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  • Research
  • Open Access

Placenta-derived exosomes continuously increase in maternal circulation over the first trimester of pregnancy

  • 1,
  • 1,
  • 2,
  • 1, 2, 3,
  • 1,
  • 1, 2 and
  • 1, 2Email author
Journal of Translational Medicine201412:204

https://doi.org/10.1186/1479-5876-12-204

  • Received: 14 May 2014
  • Accepted: 10 July 2014
  • Published:

Abstract

Background

Human placenta releases specific nanovesicles (i.e. exosomes) into the maternal circulation during pregnancy, however, the presence of placenta-derived exosomes in maternal blood during early pregnancy remains to be established. The aim of this study was to characterise gestational age related changes in the concentration of placenta-derived exosomes during the first trimester of pregnancy (i.e. from 6 to 12 weeks) in plasma from women with normal pregnancies.

Methods

A time-series experimental design was used to establish pregnancy-associated changes in maternal plasma exosome concentrations during the first trimester. A series of plasma were collected from normal healthy women (10 patients) at 6, 7, 8, 9, 10, 11 and 12 weeks of gestation (n = 70). We measured the stability of these vesicles by quantifying and observing their protein and miRNA contents after the freeze/thawing processes. Exosomes were isolated by differential and buoyant density centrifugation using a sucrose continuous gradient and characterised by their size distribution and morphology using the nanoparticles tracking analysis (NTA; Nanosight™) and electron microscopy (EM), respectively. The total number of exosomes and placenta-derived exosomes were determined by quantifying the immunoreactive exosomal marker, CD63 and a placenta-specific marker (Placental Alkaline Phosphatase PLAP).

Results

These nanoparticles are extraordinarily stable. There is no significant decline in their yield with the freeze/thawing processes or change in their EM morphology. NTA identified the presence of 50–150 nm spherical vesicles in maternal plasma as early as 6 weeks of pregnancy. The number of exosomes in maternal circulation increased significantly (ANOVA, p = 0.002) with the progression of pregnancy (from 6 to 12 weeks). The concentration of placenta-derived exosomes in maternal plasma (i.e. PLAP+) increased progressively with gestational age, from 6 weeks 70.6 ± 5.7 pg/ml to 12 weeks 117.5 ± 13.4 pg/ml. Regression analysis showed that weeks is a factor that explains for >70% of the observed variation in plasma exosomal PLAP concentration while the total exosome number only explains 20%.

Conclusions

During normal healthy pregnancy, the number of exosomes present in the maternal plasma increased significantly with gestational age across the first trimester of pregnancy. This study is a baseline that provides an ideal starting point for developing early detection method for women who subsequently develop pregnancy complications, clinically detected during the second trimester. Early detection of women at risk of pregnancy complications would provide an opportunity to develop and evaluate appropriate intervention strategies to limit acute adverse sequel.

Keywords

  • Exosomes
  • Pregnancy
  • Placenta
  • Fetal-maternal exchange

Background

The placenta plays a pivotal role in mediating maternal adaptation to pregnancy as well as regulating fetal growth and development. Pregnancy-induced changes are affected by the release of soluble autacoids as early as 6 to 8 weeks of gestation [1, 2] and the invasion of placental cells into the maternal tissues to modify maternal immune, cardiovascular and metabolic activities. Recently, we and others [37] have identified an additional pathway by which the placenta communicates with the maternal system to induce changes during pregnancy-placental exosomal signalling.

Exosomes are bilipid membrane-bound nanovesicles (50–120 nm diameter) that are actively released (via exocytosis) from cells into the extracellular space and body fluids under physiological and pathophysiological conditions [8]. Their molecular cargo of proteins, microRNAs, mRNAs and lipids appear to be selectively packaged by the late endosomal system to regulate the phenotype of target cells [3, 4, 6]. Recent studies have highlighted the putative utility of tissue-specific nanovesicles (e.g. exosomes) in the diagnosis of disease onset and treatment monitoring [4, 9, 10].

Previously, we have established that placental cells release exosomes in response to changes in the extracellular milieu (including oxygen tension and glucose concentration) and that placental cell-derived exosomes regulate target cell migration and invasion [3, 4]. In addition, we have identified placental-derived exosomes in maternal blood and reported that the concentration of placental exosomes in the maternal blood increases during normal, healthy pregnancy [7]. During early placentation, the cytotrophoblast cells form a highly invasive extravillous trophoblast that can migrate into the decidua and invade the first third of the myometrium, inducing remodelling of spiral arterioles to produce low-resistance vascular system, essential for fetal development [11]. The relative reduction of utero-placental flow caused by abnormal placentation triggers the development of placental originated diseases such as preeclampsia. Available data suggest that the concentrations of placental-derived exosomes in the maternal blood could be a potential marker of abnormal placentation [12, 13].

Early detection of disease risk and onset is the first step in implementing efficacious treatment and improving patient outcome. To date, the concentration profile of placenta-derived exosomes in the maternal blood during first trimester has not been established. Until this profile is defined, the utility of placental exosomes as an early biomarker for placental dysfunction will remain equivocal. In this study, therefore, a time-series experimental design was used to test the hypothesis that the concentration of placental exosomes in the maternal plasma of normal healthy women changes during the early pregnancy state (i.e. 6–12 weeks).

Methods

Patient selection and sample collection

A time-series experimental design was used to establish the variation in plasma exosome characteristics during normal pregnancy. All experimental procedures were conducted within an ISO17025 accredited (National Association of Testing Authorities, Australia) research facility. All data were recorded within a 21 CERF part 11 compliant electronic laboratory notebook (Iris note, Redwood City, CA, USA). Plasma samples were collected from 10 women during their first trimester of pregnancy. All patients were enrolled with informed consent and underwent routine obstetrical care at the Hospital Parroquial de San Bernardo (Santiago, Chile). Estimation of gestational age was made based on the first day of their last menstrual period and confirmed by transvaginal ultrasound at the recruitment (i.e. 6 weeks). Each patient, gave consent to have weekly blood sample collection between 6 and 12 weeks of gestation (n = 70, 10 patients with weekly blood collection at 6, 7, 8, 9, 10, 11 and 12 weeks of pregnancy). The protocol of the study was approved by the Institutional Review Board of the Universidad de los Andes (Santiago, Chile). Obstetrical history and physical findings were recorded regarding previous spontaneous abortions, course of previous pregnancies, hypertension, gestational diabetes and preeclampsia. Peripheral venous blood samples were collected in EDTA treated tubes (BD Vacutainer® Plus plastic plasma tube) from which plasma samples were obtained by centrifugation at 2000 × g at 4°C for 10 min. The plasma samples were stored in aliquots at −80°C until analysed (not more than three months).

Exosome isolation

Exosomes were isolated as previously described [3, 4, 7, 14]. Briefly, plasma from each patient was utilised to isolate exosomes. Plasma (2.5 ml) was diluted with equal volume of PBS (pH 7.4) and exosomes were isolated through differential centrifugation, microfiltration and buoyant density ultracentrifugation. Centrifugation was initially performed at 2,000 × g at 4°C for 30 min (Thermo Fisher Scientific Ins., Asheville, NC, USA, Sorvall®, high speed microcentrifuge, fixed rotor angle: 90°) followed by 12,000 × g at 4°C for 45 min to sediment cell nuclei, mitochondria and debris. The supernatant fluid (~5 ml) was transferred to an ultracentrifuge tube (Ultracrimp tubes, Thermo Fisher Scientific Ins., Asheville, NC, USA) and was centrifuged at 200,000 × g at 4°C for 2 h (Thermo Fisher Scientific Ins., Asheville, NC, USA, Sorvall®, T-8100, fixed angle ultracentrifuge rotor). The pellet was suspended in PBS (5 ml) and filtered through a 0.22 μm filter (SteritopTM, Millipore, Billerica, MA, USA). The filtrate was centrifuged at 200,000 × g at 4°C for 70 min (Thermo Fisher Scientific Ins., Asheville, NC, USA, Sorvall®, T-8100, fixed angle ultracentrifuge rotor) and the pellet resuspended in 2.5 M sucrose (4 ml).

