.Amino acids promote human blastocyst development  in vitro . Hum Reprod2001;16:749 e 56.[10] Lane M, Gardner DK. Differential regulation of mouse embryo developmentand viability by amino acids. J Reprod Fertil 1997;109:153 e 64.[11] Lane M, Gardner DK. Ammonium induces aberrant blastocyst differentiation,metabolism, pH regulation, gene expression and subsequently alters fetaldevelopment in the mouse. Biol Reprod 2003;69:1109 e 17.[12] Zhao JZ, Zhou W, Zhang W, Ge HS, Huang XF, Lin JJ.  In vitro  maturation andfertilization of oocytes from unstimulated ovaries in infertile women withpolycystic ovary syndrome. Fertil Steril 2009;91:2568 e 71.[13] Wei Z, Cao Y, Cong L, Zhou P, Zhang Z, Li J. Effect of metformin pretreatmenton pregnancy outcome of   in vitro  matured oocytes retrieved from womenwith polycystic ovary syndrome. Fertil Steril 2008;90:1149 e 54.[14] Cha KY, Chung HM, Lee DR, Kwon H, Chung MK, Park LS, et al. Obstetricoutcome of patients with polycystic ovary syndrome treated by  in vitro maturation and  in vitro  fertilization-embryo transfer. Fertil Steril 2005;83:1461 e 5.[15] Quinn P, Warnes GM, Kerin JF, Kirby C. Culture factors in relation to thesuccess of human  in vitro  fertilization and embryo transfer. Fertil Steril1984;41:202 e 9.[16] Gardner DK, Lane M. Amino acids and ammonium regulate mouse embryodevelopment in culture. Biol Reprod 1993;48:377 e 85.[17] Watson AJ, De Sousa P, Caveney A, Barcroft LC, Natale D, Urquhart J, et al.Impact of bovine oocyte maturation media on oocyte transcript levels,blastocyst development, cell number, and apoptosis. Biol Reprod 2000;62:355 e 64.[18] Lane M, Gardner DK. Increase in postimplantation development of culturedmouse embryos by amino acids and induction of fetal retardation and exen-cephaly by ammonium ions. J Reprod Fertil 1994;102:305 e 12.[19] Yuan Y, Krisher RL. Effect of ammonium during  in vitro  maturation on oocytenuclear maturation and subsequent embryonic development in pigs. AnimReprod Sci 2010;117:302 e 7.[20] Hammon DS, Wang S, Holyoak GR. Ammonia concentration in bovine follic-ular  fl uid and its effect during  in vitro  maturation on subsequent embryodevelopment. Anim Reprod Sci 2000;58:1 e 8.[21] Józwik M, Józwik M, Teng C, Battaglia FC. Amino acid, ammonia and ureaconcentrations in human pre-ovulatory ovarian follicular  fl uid. Hum Reprod2006;21:2776 e 82.[22] Rooke JA, Ewen M, Mackie K, Staines ME, McEvoy TG, Sinclair KD. Effect of ammonium chloride on the growth and metabolism of bovine ovarian gran-ulosa cells and the development of ovine oocytes matured in the presence of bovine granulosa cells previously exposed to ammonium chloride. AnimReprod Sci 2004;84:53 e 71.[23] Erdogan S, FitzHarris G, Tartia AP, Baltz JM. Mechanisms regulating intracel-lular pH are activated during growth of the mouse oocyte coincident withacquisition of meiotic competence. Dev Biol 2005;286:352 e 60. Y.-H. Lin et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 17  e  20 20
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