Unconditioned commercial embryo culture media contain a large variety of non-declared proteins: a comprehensive proteomics analysis

Human Reproduction, Vol.29, No.11 pp. 2421– 2430, 2014 Advanced Access publication on August 27, 2014 doi:10.1093/humrep/deu220

ORIGINAL ARTICLE Embryology

Unconditioned commercial embryo culture media contain a large variety of non-declared proteins: a comprehensive proteomics analysis

1

Department of Molecular Biology and Genetics, Aarhus University, Aarhus C., Denmark 2The Fertility Clinic, Aarhus University Hospital, Aarhus N, Denmark 3Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark 4Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus C., Denmark *Correspondence address. Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C., Denmark. E-mail: [email protected]

Submitted on February 28, 2014; resubmitted on July 18, 2014; accepted on July 30, 2014

study question: Which non-declared proteins (proteins not listed on the composition list of the product data sheet) are present in unconditioned commercial embryo culture media? summary answer: A total of 110 non-declared proteins were identified in unconditioned media and between 6 and 8 of these were quantifiable and therefore represent the majority of the total protein in the media samples. what is known already: There are no data in the literature on what non-declared proteins are present in unconditioned (fresh media in which no embryos have been cultured) commercial embryo media. study design, size, duration: The following eight commercial embryo culture media were included in this study: G-1 PLUS and G-2 PLUS G5 Series from Vitrolife, Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium from Cook Medical and EmbryoAssist, BlastAssist, Sequential Cleav and Sequential Blast from ORIGIO. Two batches were analyzed from each of the Sydney IVF media and one batch from each of the other media. All embryo culture media are supplemented by the manufacturers with purified human serum albumin (HSA 5 mg/ml). The purified HSA (HSA-solution from Vitrolife) and the recombinant human albumin supplement (G-MM from Vitrolife) were also analyzed. participants/materials, setting, methods: For protein quantification, media samples were in-solution digested with trypsin and analyzed by liquid chromatography– tandem mass spectrometry (LC–MS/MS). For in-depth protein identification, media were albumin depleted, dialyzed and concentrated before sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gel was cut into 14 slices followed by in-gel trypsin digestion, and analysis by LC –MS/MS. Proteins were further investigated using gene ontology (GO) terms analysis.

main results and the role of chance: Using advanced mass spectrometry and high confidence criteria for accepting proteins (P , 0.01), a total of 110 proteins other than HSA were identified. The average HSA content was found to be 94% (92–97%) of total protein. Other individual proteins accounted for up to 4.7% of the total protein. Analysis of purified HSA strongly suggests that these non-declared proteins are introduced to the media when the albumin is added. GO analysis showed that many of these proteins have roles in defence pathways, for example 18 were associated with the innate immune response and 17 with inflammatory responses. Eight proteins have been reported previously as secreted embryo proteins.

limitations, reasons for caution: For six of the commercial embryo culture media only one batch was analyzed. However, this does not affect the overall conclusions.

wider implications of the findings: The results showed that the HSA added to IVF media contained many other proteins and that the amount varies from batch to batch. These variations in protein profiles are problematic when attempting to identify proteins derived from the embryos. Therefore, when studying the embryo secretome and analyzing conditioned media with the aim of finding potential biomarkers that can distinguish normal and abnormal embryo development, it is important that the medium used in the experimental and control groups is from & The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]

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Thomas F. Dyrlund 1,*, Kirstine Kirkegaard 2,3, Ebbe Toftgaard Poulsen 1, Kristian W. Sanggaard1,4, Johnny J. Hindkjær2, Jørgen Kjems 1,4, Jan J. Enghild 1,4, and Hans Jakob Ingerslev 2

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the same batch. Furthermore, the proteins present in unconditioned media could potentially influence embryonic development, gestation age, birthweight and perhaps have subsequent effects on health of the offspring.

study funding/competing interest(s): The study was supported by the Danish Agency for Science, Technology and Innovation. Research at the Fertility Clinic, Aarhus University Hospital is supported by an unrestricted grant from Merck Sharp & Dohme Corp and Ferring. The authors declare no conflicts of interest. Key words: IVF / culture media / embryo / secretome / proteomics

Introduction

Materials and Methods Media G-1 PLUS (Lot: 504328), G-2 PLUS (Lot: 504332), G-MM (Lot: 017087) and HSA-solution (Lot: 017077) were all from the G5 Series from Vitrolife. Sydney IVF Cleavage Medium (Lot: K11935 and 512913) and Sydney IVF Blastocyst Medium (Lot: K11927 and 512892) were from Cook Medical. EmbryoAssist (Lot: 13397875), BlastAssist (Lot: 13500268), Sequential Cleav (Lot: 13440675) and Sequential Blast (Lot: 13420273) were from ORIGIO.

In-solution digestion A total of 100 mg total protein of each media sample was lyophilized and re-dissolved in 25 ml 8 M urea 0.2 M Tris – HCl, pH 8.3, reduced and alkylated by adding 15 mM dithiothreitol (DTT) followed by the addition of 30 mM iodoacetamide. Both reactions were allowed to continue for 1 h. The samples were subsequently diluted five times with 0.1 M Tris– HCl pH 8.3 and digested using 1:50 w/w trypsin (Sigma) at 378C for 16 h. From each sample, 20 mg was desalted using Poros 50 R2 reverse phase column material (PerSeptive Biosystems) packed in GELoader Tips (Eppendorf), dried in a vacuum centrifuge, dissolved in 0.1% formic acid and stored at 48C before liquid chromatography– tandem mass spectrometry (LC – MS/ MS) analysis.

