The effect of oxygen on the development of preimplantation mouse embryos in vitro.
Human Blastocyst Development In Vitro Without Co-Culture Using a Sequential Culture System
Physical Culture
Systems
Culture in Microchannels Enhances In Vitro Embryonic Development of Preimplantation Mouse Embryos
Comparison of mouse embryo development in open and microdrop co-culture systems.
Media Composition
Coenzyme Q(10) in submicron-sized dispersion improves development, hatching, cell proliferation, and adenosine triphosphate content of in vitro-produced bovine embryos.
Culture of one-cell hamster embryos with water soluble vitamins: pantothenate stimulates blastocyst production.
Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture.
Potent and stage-specific action of glutathione on the development of goat early embryos in vitro.
Pyruvate prevents peroxide-induced injury of in vitro preimplantation bovine embryos.
Developmental toxicity induced during early stages of mammalian embryogenesis.
Abstracts
J Exp Zool 1978 Oct;206(1):73-80Physical Culture Systems
The effect of oxygen on the development of preimplantation mouse embryos in vitro.
Quinn P, Harlow GMThe optimal oxygen tension for development of preimplantation mouse embryos to the blastocyst stage in vitro was found to be between 2.5% and 5%. One- and two-cell embryos had a more sharply defined range of oxygen tension capable of supporting development than 8-cell and morula stages. At all stages of development, more embryos developed to the blastocyst stage under 5% O2 compared to the numbers of developing under higher oxygen tensions (20% and 40% O2). The blastocysts developing under 20% O2 had fewer blastomeres than those which developed under 5% O2. As the time required for development to the blastocyst stage in vitro increased, there were fewer blastomeres present at the blastocyst stage. These results indicate that the cleaving mouse embryo has an optimal oxygen requirement in vitro of about 5%. At higher oxygen tensions, fewer embryos develop to the blastocyst stage and in those which do develop, there are fewer cell divisions. If a gradient of oxygen tension exists across the blastomeres from the outside of the embryo to its centre, the blastomeres might be using this gradient to obtain information about their location within the embryo and respond accordingly[1]. Thus blastomeres on the outside at a higher oxygen tension would divide at a slower rate and form trophectoderm whereas those on the inside at a lower oxygen tension would divide more rapidly and contribute to the inner cell mass.
PMID: 702089, UI: 79028765
Comment:[1]I believe this to be an erroneous conclusion; 20% O2 is a more toxic environment which is non-physiologic for a developing embryo; oxidative damage (free radical activity) results in fewer and more slowly dividing cells.Why should these cells be using a non-physiologic 02 gradient for sensing? I would nor expect O2 gradients to be relevant here, as O2 concentration in some ( and probably most..?) mammalian wombs is around 7%. Furthermore, significant O2 gradients are unlikely to be established in such relatively small cellular environments (blastocyst).
Test: would the provision of antioxidant substances in the culture media equalize the development under varying O2 concentrations?
Human Blastocyst Development In Vitro Without Co-Culture Using a Sequential Culture System
B. Behr, D. Moore, J. Gebhardt, S. Hall, D. Dasig. Department GYN/OB Stanford University Medical Center, Stanford, CA.Objective: This preliminary analysis was designed to measure the percent blastocyst development of supernumerary embryos without the use of feeder cells or conditioned medium. Embryos derived from IVF that were not transferred or cryopreserved were included for this study.
Design: A retrospective analysis of embryonic development from IVF or GIFT/IVF patients who's embryos did not meet criteria for cryopreservation or embryo transfer, were cultured in a sequential fashion to asses percent blastocyst development after 120hrs of culture.
Materials and Methods: Ova were harvested from patients undergoing IVF with a standard ovarian stimulation with GnRHa/hMG. Ova were collected and cultured in 150ul droplets of P1 (Irvine Scientific) under mineral oil, in groups at 37°C under 5%CO2, 5%O2 and 90%N2 environment. Embryo transfer was performed 72hrs post harvest. Viable embryos not transferred or cryopreserved were placed into a
modified Hams F-10 (Irvine Scientific) and cultured for an additional 48hrs. Embryos that exhibited an expanded blastocele cavity and well defined inner cell mass at 120hrs were counted.Results: 109 embryos from 17 consecutive patients with supernumerary embryos were cultured. 51% (56/109) reached the expanded blastocyst stage by 120hrs of culture and patients were given the option of cryopreservation. The embryos were cryopreserved using a standard serial addition of glycerol protocol. Several patients had blastocysts develop after 120hrs of culture that were not included.