Purification of exosomes using a continuous sucrose gradient

The resuspended 200,000 g pellet in 2.5 M sucrose was added at the bottom of an ultracentrifuge tube. A continuous sucrose gradient (26 ml; 0.25-2.5 M) was made above 4 ml of exosome suspension using a Hoefer SG30 gradient maker (GE Healthcare, NSW, Australia) and centrifuged at 110,000 g for 20 h (Sorvall, SureSpin™ 630/360, Swinging-Bucket ultracentrifuge rotor). Fractions (10 in total, 3 ml each) were collected automatically using a Pulse-Free Flow Peristaltic Pump with a flow rate range of 3 ml per min (GILSON Miniplus® model 3) and the Fraction Collector (GILSON FC 203B model). The density of each fraction was determined using the refraction index with OPTi digital refractometer (Bellingham + Stanley Inc., Lawrenceville, GA, USA). The coefficient of variation (CV) was less than 8% for the density of each fraction. Fractions (3 ml each) were diluted in PBS (60 ml) and then centrifuged at 200,000 × g for 70 min. The 200,000 g pellet was resuspended in 50 μl PBS and stored at −80°C. Exosomal protein concentrations were determined by a colorimetric assay (DC™ Protein Assay, Bio-Rad Laboratories, Hercules, CA, USA) [4].

Identification of nanoparticles by nanoparticle tracking analysis (NTA)

NTA measurements were performed using a NanoSight NS500 instrument (NanoSight NTA 2.3 Nanoparticle Tracking and Analysis Release Version Build 0033) following the manufacturer’s instructions. The NanoSight NS500 instrument measured the rate of Brownian motion of nanoparticles in a light scattering system that provides a reproducible platform for specific and general nanoparticle characterization (NanoSight Ltd., Amesbury, United Kingdom). Samples were processed in duplicates and diluted with PBS over a range of concentrations to obtain between 10 and 100 particles per image (optimal ~50 particles x image) before analysing with NTA system. The samples were mixed before introducting into the chamber (temperature: 25°C and viscosity: 0.89 cP) and the camera level set to obtain image that has sufficient contrast to clearly identify particles while minimizing background noise a video recording (camera level: 10 and capture duration: 60 s). The captured videos (2 videos per sample) were then processed and analysed. A combination of high shutter speed (450) and gain (250) followed by manual focusing enabled optimum visualization of a maximum number of vesicles. We included a minimum of 200 tracks completed per video in duplicates. NTA post acquisition settings were optimized and kept constant between samples (Frames Processed: 1496 of 1496, Frames per Second: 30, camera shutter: 20 ms; Calibration: 139 nm/pixel, Blur: 3×3; Detection Threshold: 10; Min Track Length: Auto; Min Expected Size: Auto), and each video was then analyzed to give the mean, mode, and median particles size together with an estimate of the number of particles. An Excel spreadsheet (Microsoft Corp., Redmond, Washington) was also automatically generated, showing the concentration at each particle size.

Transmission electron microscopy (TEM)

For the TEM analysis, exosome pellets (as described above, 30 μg protein) were fixed in 3% (w/v) glutaraldehyde and 2% paraformaldehyde in cacodylate buffer, pH 7.3. Exosome samples were then applied to a continuous carbon grid and negatively stained with 2% uranyl acetate. The samples were examined in an FEI Tecnai 12 transmission electron microscope (FEI™, Hillsboro, Oregon, USA) in the Central Analytical Research Facility, Institute for Future Environments, Queensland University of Technology (QUT) (see Acknowledgements).

Quantification of placental cell-derived exosome

The concentration of exosomes in maternal circulation was expressed as the total immunoreactive exosomal CD63 (ExoELISA™, System Biosciences, Mountain View, CA). Briefly, 10 μg of exosomal protein was immobilised in 96-well microtiter plates and incubated overnight (binding step). Plates were washed three times for 5 min using a wash buffer solution and then incubated with exosome specific primary antibody (CD63) at room temperature (RT) for 1 h under agitation. Plates were washed and incubated with secondary antibody (1:5000) at RT 1 h under agitation. Plates were washed and incubated with Super-sensitive TMB ELISA substrate at RT for 45 min under agitation. The reaction was terminated using Stop Buffer solution. Absorbance was measured at 450 nm. The number of exosomes/ml, (ExoELISA™ kit) was obtained using an exosomal CD63 standard curve calibrated against nanoparticle tracking data (i.e. number of exosomes, NanoSight™).

For placental cell-derived exosomes, the concentration of exosomal PLAP was quantified using a commercial ELISA kit (MYBioSource MBS701995, San Diego, CA, USA) according to manufacturer’s instructions (detection range: 84–2000 pg/ml; sensitivity: 34 pg/ml; intra-assay precision within an assay: CV% < 10%; inter-assay between assays: CV% < 15%) Briefly, 10 μg of exosomal protein was added to each well of a 96-well microtitre plate and incubated at 37°C for 30 min. Plates were washed three times while shaking for 20 s and 50 μl of HRP-conjugate was added to each well and incubated at 37°C for 20 min. Plates were washed and incubated with 50 μl of substrate A and 50 μl of substrate B at 37°C for 15 min. The incubation was terminated using 50 μl of stop solution at RT for 2 min under agitation. Absorbance was measured at 450 nm. Exosomal PLAP was expressed as pg PLAP /ml plasma.

Stability of the exosomal quantification

To determine the stability of the exosomes during freeze-thaw cycles, fresh plasma (5.0 ml) from healthy women were obtained and divided into two 2.5 ml samples (A and B). Exosomes were immediately isolated from the first aliquot (A: fresh plasma) by differential and buoyant density centrifugation and then characterised by the number of exosome particles using an ELISA kit (ExoELISA™, System Biosciences, Mountain View, CA), morphologically by electron microscope, microRNA content by real time PCR and protein profiling by mass-spectrometry. Sample B plasma was stored at −80°C for 2 months (B: frozen plasma), prior to exosome isolation and characterisation. miRNA isolation: miRNA were isolated from exosome particles as we have previously described [14]. Ambion mirVana PARIS Kit (Invitrogen, USA) was used to extract exosomal total RNA from fresh and frozen plasma by following the manufacturer’s procedure. Exosomes were first lysed by adding cell disruption buffer and vortexed or pipetted vigorously. Denaturing solution was added to samples and incubated on ice for 5 min. The first two steps stabilize RNA and inactivate RNases. The lysate is then subjected to Acid-Phenol:Chloroform extraction by adding Acid-Phenol:Chloroform, vortexed and centrifuged at 10,000 × g for 5 min. Recovery of the aqueous phase obtains semi-pure RNA samples, removing most of the other cellular components. 100% ethanol was mixed and passed through a filter cartilage. The filter was washed three times and the RNA was eluted with nuclease-free water. Real-time PCR: Reverse transcription was performed using the miScript Reverse Transcription Kit (QIAGEN, Valencia, CA, USA) in a total volume of 20 μl. cDNA was synthesised from the maximum volume of exosomal RNA (12 μl) using the BIO-RAD T100™ Thermal Cycler (USA) running for 60 min at 37°C, 5 min for 95°C and 60 min for 37°C. As the control, RNase-free water was added as the RNA template. Real-time PCR was performed with miScript SYBR Green Kit (QIAGEN, Valencia, CA, USA). Forward primers (miScript primer assays, QIAGEN, Valencia, CA, USA) designed to detect the housekeeping gene, human RNU6-2 (RNU6B) was used. The reactions were performed in triplicate using the BIO-RAD iQ™5 Multicolor Real-Time PCR Detection System (USA) with the following conditions: 94°C for 3 min, 35 amplification cycles of 94°C for 45 s, 55°C for 30 s and 72°C for 30 s, 72°C for 10 min, 12°C for ∞ min. Proteomic analysis of exosomes by mass spectrometry (MS): We utilised a Liquid Chromatography (LC) and Mass Spectrometry (MS) LC/MS/MS instrumentation available within the University of Queensland Centre for Clinical Research (5500qTRAP and 5600 Triple TOF) to undertake in depth quantitative proteomic analysis of the exosome samples (isolated from fresh and frozen plasma) to determine the proteome of exosomes as we have previously published [4]. Briefly, exosomes were adjusted to 8 M urea in 50 mM ammonium bicarbonate, pH 8.5, and reduced with tris (2-carboxyethyl) phosphine (5 mM) at room temperature for 1 h. Proteins were then alkylated in 10 mM IAA for 1 h in the dark. The sample was diluted 1:10 with 50 mM ammonium bicarbonate and then digested with trypsin (20 μg) at 37°C for 18 h. The samples were dried by centrifugal evaporation to remove the acetonitrile and then redissolved in Solvent A. The digested protein samples were analysed using a 5600 Triple TOF mass spectrometer (ABSciex) to obtain initial high mass accuracy survey MS/MS data, identifing the peptides present in the samples. The in depth proteomic analysis was performed using the Information Dependent Acquisition (IDA) experiments on the 5600 Triple TOF MS and utilized an enhanced MS survey scan (m/z350–1500) followed by 50 data-dependent product ion scans of the 50 most intense precursor ions. The MS data was analysed with the Markerview software package using Principal Components Analysis (PCA) or PCA-Discriminate Analysis (PCA-DA) which compares data across multiple samples, groupings the data sets, and graphically showing the groups in a Scores plot. The Loadings plot provides valuable insight into variables that lead to sample clustering and illustrates which biomarkers are up- or down-regulated. All mass spectra were analysed using the Mascot and Protein Pilot search engines against the Swissprot-swissprot database with the species set as human (scores greater than 30). False discovery rate (FDR) was estimated using a reversed sequence database. Finally, proteins identified were submitted to bioinformatic pathway analysis (Ingenuity Pathway Analysis [IPA]; Ingenuity Systems, Mountain View, CA; http://www.ingenuity.com).

