https://orcid.org/0000-0002-0132-9858
Abstract
Ageing and parturition share common pathways, but their relationship remains poorly understood. Decidual cells undergo ageing as parturition approaches term, and these age-related changes may trigger labour. Mesenchymal stem/stromal cells (MSCs) are the predominant stem cell type in the decidua. Stem cell exhaustion is a hallmark of ageing, and thus ageing of decidual MSCs (DMSCs) may contribute to the functional changes in decidual tissue required for term spontaneous labour. Here, we determine whether DMSCs from patients undergoing spontaneous onset of labour (SOL-DMSCs) show evidence of ageing-related functional changes compared with those from patients not in labour (NIL-DMSCs), undergoing Caesarean section. Placentae were collected from term (37–40 weeks of gestation), SOL (n = 18) and NIL (n = 17) healthy patients. DMSCs were isolated from the decidua basalis that remained attached to the placenta after delivery. DMSCs displayed stem cell-like properties and were of maternal origin. Important cell properties and lipid profiles were assessed and compared between SOL- and NIL-DMSCs. SOL-DMSCs showed reduced proliferation and increased lipid peroxidation, migration, necrosis, mitochondrial apoptosis, IL-6 production and p38 MAPK levels compared with NIL-DMSCs (P < 0.05). SOL- and NIL-DMSCs also showed significant differences in lipid profiles in various phospholipids (phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine), sphingolipids (ceramide, sphingomyelin), triglycerides and acyl carnitine (P < 0.05). Overall, SOL-DMSCs had altered lipid profiles compared with NIL-DMSCs. In conclusion, SOL-DMSCs showed evidence of ageing-related reduced functionality, accumulation of cellular damage and changes in lipid profiles compared with NIL-DMSCs. These changes may be associated with term spontaneous labour.
Introduction
As pregnancy approaches term, the decidua undergoes various changes to prepare for labour (Menon et al., 2016; Cox and Redman, 2017). These changes are postulated to originate from the cellular level e.g. from decidual stromal cells (DSCs) and mesenchymal stem cells (MSCs), which are predominant resident cell types in the decidua (Dunn et al., 2003; Abomaray et al., 2016). DSCs are round, epithelioid-like cells that arise following differentiation of the elongated, fibroblast-like endometrial mesenchymal cells during the secretory phase of menstrual cycle (Salamonsen et al., 2009; Okada et al., 2018). As parturition approaches, DSCs undergo various changes similar to those seen in ageing processes, e.g. senescence (Hirota et al., 2010, 2011; Cha et al., 2013; Deng et al., 2016) and inflammation (El-Azzamy et al., 2017). Studies in human and animal models show that senescent DSCs are dominant in labouring term decidua, while hindered progression of DSC senescence delays the timing of parturition (Hirota et al., 2011). Gradual accumulation of senescent DSCs stimulates labour possibly by weakening the physical strength of the decidual tissue, or by secreting senescence-associated secretory phenotype molecules (SASPs), which mostly comprise pro-inflammatory cytokines required for stimulating the contractile state of the myometrium during labour (Norwitz et al., 2015; Cha and Aronoff, 2017).
MSCs are multipotent stem cells capable of self-regeneration and differentiation into various mesenchymal cell lineages, including adipocytes, osteoblasts and chondrocytes. In culture, MSCs are characterised by their adherence to plastic surface, and expression of an MSC-specific combination of surface markers, as defined by the International Society for Cellular Therapy (ISCT) (Dominici et al., 2006). In the decidua, MSCs play an important role in rejuvenating the DSC population. Since labour is associated with ageing of DSCs, we propose that decidual MSCs (DMSCs) may also be affected by the ageing process, and this will be reflected in reduced functionality at term. The decline in numbers or the loss of function of stem cells is called stem cell exhaustion and is a hallmark of ageing (Lopez-Otin et al., 2013). Reduced functionality of DMSCs would limit their ability to replenish aged DSCs, and thus may contribute to the accumulation of senescent DSCs in the labouring decidua. Consequently, the attachment between the decidual tissue and its adjacent gestational tissues, e.g. placenta, may be weakened, thereby facilitating the delivery of the neonate and the placenta during labour at term. Indirect evidence that stem cells play an important role in maintaining tissue strength and integrity comes from studies of muscle cells, where stem cell exhaustion leads to loss of tissue strength during the ageing process (Goldspink, 2007, 2012; Mendelsohn and Larrick, 2016).
Therefore, this study aims to determine whether DMSCs from term (37–40 weeks of gestation) placentae of patients undergoing spontaneous onset of labour (SOL) show evidence of ageing-related functional changes compared with DMSCs from term placentae of patients, not in labour (NIL), undergoing Caesarean section.