Albumin depletion A total of 5 ml (25 mg serum albumin) Sydney IVF Cleavage Medium (Cook Medical, Lot: K11935) or 0.5 ml (25 mg serum albumin) G-MM recombinant human serum albumin (HSA) supplement (Vitrolife, Lot: 017087) was diluted four times in phosphate-buffered saline, 5 mM ethylenediaminetetraacetic acid, pH 7.4 (loading buffer) and applied to a 1 ml column containing the recombinant albumin-binding domain of streptococcal protein G (Kronvall et al., 1979) equilibrated in loading buffer. To prevent exceeding the albumin-binding capacity of the column (30 mg/ml), each diluted medium sample was divided in five fractions and each fraction was depleted separately. The flow through was collected for each run, and the column regenerated in between using wash buffer (20 mM Na-citrate, 150 mM NaCl, pH 2.5). Flow through for all runs were pooled for each media and dialyzed overnight into 10 mM Tris – HCl, pH 7.4. The depleted and dialyzed media was then concentrated using a 15 ml Centriprep 10 K centrifugal filter (Merck Millipore

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Infertility is a growing problem worldwide and in some countries up to 4.5% of a cohort of children are conceived with the help of fertility treatments (Ferraretti et al., 2013). Since only 25–30% of human embryos implant, the selection of which embryo (or embryos) to be transferred to the uterus is a critical step in IVF treatments in order to maximize the probability of pregnancy. Today, the most common method to assess the developmental and viability potential of embryos is the assessment of embryo morphology at discrete time points, a method that has several limitations. Some of these limitations, in particular the restricted, time-dependent observations and potentially subjective classification, have been overcome with the introduction of time-lapse photography (Meseguer et al., 2011; Sundvall et al., 2013). Nevertheless, the expected benefits of morpho-kinetic analysis in terms of improved pregnancy rate have yet to be documented (Kirkegaard et al., 2013). Furthermore, methods that evaluate non-morphological parameters may be expected to contribute not only with additional selection criteria but also with a knowledge of other more biochemical aspects of embryo physiology and development. It is believed that selection based on both morphological assessment and non-invasive analysis may improve the success rates of IVF and facilitate single embryo transfers (Katz-Jaffe et al., 2009). Recent strategies have involved the measurement of embryonic metabolic activity such as amino acid turnover (Houghton et al., 2002), pyruvate and glucose uptake (Gardner et al., 2001, 2011), oxygen consumption (Lopes et al., 2007; Tejera et al., 2012) and the turnover of the entire metabolome (Seli et al., 2010; Hardarson et al., 2012; Vergouw et al., 2012). However, none of these strategies has resulted in clinically applicable methods. Analysis of the proteins produced and secreted by embryos throughout embryo development has been proposed to reflect the embryo’s viability (Katz-Jaffe et al., 2009). Consequently, a deeper knowledge of which proteins are secreted by the embryo may lead to a more comprehensive assessment of embryos during their preimplantation development and aid in the selection of the most viable embryos during IVF treatments. However, the identity of the proteins secreted by human embryos is largely unknown and, despite intense and promising research, results remain conflicting and no clinical validation has been performed. This is attributed to the limited starting material and technological limitations. An important source of error may be if proteins are present in the unconditioned culture media (fresh media in which no embryos have been cultured), which could lead to misinterpretation when identifying secreted proteins. If proteins are present in the media before culture, such information should necessarily be employed as a reference in the attempt to identify proteins secreted or metabolized by human embryos. Furthermore, embryo toxic factors have been identified that result in poor embryo quality and impaired developmental growth

(Dokras et al., 1993; Groebe et al., 2010). The safety of culture media therefore makes it important to know the complete composition of the embryo culture media, and it would be advisable to test all factors for their positive and negative effects on embryo development and health effects for the offspring. The purpose of the present study was to identify and quantify non-declared proteins present in media used for human embryo culture in order to provide a protein reference set.

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Ltd) to a volume of 0.5 ml before being lyophilized using a speedvac (Savant) to a final volume of 50 ml.

Protein separation by SDS – PAGE and in-gel digestion

LC –MS/MS analysis LC – MS/MS analyses were performed on an EASY-nLC II system (Thermo Scientific) connected to a TripleTOF 5600+ mass spectrometer (AB Sciex) equipped with a NanoSpray III source (AB Sciex) and operated under Analyst TF 1.6.0 control. The samples were injected, trapped and desalted isocratically on a Biosphere C18 precolumn (ID 100 mm × 2 cm, 5 mm, 120 A˚, NanoSeparation, Nieuwkoop, Netherlands). Next, the peptides were eluted from the trap column and separated on a 15 cm analytical column (75 mm i.d.), which was pulled in-house on Polymicro silica tubing by a P2000 laser puller (Sutter Instrument Co.), and packed in-house with ReproSil-Pur C18-AQ 3 mm resin (Dr Marisch GmbH, AmmerbuchEntringen, Germany). Peptides were eluted at a flow rate of 250 nl/min using a 50 min gradient from 5 to 35% phase B (0.1% formic acid and 90% acetonitrile), followed by re-equilibration for 10 min back to the starting conditions.

