ORIGINAL ARTICLES: REPRODUCTIVE SCIENCE

Composition of single-step media

used for human embryo culture
Dean E. Morbeck, Ph.D.,a,b Nikola A. Baumann, Ph.D.,b and Devin Oglesbee, Ph.D.b
a
Department of Obstetrics and Gynecology, Mayo Clinic, Rochester, Minnesota; and b Department of Laboratory Medicine
and Pathology, Mayo Clinic, Rochester, Minnesota

Objective: To determine compositions of commercial single-step culture media and test with a murine model whether differences in
composition are biologically relevant.
Design: Experimental laboratory study.
Setting: University-based laboratory.
Animal(s): Inbred female mice were superovulated and mated with outbred male mice.
Intervention(s): Amino acid, organic acid, and ions content were determined for single-step culture media: CSC, Global, G-TL, and 1-
Step. To determine whether differences in composition of these media are biologically relevant, mouse one-cell embryos were cultured
for 96 hours in each culture media at 5% and 20% oxygen in a time-lapse incubator.
Main Outcome Measure(s): Compositions of four culture media were analyzed for concentrations of 30 amino acids, organic acids,
and ions. Blastocysts at 96 hours of culture and cell cycle timings were calculated, and experiments were repeated in triplicate.
Result(s): Of the more than 30 analytes, concentrations of glucose, lactate, pyruvate, amino acids, phosphate, calcium, and magnesium
varied in concentrations. Mouse embryos were differentially affected by oxygen in G-TL and 1-Step.
Conclusion(s): Four single-step culture media have compositions that vary notably in pyruvate, lactate, and amino acids. Blastocyst
development was affected by culture media and its interaction with oxygen concentration. (Fertil SterilÒ 2017;107:1055-60. Ó2017 by
American Society for Reproductive Medicine.)
Key Words: Culture media, embryo culture, mouse embryo assay, quality control
Discuss: You can discuss this article with its authors and with other ASRM members at https://www.fertstertdialog.com/users/
16110-fertility-and-sterility/posts/14316-23139

A
ssisted reproductive technol- new single-step media from companies culture media on assisted reproduc-
ogy (ART) success, defined as that previously only offered sequential tive technology are limited by a lack
delivery of a healthy child, de- media (4). A new culture medium can of data (1, 6, 7).
pends importantly on quality of the be introduced to the market without To address this gap, we analyzed the
embryo culture environment. The extensive clinical testing and without composition of single-step culture me-
foundation of this environment is the disclosure of detailed composition. dia from four suppliers and determined
culture medium, yet detailed composi- Furthermore, although media composi- whether differences in media composi-
tion of this medium is not provided by tion varies considerably (5), the scienti- tion were biologically relevant by
manufacturers, despite requests from fic rationale for different formulations culturing single-cell murine embryos to
the embryology community (1, 2). is rarely provided. the blastocyst stage. Cultures were per-
Routine time-lapse imaging for Identification of the best and formed with reduced (5%) and ambient
embryo culture introduced a paradigm safest conditions for embryo culture (20%) oxygen, because we had previ-
shift for embryo culture, because more to support optimal embryo develop- ously demonstrated medium
oxygen
information is obtained with less ment remains elusive. Assisted repro- interactions that altered mouse embryo
handling compared with conventional ductive technology laboratories are development (5).
culture (3). This shift favors single- faced with choices without having
step media over sequential media and detailed compositional analysis.
MATERIALS AND METHODS
in turn has led to the introduction of Details of the long-term effects of
Culture Media Analysis
Received September 24, 2016; revised January 2, 2017; accepted January 13, 2017; published online
February 24, 2017.
Culture media sources. Culture media
D.E.M. has nothing to disclose. N.A.B. has nothing to disclose. D.O. has nothing to disclose. were purchased in April and May
Supported by a grant from Mayo Clinic Department of Obstetrics and Gynecology. 2015 from four suppliers and included
Reprint requests: Dean E. Morbeck, Ph.D., Fertility Associates, 7 Ellerslie Racecourse Drive, Remeura,
Auckland 1051, New Zealand (E-mail: deanmorbeckphd@gmail.com). Global (Global-Total, LifeGlobal, IV-
FOnline), CSC (Irvine Scientific), G-TL
Fertility and Sterility® Vol. 107, No. 4, April 2017 0015-0282/$36.00
Copyright ©2017 American Society for Reproductive Medicine, Published by Elsevier Inc.
(Vitrolife), and 1-Step (Origio). Global
http://dx.doi.org/10.1016/j.fertnstert.2017.01.007 is provided with 10 mg/mL human

