Composition of commercial media used for human embryo culture Dean E. Morbeck, Ph.D.,a,b Rebecca L. Krisher, Ph.D.,c Jason R. Herrick, Ph.D.,c Nikola A. Baumann, Ph.D.,b Dietrich Matern, M.D., Ph.D.,b and Thomas Moyer, Ph.D.b a Department of Obstetrics and Gynecology and b Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota; and c National Foundation for Fertility Research, Lone Tree, Colorado

Objective: To determine the composition of commercially available culture media and test whether differences in composition are biologically relevant in a murine model. Design: Experimental laboratory study. Setting: University-based laboratory. Animal(s): Cryopreserved hybrid mouse one-cell embryos were used in experiments. Intervention(s): Amino acid, organic acid, ions, and metal content were determined for two different lots of media from Cook, In Vitro Care, Origio, Sage, Vitrolife, Irvine CSC, and Global. To determine whether differences in the composition of these media are biologically relevant, mouse one-cell embryos were thawed and cultured for 120 hours in each culture media at 5% and 20% oxygen in the presence or absence of protein in an EmbryoScope time-lapse incubator. Main Outcome Measure(s): The compositions of seven culture media were analyzed for concentrations of 39 individual amino acids, organic acids, ions, and elements. Blastocyst rates and cell cycle timings were calculated at 96 hours of culture, and the experiments were repeated in triplicate. Result(s): Of the 39 analytes, concentrations of glucose, lactate, pyruvate, amino acids, phosphate, calcium, and magnesium were present in variable concentrations, likely reflecting differences in the interpretation of animal studies. Essential trace elements, such as copper and zinc, were not detected. Mouse embryos failed to develop in one culture medium and were differentially affected by oxygen in two other media. Conclusion(s): Culture media composition varies widely, with differences in pyruvate, lactate, and amino acids especially notable. Blastocyst development was culture media dependent and Use your smartphone showed an interaction with oxygen concentration and presence of protein. (Fertil SterilÒ to scan this QR code 2014;102:759–66. Ó2014 by American Society for Reproductive Medicine.) and connect to the Key Words: Culture media, quality control, mouse embryo assay, embryo culture Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/morbeckd-commercial-media-composition/

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umans conceived in vitro have an increased incidence of low birth weight (1), preterm birth, and perinatal complications (2). Low birth weight is associated with increased risk for type 2 diabetes (3), hypertension, and cardiovascular disease (4). The underlying etiology of low birth weight in children conceived in vitro remains unclear, obscured by multiple factors, including infertility,

increased incidence of multiple births, and genetic background (5). Thus, the popularity of assisted reproductive technology (ART) and the potential for health complications compels further investigations into which aspects of the in vitro culture environment are most influential. In vivo, the composition of the early embryo's environment (oviductal and uterine fluids) is largely

Received April 12, 2014; revised May 16, 2014; accepted May 27, 2014; published online July 4, 2014. D.E.M. has nothing to disclose. R.L.K. has nothing to disclose. J.R.H. has nothing to disclose. N.A.B. has nothing to disclose. D.M. has nothing to disclose. T.M. has nothing to disclose. Supported by a grant from the Mayo Clinic Department of Laboratory Medicine and Pathology and an equipment loan (EmbryoScope) from Unisense Fertilitech. Reprint requests: Dean E. Morbeck, Ph.D., Department of Obstetrics and Gynecology, Mayo Clinic, Charlton 3A, Rochester, Minnesota 55905 (E-mail: [email protected]). Fertility and Sterility® Vol. 102, No. 3, September 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.05.043 VOL. 102 NO. 3 / SEPTEMBER 2014

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determined by the composition of the maternal diet (6). In contrast, the environment of an IVF embryo is the culture medium. Epidemiologic studies demonstrating the effects of maternal nutrition on birth weight and risks for metabolic and cardiovascular disease (7) support the hypothesis that the composition of culture media may affect offspring health (8, 9). Although reports on the association between culture media and birth weight in humans are conflicting (10, 11), studies in mammalian animal models demonstrate strong relationships between media composition and multiple aspects of embryonic, fetal, and offspring health (12). Types and concentrations of nutrients provided to preimplantation 759