Protein identification The collected MS files were converted to Mascot generic format (MGF) using the AB SCIEX MS Data Converter beta 1.3 (AB SCIEX) and the ‘proteinpilot MGF’ parameters. The peak lists were used to interrogate the Swiss-Prot (version 2014_01, 542 258 sequences) Homo sapiens (20 273 sequences) database or the Swiss-Prot Fungi (31 056 sequences) database using Mascot 2.3.02 (Matrix Science) (Perkins et al., 1999). Trypsin was employed as enzyme allowing one missed cleavage. Propionamide was entered as a fixed modification for in-gel digests and carbamidomethyl for in-solution digests, whereas oxidation of methionine, and hydroxylation of proline were entered as variable modifications. The mass accuracy of the precursor and product ions were 10 ppm and 0.2 da, and the instrument setting was specified as ESI-QUAD-TOF. The significance threshold ( p) was set at 0.01 and the ion score expect cut-off at 0.005. Mascot results were parsed using MS Data Miner v. 1.2.2 (Dyrlund et al., 2012) accepting only protein hits identified based on three or more unique peptides with expected ion values equal to or lower than 0.005. All keratin contaminants were removed from the final protein lists. MS Data Miner was further used to extract Gene Ontology (GO) information for all identified proteins. Note that since proteins are annotated with several GO terms, they can be represented in multiple GO summary categories.

Proteins were quantified based on the average MS signal response for the three most intense tryptic peptides for each protein and expressed as percentage of total protein (Silva et al., 2006). In short, the MS files were searched as listed above and the Mascot .dat result files were used to generate a spectral library in Skyline v. 2.1.0.4936 (MacLean et al., 2010) using the human sequences from Swiss-Prot as background proteome. Employing the same parameters as for the Mascot search, the three most abundant peptides for each protein were manually chosen from all peptides available in the spectral library. After data import, the chromatographic traces (extracted ion chromatograms) were manually inspected and adjusted where needed to correct wrongfully assigned peaks. Proteins identified with ,3 peptides were not included in the quantification. The relative abundance and standard deviation of proteins quantified in all four of the technical replicates was calculated as the average MS intensity for the three peptides for each protein divided by the sum of the average signal for all quantified proteins in the sample.

Results To examine the extent of non-declared proteins present in media used for human embryo culture, eight different embryo culture media from three different suppliers were collected and the most abundant proteins present in the media were quantified using MS. Other than HSA, between 6 and 8 proteins were quantifiable in all media samples, with relative protein amounts ranging from 0.01 to 4.7% (Table I). The average amount of HSA present in the samples was 94.5%. Furthermore, to examine batch-to-batch variations in protein amounts, two different batches of the Sydney IVF Cleavage Medium and two of the Sydney IVF Blastocyst Medium were included in the analysis. Relative protein amounts were observed to differ with up to 1-fold indicating batch-to-batch variation (Table I). In the purified HSA solution (HSA-solution), 18 proteins were quantifiable. These included all the proteins quantified in the other embryo culture media as well as attractin and thyroxine-binding globulin. In order to identify low abundance proteins present in the medium, 5 ml Sydney IVF Cleavage Medium was albumin depleted before protein separation by SDS–PAGE and identification by LC–MS/MS analysis (Supplementary data, Fig. S1). Using high confidence criteria, a total of 111 proteins were identified (Table II). A GO annotation analysis was performed on the proteins based on a series of summary categories (GO slim categories), including molecular function and biological processes. Based on biological process, the proteins grouped into three major groups: inflammatory response, innate immune response and response to peptide hormone stimulus. For the molecular function, five large groups were identified: enzyme inhibitor activity, ion binding, peptidase activity, receptor activity and transporter activity (to access these data, see below). Finally, 0.5 ml (25 mg total protein) recombinant human albumin supplement, G-MM from Vitrolife, was albumin depleted and analyzed as above. No human or yeast proteins were identified in the supplement media except for HSA. The MS proteomics data and further MS data have been deposited with the ProteomeXchange Consortium (http://proteomecentral .proteomexchange.org) via the PRIDE partner repository (Vizcaino et al., 2013) with the dataset identifier PXD001174.

Discussion This study demonstrates the presence of 110 proteins other than HSA in unconditioned media where only human serum albumin is declared to be

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The albumin-depleted samples were boiled for 5 min in sodium dodecyl sulfate (SDS) sample buffer containing 50 mM DTT, separated by SDS polyacrylamide gel electrophoresis (SDS – PAGE) using a 5 – 15% (w/v) gel (Bury, 1981) and stained using Coomassie blue (RAPIDstain, G-Biosciences). The gel was cut into 14 slices, and these were washed three times in milli-Q water and incubated two times 15 min in 130 ml 50% acetonitrile, dehydrated in 130 ml acetonitrile for 15 min and equilibrated in 150 ml 0.1 M NH4HCO3 for 5 min before 150 ml acetonitrile was added. After 15 min the supernatants were removed and the gel pieces were lyophilized for 20 min. In-gel digestions were performed by digesting the gel pieces with trypsin (Sigma) using a 1:50 (w/w) ratio in 50 mM NH4HCO3 at 378C for 16 h. The resulting peptides were desalted using Poros 50 R2 reverse phase column material (PerSeptive Biosystems) packed in GELoader Tips (Eppendorf), dried in a vacuum centrifuge, dissolved in 0.1% formic acid and stored at 48C before LC – MS/MS analysis.