VOL. 107 NO. 4 / APRIL 2017 1055

ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE

serum albumin (HSA), whereas the other three media con- Data for precise cell division timings using time-lapse imag-
tained 5 mg/mL HSA. ing were obtained; all annotations were performed by one
Amino acid and organic acid analysis. Amino acids were technician.
quantified in duplicate (5). Organic acids (lactate, pyruvate,
and citrate) were prepared as oximated silyl-derivatives for
stable isotope dilution for capillary gas chromatography– Data and Statistical Analysis
mass spectrometry analysis. Briefly, 100 mL of each sample Developmental and time-lapse data were analyzed using a
was diluted 1:1 into isotopic internal standard mix (contain- one-way analysis of variance (ANOVA) with Tukey's test
ing D4-citrate, D4-lactate, 13C2-pyruvate), followed by oxima- for pairwise comparisons of blastocyst development and cell
tion with pentafluorobenzyl hydroxylamine. Organic oximes division timings. Analyses were performed in two steps. First,
were extracted with ethyl acetate and derivatized with N,O,- a 4
2 factorial ANOVA was used to compare blastocyst
bis-(trimethylsilyl) trifluoroacetamide and 1% trimethyl- development and timings for four media at low and high ox-
chlorosilane to form silyl-derivatives. Oximes (1 mL) were ygen. Second, because the primary treatment effect observed
injected into an Agilent 6890/5973 gas chromatograph– was a media
oxygen interaction, a Student t test ANOVA
mass spectrometer using a splitless/split combination (50:1 was used to compare blastocyst development and timings be-
split mode). Quantification of analytes was performed by tween low and high oxygen within each medium. Statistical
comparing measured peak areas of quantifying ions against analyses were performed using JMP statistical software
unique quantifying ions of known concentrations of (SAS Institute).
isotope-labeled internal standards. Coefficients of variation
for citrate, lactic acid, and pyruvate at concentrations
measured were 6.8%, 1.9%, and 2.1%, respectively. RESULTS
Inorganic ion analysis. Calcium (colorimetric o-cresolphtha- Media Composition
lein complexone), chloride (ion-selective electrode [ISE]), po-
Two of the culture media (G-TL and 1-Step) were only avail-
tassium (ISE module), magnesium (colorimetric), sodium
able presupplemented with protein (HSA), therefore all
(indirect ISE), and phosphorus (photometric) were quantified
experiments used presupplemented media. Protein supple-
in duplicate using Roche Cobas reagents and chemistry ana-
mentation constitutes addition of undefined components,
lyzers (Cobas 6000 c501 or Cobas 8000 ISE, c701, c502 mod-
but our analysis represents only components reported by
ules) as previously described (5).
manufacturers or known components of albumin prepara-