Conclusion: This report demonstrates that implementing a simple system of sequential culture generated acceptable blastocyst development (51%) with ``left over'' embryos without the use of feeder cells or conditioned medium. Recognizing the deferential metabolic requirements of early and late cleavage stage embryos has enabled the implementation of a glucose/phosphate-free simple culture medium (P1) for up to 72hrs of culture and a complex glucose containing medium (Mod. Hams F-10) for subsequent blastocyst development. Five patients have had thawed blastocyststransferred with 3 resulting in clinical pregnancy. The role of specific medium components and their relevancy to preimplantation embryo metabolism and the implications of blastocyst cryopreservation will be discussed.
Theriogenology: January 1 2001 Vol.55 No.1, page 241
Culture in Microchannels Enhances In Vitro Embryonic Development of Preimplantation Mouse Embryos
S. Raty1, J.A. Davis1, D.J Beebe1,2 , S.L. Rodrireuz-Zas1, and M.B. Wheeler1
1University of Illinois at Urbana-Champaign, Urbana, IL, USA,2 University of Wsconsin-Madison, Madison,WI,USA
Values on the same row differ at *P<0.05, or **P<0.01.
Table 1: Least Square means of the percentages of embryos at a particular stage of development and time point. Stage of Development Microchannels (n=170) Control(droplet)(n=170) Standard error(%) 16-cell/morula @ 24hr 23.5(n=40)* 47(n=8) 6.2 Blastocysts @ 48hr 17.6(n=30)** 24(n=4) 2.4 Blastocysts @ 72hr 72.9(n=117)** 42.9(n=73) 4.6 Hatched Blastocysts @ 72hr 4.1(n=7) 0(n=0) 1.2 Hatched Blastocysts @ 96hr 26.5(n=45)** 8.8(n=15) 3.2 Degenerated Embryos @ 96hr 20.0(n=34)** 42.9(n=73) 4.6
Quotes & Notes:This was a student poster presentation at the 2001 IETS meeting, so far the abstract is unavailable through pubmed.
Comment:
This could have significant benefit throughout the entire IVF industry if the results carry over to human embryos and are repeatable.
This study essentally demonstrates the potential benefit of maintaining blastocysts in a constant stream of fresh media, with a gradual rather than abrupt transtion between G1 and G2 media, in which primary energy substrates differ. See next reference.
Theriogenology 1998 Jan 1;49(1):83-102
Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture.
Gardner DKColorado Center for Reproductive Medicine, Denver 80110, USA.
Along with the transition from maternal to embryonic genome control the mammalian preimplantation embryo undergoes significant changes in its physiology during development. Concomitant with these changes are altering patterns of nutrient uptake and differences in the subsequent fate of such nutrients. The most significant nutrients to the developing mammalian preimplantation embryo are carbohydrates and amino acids, which serve not only to provide energy but also to maintain embryo function by preventing cellular stress induced by suboptimal culture conditions in vitro. It is subsequently proposed that optimal development of the mammalian embryo in culture requires the use of two or more media, each designed to cater for the changing requirements of the embryo. Importantly, culture conditions that maintain the early embryo are not ideal for the embryo post-compaction, and conditions that support excellent development and differentiation of the blastocyst can actually be inhibitory to the zygote. A marker of in vitro-induced cellular stress to the embryo is the relative activity of the metabolic pathways used to generate energy for development. Quantification of embryo energy metabolism may therefore serve as a valuable marker of embryo development and viability.
PMID: 10732123Comment:
An abrupt transition in the composition of energy substrates in the culture media has the potential to induce cellular stress. See preceding reference.
Hum Reprod 2000 Jan;15(1):157-64
Culture of one-cell hamster embryos with water soluble vitamins: pantothenate stimulates blastocyst production.
McKiernan SH, Bavister BDThe effects of water-soluble vitamins, singly or in combinations, on development of hamster 1-cell embryos were examined in a protein-free, chemically defined culture medium, HECM-6. Pantothenate significantly stimulated blastocyst development compared to the vitamin-free control and to every other single vitamin, except thiamine. Ascorbic acid, biotin, choline, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine had no detectable stimulation or inhibition on cleavage stage development or morula/blastocyst formation. When combinations of vitamins were tested, embryo development was either unchanged or significantly greater than in the control, but never significantly greater than development with pantothenate alone. A dose response to pantothenate indicated that 3 mol/l was the optimum concentration. After embryo transfer, the percentage of live fetuses recovered per 100 1-cell embryos cultured in HECM-6 plus pantothenate (now designated HECM-9) was 24%, significantly higher than the 11% recovered from 100 1-cell embryos cultured in HECM-6 alone. This is the first report to show a stimulatory effect of a single vitamin on in-vitro development of preimplantation embryos in any mammalian species.