Protein quantification

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Table I Human proteins quantified in embryo culture medium. Accession

Protein name

Lot #:

Vitrolife

Cook medical (Sydney IVF)

............................................... ..................................................................

ORIGIO

............................................................................

HSA Solution

G-1 Plus

G-2 Plus

Cleavage

Blastocyst

Cleavage L2

Blastocyst L2

EmbryoAssist

BlastAssist

Sequential Cleav

Sequential Blast

017077

504328

504332

512913

512892

K11935

K11927

13397875

13500268

13440675

13420273

.......................................................................................................................................................................................................................................................... P02768

Serum albumin*

96.8 + 0.9

92.0 + 1.4

96.7 + 1.0

94.2 + 1.4

96.5 + 0.6

94.5 + 0.5

95.3 + 0.8

95.6 + 0.3

92.9 + 1.1

94.5 + 1.3

92.4 + 0.6

P00738

Haptoglobin*

1.5 + 0.3

1.5 + 0.3

1.0 + 0.3

2.3 + 0.8

1.0 + 0.2

2.1 + 0.4

2.0 + 0.3

1.6 + 0.1

2.5 + 0.5

2.2 + 0.7

2.4 + 0.2

P02790

Hemopexin

0.6 + 0.3

4.7 + 1.9

1.4 + 0.5

1.5 + 0.4

1.1 + 0.2

1.6 + 0.4

1.2 + 0.2

1.5 + 0.1

3.0 + 0.8

1.4 + 0.3

3.3 + 0.8

P43652

Afamin*

0.3 + 0.03

0.8 + 0.5

0.3 + 0.04

0.4 + 0.1

0.5 + 0.1

0.7 + 0.2

0.7 + 0.3

0.7 + 0.2

0.4 + 0.1

0.5 + 0.1

0.5 + 0.1

P04217

Alpha-1B-glycoprotein

0.2 + 0.02

0.3 + 0.1

0.1 + 0.04

0.9 + 0.2

0.6 + 0.1

0.5 + 0.2

0.6 + 0.2

0.3 + 0.1

0.5 + 0.1

0.6 + 0.1

0.7 + 0.1

P02749

Beta-2-glycoprotein 1

0.2 + 0.1

0.2 + 0.02

0.2 + 0.1

P69905

Hemoglobin subunit alpha

0.1 + 0.03

0.2 + 0.1

P68871

Hemoglobin subunit beta

0.1 + 0.02

0.5 + 0.1

0.2 + 0.03

Q96PD5

N-acetylmuramoyl-L-alanine amidase

0.1 + 0.00

P02765

Alpha-2-HS-glycoprotein

0.1 + 0.04

P02766

Transthyretin

0.1 + 0.04

P25311

Zinc-alpha-2-glycoprotein

O75882

Attractin

0.02 + 0.001

P02787

Serotransferrin*

0.02 + 0.000

P02763

Alpha-1-acid glycoprotein 1

0.02 + 0.01

P01042

Kininogen-1

0.01 + 0.01

P02750

Leucine-rich alpha-2-glycoprotein

0.01 + 0.004

P05543

Thyroxine-binding globulin

0.00 + 0.002

0.03 + 0.003

0.3 + 0.02 0.1 + 0.02 0.1 + 0.04 0.05 + 0.01 0.3 + 0.2

0.2 + 0.03

0.3 + 0.02

0.2 + 0.1

0.1 + 0.02

0.3 + 0.1

0.5 + 0.1

0.5 + 0.1

0.1 + 0.03

0.1 + 0.03

0.2 + 0.1

0.1 + 0.03

0.1 + 0.01 0.3 + 0.1 0.1 + 0.03

0.1 + 0.03 0.1 + 0.02

0.1 + 0.05

0.2 + 0.1 0.2 + 0.01

0.2 + 0.1 0.1 + 0.02

0.1 + 0.02

The relative abundance (expressed as a percentage of total protein + SD) of proteins quantified in all four of the technical replicates is shown. Proteins which have previously been reported as secreted by embryos are marked with a *. L2 denotes lot 2.

Dyrlund et al.

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Embryo culture media contain non-declared proteins

Table II Human proteins identified in Sydney IVF Cleavage Medium (Lot: K11935).

Table II Continued Accession

Accession

Protein name

........................................................................................