Statistical analysis

Data are presented as mean ± SEM, with n = 10 different patients per group (i.e. 6, 7, 8, 9, 10, 11, 12 weeks). The effect of gestational age on number of exosome particles and placental-derived exosomes were assessed by two-way ANOVA, with variance partitioned between gestational age and subject. Statistical difference between group means was assessed by Dunn’s test to compare each treatment to the control group where the data distribution approximates normality and by Mann–Whitney U-test for distribution independent data analysis. Two group means were statistically assessed by Student’s t-test. Statistical significance was defined as p < 0.05.

Results

Exosome characterisation

Maternal plasma exosomes isolated by differential and sucrose density gradient centrifugation were characterised by a buoyant density of 1.122 to 1.197 g/ml (fractions 4 to 7) (Figure 1A-D). Nanoparticle tracking analysis showed a particle size distribution of 200,000 × g pellet (Figure 1A) ranging from 30 to 300 nm in diameter corresponding to microsomal fraction (including exosomes particles) with an average of 147 ± 71 nm (mean ± SD) (Figure 1B). After the sucrose continuous gradient, we mixed the enriched exosomal fractions (1.122 to 1.197 g/ml) (Figure 1C) and obtained a particle size distribution ranged from 50 to 140 nm in diameter, with an average of 98 ± 39 nm (mean ± SD) (Figure 1D). Electron microscopy revealed the presence of spherical vesicles, with a typical cup-shape and diameters ranging from 30 to 120 nm (Figure 1D, insert).
Figure 1
Figure 1

Characterisation of exosome from maternal circulation. Exosome were isolated from women uncomplicated pregnancies during first trimester by differential and buoyant density centrifugation (see Methods). (A) Flow chart for the exosome purification procedure based on differential ultracentrifugation. (B) Representative particles size distribution of microsomal fraction. (C) Flow chart for the exosome purification procedure based on sucrose continuous gradient (exosome enriched fractions in yellow 4–7). (D) Representative particles size distribution of enriched exosomal fractions (fraction 4–7 were mixed). Insert: Representative electron micrograph exosome fractions (pooled enriched exosome population from fractions 4 to 7), Scale bar 200 nm.

The stability of exosomes after a freeze and thaw cycle was evaluated using fresh and frozen plasma. No significant difference was observed using fresh or frozen plasma in exosome quantification, exosomal marker expression, microRNA expression or protein content (Figure 2A-D, Table 1).
Figure 2
Figure 2

Characteristics of exosomes isolated from plasma immediately after phlebotomy () and after 30 days stored at −80°C (). (A) Number of exosome particles. (B) Exosomes characterization. b1: electron microscope (scale bar 100 nm) and b2: Western blot for CD63 (exosomal marker); lane 1: Fresh and lane 2: stored. (C) Expression of miRNA RNU6B in exosomes. (D) Venn diagram of proteins identified in fresh and stored exosomes.

Table 1

Common proteins identified in exosomes isolated from fresh plasma and after freeze/thawing cycles

Protein ID

Symbol

Entrez gene name

Location

Type(s)

A2MG_HUMAN

A2M

alpha-2-macroglobulin

Extracellular Space

transporter

A2ML1_HUMAN

A2ML1

alpha-2-macroglobulin-like 1

Cytoplasm

other

ACACA_HUMAN

ACACA

acetyl-CoA carboxylase alpha

Cytoplasm

enzyme

ACTN3_HUMAN

ACTN3

actinin, alpha 3

Plasma Membrane

other

ADAL_HUMAN

ADAL

adenosine deaminase-like

Cytoplasm

enzyme

ATS16_HUMAN

ADAMTS16

ADAM metallopeptidase with thrombospondin type 1 motif, 16

Extracellular Space

other

ATS9_HUMAN

ADAMTS9

ADAM metallopeptidase with thrombospondin type 1 motif, 9

Extracellular Space

peptidase

DSRAD_HUMAN

ADAR

adenosine deaminase, RNA-specific

Nucleus

enzyme

ADCY7_HUMAN

ADCY7

adenylate cyclase 7

Plasma Membrane

enzyme

KFA_HUMAN

AFMID

arylformamidase

Nucleus

enzyme

ANGT_HUMAN

AGT

angiotensinogen (serpin peptidase inhibitor, clade A, member 8)

Extracellular Space

growth factor

ALBU_HUMAN

ALB

albumin

Extracellular Space

transporter

AMZ1_HUMAN

AMZ1

archaelysin family metallopeptidase 1

Other

peptidase

ANK2_HUMAN

ANK2

ankyrin 2, neuronal

Plasma Membrane

other

ANKAR_HUMAN

ANKAR

ankyrin and armadillo repeat containing

Nucleus

transcription regulator

AKD1B_HUMAN

ANKDD1B

ankyrin repeat and death domain containing 1B

Other

other

ANKL1_HUMAN

ANKLE1

ankyrin repeat and LEM domain containing 1

Other

other

ANR12_HUMAN

ANKRD12

ankyrin repeat domain 12

Nucleus

other

ANR26_HUMAN

ANKRD26

ankyrin repeat domain 26

Nucleus

transcription regulator

ANKUB_HUMAN

ANKUB1

ankyrin repeat and ubiquitin domain containing 1

Other

other

APOA1_HUMAN

APOA1

apolipoprotein A-I

Extracellular Space

transporter

APOB_HUMAN

APOB

apolipoprotein B

Extracellular Space

transporter

APOL1_HUMAN

APOL1

apolipoprotein L, 1

Extracellular Space

transporter

APOP1_HUMAN

APOPT1

apoptogenic 1, mitochondrial

Cytoplasm

other

DP13B_HUMAN

APPL2

adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2

Cytoplasm

other

RHG15_HUMAN

ARHGAP15

Rho GTPase activating protein 15

Cytoplasm

other

RHG08_HUMAN

ARHGAP8/PRR5-ARHGAP8

Rho GTPase activating protein 8

Cytoplasm

other

ARHGB_HUMAN

ARHGEF11

Rho guanine nucleotide exchange factor (GEF) 11

Cytoplasm

other

ASPM_HUMAN

ASPM

asp (abnormal spindle) homolog, microcephaly associated (Drosophila)

Nucleus

other

ATG2B_HUMAN

ATG2B

autophagy related 2B

Other

other

AT2A3_HUMAN

ATP2A3

ATPase, Ca++ transporting, ubiquitous

Cytoplasm

transporter

RENR_HUMAN

ATP6AP2

ATPase, H + transporting, lysosomal accessory protein 2

Cytoplasm

transporter

ATR_HUMAN

ATR

ataxia telangiectasia and Rad3 related

Nucleus

kinase

B4GT7_HUMAN

B4GALT7

xylosylprotein beta 1,4-galactosyltransferase, polypeptide 7

Cytoplasm

enzyme

BEND4_HUMAN

BEND4

BEN domain containing 4

Other

other

OSTCN_HUMAN

BGLAP

bone gamma-carboxyglutamate (gla) protein

Extracellular Space

other

BLM_HUMAN

BLM

Bloom syndrome, RecQ helicase-like

Nucleus

enzyme

BRCA2_HUMAN

BRCA2

breast cancer 2, early onset

Nucleus

transcription regulator

BRPF1_HUMAN

BRPF1

bromodomain and PHD finger containing, 1

Nucleus

transporter

CS068_HUMAN

C19orf68

chromosome 19 open reading frame 68

Other

other

CA174_HUMAN

C1orf174

chromosome 1 open reading frame 174

Nucleus

other

CA228_HUMAN

C1orf228

chromosome 1 open reading frame 228

Other

other

C1QC_HUMAN

C1QC

complement component 1, q subcomponent, C chain

Extracellular Space

other

CO3_HUMAN

C3

complement component 3

Extracellular Space

peptidase

CO4A_HUMAN

C4A/C4B

complement component 4B (Chido blood group)