ORIGINAL ARTICLE: ASSISTED REPRODUCTION embryos vary among brands of culture media, but the only information provided by manufacturers is a list of ingredients. Unless detailed descriptions of the composition of these media are disclosed, it is impossible to determine which components might be responsible for differences in preimplantation development or birth weight, or to generate hypotheses for future study. To address this gap in available information, we performed a systematic analysis of the composition of culture media from seven suppliers. We then determined whether observed differences in media composition were biologically relevant for blastocyst development using mouse embryos, the standard model used for development of media used for human embryo culture. Two components of culture that introduce additional sources of variation are oxygen and protein. Because oxygen is a metabolic nutrient, and the amount of oxygen used varies in clinical practice, we compared mouse embryo development at both reduced (5%) and ambient (20%) oxygen to determine whether there were any media  oxygen interactions. Studies were also conducted with and without protein supplementation, because protein supplements, which are highly heterogeneous (13), perform many nonspecific functions and carry many undefined components that may mask differences in culture media.

MATERIALS AND METHODS Culture Media Analysis Culture media sources. Culture media (two lots per medium) were purchased from seven suppliers and analyzed. Two single-step (‘‘Global,’’ LifeGlobal, IVFOnline; CSC [‘‘Irvine’’], Irvine Scientific) and five sequential (Sydney IVF Cleavage and Blastocyst Media [‘‘Cook’’], Cook Medical; IVC1 and IVC3 [‘‘IVC’’], In Vitro Care; ISM1 and BlastAssist [‘‘Origio’’], Origio; Sage Quinn's Advantage Cleavage and Quinn's Advantage Blastocyst Medium [‘‘Sage’’], Cooper Surgical; and G1v5 and G2v5 [‘‘Vitrolife’’], Vitrolife) media were analyzed. All media were protein-free, except Cook and Origio, which are only available with human serum albumin (HSA). Amino acid and organic acid analysis. Amino acids (AAs) and organic acids were quantified in media using established methods for liquid chromatography–tandem mass spectrometry and gas chromatography–mass spectrometry, respectively (14, 15). Elemental analysis. Calcium (colorimetric o-cresolphthalein complexone), chloride (ion selective electrode [ISE]), potassium (ISE module), magnesium (colorimetric), sodium (indirect ISE), and phosphorus (photometric) were quantified using Roche Cobas chemistry analyzers (Cobas 6000 c501 or Cobas 8000 ISE, c701, c502 modules) and Roche Cobas reagents. Samples were serially diluted, and analyte recovery after dilution was verified. In samples in which the analyte concentration was at the low end of the analytic measuring range (magnesium, phosphorus), spiked recovery of analyte was performed to verify accurate measurement in the sample matrix. Samples for trace metal analyses (aluminum, chromium, cobalt, copper, iron, manganese, selenium, and zinc) were collected in polyethylene containers demonstrated to be free of metal contamination. 760

Quantification was performed by inductively coupled plasma mass spectrometry on samples diluted in 1% ultra-pure nitric acid. Calibration was performed in the same matrix using reference materials acquired from Venture Analytics. Other analytes. Glucose (hexokinase) was quantified using Roche Cobas chemistry analyzers (Cobas 6000 c501 or Cobas 8000 ISE, c701, c502 modules) and Roche Cobas reagents. Insulin was measured with a Roche Cobas e immunoassay analyzer. Lactate was assayed by lactate oxidase/peroxidase (dry slide chemistry) on a Vitros 350 chemistry analyzer (Ortho Clinical Diagnostics). Samples were serially diluted, and analyte recovery after dilution was verified. In samples in which the analyte concentration was at the low end of the analytic measuring range (glucose, insulin), spiked recovery of analyte was performed to verify accurate measurement in the sample matrix.