Protein name

Fumarylacetoacetase

ADP-ribosyl cyclase 2

Q92820

Gamma-glutamyl hydrolase

P43652*

Afamin

P06396

Gelsolin

P02763

Alpha-1-acid glycoprotein 1

P78417

Glutathione S-transferase omega-1

P19652

Alpha-1-acid glycoprotein 2

P04406

Glyceraldehyde-3-phosphate dehydrogenase

P01011

Alpha-1-antichymotrypsin

P11217

Glycogen phosphorylase, muscle form

P01009

Alpha-1-antitrypsin

P00738*

Haptoglobin

P04217

Alpha-1B-glycoprotein

P00739*

Haptoglobin-related protein

P08697

Alpha-2-antiplasmin

P69905

Hemoglobin subunit alpha

P02765

Alpha-2-HS-glycoprotein

P68871

Hemoglobin subunit beta

P15144

Aminopeptidase N

P02042

Hemoglobin subunit delta

P12821

Angiotensin-converting enzyme

P02790

Hemopexin

P01019

Angiotensinogen

Q04756

Hepatocyte growth factor activator

P01008

Antithrombin-III

P01876

Ig alpha-1 chain C region

P02647*

Apolipoprotein A-I

P01877

Ig alpha-2 chain C region

P02652

Apolipoprotein A-II

P01857

Ig gamma-1 chain C region

P05090

Apolipoprotein D

P01859

Ig gamma-2 chain C region

Q13790

Apolipoprotein F

P01834

Ig kappa chain C region

O95445

Apolipoprotein M

P0CG05

Ig lambda-2 chain C regions

P05089

Arginase-1

P01591

Immunoglobulin J chain

O75882

Attractin

B9A064

Immunoglobulin lambda-like polypeptide 5

P02749

Beta-2-glycoprotein 1

Q14624

Inter-alpha-trypsin inhibitor heavy chain H4

Q96KN2

Beta-Ala-His dipeptidase

P14923

Junction plakoglobin

P43251

Biotinidase

P29622

Kallistatin

P00915

Carbonic anhydrase 1

P01042

Kininogen-1

Q96IY4

Carboxypeptidase B2

P02750

Leucine-rich alpha-2-glycoprotein

P15169

Carboxypeptidase N catalytic chain

P51884

Lumican

P22792

Carboxypeptidase N subunit 2

Q16853

Membrane primary amine oxidase

P31944

Caspase-14

P01033

Metalloproteinase inhibitor 1

P07339

Cathepsin D

P08571

Monocyte differentiation antigen CD14

P16070

CD44 antigen

P12882

Myosin-1

P43121

Cell surface glycoprotein MUC18

Q9UKX2

Myosin-2

P00450

Ceruloplasmin

Q9Y623

Myosin-4

P06276

Cholinesterase

P12883

Myosin-7

P10909

Clusterin

Q96PD5

N-acetylmuramoyl-L-alanine amidase

Q9NZP8

Complement C1r subcomponent-like protein

O95497

Pantetheinase

P01024

Complement C3

Q6UXB8

Peptidase inhibitor 16

P0C0L5

Complement C4-B

Q06830*

Peroxiredoxin-1

P08185

Corticosteroid-binding globulin

P04180

Phosphatidylcholine-sterol acyltransferase

P15924

Desmoplakin

P80108

Phosphatidylinositol-glycan-specific phospholipase D

Q01459

Di-N-acetylchitobiase

P00558

Phosphoglycerate kinase 1

P27487

Dipeptidyl peptidase 4

P05155

Plasma protease C1 inhibitor

P17813

Endoglin

P05154

Plasma serine protease inhibitor

P00533*

Epidermal growth factor receptor

P07359

Platelet glycoprotein Ib alpha chain

P61916

Epididymal secretory protein E1

Q6UX71

Plexin domain-containing protein 2

Q9UGM5

Fetuin-B

P41222

Prostaglandin-H2 D-isomerase

P04075

Fructose-bisphosphate aldolase A

P02760

Protein AMBP

Continued

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P16930

Q10588

........................................................................................

Continued

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Table II Continued Accession

Protein name

........................................................................................ Q92954

Proteoglycan 4

Q12913

Receptor-type tyrosine-protein phosphatase eta

P02753

Retinol-binding protein 4

P02787*

Serotransferrin

P29508

Serpin B3

P02768*

Serum albumin

P04278

Sex hormone-binding globulin

Q7Z7G0

Target of Nesh-SH3

P05543

Thyroxine-binding globulin

P37837

Transaldolase

Q15582

Transforming growth factor-beta-induced protein ig-h3

P02766

Transthyretin Triosephosphate isomerase

Q6EMK4

Vasorin

P02774

Vitamin D-binding protein

P54289

Voltage-dependent calcium channel subunit alpha-2/ delta-1

P12955

Xaa-Pro dipeptidase

P25311

Zinc-alpha-2-glycoprotein

Only proteins identified with three unique peptides were accepted. Proteins which have previously been reported as secreted by embryos or as a potential biomarker for positive implantation are marked with *.