Extracellular Space

other

C4BPA_HUMAN

C4BPA

complement component 4 binding protein, alpha

Extracellular Space

other

CI078_HUMAN

C9orf78

chromosome 9 open reading frame 78

Other

other

CAH3_HUMAN

CA3

carbonic anhydrase III, muscle specific

Cytoplasm

enzyme

CABIN_HUMAN

CABIN1

calcineurin binding protein 1

Nucleus

other

CAND1_HUMAN

CAND1

cullin-associated and neddylation-dissociated 1

Cytoplasm

transcription regulator

CAN1_HUMAN

CAPN1

calpain 1, (mu/I) large subunit

Cytoplasm

peptidase

CAN2_HUMAN

CAPN2

calpain 2, (m/II) large subunit

Cytoplasm

peptidase

CASC5_HUMAN

CASC5

cancer susceptibility candidate 5

Nucleus

other

C8AP2_HUMAN

CASP8AP2

caspase 8 associated protein 2

Nucleus

transcription regulator

CC154_HUMAN

CCDC154

coiled-coil domain containing 154

Other

other

CC171_HUMAN

CCDC171

coiled-coil domain containing 171

Other

other

CCD30_HUMAN

CCDC30

coiled-coil domain containing 30

Other

other

CCD37_HUMAN

CCDC37

coiled-coil domain containing 37

Other

other

CCD80_HUMAN

CCDC80

coiled-coil domain containing 80

Nucleus

other

CCHCR_HUMAN

CCHCR1

coiled-coil alpha-helical rod protein 1

Cytoplasm

other

CENPH_HUMAN

CENPH

centromere protein H

Nucleus

other

CP135_HUMAN

CEP135

centrosomal protein 135 kDa

Cytoplasm

other

CFAH_HUMAN

CFH

complement factor H

Extracellular Space

other

CHD4_HUMAN

CHD4

chromodomain helicase DNA binding protein 4

Nucleus

enzyme

CHD9_HUMAN

CHD9

chromodomain helicase DNA binding protein 9

Cytoplasm

other

ACHG_HUMAN

CHRNG

cholinergic receptor, nicotinic, gamma (muscle)

Plasma Membrane

transmembrane receptor

CHSTB_HUMAN

CHST11

carbohydrate (chondroitin 4) sulfotransferase 11

Cytoplasm

enzyme

CHSS3_HUMAN

CHSY3

chondroitin sulfate synthase 3

Cytoplasm

enzyme

CILP1_HUMAN

CILP

cartilage intermediate layer protein, nucleotide pyrophosphohydrolase

Extracellular Space

phosphatase

CLNK_HUMAN

CLNK

cytokine-dependent hematopoietic cell linker

Cytoplasm

other

CLUS_HUMAN

CLU

clusterin

Cytoplasm

other

CMBL_HUMAN

CMBL

carboxymethylenebutenolidase homolog (Pseudomonas)

Cytoplasm

enzyme

CNO6L_HUMAN

CNOT6L

CCR4-NOT transcription complex, subunit 6-like

Cytoplasm

enzyme

COPA1_HUMAN

COL25A1

collagen, type XXV, alpha 1

Cytoplasm

other

CROCC_HUMAN

CROCC

ciliary rootlet coiled-coil, rootletin

Plasma Membrane

other

CSRN1_HUMAN

CSRNP1

cysteine-serine-rich nuclear protein 1

Nucleus

transcription regulator

DIAC_HUMAN

CTBS

chitobiase, di-N-acetyl-

Cytoplasm

enzyme

CUL9_HUMAN

CUL9

cullin 9

Cytoplasm

other

CWC25_HUMAN

CWC25

CWC25 spliceosome-associated protein homolog (S. cerevisiae)

Other

other

CP1A2_HUMAN

CYP1A2

cytochrome P450, family 1, subfamily A, polypeptide 2

Cytoplasm

enzyme

CP51A_HUMAN

CYP51A1

cytochrome P450, family 51, subfamily A, polypeptide 1

Cytoplasm

enzyme

DAPL1_HUMAN

DAPL1

death associated protein-like 1

Other

other

DCAF6_HUMAN

DCAF6

DDB1 and CUL4 associated factor 6

Nucleus

transcription regulator

DCR1B_HUMAN

DCLRE1B

DNA cross-link repair 1B

Nucleus

enzyme

DCSTP_HUMAN

DCSTAMP

dendrocyte expressed seven transmembrane protein

Plasma Membrane

other

DCX_HUMAN

DCX

doublecortin

Cytoplasm

other

DDX51_HUMAN

DDX51

DEAD (Asp-Glu-Ala-Asp) box polypeptide 51

Other

enzyme

DEN2D_HUMAN

DENND2D

DENN/MADD domain containing 2D

Cytoplasm

other

DESM_HUMAN

DES

desmin

Cytoplasm

other

DGAT1_HUMAN

DGAT1

diacylglycerol O-acyltransferase 1

Cytoplasm

enzyme

DGC14_HUMAN

DGCR14

DiGeorge syndrome critical region gene 14

Nucleus

other

DHX30_HUMAN

DHX30

DEAH (Asp-Glu-Ala-His) box helicase 30

Nucleus

enzyme

DIP2B_HUMAN

DIP2B

DIP2 disco-interacting protein 2 homolog B (Drosophila)

Cytoplasm

other

DMXL1_HUMAN

DMXL1

Dmx-like 1

Extracellular Space

other

DYH17_HUMAN

DNAH17

dynein, axonemal, heavy chain 17

Cytoplasm

other

DYH2_HUMAN

DNAH2

dynein, axonemal, heavy chain 2

Other

other

DYH3_HUMAN

DNAH3

dynein, axonemal, heavy chain 3

Extracellular Space

enzyme

DYH5_HUMAN

DNAH5

dynein, axonemal, heavy chain 5

Cytoplasm

enzyme

DNJC7_HUMAN

DNAJC7

DnaJ (Hsp40) homolog, subfamily C, member 7

Cytoplasm

other

DOP1_HUMAN

DOPEY1

dopey family member 1

Cytoplasm

other

DSCAM_HUMAN

DSCAM

Down syndrome cell adhesion molecule

Plasma Membrane

other

DUS3L_HUMAN

DUS3L

dihydrouridine synthase 3-like (S. cerevisiae)

Other

other

DYHC2_HUMAN

DYNC2H1

dynein, cytoplasmic 2, heavy chain 1

Cytoplasm

other

COE2_HUMAN

EBF2

early B-cell factor 2

Nucleus

other

EBP_HUMAN

EBP

emopamil binding protein (sterol isomerase)

Cytoplasm

enzyme

EIF3C_HUMAN

EIF3C

eukaryotic translation initiation factor 3, subunit C

Other

translation regulator

ENPP1_HUMAN

ENPP1

ectonucleotide pyrophosphatase/phosphodiesterase 1

Plasma Membrane

enzyme

ENPP5_HUMAN

ENPP5

ectonucleotide pyrophosphatase/phosphodiesterase 5 (putative)

Extracellular Space

enzyme

PERE_HUMAN

EPX

eosinophil peroxidase

Cytoplasm

enzyme

EXOS1_HUMAN

EXOSC1

exosome component 1

Nucleus

enzyme

F150A_HUMAN

FAM150A

family with sequence similarity 150, member A

Other

other

F196B_HUMAN

FAM196B

family with sequence similarity 196, member B

Other

other

F208B_HUMAN

FAM208B

family with sequence similarity 208, member B

Other

other

YV021_HUMAN

FAM230B

family with sequence similarity 230, member B (non-protein coding)

Extracellular Space

other

FA78B_HUMAN

FAM78B

family with sequence similarity 78, member B

Other

other

FBF1_HUMAN

FBF1

Fas (TNFRSF6) binding factor 1

Nucleus

other

FIBA_HUMAN

FGA

fibrinogen alpha chain

Extracellular Space

other

FIBB_HUMAN

FGB

fibrinogen beta chain

Extracellular Space

other

FR1OP_HUMAN

FGFR1OP

FGFR1 oncogene partner

Cytoplasm

kinase

FGRL1_HUMAN

FGFRL1

fibroblast growth factor receptor-like 1

Plasma Membrane

transmembrane receptor

FIBG_HUMAN

FGG

fibrinogen gamma chain

Extracellular Space

other

FHAD1_HUMAN

FHAD1

forkhead-associated (FHA) phosphopeptide binding domain 1

Other

other

FIGL2_HUMAN

FIGNL2

fidgetin-like 2

Other

other

FLNB_HUMAN

FLNB

filamin B, beta

Cytoplasm

other

FINC_HUMAN

FN1

fibronectin 1

Extracellular Space

enzyme

FRMD3_HUMAN

FRMD3

FERM domain containing 3

Other

other

G6PC2_HUMAN

G6PC2

glucose-6-phosphatase, catalytic, 2

Cytoplasm

phosphatase

GAK_HUMAN

GAK

cyclin G associated kinase

Nucleus

kinase

GSH0_HUMAN

GCLM

glutamate-cysteine ligase, modifier subunit

Cytoplasm

enzyme

GCN1L_HUMAN

GCN1L1

GCN1 general control of amino-acid synthesis 1-like 1 (yeast)