Other analytes. Glucose (hexokinase) was quantified in tions (e.g., octanoate). Manufacturer-reported components
duplicate using Roche Cobas chemistry analyzers (Cobas that were not analyzed include ethylenediaminetetraacetic
6000 c501 or Cobas 8000 ISE, c701, c502 modules), and acid, gentamicin, dipeptide forms of glutamine, calcium
lactate was assayed by lactate oxidase/peroxidase (dry slide pantothenate, pyridoxine, riboflavin, thiamine, sodium bicar-
chemistry) on a Vitros 350 chemistry analyzer (Ortho Clinical bonate, and sodium hyaluronate. Age of culture media may
Diagnostics) as previously described (5). pH and osmolality impact composition (9), therefore all analyses were performed
were determined as previously described (8). within 60 days of media manufacture date.
Glucose and organic acids. Pyruvate, lactate, and glucose
Mouse Embryo Assay are the primary energy substrates for preimplantation em-
bryos. All four media contained these carbohydrates, but con-
The Mayo Clinic Institutional Animal Care and Use Commit- centrations differed (Table 1). Global and 1-Step had similar
tee approved procedures involving animals. Mice were concentrations of all three substrates. G-TL had a fivefold
obtained from Charles River Laboratories. Six- to nine- higher concentration of glucose and twofold higher concen-
week-old FVB mice were superovulated with 5 IU of intraper- trations of pyruvate and lactate than Global. The ratio of py-
itoneal pregnant mare serum (National Hormone and Peptide ruvate to lactate was similar for all four media.
Program), followed 48 hours later with 5 IU of intraperitoneal
hCG (APP Pharmaceuticals). Females were caged individually
with male CF1 mice overnight, and mating was confirmed by
TABLE 1
presence of a vaginal plug. Cumulus surrounded zygotes were
isolated from oviducts obtained 18 hours after hCG Concentrations of glucose and organic acids, and the lactate to
administration. pyruvate (L/P) ratio in embryo culture media.
After denuding zygotes with hyaluronidase (250 u/mL; Medium
H3506, Sigma Chemical), embryos were transferred individu-
Variable Global CSC G-TL 1-Step
ally into 25 mL of media in an EmbryoSlide (Unisense Fertil-
itech), and each slide was inserted into an EmbryoScope Glucose (mM) 0.18 0.47 0.97 0.19
Citrate (mM) 0 0.02 0.01 0
(Unisense Fertilitech). A 4
2 factorial study was performed Octanoate (mM) 0.681 0.324 0.344 0.355
with four media at 5% and 20% oxygen. All experiments were Lactate (mM) 4.9 5.71 10.01 4.35
performed in triplicate at 37 C and 6.2% CO2, with 10 to 11 Pyruvate (mM) 0.24 0.28 0.55 0.22
embryos for each protein/condition combination. Expanded L/P ratio 20 21 18 20
Morbeck. Composition of continuous culture media. Fertil Steril 2017.
blastocyst at 96 hours of culture was the primary endpoint.