present. Eight of these proteins have previously been suggested as biomarkers of embryonic viability (Table II). This finding therefore has implications for the interpretation of studies of the human secretome performed on IVF embryos. Previous proteomic studies of conditioned embryo media have focused on identifying proteins secreted into the surrounding media. In a first study, Katz-Jaffe et al. used MS to identify biomarkers for embryo viability. Their analysis of individual human embryos showed that protein expression profiles could be related to morphology and thus embryo viability (Katz-Jaffe et al., 2006a,b). In a follow-up study, the same group analyzed the human embryo secretome. They identified ubiquitin as a potential biomarker which was up-regulated in developing blastocysts compared with degenerating embryos (Katz-Jaffe et al., 2006a,b). However, up-regulation of ubiquitin has not been correlated with pregnancy rates in IVF trials. In addition, protein microarray studies have correlated the concentration of proteins, such as C-X-C motif chemokine 13, stem-cell factor, macrophage-stimulating protein-alpha, TRAILR3, MIP-1b, soluble tumor necrosis factor receptor 1, Interleukin-10, Insulin-like growth factor I, Interleukin-1 receptor type 1, Eotaxin-3 and NT-4 to embryo viability (Dominguez et al., 2008). Microarray studies are, however, limited by their high costs, and the dependence on the existence of antibodies and their sensitivity, which means that samples from individual embryos cannot be measured (Katz-Jaffe et al., 2009). Other studies have focused on individual proteins, identifying the secretion of platelet activation factor (O’Neill, 1985), leptin (Gonzalez et al., 2000), acrogranin (Diaz-Cueto et al., 2000) and soluble human leukocyte antigen G (Sher et al., 2005). In total, 84 proteins have been identified as secreted by mammalian embryos, 41 of which are from human embryos (Table III).

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P60174

Our results show that even though HSA is the only protein listed as being added to embryo culture media, many other proteins are also present in the media with other proteins on average adding up to 5.5% of the total (ranging from 3.3 to 8.0%). In some samples, single proteins accounted for up to 4.7% of the total protein, making them a significant constituent of the media. With modern plasma fractionation methods, HSA can only be purified to 99% purity (Burnouf, 2007) and the 110 other identified proteins are most likely impurities in the HSA added to the embryo media. Of the identified proteins, all proteins except Myosin-1 and Myosin-4 have also been identified in human plasma (data not shown). The proteins are hence likely co-purified with serum albumin by specific or non-specific binding to serum albumin. Analysis of purified HSA from Vitrolife (HSA-solution) also showed that the proteins identified in the embryo culture media are also present in the HSAsolution. This indicates that the HAS added to IVF media is the source of contaminating proteins, and Vitrolife has confirmed that the serum albumin in their culture media is the same as in the HSA-solution. The presence of proteins other than HSA in unconditioned media makes it difficult to determine if a protein present in conditioned embryo media is truly a secreted protein. Quantitative measurements therefore have to be taken into account when analyzing the embryo’s secretome. Previous studies have shown that not all serum albumin preparations are alike (Xiao and Isaacs, 2012) and batch to batch variation in IVF embryo media is to be expected. This was examined here by analyzing two different batches of the Sydney IVF Cleavage Medium and two batches of the Sydney IVF Blastocyst Medium. Batch to batch variation in protein amounts was observed, and the relative protein amount of haptoglobin was found to vary by up to 1-fold between two batches of the Sydney IVF Blastocyst Medium (Table I). Accordingly, it is important that experimental and control media are from the same batch when studying the embryo secretome and when analyzing conditioned embryo media with the aim of finding potential biomarkers that can distinguish normal and abnormal embryonic development. Furthermore, batch or lot numbers should preferentially be included when publishing IVF data as variations and differences in results might be a result of variations in the media. Of the 84 proteins previously identified as secreted by embryos, the following eight are also identified in this study: afamin, apolipoprotein A-I, epidermal growth factor receptor, haptoglobin, haptoglobin-related protein, peroxiredoxin-1, serotransferrin and serum albumin (Table II). These proteins have been shown to be increased in conditioned media compared with control media, but it is uncertain from the studies whether or not the control media are from the same batch as the conditioned embryo media. Afamin and haptoglobin were quantifiable in all analyzed media samples indicating that these proteins are present in high amounts in unconditioned embryo culture media: These two proteins were found to differ by up to 70 and 95%, respectively, between two batches, therefore indicating high differences between batches and making it easy to incorrectly identify these proteins as potentially secreted or taken up by embryos. Serotransferrin was also found to be among the most abundant proteins in three of the tested media. Regardless, it is important to know that these proteins are present in the media before embryo incubation. IVF medium is a valuable source for proteins that can be used as biomarkers in IVF. The focus has been on secreted proteins, but the proteins already present in the medium can be equally valuable in the pursuit for biomarkers. These proteins may be necessary for embryo development,

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Embryo culture media contain non-declared proteins

Table III List of proteins in the literature which have been identified in conditioned culture media from embryos. Protein name

Regulation

Species

Reference

............................................................................................................................................................................................. 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF)



Murine

2,3-bisphosphoglycerate mutase



Murine

Beardsley et al. (2010)

2′ -5′ oligoadenylate synthetase 1C



Murine

Beardsley et al. (2010)

25-Hydroxyvitamin D3 1a-hydroxylase



Porcine

Powell et al. (2010)

Acrogranin



Murine

Diaz-Cueto et al. (2000)

Afamin



Human

Mains et al. (2011)

Agouti-related protein

+

Human

Cortezzi et al. (2011)

Albumin



Porcine

Powell et al. (2010)