Cytoplasm

translation regulator

CXB1_HUMAN

GJB1

gap junction protein, beta 1, 32 kDa

Plasma Membrane

transporter

GLRA2_HUMAN

GLRA2

glycine receptor, alpha 2

Plasma Membrane

ion channel

GMEB1_HUMAN

GMEB1

glucocorticoid modulatory element binding protein 1

Nucleus

transcription regulator

GOGA3_HUMAN

GOLGA3

golgin A3

Cytoplasm

transporter

AATC_HUMAN

GOT1

glutamic-oxaloacetic transaminase 1, soluble

Cytoplasm

enzyme

GRID2_HUMAN

GRID2

glutamate receptor, ionotropic, delta 2

Plasma Membrane

ion channel

GSAP_HUMAN

GSAP

gamma-secretase activating protein

Cytoplasm

peptidase

GSAS1_HUMAN

GSN-AS1

GSN antisense RNA 1

Other

other

GSHB_HUMAN

GSS

glutathione synthetase

Cytoplasm

enzyme

HERC1_HUMAN

HERC1

HECT and RLD domain containing E3 ubiquitin protein ligase family member 1

Cytoplasm

other

HES1_HUMAN

HES1

hes family bHLH transcription factor 1

Nucleus

transcription regulator

HILS1_HUMAN

HILS1

histone linker H1 domain, spermatid-specific 1, pseudogene

Nucleus

other

HIP1_HUMAN

HIP1

huntingtin interacting protein 1

Cytoplasm

other

HJURP_HUMAN

HJURP

Holliday junction recognition protein

Nucleus

other

HPTR_HUMAN

HPR

haptoglobin-related protein

Extracellular Space

peptidase

5HT2A_HUMAN

HTR2A

5-hydroxytryptamine (serotonin) receptor 2A, G protein-coupled

Plasma Membrane

G-protein coupled receptor

I23O2_HUMAN

IDO2

indoleamine 2,3-dioxygenase 2

Cytoplasm

enzyme

GILT_HUMAN

IFI30

interferon, gamma-inducible protein 30

Cytoplasm

enzyme

IGHA1_HUMAN

IGHA1

immunoglobulin heavy constant alpha 1

Extracellular Space

other

IGHG1_HUMAN

IGHG1

immunoglobulin heavy constant gamma 1 (G1m marker)

Extracellular Space

other

IGHM_HUMAN

IGHM

immunoglobulin heavy constant mu

Plasma Membrane

transmembrane receptor

IGJ_HUMAN

IGJ

immunoglobulin J polypeptide, linker protein for immunoglobulin alpha and mu polypeptides

Extracellular Space

other

IGKC_HUMAN

IGKC

immunoglobulin kappa constant

Extracellular Space

other

KV401_HUMAN

IGKV4-1

immunoglobulin kappa variable 4-1

Extracellular Space

other

LAC1_HUMAN

IGLC1

immunoglobulin lambda constant 1 (Mcg marker)

Cytoplasm

other

LAC2_HUMAN

IGLC2

immunoglobulin lambda constant 2 (Kern-Oz- marker)

Extracellular Space

other

IHH_HUMAN

IHH

indian hedgehog

Extracellular Space

enzyme

RED_HUMAN

IK

IK cytokine, down-regulator of HLA II

Extracellular Space

cytokine

IL1AP_HUMAN

IL1RAP

interleukin 1 receptor accessory protein

Plasma Membrane

transmembrane receptor

IRPL2_HUMAN

IL1RAPL2

interleukin 1 receptor accessory protein-like 2

Plasma Membrane

transmembrane receptor

IL26_HUMAN

IL26

interleukin 26

Extracellular Space

cytokine

INCE_HUMAN

INCENP

inner centromere protein antigens 135/155 kDa

Nucleus

other

IQCF6_HUMAN

IQCF6

IQ motif containing F6

Other

other

JARD2_HUMAN

JARID2

jumonji, AT rich interactive domain 2

Nucleus

transcription regulator

KTNB1_HUMAN

KATNB1

katanin p80 (WD repeat containing) subunit B 1

Cytoplasm

enzyme

KCND2_HUMAN

KCND2

potassium voltage-gated channel, Shal-related subfamily, member 2

Plasma Membrane

ion channel

KCNQ5_HUMAN

KCNQ5

potassium voltage-gated channel, KQT-like subfamily, member 5

Plasma Membrane

ion channel

KDM2B_HUMAN

KDM2B

lysine (K)-specific demethylase 2B

Nucleus

other

KDM5A_HUMAN

KDM5A

lysine (K)-specific demethylase 5A

Nucleus

transcription regulator

TALD3_HUMAN

KIAA0586

KIAA0586

Cytoplasm

other

K1161_HUMAN

KIAA1161

KIAA1161

Nucleus

other

KI13A_HUMAN

KIF13A

kinesin family member 13A

Cytoplasm

transporter

KIF19_HUMAN

KIF19

kinesin family member 19

Extracellular Space

enzyme

KIRR1_HUMAN

KIRREL

kin of IRRE like (Drosophila)

Plasma Membrane

other

KLC2_HUMAN

KLC2

kinesin light chain 2

Cytoplasm

other

KLRF1_HUMAN

KLRF1

killer cell lectin-like receptor subfamily F, member 1

Plasma Membrane

transmembrane receptor

LDB1_HUMAN

LDB1

LIM domain binding 1

Nucleus

transcription regulator

LHPL3_HUMAN

LHFPL3

lipoma HMGIC fusion partner-like 3

Other

other

LIPC_HUMAN

LIPC

lipase, hepatic

Extracellular Space

enzyme

YP023_HUMAN

LOC100128265

uncharacterized LOC100128265

Other

other

LRP1B_HUMAN

LRP1B

low density lipoprotein receptor-related protein 1B

Plasma Membrane

transmembrane receptor

LTBP2_HUMAN

LTBP2

latent transforming growth factor beta binding protein 2

Extracellular Space

other

LY75_HUMAN

LY75

lymphocyte antigen 75

Plasma Membrane

transmembrane receptor

MACD1_HUMAN

MACROD1

MACRO domain containing 1

Cytoplasm

enzyme

MANF_HUMAN

MANF

mesencephalic astrocyte-derived neurotrophic factor

Extracellular Space

other

MLP3A_HUMAN

MAP1LC3A

microtubule-associated protein 1 light chain 3 alpha

Cytoplasm

other

MAP4_HUMAN

MAP4

microtubule-associated protein 4

Cytoplasm

other

MA7D3_HUMAN

MAP7D3

MAP7 domain containing 3

Cytoplasm

other

MBD5_HUMAN

MBD5

methyl-CpG binding domain protein 5

Nucleus

other

MDN1_HUMAN

MDN1

MDN1, midasin homolog (yeast)

Nucleus

other

MEX3B_HUMAN

MEX3B

mex-3 RNA binding family member B

Other

kinase

MFNG_HUMAN

MFNG

MFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase

Cytoplasm

enzyme

MKL1_HUMAN

MKL1

megakaryoblastic leukemia (translocation) 1

Nucleus

transcription regulator

MRE11_HUMAN

MRE11A

MRE11 meiotic recombination 11 homolog A (S. cerevisiae)

Nucleus

enzyme

RM32_HUMAN

MRPL32

mitochondrial ribosomal protein L32

Cytoplasm

translation regulator

MYBA_HUMAN

MYBL1

v-myb avian myeloblastosis viral oncogene homolog-like 1

Nucleus

transcription regulator

MYO15_HUMAN

MYO15A

myosin XVA

Cytoplasm

other

MYO3A_HUMAN

MYO3A

myosin IIIA

Cytoplasm

kinase

MYO6_HUMAN

MYO6

myosin VI

Cytoplasm

other

ULA1_HUMAN

NAE1

NEDD8 activating enzyme E1 subunit 1

Cytoplasm

enzyme

NUCL_HUMAN

NCL

nucleolin

Nucleus

other

NCOA2_HUMAN

NCOA2

nuclear receptor coactivator 2

Nucleus

transcription regulator

NEBU_HUMAN

NEB

nebulin

Cytoplasm

other

NEDD4_HUMAN

NEDD4

neural precursor cell expressed, developmentally down-regulated 4, E3 ubiquitin protein ligase