PMID: 10611206, UI: 20079210Quotes & Notes:Pending
Comment:Pantothenate may stimulate the production of Acetyl-CoA, which is an important substrate for acetylation reactions.The acetylated form of histone protein H1 has recently been shown to be of importance in nuclear reprogramming. Histone acetylation has been correlated with transcriptional activity. See "Chromatin Remodeling" section.
The commonly used G2.2 media for IVF does contain calcium pantothenate. What concentration I do not know.Also see this reference.
Mol Reprod Dev 2000 Sep;57(1):48-54
Potent and stage-specific action of glutathione on the development of goat early embryos in vitro.
Lee CS, Koo DB, Fang N, Lee Y, Shin ST, Park CS, Lee KKThe effect of glutathione (GSH) addition on the development of 1- or 2-cell goat early embryos in vitro was examined. Embryos were collected from superovulated Korean black goat (Capra hircus aegagrus) and cultured for 6 days in synthetic oviduct fluid medium supplemented with either bovine serum albumin (BSA) or serum. Without GSH addition, almost all embryos could not develop beyond 8- to 16-cell block. However, GSH addition greatly improved in vitro development of early embryos to blastocyst stage, and its action was highly dependent on the presence and source of proteins supplemented into the culture medium. Among the protein-supplemented cultures, GSH effect was most prominent in 10% FBS-supplemented culture, in which the proportion (91%) of blastocysts developed from early embryos was much higher than that of BSA- (42-64% depending on its content) or goat serum (GS)-supplemented cultures (21%), or even than that of somatic cell-supported co-culture (60%). As well as in terms of the morphological development, mean cell number of blastocysts (185 +/- 12) developed from FBS condition was significantly higher than that of blastocysts developed from any other culture conditions and moreover comparable to that of blastocysts developed in vivo (190 +/- 9). The viability of these blastocysts was finally confirmed by their term development (6/12) from embryo transfer. To delineate action time of GSH during embryo development, GSH was treated at 1-day intervals through 6-days culture periods excepting the last day. In the GSH-treated embryos at day 3 of culture, which corresponds to the time of in vitro 8- to 16-cell block stage, the proportion of blastocyst was markedly increased up to 77% of cultured embryos and conversely that of the arrested embryos was decreased to 7%. In the embryos treated later, however, their developmental potency decreased abruptly. Therefore, these results clearly demonstrated that GSH could greatly improve the in vitro development of goat early embryos by specifically acting on the 8- to 16-cell block stage during in vitro development, suggesting that GSH may be one of the important regulators on the development of goat embryos in vivo.
Copyright 2000 Wiley-Liss, Inc.
PMID: 10954855, UI: 20413442Quotes & Notes:Pending
Comment:Seems like a reasonable addition to G1 media....
Mean cell number is the best single criterion to apply in embryo scoring.
Biol Reprod 1999 Aug;61(2):541-7
Coenzyme Q(10) in submicron-sized dispersion improves development, hatching, cell proliferation, and adenosine triphosphate content of in vitro-produced bovine embryos.
Stojkovic M, Westesen K, Zakhartchenko V, Stojkovic P, Boxhammer K, Wolf ECoenzyme Q(10) (CoQ(10)) is an essential component of the plasma membrane ion transporter (PMIT) system and of the electron transport chain in the inner mitochondrial membrane. Because of its intrinsic functions in cell growth and energy metabolism (ATP synthesis), and its protective effects against oxidative stress, CoQ(10) is a good candidate for supporting growth of cells in culture. However, because of its quinone structure, CoQ(10) is extremely lipophilic and practically insoluble in water. We used a specific technology to prepare a submicron-sized dispersion of CoQ(10), inhibiting re-crystallization by a stabilizer. This dispersion, which exhibits a very large specific surface area for drug dissolution, was tested as a supplement for the in vitro culture of bovine embryos in a chemically defined system. The rate of early cleavage of embryos (5- to 8-cell stages) was evaluated 66 h postinsemination (hpi) and was highest in medium supplemented with 30 or 100 microM CoQ(10) (66.5 +/- 0.8% and 68.7 +/- 1.1%, respectively) and lowest in 10 microM CoQ(10) (55.3 +/- 0.8%). The proportions of oocytes developing to blastocysts by 186 hpi were 19.0 +/- 0.6% and 25.2 +/- 0.3% in medium supplemented with 10 microM and 30 microM CoQ(10), respectively, and were significantly (p < 0.001) higher than those obtained with the equivalent amounts of stabilizer (9.9 +/- 0.4% and 11.3 +/- 0.4%). In the presence of 30 microM CoQ(10), significantly (p < 0.001) more blastocysts hatched by 210 hpi than in the equivalent amount of stabilizer (31.8 +/- 1.3 vs. 8.4 +/- 2.2). Expanded blastocysts produced in the presence of 30 microM CoQ(10) had significantly (p < 0.01) more inner cell mass cells and trophectoderm cells, and a significantly (p < 0.001) increased ATP content as compared to expanded blastocysts produced in the presence of the corresponding amount of stabilizer. Our results show that noncrystalline CoQ(10) in submicron-sized dispersion supports the development and viability of bovine embryos produced in a chemically defined culture system.