Apolipoprotein A-1



Human

Mains et al. (2011)

Arginine/serine-rich coiled-coil protein 2

+

Human

Cortezzi et al. (2011)

Ataxia-telangiectasia mutated protein



Porcine

Powell et al. (2010)

b-1 adrenergic receptor

O’Neill (1985)

Powell et al. (2010)



Mammalian

Gilchrist et al. (2008)

Bone morphogenetic protein 6 (BMP6)



Mammalian

Gilchrist et al. (2008)

Brefeldin A resistant Arf-guanine nucleotide exchange factor 1b

+

Human

Cortezzi et al. (2011)

Calreticulin



Murine

Beardsley et al. (2010)

cDNA FLJ32614 fis, clone STOMA2000121, highly similar to Homo sapiens kynurenine aminotransferase III

4

Human

Cortezzi et al. (2011)

cDNA FLJ51265, moderately similar to Beta-2-glycoprotein 1 (Beta-2-glycoprotein I)

4

Human

Cortezzi et al. (2011)

cDNA FLJ51378, highly similar to Homo sapiens NYD-SP28 protein (NYD-SP28), mRNA

4

Human

Cortezzi et al. (2011)

cDNA FLJ57033, highly similar to Antigen peptide transporter 2

+

Human

Cortezzi et al. (2011)

CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase, mitochondrial

+

Human

Cortezzi et al. (2011)

Chemokine ligand 13 (CXCL13)



Human

Dominguez et al. (2008)

Cocaine- and amphetamine-regulated transcript protein

+

Human

Cortezzi et al. (2011)

Connective tissue growth factor



Porcine

Powell et al. (2010)

Cortactin-binding protein 2



Porcine

Powell et al. (2010)

C-type lectin domain family 14 member A

4

Human

Cortezzi et al. (2011)

Dystrophin



Porcine

Powell et al. (2010)

Eotaxin-3

*

Human

Dominguez et al. (2008)

Epidermal growth factor receptor



Porcine

Powell et al. (2010)

Fatty acid synthase



Porcine

Powell et al. (2010)

F-box domain-containing protein



Murine

Beardsley et al. (2010)

Glutaminase



Porcine

Powell et al. (2010)

Glycogenin 1



Murine

Beardsley et al. (2010)

Granulocyte-macrophage colony-stimulating factor (GM-CSF)



Human

Dominguez et al. (2008)

Growth differentiation factor 9 (GDF9)

?

Murine

Yeo et al. (2008)

Haptoglobin



Human

Mains et al. (2011)

Haptoglobin-related protein

+

Human

Cortezzi et al. (2011)

Heat shock protein HSP 90-alpha



Murine

Beardsley et al. (2010)

HP protein

4

Human

Cortezzi et al. (2011)

Human leukocyte antigen G (sHLA-G)



Human

Rebmann et al. (2010) and Sher et al. (2005)

Insulin-like growth factor 1 (IGF-1)

*

Human

Dominguez et al. (2008)

Insulin-like growth factor II (IGF-II)



Bovine

Winger et al. (1997)

Interleukin-1 receptor type 1 (IL-1R)

*

Human

Dominguez et al. (2008)

Interleukin-10 (IL-10)



Human

Dominguez et al. (2008)

IS10-right transposase



Porcine

Powell et al. (2010)

Continued

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Porcine

Bone morphogenetic protein 15 (BMP15)

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Dyrlund et al.

Table III Continued Protein name

Regulation

Species

Reference

............................................................................................................................................................................................. 

Porcine

Powell et al. (2010)

Lactate dehydrogenase B



Murine

Beardsley et al. (2010)

Leptin



Human

Gonzalez et al. (2000)

LIM/homeobox protein Lhx9

4

Human

Cortezzi et al. (2011)

Lipocalin-1



Human

McReynolds et al. (2011)

Macrophage inflammatory protein (MIP-1b)



Human

Dominguez et al. (2008)

Macrophage-stimulating protein-alpha (MSP-a)



Human

Dominguez et al. (2008)

Mannose-6-phosphate/insulin-like growth factor II receptor



Porcine

Powell et al. (2010)

Neurotrophin-4 (NT-4)

*

Human

Dominguez et al. (2008)

NLR family, pyrin domain containing 5



Murine

Beardsley et al. (2010)

Nuclear export factor CRM1



Porcine

Powell et al. (2010)

Oviductal glycoprotein 1



Murine

Beardsley et al. (2010)

Peptidyl arginine deiminase, Type VI



Murine

Beardsley et al. (2010)

Peptidylprolyl isomerase A



Murine

Beardsley et al. (2010)

Peroxiredoxin 1



Murine

Beardsley et al. (2010)

Phosphatidylethanolamine binding protein 1



Murine

Beardsley et al. (2010)

Prolyl 4-hydroxylase, beta polypeptide (ERp59)



Murine

Beardsley et al. (2010)

Protein disulfide-isomerase A3 (ERp61)



Murine

Beardsley et al. (2010)

Protein Jumonji

+

Human

Cortezzi et al. (2011)

Receptor-type tyrosine-protein phosphatase S

4

Human

Cortezzi et al. (2011)

Ryanodine receptor



Porcine

Powell et al. (2010)