Mouse Embryo Assay Cryopreserved one-cell in vivo–fertilized mouse embryos from F1 hybrid mice (bcl/B6  B6/bcl) were obtained from Embryotech Laboratories. After thawing (survival rate >98%), embryos were transferred to 25 mL of media in individual wells in an EmbryoSlide (Unisense Fertilitech), and each slide was inserted into an EmbryoScope (Unisense Fertilitech). Two sets of media were not available without protein, and it is known that protein can obscure the nature of culture media; therefore, SICM/SIBM and ISM1/BA were included in the study but not included in the statistical analysis of developmental data. Thus a 5  2  2 factorial study was performed with five culture media (Global, Irvine, IVC, Sage, and Vitrolife) with or without protein (5 mg/mL HSA; IVFOnline), and cultured at 20% or 5% oxygen. All experiments were performed in triplicate at 37 C and 6.2% CO2 (pH 7.2– 7.3 for each media), with 10 to 11 embryos for each media/ condition combination. Expanded blastocyst at 96 hours of culture was the primary endpoint. Data for precise cell division timings using time-lapse imaging were also obtained and are presented in the online supplement.

Data and Statistical Analysis Developmental and time-lapse data were analyzed using a one-way analysis of variance (ANOVA) with Tukey's test for pair-wise comparisons of blastocyst development. Analyses were performed in three steps. First, a 2  7 factorial ANOVA was used to compare blastocyst development and timings for all seven media containing protein at low and high oxygen. Second, a 2  2  5 factorial ANOVA was used to compare blastocyst development for the five media available without protein, tested with and without protein at low and high oxygen. Statistical analyses were performed using JMP statistical software (SAS Institute).

RESULTS Media Composition Because Cook and Origio were not available without protein, data from these media are presented despite the potential presence of contaminants from the protein. In all of the VOL. 102 NO. 3 / SEPTEMBER 2014

Fertility and Sterility® protein-free media analyzed the reagents listed agreed with the reagents found, with the exception of the Sage media series. In both of the Sage media analyzed, aluminum, iron, and manganese were found but are not listed as media components. Two lots of each media were independently analyzed; results of one lot are presented in Tables 1–3 and the second lot in Supplemental Tables 1–3 (available online). Media composition was consistent between the two lots tested. Spiked recovery of analyte into media was performed to confirm that there were no matrix effects caused by measuring analyte in a nonserum matrix. Mean recoveries of spiked analytes were between 99% and 106%, indicating that analyte could be accurately measured in the medium matrix. For analytes with concentrations within the analytical measuring range, serial dilution of samples also demonstrated acceptable recovery.

metals. An exception was the presence of aluminum and manganese in Sage. The only media containing a physiologic iron concentration was Origio BlastAssist (57 mg/L); all other media were iron deficient. None of the media contained physiologically significant concentrations of chromium, cobalt, copper, selenium, and zinc (Table 3). Insulin. Most notably, Origio BlastAssist contained 7885 mIU/mL insulin. The only other media in which insulin was detected was the Cook media series (0.01 mIU/mL).

Embryo Development To determine whether differences in media composition were biologically relevant, we cultured mouse zygotes and used expanded blastocyst formation at 96 hours as a primary endpoint and cell cycle timings as secondary measures (cell cycle timings are presented in the Supplemental Material, Supplemental Fig. 1, and Supplemental Tables 4 and 5). Of the two media only available with protein, Cook resulted in embryo development to blastocyst that was similar to embryos in Global, Irvine, and Vitrolife, with protein in both 5% and 20% O2 (74.2%  8.0% and 83.9%  6.7%, respectively). In contrast, most embryos cultured in Origio in either O2 concentration arrested at the two- to four-cell stage, and only 5% developed to expanded blastocyst. Because oxygen concentrations during embryo culture and type of protein supplementation vary among IVF clinics and because protein is a known chelator and antioxidant, we compared mouse embryo development with and without protein in 5% and 20% oxygen in a 2  2  5 factorial model. Culture media alone was significant for the model, and overall significantly fewer embryos developed to the blastocyst stage in Sage and IVC (P< .05; Fig. 1A). As independent variables, protein (P¼ .06) and oxygen (P¼ .66) alone did not affect blastocyst formation; however, the media  oxygen interaction (Fig. 1B) and the media  protein interaction (Fig. 1C) were significant for expanded blastocyst formation in the 2  2  5 factorial model. Embryo development to the blastocyst stage was similar for Global, Irvine, and Vitrolife and was not affected by presence of protein or O2 concentration. There was an interaction of media with protein and oxygen for both Sage and IVC. Whereas embryos in Sage developed to blastocysts similar to Global, Irvine, and Vitrolife at 5% O2, development to the blastocyst stage was compromised at 20% O2 (Fig. 1B). In contrast, embryos in IVC in 20% O2 developed