1056 VOL. 107 NO. 4 / APRIL 2017

 Fertility and Sterility®

Two organic acids that are not essential components of

TABLE 3
preimplantation embryo development are octanoate and cit-
rate. Octanoate is an additive in HSA preparations, with an Electrolytes in embryo culture media.
expected concentration of 400 uM for medium containing
5 mg/mL HSA. Global contains 10 mg/mL HSA and was the Medium
only medium with octanoate above 400 uM (Table 1). Citrate, Electrolyte Global CSC G-TL 1-Step
a proposed embryotrophic factor (10), was present in CSC and Calcium (Ca) (mM) 1.6 1.9 1.0 2.1
G-TL at low concentrations (0.01–0.02 mM). Phosphorus (mM) 0.22 0.15 0.25 0.34
Potassium (mM) 2.8 2.8 5.5 2.9
Amino acids. Concentrations of essential and nonessential Chloride (mM) 108 112 106 112
amino acids of Global and CSC (Table 2) were similar, as pre- Sodium (mM) 138 138 137 132
viously demonstrated (5). G-TL and 1-Step differed from Magnesium (Mg) (mM) 0.24 0.78 1.62 1.78
Global and CSC; most notably for G-TL was lack of glutamate, Ca/Mg ratio 7.1 2.4 0.6 1.2
Morbeck. Composition of continuous culture media. Fertil Steril 2017.
low aspartate, and addition of taurine (40 uM), and for both,
low but detectable concentrations of glutamine, a labile
amino acid supplied as a dipeptide. Another notable differ- TL to a high of 7.1 in Global. Remaining electrolytes were
ence for G-TL and 1-Step was an overall increase in amino relatively similar, except that potassium was twofold higher
acid concentration. Compared with Global and CSC, nine in G-TL than in the other three media.
essential amino acids were higher in G-TL and 1-Step.
Excluding glutamine, which is present as a dipeptide and
not quantified in this study, five nonessential amino acids Mouse Embryo Development
were present at higher concentrations in G-TL, with glycine, pH and osmolality. When comparing embryo development in
proline, serine, and taurine more than twofold higher. The different culture media, accounting for pH and osmolality is
summed concentration of amino acids was 1,741, 1,863, important. All mouse embryo testing was performed at a
2,250, and 2,075 uM for Global, CSC, G-TL, and 1-Step, CO2 setting of 6.2%, which yielded pH of 7.30, 7.32, 7.35,
respectively. and 7.25 for Global, CSC, G-TL, and 1-Step, respectively.
Electrolytes. Ionic electrolytes varied among the four media Global, CSC, and 1-Step recommend CO2 settings that yield
(Table 3). Calcium and magnesium concentrations differed pH of 7.3  0.1, whereas G-TL recommends a CO2 setting of
most. Calcium in G-TL (1.0 mM) was approximately 50% of 6.0%, with no recommended pH target. Osmolalities of the
that found in Global, CSC, and 1-Step. Magnesium in G-TL media were similar at 266, 265, 269, and 262 mOsm/kg for
and 1-Step was two- to sixfold higher than in CSC and Global. Global, CSC, G-TL, and 1-Step, respectively.
The Ca/Mg ratio differed considerably, from a low of 0.6 in G- Blastocyst development. Overall, main effects of culture me-
dia (P¼ .35) or oxygen (P¼ .75) were not significant; however,
the medium
oxygen interaction was significant (P< .05).
TABLE 2 Therefore we performed oxygen concentration subanalyses
for blastocyst rate and cell cycle timings for each culture me-
Amino acid concentrations in embryo culture media. dium (Fig. 1). Oxygen concentration did not affect number of
Medium blastocysts for either Global or CSC but differentially affected
blastocyst formation in G-TL and 1-Step. Blastocyst forma-
Amino acid Global CSC G-TL 1-Step
tion was lower (P< .05) with 20% oxygen for G-TL and lower
Essential (mM) (P< .05) with 5% oxygen for 1-Step (Fig. 1).
Arg 278 292 324 336
Cys 32 34 26 28 Time-lapse analysis. Cell cycle timings were monitored to
His 76 80 89 90 determine whether temporal changes in embryo growth
Ile 182 199 215 204
Leu 177 188 204 206
were associated with blastocyst development. The three media
Lys 154 168 182 182 that performed similarly in 5% oxygen (Global, CSC, G-TL)
Met 44 50 54 54 had similar embryo growth patterns (Supplemental Fig. 1A–
Phe 79 83 91 92 C, available online), whereby 5% oxygen resulted in faster
Thr 162 176 184 204
Trp 18 20 21 23 development until the postcompaction stage, when develop-
Tyr 69 75 80 83 ment to the blastocyst stage was faster in 20% oxygen. A con-
Val 163 174 200 196 trasting pattern of embryo development was observed for 1-
Nonessential (mM)
Step: embryo growth was slower in 5% oxygen
Ala 46 48 63 38
Asn 42 46 40 36 (Supplemental Fig. 1D), and the rate of development during
Asp 42 43 12 58 postcompaction was similar between the two oxygen
Glu 40 41 0 49 concentrations.
Gln 0 0 10 36
Gly 42 44 185 48 For G-TL and 1-Step, the two media differentially
Pro 55 60 126 66 affected by oxygen, timing of development was medium-
Ser 40 42 96 46 specific, with differences in development for G-TL appearing
Tau 0 0 48 0 primarily at the four-cell stage and beyond, whereas differ-
Morbeck. Composition of continuous culture media. Fertil Steril 2017.
ences for 1-Step were most evident from the two- to four-