Serotransferrin



Human

Mains et al. (2011)

Soluble TNF receptor 1 (sTNFR1)



Human

Dominguez et al. (2008)

Sperm-associated antigen 6

+

Human

Cortezzi et al. (2011)

Spindlin isoform 1



Murine

Beardsley et al. (2010)

Stem-cell factor (SCF)



Human

Dominguez et al. (2008)

TNF-related apoptosis-inducing ligand receptor 3 (TRAILR3)



Human

Dominguez et al. (2008)

Transducin-like enhancer of split 6



Murine

Beardsley et al. (2010)

Transforming growth factor b 1 (TGFb1)



Mammalian

Gilchrist et al. (2008)

Transforming growth factor b 2 (TGFb2)



Mammalian

Gilchrist et al. (2008)

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma polypeptide



Murine

Beardsley et al. (2010)

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta/delta polypeptide



Murine

Beardsley et al. (2010)

Ubiquitin



Human

Katz-Jaffe et al. (2006a,b)

Ubiquitin carboxyl-terminal hydrolase 20

4

Human

Cortezzi et al. (2011)

UDP-galactose-4-epimerase

+

Human

Cortezzi et al. (2011)

Uncharacterized protein C13orf27

+

Human

Cortezzi et al. (2011)

Uncharacterized protein QRICH2

+

Human

Cortezzi et al. (2011)

Uncharacterized protein TSGA10

4

Human

Cortezzi et al. (2011)

Zinc finger, BED domain containing 3



Murine

Beardsley et al. (2010)

The regulation symbols are as follows:  or  denotes if a protein is increased or decreased compared with control samples, + or 4 denotes if the protein was detected in a positive or negative implantation group. *Indicates that the observed regulation was not significant.

and the uptake or degradation of specific proteins might correlate to a certain embryo development or lack thereof. The 111 proteins identified in the Sydney IVF Cleavage Medium therefore have to be evaluated for their potential to differentiate embryos success rates, as all these proteins can be potential biomarkers. Additionally, the safety of these

proteins in culture media for the offspring should be evaluated. Recent studies indicate a correlation between birthweight and embryo culture media (Dumoulin et al., 2010) and that the protein source might influence birthweight (Zhu et al., 2014). However, since this protein source comparison was based on G1-Plus versus G1 supplemented with

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Kelch-like ECH-associated protein 1

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Embryo culture media contain non-declared proteins

Supplementary data Supplementary data are available at http://humrep.oxfordjournals.org/.

Acknowledgements The authors thank the PRIDE team for assistance in data management in the ProteomeXchange Consortium.

Authors’ roles T.F.D.: contributed to the design of the study, the acquisition and interpretation of the data and in writing of the manuscript draft. K.K.: contributed to the interpretation of the data and in writing the manuscript draft. E.T.P.: contributed to the design of the study, the acquisition and interpretation of the data and edited the final version of the manuscript. K.W.S., J.J.H., J.K. and H.J.I.: contributed in editing of the final version of the manuscript. J.J.E.: contributed to the design of the study and edited the final version of the manuscript.

Funding This study was supported by the Danish Agency for Science, Technology and Innovation. Research at the Fertility Clinic, Aarhus University Hospital is supported by an unrestricted grant from MSD and Ferring.

Conflict of interest None declared.

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HSA-solution, and since Vitrolife uses the same serum albumin in G1-Plus media as in the HSA-solution, the conclusions drawn by Zhu et al. (2014) seem to be more related to batch to batch variation or factors not related to the IVF treatment than to different protein sources. A GO terms analysis of the proteins identified in the unconditioned media reveal a large number of proteins with defence responses roles, including 18 associated with the innate immune response and 17 with inflammatory responses. Furthermore, 15 proteins are annotated with transporter activity, 26 with ion binding function and 10 with receptor activity. Finally, 13 proteins have terms corresponding to peptidase activity, 22 have enzyme inhibitor activity and 10 have hormone like or hormone modulating properties. If the proteins from the media are taken up by the embryo they could have a wide range of effects on the development, and any protein secreted by the embryo could likewise interact with the proteins present in the media, thereby hindering their function. Further analysis is necessary to determine the effect of the additional non-declared proteins on aspects such as gestation age, birthweight and effects on the health of offspring. In order to identify a medium that is optimal for analyzing secreted embryo proteins, the recombinant human serum albumin supplement, G-MM from Vitrolife, was tested for proteins other than serum albumin. As expected, no other human proteins were identified in the media. The HSA is expressed in yeast cells but this analysis could likewise not identify any yeast proteins. The recombinant human serum albumin supplement seems to be free of contaminating proteins and is hence an ideal option for analyzing secreted embryo proteins when combined with protein-free media as well as for analyzing how the additional proteins influence embryo development. In conclusion, this study has highlighted that the HSA added to IVF media contains many other proteins consequently making it more difficult to analyze the embryo’s secretome. More notably, the proteins present in the unconditioned medium have been overlooked in previous IVF studies as a source of potential biomarkers that can distinguish normal and abnormal embryonic development based on which proteins are metabolized by human embryos. Finally, more research is needed into the effect of the identified proteins on embryonic development, and gestation age and birthweight.

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