Glucose and organic acids. Glucose was present in all media except IVC and ranged from 0.1 to 1.0 mM for single-step or cleavage-stage media, and from 1.0 to 3.2 mM for blastocyststage media (Table 1). Citrate was present in low concentrations (0.003 to 0.16 mM) in just over half of the media analyzed (Table 1). Lactate and pyruvate, two primary energy substrates for early embryos, varied considerably among single-step, cleavage-stage, and blastocyst-stage media (Table 1). The ratio of lactate to pyruvate (L:P) ranged from 5 to 126 for cleavage-stage media and from 1.2 to 105 for blastocyst-stage media. Single-step media (Global, Irvine) had similar L:P ratios of 24 and 33, respectively. Amino acids. Glutamine, a labile AA, is supplied in all media except Origio ISM1 as a dipeptide (alanyl- or glycylglutamine) and was not quantified in this analysis owing to method limitations. Concentrations of most AAs were similar among media types, with a few notable exceptions (Table 2). For instance, Cook had 30-fold higher concentrations of taurine and glycine than found in the other media, and Origio ISM1, in addition to containing glutamine, contained both nonessential and essential AAs and increased glycine. The two single-step media, Global and Irvine, had very similar concentrations of all AAs tested. Elements. Concentrations of Cl and Na were similar among media, but Ca (1.1–2.2 mM), K (2.8–8.4 mM), Mg (0.2– 2.0 mM), and P (0–1.1 mM) were markedly different (Table 3). Trace metals were only present in media presupplemented with protein, a known carrier of trace and heavy

TABLE 1 Concentrations of glucose, organic acids, and the lactate/pyruvate ratio in embryo culture media. IVFOnline

Irvine

Vitrolife

Sage

Cook

In Vitro Care

Origio

Variable

Global

CSC

G1

G2

QACM

QABM

SICM

SIBM

IVC1

IVC3

ISM1

BA

Glucose (mM) Citrate (mM) Lactate (mM) Pyruvate (mM) L:P ratio

0.2 0 4.8 0.20 24

0.5 0.01 5.6 0.17 33

0.5 0.08 10.8 0.30 36

3.4 0.08 6.0 0.07 86

0.1 0 3.9 0.52 7.5

2.8 0.16 3.9 0.07 56

0.3 0 1.8 0.36 5.0

3.1 0 1.8 0.31 5.9

0 0 10.1 0.08 126

2.7 0.16 9.4 0.09 105

1.0 0.02 3.2 2.0 18.5

1.0 0.003 2.4 0.17 1.2

Note: QACM ¼ Quinn's Advantage Cleavage media; QABM ¼ Quinn's Advantage Blastocyst media; SICM ¼ Sydney IVF Cleavage media; SIBM ¼ Sydney IVF Blastocyst media; BA ¼ Blastassist. Morbeck. Composition of embryo culture media. Fertil Steril 2014.

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TABLE 2 Amino acid concentrations in embryo culture media (mM). IVFOnline

Irvine

Global

CSC

G1

G2

328 52 111 221 230 232 51 112 216 28 100 233

281 46 105 202 214 223 53 106 195 26 95 215

0 0 0 0 0 0 0 0 0 0 0 0

360 54 121 249 265 260 63 125 242 30 114 256

0 0 0 0 0 0 0 0 0 0 0 0

65 52 55 54 0 63 51 58 0

62 57 47 46 0 58 48 55 0

148 126 0 0 0 135 112 127 131

151 129 0 0 0 141 113 130 0

0 112 93 0 0 119 93 107 122

Variable Essential Arg Cys His Ile Leu Lys Met Phe Thr Trp Tyr Val Nonessential Ala Asn Asp Glu Gln Gly Pro Ser Tau