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ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE
FIGURE 1
100%
90%
80%
* In contrast to media optimized via a mouse model (Global
Blastocysts at 96h (%)
70%
60%
50%
40%
30%
20%
10%
0%
Global CSC G-TL
Development of individually cultured one-cell mouse embryos to the
expanded blastocyst stage at 96 hours in four commercially available
culture media containing protein. Embryo development was
compared in 5% vs. 20% oxygen (n ¼ 60–66 embryos per media).
*P<.05 within medium type.
Morbeck. Composition of continuous culture media. Fertil Steril 2017.
cell stage. Embryos cultured in 1-Step at 5% oxygen remained
at the two-cell stage for the least amount of time of all treat-
ments (22.3  1.3 hours vs. 23.8  3.3 hours for 5% vs. 20%
oxygen, respectively; P< .05), yet spent more time as three-
cell embryos at 5% oxygen (2.6  4.5 hours vs. 0.9 
0.7 hours; P< .05). In contrast, the duration of the four-cell
stage, which was 10.3 hours for all treatments combined,
was differentially affected by oxygen in 1-Step and G-TL.
Embryos at 5% oxygen in 1-Step divided more rapidly to
the five-cell stage than in 20% (9.1  2.7 hours vs. 10.2 
1.0 hours at 20% oxygen; P< .05).
DISCUSSION
A shift in clinical practice has led to the introductions of new
single-step culture media, but formulations are not provided
by manufacturers. We present compositions of two new cul-
ture media along with two single-step media previously stud-
ied (5) and compare mouse embryo development among the
media at 5% and 20% oxygen concentrations.
Our findings confirm that Global and CSC are similar and
primarily based on simplex optimized KSOM developed by
Biggers’ group (for review, see reference [11]). In contrast,
G-TL is a nutrient-rich medium that more closely reflects
reproductive-tract composition-based formulations of the
sequential G-Series media (12, 13). The formulation of G-TL
combines features of sequential culture media developed by
Gardner. Sage 1-Step medium also combines features of its
predecessor sequential series, which we previously showed
was similar to G-Series media (5). Despite the historical sim-
ilarities between Sage and G sequential media, 1-Step is
similar to Global and CSC, although concentrations of inor-
ganic ions are different and share many characteristics with
the Sage sequential series (5).
Mouse embryos were used to test the ability of each me-
dium to support development to the blastocyst stage under
different oxygen concentrations. Global and CSC yielded
blastocyst formation rates that were not affected by oxygen
1058
concentrations. Global tended to have a lower blastocyst per-
centage (60%), likely owing to effects of higher octanoate pre-
sent within the 10 mg/mL albumin in Global Total. We
5% O2
* demonstrated that mouse embryo development is slowed by
20% O2
octanoate in a dose-dependent manner (14, 15).
and CSC), embryo development in G-TL and 1-Step was
affected by oxygen concentration. The reason for this differ-
ence may be species-specific, because the G-Series medium
was developed on the basis of concentrations of nutrients in
the human fallopian tube (12). We showed that mouse em-
bryos cultured with Sage Sequential media were also affected
by oxygen concentration, though 20% oxygen yielded lower
1-Step blastocyst development than 5% oxygen (5), opposite of what
was observed for 1-Step in the present study.
Although culture media for human embryos has been
developed using mouse embryos, one limitation of the present
study is the lack of assessment of the effect of differences in
composition directly on human embryos. We cannot account
for batch-to-batch variation, and we were unable to measure
all analytes, a problem that could be addressed in the future
using a metabolomic/proteomic approach (16).
Glucose, Pyruvate, and Lactate
Preimplantation embryos transition from a preference for py-
ruvate during the precompaction stage to glucose during the
postcompaction stage (12, 17). This reflects low glucose and
high pyruvate and lactate at midcycle in the human oviduct
(12, 18, 19) and high glucose with low pyruvate in the
uterus (12). This shift in energy metabolism presents single-
step continuous media with a challenge to meet these diverse
needs with one formulation. Manufacturers seem to have
settled on a consistent ratio of lactate to pyruvate, a ratio
that reflects the in vivo environment of the oviduct.
Although glucose is not an absolute requirement for pre-
compaction development, it is used by postcompaction em-
bryos and blastocysts. The simplex optimization process
used to develop what is now Global yielded a concentration
of 0.2 mM glucose (20, 21). Contrary to claims that glucose
inhibits embryo development, Gardner demonstrated that
addition of amino acids effectively alleviates any adverse
effects (22). These observations could underlie the rationale
to increase glucose to provide more energy for
metabolically active compacting embryos and blastocysts.
Amino Acids
The most significant change from earlier versions of sequen-
tial media is the addition of both nonessential and essential
amino acids to the single-step media (G-TL and 1-Step). G-
TL and 1-Step now have higher overall amino acid concentra-
tions than Global, but this difference may be the result of a
protein supplementation dilution effect. The protein-free
version of Global had amino acid concentrations that were
10% higher than observed in this study for Global Total,
which contains 10 mg/mL HSA (5). G-TL differs from the
other three media by increasing the osmolytic amino acids
glycine, proline, and taurine and by the absence of aspartate
VOL. 107 NO. 4 / APRIL 2017