Cytoplasm

enzyme

NHS_HUMAN

NHS

Nance-Horan syndrome (congenital cataracts and dental anomalies)

Nucleus

other

NOA1_HUMAN

NOA1

nitric oxide associated 1

Cytoplasm

other

NRX3A_HUMAN

NRXN3

neurexin 3

Other

transporter

NSD1_HUMAN

NSD1

nuclear receptor binding SET domain protein 1

Nucleus

transcription regulator

NSN5C_HUMAN

NSUN5P2

NOP2/Sun domain family, member 5 pseudogene 2

Other

other

NET5_HUMAN

NTN5

netrin 5

Other

other

NUD15_HUMAN

NUDT15

nudix (nucleoside diphosphate linked moiety X)-type motif 15

Cytoplasm

phosphatase

OBSCN_HUMAN

OBSCN

obscurin, cytoskeletal calmodulin and titin-interacting RhoGEF

Cytoplasm

kinase

OCEL1_HUMAN

OCEL1

occludin/ELL domain containing 1

Other

other

ODFP2_HUMAN

ODF2

outer dense fiber of sperm tails 2

Cytoplasm

other

NOE2_HUMAN

OLFM2

olfactomedin 2

Cytoplasm

other

OPN4_HUMAN

OPN4

opsin 4

Plasma Membrane

G-protein coupled receptor

OR4K1_HUMAN

OR4K1

olfactory receptor, family 4, subfamily K, member 1

Plasma Membrane

G-protein coupled receptor

PALB2_HUMAN

PALB2

partner and localizer of BRCA2

Nucleus

other

PAR3L_HUMAN

PARD3B

par-3 family cell polarity regulator beta

Plasma Membrane

other

PARP4_HUMAN

PARP4

poly (ADP-ribose) polymerase family, member 4

Cytoplasm

enzyme

PCDH8_HUMAN

PCDH8

protocadherin 8

Plasma Membrane

other

PCLO_HUMAN

PCLO

piccolo presynaptic cytomatrix protein

Cytoplasm

transporter

PEAK1_HUMAN

PEAK1

pseudopodium-enriched atypical kinase 1

Plasma Membrane

kinase

PEG10_HUMAN

PEG10

paternally expressed 10

Nucleus

other

PER3_HUMAN

PER3

period circadian clock 3

Nucleus

other

PFD6_HUMAN

PFDN6

prefoldin subunit 6

Cytoplasm

other

PIGS_HUMAN

PIGS

phosphatidylinositol glycan anchor biosynthesis, class S

Cytoplasm

enzyme

P3C2A_HUMAN

PIK3C2A

phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2 alpha

Cytoplasm

kinase

SOX_HUMAN

PIPOX

pipecolic acid oxidase

Cytoplasm

enzyme

PLCD3_HUMAN

PLCD3

phospholipase C, delta 3

Cytoplasm

enzyme

PLXA4_HUMAN

PLXNA4

plexin A4

Plasma Membrane

transmembrane receptor

PNKD_HUMAN

PNKD

paroxysmal nonkinesigenic dyskinesia

Nucleus

other

PNKP_HUMAN

PNKP

polynucleotide kinase 3'-phosphatase

Nucleus

kinase

DPOLQ_HUMAN

POLQ

polymerase (DNA directed), theta

Nucleus

enzyme

PMGT1_HUMAN

POMGNT1

protein O-linked mannose N-acetylglucosaminyltransferase 1 (beta 1,2-)

Cytoplasm

enzyme

PPIG_HUMAN

PPIG

peptidylprolyl isomerase G (cyclophilin G)

Nucleus

enzyme

PP12C_HUMAN

PPP1R12C

protein phosphatase 1, regulatory subunit 12C

Cytoplasm

phosphatase

PPT2_HUMAN

PPT2

palmitoyl-protein thioesterase 2

Cytoplasm

enzyme

PREB_HUMAN

PREB

prolactin regulatory element binding

Nucleus

transcription regulator

PPCEL_HUMAN

PREPL

prolyl endopeptidase-like

Other

peptidase

PRG4_HUMAN

PRG4

proteoglycan 4

Extracellular Space

other

PRP31_HUMAN

PRPF31

pre-mRNA processing factor 31

Nucleus

other

PRC2A_HUMAN

PRRC2A

proline-rich coiled-coil 2A

Cytoplasm

other

PSB3_HUMAN

PSMB3

proteasome (prosome, macropain) subunit, beta type, 3

Cytoplasm

peptidase

PRS7_HUMAN

PSMC2

proteasome (prosome, macropain) 26S subunit, ATPase, 2

Nucleus

peptidase

PTPRM_HUMAN

PTPRM

protein tyrosine phosphatase, receptor type, M

Plasma Membrane

phosphatase

PTTG3_HUMAN

PTTG3P

pituitary tumor-transforming 3, pseudogene

Other

other

PZP_HUMAN

PZP

pregnancy-zone protein

Extracellular Space

other

RAB10_HUMAN

RAB10

RAB10, member RAS oncogene family

Cytoplasm

enzyme

RB3GP_HUMAN

RAB3GAP1

RAB3 GTPase activating protein subunit 1 (catalytic)

Cytoplasm

other

RAB6A_HUMAN

RAB6A

RAB6A, member RAS oncogene family

Cytoplasm

enzyme

RAB8B_HUMAN

RAB8B

RAB8B, member RAS oncogene family

Cytoplasm

enzyme

RGPA2_HUMAN

RALGAPA2

Ral GTPase activating protein, alpha subunit 2 (catalytic)

Cytoplasm

other

RBM23_HUMAN

RBM23

RNA binding motif protein 23

Nucleus

other

REG1A_HUMAN

REG1A

regenerating islet-derived 1 alpha

Extracellular Space

growth factor

RELN_HUMAN

RELN

reelin

Extracellular Space

peptidase

RFC4_HUMAN

RFC4

replication factor C (activator 1) 4, 37 kDa

Nucleus

other

RFX8_HUMAN

RFX8

RFX family member 8, lacking RFX DNA binding domain

Other

other

RMND1_HUMAN

RMND1

required for meiotic nuclear division 1 homolog (S. cerevisiae)

Cytoplasm

other

RNF17_HUMAN

RNF17

ring finger protein 17

Cytoplasm

other

RN213_HUMAN

RNF213

ring finger protein 213

Cytoplasm

enzyme

RN219_HUMAN

RNF219

ring finger protein 219

Other

other

FTM_HUMAN

RPGRIP1L

RPGRIP1-like

Cytoplasm

other

RL29_HUMAN

RPL29

ribosomal protein L29

Cytoplasm

other

RL37_HUMAN

RPL37

ribosomal protein L37

Cytoplasm

other

KS6A4_HUMAN

RPS6KA4

ribosomal protein S6 kinase, 90 kDa, polypeptide 4

Cytoplasm

kinase

RTKN_HUMAN

RTKN

rhotekin

Cytoplasm

other

RYR2_HUMAN

RYR2

ryanodine receptor 2 (cardiac)

Plasma Membrane

ion channel

SAMD8_HUMAN

SAMD8

sterile alpha motif domain containing 8

Cytoplasm

other

SASH1_HUMAN

SASH1

SAM and SH3 domain containing 1

Extracellular Space

other

UTER_HUMAN

SCGB1A1

secretoglobin, family 1A, member 1 (uteroglobin)

Extracellular Space

cytokine

SCUB3_HUMAN

SCUBE3

signal peptide, CUB domain, EGF-like 3

Plasma Membrane

other

SPB9_HUMAN

SERPINB9

serpin peptidase inhibitor, clade B (ovalbumin), member 9

Cytoplasm

other

SET1A_HUMAN

SETD1A

SET domain containing 1A

Nucleus

ion channel

SHAN1_HUMAN

SHANK1

SH3 and multiple ankyrin repeat domains 1

Cytoplasm

other

SHAN3_HUMAN

SHANK3

SH3 and multiple ankyrin repeat domains 3

Plasma Membrane

other

CTL1_HUMAN

SLC44A1

solute carrier family 44 (choline transporter), member 1

Plasma Membrane

transporter

SNTAN_HUMAN

SNTN

sentan, cilia apical structure protein

Other

other

SOLH1_HUMAN

SOHLH1

spermatogenesis and oogenesis specific basic helix-loop-helix 1

Cytoplasm

transcription regulator

SPAG7_HUMAN

SPAG7

sperm associated antigen 7

Nucleus

other

SPA2L_HUMAN

SPATA2L

spermatogenesis associated 2-like

Other

other

CYTSB_HUMAN

SPECC1

sperm antigen with calponin homology and coiled-coil domains 1

Nucleus

other

SPO11_HUMAN

SPO11

SPO11 meiotic protein covalently bound to DSB

Nucleus

enzyme

SPTN5_HUMAN

SPTBN5

spectrin, beta, non-erythrocytic 5

Plasma Membrane

other

SRGP2_HUMAN

SRGAP2

SLIT-ROBO Rho GTPase activating protein 2

Cytoplasm

other

SRG2C_HUMAN

SRGAP2C

SLIT-ROBO Rho GTPase activating protein 2C

Other

other

SIA7B_HUMAN

ST6GALNAC2

ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 2

Cytoplasm

enzyme

STXB1_HUMAN

STXBP1

syntaxin binding protein 1

Cytoplasm

transporter

SP20H_HUMAN

SUPT20H

suppressor of Ty 20 homolog (S. cerevisiae)