Vitrolife

Sage QACM

Cook

QABM

In Vitro Care

Origio

SICM

SIBM

IVC1

IVC3

ISM1

BA

313 54 102 209 227 223 56 106 210 28 100 224

25 2 8 17 18 18 4 8 18 2 12 17

252 32 86 169 182 174 43 86 172 22 114 179

0 0 0 0 0 0 0 0 0 0 0 0

590 9 188 388 408 417 100 200 374 51 186 428

138 42 99 147 158 148 89 90 81 100 70 356

124 38 54 208 217 179 54 104 211 21 91 225

0 124 104 0 0 131 103 123 120

135 88 81 90 30 6647 85 92 6489

135 84 85 87 26 4815 80 89 6380

0 0 0 0 0 0 0 0 0

136 113 95 103 0 121 99 109 0

338 73 6 1 778 1760 82 96 296

124 104 578 102 0 701 96 113 0

Note: Abbreviations as in Table 1. Morbeck. Composition of embryo culture media. Fertil Steril 2014.

highlight the need to understand what we are ‘‘feeding’’ preimplantation embryos. Human embryos are not available in sufficient numbers to conduct the multifactorial experiments necessary to determine specific substrate preferences or optimize culture conditions. Instead, data from murine embryo research have been extrapolated to humans, such that commercially available media for human embryos are almost exclusively proprietary modifications of media originally designed for mouse embryos (17–19). Similarly, studies of the effects of culture conditions on the

to blastocysts with similar success to other media in 20% O2, whereas blastocyst development was lower in 5% O2. Addition of protein had little effect on blastocyst development for all media except IVC and Sage, for which lack of protein resulted in significantly fewer blastocysts (Fig. 1C).

DISCUSSION Conflicting reports of culture media–induced differences in birth weight of infants conceived using ART (8–11, 16)

TABLE 3 Electrolytes and metals in embryo culture media. IVFOnline

Irvine

Vitrolife

Sage

Cook

In Vitro Care

Origio

Variable

Global

CSC

G1

G2

QACM

QABM

SICM

SIBM

IVC1

IVC3

ISM1

BA

Calcium (mM) Phosphorus (mM) Potassium (mM) Chloride (mM) Sodium (mM) Magnesium (mM) Copper (mg/L) Iron (mg/L) Selenium (mg/L) Zinc (mg/L) Aluminum (mg/L) Chromium (mg/L) Cobalt (mg/L) Manganese (mg/L)

1.9 0.4 2.9 112 137 0.2 0 0 0 0 1 0.4 0.1 0.4

1.9 0.2 2.8 112 135 0.8 0 4 0 0 0 0.7 0.2 0.8

1.1 0.3 5.8 124 152 1.7 0 0 0 0 1 0.1 0.3 0.5

1.1 0.3 5.8 127 149 1.7 0 3 0 0 1 0.2 0.2 0.6

2.2 0 4.7 118 132 1.8 0 1 0 0 18 0.2 0.2 3.6

2.2 0.3 4.9 113 132 1.8 0 2 0 0 21 0.2 0.2 3.6

1.1 0.2 4.8 122 140 1.5 0 11 3 0 1 0.3 0.3 0.5

1.1 0.4 5.0 121 136 1.5 0 9 3 0 1 0.3 0.2 0.5

2.1 0 4.6 107 142 0.2 0 5 0 0 2 0.7 0.2 0.6

1.9 0.3 4.9 108 143 0.2 0 5 0 0 6 0.9 0.2 0.6

1.8 0.3 8.4 116 131 0.9 0 9 4 0 5 0.2 0.5 1.4

1.4 1.1 5.3 114 144 0.8 0 57 4 0 16 1.2 0.6 0.8

Note: Abbreviations as in Table 1. Morbeck. Composition of embryo culture media. Fertil Steril 2014.

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FIGURE 1

Morbeck. Composition of embryo culture media. Fertil Steril 2014.

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FIGURE 1 Continued Development of individually cultured one-cell mouse embryos to the expanded blastocyst stage at 96 hours in commercially available culture media. Analysis of (A) all experimental treatments combined (with and without protein at 5% and 20% oxygen, n ¼ 120–132 embryos per media), (B) comparison of 5% vs. 20% oxygen (n ¼ 60–66 embryos per media), and (C) comparison of culture with or without protein (n ¼ 60–66 embryos per media). Values with different letters are statistically different, P

Composition of commercial media used for human embryo culture.

To determine the composition of commercially available culture media and test whether differences in composition are biologically relevant in a murine...
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