and glutamate, two amino acids that were removed from the
G-Series media in its third generation. Replacement of gluta-
mine with a dipeptide form virtually eliminates the formation
of toxic levels of ammonium in culture (23), even though we
detected low concentrations of free glutamine in G-TL and 1-
Step. Thus, ammonium accumulation does not seem to be a
concern when performing continuous, undisturbed embryo
culture (4, 23, 24).
Calcium, Magnesium, and Potassium
The two new culture media, G-TL and 1-Step, maintain the
concentrations and ratio of Ca/Mg as is present in their prede-
cessors (G1/G2 and Sage Cleavage/Blastocyst, respectively),
concentrations and ratios that contrast to Global and CSC.
The basis for high magnesium in G-TL and the G-Series media
may be a study of tubal fluid from seven patients (1.4 mM
[25]), as well as observation that many cell types, including
hamster zygotes, benefit from low and constant calcium levels
(26, 27), with magnesium inhibiting calcium uptake and
release. The low magnesium concentration first appeared in
Quinn's HTF medium (28), on the basis of results from a
study of tubal fluid from two patients (0.19 mM [18]).
Global is unique among these four media in that it contains
the lowest magnesium concentration at 0.2 mM, which is
significantly lower than serum levels. It seems that the
concentrations of magnesium and calcium in Global reflect
the development of KSOM as it relates to Lopata et al.’s
work (18). A benefit of low magnesium may arise if media
composition favors glycolysis, because the presence of
cation chelating agents such as ethylenediaminetetraacetic
acid decreases the activity of glycolytic enzymes that are
detrimental during the cleavage stage (29). Good-quality blas-
tocyst rates for human embryos were similar (52.1% vs.
52.8%) for media that contained 0.2 or 1.2 mM magnesium,
respectively, suggesting that human oocytes and embryos
do not require low magnesium for optimal development
(30). Global and G-TL differ markedly in their approach to
the Ca/Mg ratio, with a 10-fold difference. CSC and 1-Step
provide compromises between these two extremes, though
1-Step contains the highest concentrations of both ions at a
nearly 1:1 ratio. The significance of the difference in Ca/Mg
ratios is unknown but could be critical during fertilization,
indicating that care should be taken if these media are used
for insemination/ICSI.
Potassium is unique among inorganic ions in that human
and animal oviduct fluid contains high concentrations rela-
tive to serum (25, 31, 32). Of the media studied, G-TL has
the highest potassium concentration, matching serum
levels. The original simplex optimization, which did not
optimize potassium concentrations, resulted in medium
(SOM) with a low potassium concentration (1.2 mM [20]).
Subsequent observations showed that this low potassium
concentration resulted in an abnormally low intracellular K/
Na ratio (33), prompting the modification of SOM to yield
KSOM containing 2.8 mM potassium (34). Although there
are conflicting reports of effects of potassium on embryo
development (35, 36), the evidence for providing low
potassium (<4 mM) is lacking and warrants further study.
VOL. 107 NO. 4 / APRIL 2017
Fertility and Sterility®
Clinical Significance
Unlike most medical devices, a new culture medium does not
require clinical trials as long as it is based on components that
are present in medium currently approved by the US Food and
Drug Administration. All four culture media tested here were
introduced to the market with little or no prior clinical testing,
and currently there are no full published articles with human
embryos comparing any of these media directly. The effect of
culture media composition on clinical outcomes has been re-
viewed extensively, with insufficient evidence to draw con-
clusions (6, 7), although a recent randomized, controlled
trial showed significant superiority in terms of live birth for
a complex sequential system vs. HTF, an early generation
medium that lacks amino acids (37). Only two of the
continuous media have been tested against sequential
media systems, and neither Global (38, 39) nor G-TL (4)
resulted in an improvement in live birth rates.
In summary, in this work we illustrate significant
compositional differences among four single-step, contin-
uous culture media, differences that are not supported by
published evidence or clinical trials. We used a mouse model
to demonstrate that the differences observed affect embryo
development, particularly with respect to differences in ox-
ygen concentrations. We concur with Sunde and et al. (1)
that the development, testing, and documentation of culture
media for human clinical use should be thorough and
transparent.
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VOL. 107 NO. 4 / APRIL 2017
 Fertility and Sterility®

SUPPLEMENTAL FIGURE 1

A 90
Global
B 90
CSC
80 80

70 70

60 60
**
Time in Culture (h)

Time in Culture (h)

*
50 50
5% 5%
*
40 20% 40
20%

30 30

20 20

10 10

0 0
2C 3C 4C 5C 8C Blastocyst 2C 3C 4C 5C 8C Blastocyst
(tSB) (tSB)

C 90 D 90
G-TL 1-Step
80 80

70 70

60 ** 60
Time in Culture (h)

Time in Culture (h)

***
50 50

5% * 5%
40 40
20% 20%

30 30

20 20

10 10

0 0
2C 3C 4C 5C 8C Blastocyst 2C 3C 4C 5C 8C Blastocyst
(tSB) (tSB)

Cell division timings for embryos cultured in four different single-step culture media at both 5% and 20% oxygen. (A) Global, (B) CSC, (C) G-TL, (D)
1-Step. *P<.05, **P<.01, ***P<.001.
Morbeck. Composition of continuous culture media. Fertil Steril 2017.

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