PMID: 10411538, UI: 99339862Quotes & Notes:Pending
Comment:The early embryo maintains proper intracellular pH through the action of H+/Na+ antiporter, which is ATP driven.Stress to the early embryo may deplete critical ATP reserves, a plausible factor contributing to incomplete nuclear reprogramming, especially when viewed from within the conceptual framework of enhancing "chromatin fluidity".
Increasing intracellular energy stores in the form of ATP could similarly potentiate successful nuclear reprogramming, as well as enhancing blastocyst development as demonstrated in this article.
One should strongly consider adding this form of CoQ10 to all stages of cellular culture ( donor cell, early embryo, and blastocyst)
Mol Reprod Dev 1999 Feb;52(2):149-57
Pyruvate prevents peroxide-induced injury of in vitro preimplantation bovine embryos.
Morales H, Tilquin P, Rees JF, Massip A, Dessy F, Van Langendonckt AUnite des Sciences Veterinaires, Universite catholique de Louvain,Louvain-la-Neuve, Belgium.
The impact of oxidative stress on the in vitro development of bovine embryos in synthetic oviduct fluid medium (mSOF) was assessed by using H2O2 as a stress inducer. In a preliminary experiment, a chemiluminescent method was used to measure the antioxidative capacity of the mSOF culture medium. Pyruvate was the mSOF component displaying the highest H2O2 degrading ability. Essential and nonessential amino acids also significantly reduced the H2O2 concentration, whereas lactate and glutamine were ineffective. The effect on further development of a short exposure of zygotes, 9-16-cell stage embryos and blastocysts to 0 M; 10(-7) M ; 10(-6) M, and 10(-5) M H2O2 in pyruvate-free mSOF was evaluated. Developmental rates of the H2O2-treated zygotes to the 5-8-cell or blastocyst stages and survival of H2O2-treated blastocysts were reduced in a dose-dependent manner whereas the 9-16-cell embryos were unaffected by those treatments. Blastocysts treated with H2O2 also tended to have lower numbers of bisbenzimide-stained nuclei and showed increased nuclear fragmentation. Including pyruvate in the mSOF culture medium during a 10(-5) M H2O2 pulse highly reduced the H2O2 concentration as measured by chemiluminescence and improved zygote and blastocyst development, but failed to prevent blastocyst nuclei degradation. These experiments suggest that bovine embryos show developmental change in sensitivity to exogenous H2O2, the 9-16-cell embryos being more resistant than zygotes and blastocysts and that H2O2 and its toxic effects can be attenuated by including pyruvate in the medium.
PMID: 9890745
Quotes & Notes:Pending
Comment:Human embryos are commonly cultured in a two stage G1 and G2 media. G1 contains pyruvate, which at early stages is the primary energy substrate of the developing embryo. The embryonic metabolism shifts to utilize glucose as the primary energy substrate at about the blastocyst stage. Pyruvate is retained in G2.2 media.
Hum Reprod 1996 Oct;11(10):2223-9
Comparison of mouse embryo development in open and microdrop co-culture systems.
Sherbahn R, Frasor J, Radwanska E, Binor Z, Wood-Molo M, Hibner M, Mack S, Rawlins RG
Department of Obstetrics and Gynecology, Rush University Medical Center, Chicago, IL, USA.Co-culture with numerous cell lines has been shown to improve in-vitro embryo development. It is usually performed in open culture without an oil overlay, or in relatively large volumes of medium (e.g. 0.5 ml) under oil. We compared the efficacy of open and microdrop co-culture systems using human endometrial and tubal cell lines and mouse zygotes. Although the mean pH values of the media from the tubal cell cultures (both open and oil-covered) decreased significantly over 5 days of culture, this did not appear to impair embryo development. Both co-culture and microdrop culture significantly improved blastocyst and hatching blastocyst formation rates. The combination of the two techniques (microdrop and co-culture) demonstrated the highest blastocyst formation and hatching blastocyst formation rates, as well as the highest mean cell numbers in hatching blastocysts. Co-culture in a microdrop is a superior system for mouse embryo culture.
PMID: 8943534, UI: 97098918Quotes & Notes:Pending
Comment:Cell number has been shown to be one of the best predictors of blastocyst quality see here