Nucleus

other

SPT6H_HUMAN

SUPT6H

suppressor of Ty 6 homolog (S. cerevisiae)

Nucleus

transcription regulator

SVEP1_HUMAN

SVEP1

sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1

Cytoplasm

other

SYNJ1_HUMAN

SYNJ1

synaptojanin 1

Cytoplasm

phosphatase

TADA3_HUMAN

TADA3

transcriptional adaptor 3

Nucleus

transcription regulator

TBX20_HUMAN

TBX20

T-box 20

Nucleus

transcription regulator

TDRD1_HUMAN

TDRD1

tudor domain containing 1

Cytoplasm

other

TET1_HUMAN

TET1

tet methylcytosine dioxygenase 1

Nucleus

other

THMS1_HUMAN

THEMIS

thymocyte selection associated

Cytoplasm

other

TLK2_HUMAN

TLK2

tousled-like kinase 2

Cytoplasm

kinase

TM131_HUMAN

TMEM131

transmembrane protein 131

Extracellular Space

other

T132C_HUMAN

TMEM132C

transmembrane protein 132C

Other

other

T151A_HUMAN

TMEM151A

transmembrane protein 151A

Other

other

TM232_HUMAN

TMEM232

transmembrane protein 232

Other

other

TNFA_HUMAN

TNF

tumor necrosis factor

Extracellular Space

cytokine

TPD54_HUMAN

TPD52L2

tumor protein D52-like 2

Cytoplasm

other

TRML4_HUMAN

TREML4

triggering receptor expressed on myeloid cells-like 4

Other

other

TRI32_HUMAN

TRIM32

tripartite motif containing 32

Nucleus

transcription regulator

TRI65_HUMAN

TRIM65

tripartite motif containing 65

Other

other

TARA_HUMAN

TRIOBP

TRIO and F-actin binding protein

Nucleus

other

TRIPB_HUMAN

TRIP11

thyroid hormone receptor interactor 11

Cytoplasm

transcription regulator

TROAP_HUMAN

TROAP

trophinin associated protein

Cytoplasm

peptidase

TRPC5_HUMAN

TRPC5

transient receptor potential cation channel, subfamily C, member 5

Plasma Membrane

ion channel

TSG13_HUMAN

TSGA13

testis specific, 13

Other

other

TTC12_HUMAN

TTC12

tetratricopeptide repeat domain 12

Other

other

TITIN_HUMAN

TTN

titin

Cytoplasm

kinase

GCP6_HUMAN

TUBGCP6

tubulin, gamma complex associated protein 6

Cytoplasm

other

TRXR3_HUMAN

TXNRD3

thioredoxin reductase 3

Cytoplasm

enzyme

UBQLN_HUMAN

UBQLNL

ubiquilin-like

Other

other

UCKL1_HUMAN

UCKL1

uridine-cytidine kinase 1-like 1

Cytoplasm

kinase

UGDH_HUMAN

UGDH

UDP-glucose 6-dehydrogenase

Nucleus

enzyme

USP9X_HUMAN

USP9X

ubiquitin specific peptidase 9, X-linked

Plasma Membrane

peptidase

UTRO_HUMAN

UTRN

utrophin

Plasma Membrane

transmembrane receptor

VP13C_HUMAN

VPS13C

vacuolar protein sorting 13 homolog C (S. cerevisiae)

Cytoplasm

other

WAC_HUMAN

WAC

WW domain containing adaptor with coiled-coil

Nucleus

other

WDR1_HUMAN

WDR1

WD repeat domain 1

Extracellular Space

other

WDR35_HUMAN

WDR35

WD repeat domain 35

Cytoplasm

other

WDR43_HUMAN

WDR43

WD repeat domain 43

Nucleus

other

WFDC3_HUMAN

WFDC3

WAP four-disulfide core domain 3

Extracellular Space

other

YIPF1_HUMAN

YIPF1

Yip1 domain family, member 1

Cytoplasm

other

NIPA_HUMAN

ZC3HC1

zinc finger, C3HC-type containing 1

Nucleus

other

ZFHX4_HUMAN

ZFHX4

zinc finger homeobox 4

Extracellular Space

other

ZF64B_HUMAN

ZFP64

ZFP64 zinc finger protein

Nucleus

other

ZN132_HUMAN

ZNF132

zinc finger protein 132

Nucleus

other

ZNF14_HUMAN

ZNF14

zinc finger protein 14

Nucleus

transcription regulator

ZN215_HUMAN

ZNF215

zinc finger protein 215

Nucleus

transcription regulator

Z286B_HUMAN

ZNF286B

zinc finger protein 286B

Other

other

ZN345_HUMAN

ZNF345

zinc finger protein 345

Nucleus

transcription regulator

ZN532_HUMAN

ZNF532

zinc finger protein 532

Other

other

ZN561_HUMAN

ZNF561

zinc finger protein 561

Nucleus

other

ZN624_HUMAN

ZNF624

zinc finger protein 624

Nucleus

other

ZNF74_HUMAN

ZNF74

zinc finger protein 74

Nucleus

other

List of common exosomal proteins are presented as Protein ID, Symbol, Entrez Gene Name, Location and type. No significant differences were observed en exosomal protein content from fresh or frozen plasma (coefficient of variation < 5%) after different freeze thawing cycle from the same sample.

Placenta-derived exosome increased during first trimester in normal pregnancy

Pooled exosome-containing fractions (i.e. fractions 4 to 7) were further characterised by determining the number of exosome (NEP) and exosomal PLAP concentration in the serial samples of maternal plasma obtained during first trimester of pregnancy (i.e. 6–12 weeks).

The gestational age variation in plasma exosome number was analysed by two-way ANOVA with the variance partitioned between gestational age and subject. A significantly effect of gestational age was identified (n = 69, one missing value, p < 0.005). A post-hoc multiple range test was used to identify statistically significant (p <0.05) differences between pairwise comparisons (Figure 3A). In addition, a significant effect of subject was identified (n = 69, one missing value, p < 0.05) (Figure 3B). In addition, NEP and gestational age (i.e. 6–12 weeks) displayed a significant positive linear relationship (r2 = 0.202, p < 0.001, n = 69, one missing value).
Figure 3
Figure 3

Exosome profiling across first trimester pregnancy. Enriched exosomal population (i.e. number of exosome particles) and placenta-derived exosomes (i.e. exosomal PLAP) were quantified in in peripheral plasma of women in the first trimester of pregnancy by ELISA. (A) exosomes as particles per ml plasma. (B) individual variation in exosome number for each week (C) exosomal PLAP during first trimester of pregnancy (i.e. 6–12 weeks). (D) individual variation in exosomal PLAP for each week. Data are presented as aligned dot plot and values are mean ± SEM. In A, two-way ANOVA **p = 0.0048, Dunn’s post-hoc test analysis = *p < 0.05 6 vs. 7 weeks and p < 0.005: 6 vs. 12 weeks. In C, two-way ANOVA ***p < 0.0001, Dunn’s post-hoc test analysis = *p < 0.05 6 vs. 9 and 10 weeks, p < 0.005: 6 vs. 11 and 12 weeks, and p < 0.005: 8 vs. 11 and 12 weeks.

To assess gestational variation in placenta-derived exosomes, exosomal immunoreative (IR) PLAP was quantified using a commercial ELISA kit (see Methods). IR exosomal PLAP concentrations were analysed by two-way ANOVA with the variance partitioned between gestational age and subject. A significant effect of gestational age was identified (p < 0.0001, n = 69, one missing value) (Figure 3C). A post-hoc multiple range test was used to identify statistically significant (p <0.05) differences between pairwise comparisons (Figure 3D). No significant effect of patient on exosomal PLAP concentration was identified (p = 0.123). Immunoreactive exosomal PLAP concentration and gestational age displayed a significant positive linear linear relationship (r2 = 0.711, p < 0.001, n = 69, one missing value).

Specific placental-derived exosomes

Exosomal PLAP concentration and exosome number were subjected to linear regression analysis. The fitted linear model was described by the following equation: plasma exosomal PLAP pg/ml = 85.6 + 5.47 × 10−11 × exosome number/ml (p < 0.006, n = 69, one missing pair). The coefficient of determination (r2) was 10.8 (Figure 4A).
Figure 4
Figure 4

Contribution of placental-derived exosomes into maternal circulation. (A) Relationship between exosomal PLAP and exosomes (particles per ml plasma) across first trimester of pregnancy (i.e. 6–12 weeks represented by colours). (B) Ratio of specific placental exosome and exosomes. In A, values are mean ± SEM, Linear correlation (−). In B, Data are presented as aligned dot plot and values are mean ± SEM, two-way ANOVA p > 0.05.

To estimate changes in the relative contribution of placental exosomes within the total exosomes present in maternal plasma and identify changes over the gestational age, the apparent PLAP content per 109 exosome (PLAP ratio) was determined. Overall PLAP ratio averaged 2.01 ± 0.33 × 10−9 exosomal PLAP (pg) per exosome. The effects of gestational age on PLAP ratio were assessed by Kruskal-Wallis one-way ANOVA. No significant effect of gestational age on PLAP ratio was identified (p = 0.06) (Figure 4B).

Discussion

Currently, there are no proven means of identifying presymptomatic women who subsequently develop complications of pregnancy during early pregnancy. Most women who are triaged into high-risk clinical units based on previous poor obstetric history ultimately have uncomplicated pregnancies. Available evidence supports the hypothesis that the aetiology of pregnancy complications begins during 1st trimester [15, 16]. If this is the case, profile of placenta-derived biomarkers during early pregnancy may be common between women with risk of developing pregnancy complications. Identification of such characteristics would provide opportunity to develop clinically useful early pregnancy screening tests.

Previously we have established that normal pregnancy is associated with the increase of exosomes into maternal plasma and the concentration of placenta-derived exosomes increases by 6-fold in uncomplicated healthy pregnancy during the first to third trimester [7] , however, the exosome profile in early pregnancy (i.e. from 6 to 12 weeks) remained to be established. The aim of this study was to characterise placenta-derived exosomes in maternal plasma over the first trimester of pregnancy and observe inter-subject variations in the exosome concentration. Weekly collected blood samples (from 6 to 12 weeks) were collected from normal healthy women to isolate and characterise the exosomes. The presence of exosomes were confirmed by: size (50–120 nM), and buoyant density (1.122- 1.197 g/ml). Endosomal (CD63) and placental (PLAP) antigens were identified in maternal plasma from as early as sixth week of pregnancy. The number of exosomes present in the maternal plasma increased progressively during the first trimester, as well as the exosomal PLAP concentration.

We isolated exosomes from the maternal plasma by differential and buoyant density centrifugation using a sucrose continuous gradient [7, 17]. The purification of exosomes from plasma and other biological fluids is not trivial, however, the use of an automatic system for fraction collection after the sucrose continuous gradient enable a high-reproducibility density, and decreasing the coefficient of variation between samples. In addition, using purification method based on the density of exosomes discards vesicles with the same size of exosomes with no endosomal origin, increasing the purity of exosome samples.

Previous studies have established that extracellular vesicles, including exosomes are released under physiological and pathophysiological conditions as well as during gestation [18]. The release of these vesicles is increased during pregnancy in response to different pathological conditions, presumably due to exosomal secretion from the placental trophoblast cells to the maternal peripheral circulation [19, 20]. In this study, we have established that exosomes are very stable when stored at −80°C. We obtained similar exosome yield from fresh and stored samples (i.e. plasma) and were able to identify gestational age differences in plasma exosome number in samples stored in long term. The isolation of exosomes from stored biofluids is the normal rather than the exception. These results are consistent with those of other studies [21, 22] suggesting that the exosomal content is protected inside these vesicles, highlighting the potential use of exosomes as biomarker for their high stability under different conditions.

As exosomes carry different kinds of protein, mRNA and miRNA [23], engaging in cell-to-cell communication, it is likely that they play an important role in modifying the maternal physiological state to maintain a successful pregnancy [24]. Interestingly, in this study we found that placental-derived exosomes increased systematically during the first trimester as early as sixth week of pregnancy when the intervillous circulation is not fully established. However, it has been observed that communication between placental and fetal circulation occurs at the beginning of the fourth week post conception [25]. Moreover, the lacunar spaces are formed in the trophoblast from as early as nine days post-ovulation and maternal blood flows into the trophoblast lacunae between ten and eleven days after fecundation. In addition, it has been reported that the intervillous blood flow is present in an early stage (i.e. < seventh week) [26] and increases gradually from fourth week during the first trimester of pregnancy [27].

Trophoblast plugs occlude the spiral arteries to prevent the contact of maternal blood flow into the intervillous space, however, at the same time trophoblast plug are in contact with the maternal blood, and could releases soluble proteins (e.g. human chorionic gonadotropin, hCG) and vesicles (e.g. nanovesicles) into maternal circulation. Interesting to highlight that hCG can be measured in maternal plasma as early as 4 weeks of gestation, confirming the presence of molecules released from the trophoblast in early pregnancy. Moreover, β-hCG and pregnancy-associated plasma protein A (PAPP-A) have been measured in maternal plasma as early as 6 weeks of gestation [28].

Specific placental-derived exosomes were quantified in the maternal circulation using the immunoreactive placental protein PLAP. Recent studies have demonstrated the presence of exosomes-PLAP+ive only in peripheral circulation of pregnant women [7, 29]. PLAP is an integral membrane protein (enzyme) unique to the placenta (it has also been observed in some gynaecologic cancers), produced mainly by syncytiotrophoblast [30, 31]. Nevertheless, PLAP expression has been found in primary trophoblast cytotrophoblast cells [7] and ED27 trophoblast-like cells, both isolated from first trimester chorionic villi, and also in JEG-3 cells (a extravillous trophoblast model) [32]. In addition, using immunohistochemistry stain for PLAP, the majority of chorionic trophoblastic cells were positive for PLAP [33]. During the first trimester of pregnancy, the release of placental exosomes into the maternal blood may result from extravillous trophoblast and/or syncytiotrophoblast cells; however, while a definitive answer awaits further investigation, it is of relevance to note that fetal cells are present in maternal blood from 4 weeks of pregnancy and that trophoblast cells invade the decidua and myometrium from the time of implantation. Thus, a cellular and exosomal pathway exists for delivery into the maternal circulation.

Recently, several attempts and techniques were undertaken to determine and characterize the exosomal content in different biological fluids including normal human blood plasma [3436]. As, the content of these released exosomes are placenta- specific [37], studying these nanovesicles is excellent method to understand the different processes occurring during embryo/fetal development and the feto-maternal interaction. Exosome analysis provides diagnostic and therapeutic potential, and biomarker opportunities for the early detection of diseases [3840]. To date, several research studies have been performed to identify the morphologic and proteomic characteristics of exosomes released from the placental extravilous trophoblast cells and expression profile of these exosomal contents relates to common pregnancy conditions [8, 41, 42]. However, all these studies considered the late second or third trimester of pregnancy plasma samples for analysis.

Conclusions

In conclusion, this study present longitudinal data on placental-derived exosomes in the first trimester of pregnancy, starting from as early as 6 weeks after implantation. Early detection of women at risk of complications of pregnancy would provide opportunity to evaluate appropriate intervention strategies to limit acute adverse squeal. The rationale for developing early pregnancy screening tests is not only for the management of the contemporaneous pregnancy but also to optimise lifelong and intergenerational health. If this can be achieved, it will provide an opportunity for early assignment of risk and the implementation of an alternative clinical management strategy to improve outcome for both the mother and baby.

Declarations

Acknowledgements

We acknowledge the assistance of Dr. Jamie Riches and Dra. Rachel Hancock of the Central Analytical Research Facility, Institute for Future Environments, Queensland University of Technology (QUT) for the electron microscope analyses. This project was supported, in part by funding from Therapeutics Innovation Australia.

Authors’ Affiliations

(1)
UQ Centre for Clinical Research, Centre for Clinical Diagnostics, Royal Brisbane and Women’s Hospital, University of Queensland, Building 71/918, Herston, QLD 4029 Queensland, Australia
(2)
Department of Obstetrics and Gynaecology, Faculty of Medicine, Universidad de los Andes, Santiago, Chile
(3)
Department of Obstetrics and Gynaecology, Perinatal unit, Clinica Dávila, Santiago, Chile

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© Sarker et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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