Materials and methods
Tissue collection
Term placentae (37–40 weeks of gestation) from patients undergoing SOL + vaginal delivery (n = 18) and NIL + elective C-section delivery (n = 17) were collected by research midwives at the Royal Women’s Hospital, Parkville, Australia. Labour was defined as spontaneous if the contractions occurred naturally without induction and completed without pharmacological augmentation. Further exclusion criteria related to factors that affect placental function or interfere with particular stem cell function. Placental samples were not collected from donors >35 years of age (advanced maternal age) and with a BMI >30 kg/m2 (obese range). Patients with multiple gestations, miscarriage, polycystic ovary syndrome, hyperthyroidism, gastric medicine and aspirin consumption, as well as major foetal and placental abnormalities were omitted from this study. Patients with breech birth and alcohol consumption were excluded from the lipid peroxidation assay. The project was approved by the Royal Women’s Hospital Research and Ethics Committee, VIC, Australia (Projects 10/49 and 12/42). All patients have provided written consent prior to donating their samples.
Isolation, characterisation and culture maintenance of primary cells
Isolation, characterisation (i.e. flow cytometry analyses, in-vitro adipogenic and osteogenic differentiation assays and fluorescence in-situ hybridisation (FISH) analyses) and culture maintenance of DMSCs were performed according to the published methods (Kusuma et al., 2018). Primary cells of maternal origin that were consistent with the ISCT criteria for MSCs (Dominici et al., 2006) were referred as DMSCs. The standard culture medium used was α-MEM complete, which consists of α-MEM (Sigma Aldrich, USA) supplemented with 10% FCS (Gibco, USA), 1% l-glutamine (Life Technologies, USA) and 1% pen/strep (Sigma Aldrich, USA). Most experiments were performed at passage 3–5. DMSCs were used up to passage 5 since subsequent generations do not reliably maintain their stem cell-like properties (Portmann-Lanz et al., 2006; Soncini et al., 2007).
Functional assays
xCELLigence real-time proliferation assay.
xCELLigence system (ACEA Biosciences, Inc., USA) recorded real-time changes in the electrical impedance resulting from cellular events detected by the microelectrode of the xCELLigence E-plate. These changes were reported as arbitrary cell indices. The growth profile of DMSCs was established using the xCELLigence system as previously described (Abumaree et al., 2017; Alshabibi et al., 2017, 2018) with following modifications. SOL- and NIL-DMSCs (n = 10 per group) were plated into a 96-well E-plate in triplicate (1 × 104 cells/100 µl α-MEM complete/well), then incubated at 37°C, 5% CO2 for 400 h without a medium change. Cell index was measured in real time every 5 min for the first 24 h, and subsequently, every 1 h using RTCA Software 1.2.1. Cell index of SOL- and NIL-DMSCs was compared at various time points in attachment and proliferation phase. Proliferation was examined by measuring cell indices at the 120 h time point, and then they were normalised to the indices at 15 h, which was designated as the start of proliferation phase.
Cell viability measurement.
DMSCs were prepared, plated and incubated as described for the xCELLigence real-time proliferation assay, but in a standard 96-well plate. At 250 h, which was designated as the start of survival phase of DMSCs in the xCELLigence real-time proliferation assay, cells were assessed for their viability using Trypan blue exclusion test (Strober, 1997). The numbers of viable SOL- and NIL-DMSCs were compared.
xCELLigence real-time migration assay.
For the migration assay, the xCELLigence system (ACEA Biosciences, Inc., USA) utilises 16-well CIM-plate consisting of two chambers (upper and lower) separated by a polyethylene terephthalate membrane with porous size of 8 µm. Microelectrodes were embedded to the membrane at the lower chamber side. The migration of DMSCs was evaluated using the xCELLigence system as previously described (Abumaree et al., 2017; Alshabibi et al., 2017, 2018) with following modifications. The α-MEM complete medium (165 µl/well) and α-MEM alone (50 µl/well) were added to the lower and upper chamber of the CIM-plate, respectively. SOL- and NIL-DMSCs (n = 8 and n = 7, respectively) were plated into the upper chamber of CIM-plate (4 × 104 cells/100 µl α-MEM alone/well), then incubated at 37°C, 5% CO2 for 24 h. Cell index was measured and recorded real time every 15 min using RTCA Software 1.2.1. Cell index of 0.1 was determined as the migration starting point as per manufacturer’s recommendation.
Cell death assays.
Mitochondrial apoptosis assay
This assay was performed as described by the manufacturer (Biotium, USA) with following modifications. SOL- and NIL-DMSCs (n = 10 per group, 1.5 × 105 cells) were subjected to the JC-1 cationic dye to determine apoptotic cells. Cells were incubated at 37°C for 15 min, washed and resuspended in 500 µl PBS. The expression of JC-1 dye in DMSCs was measured using a BD LSRII flow cytometer and analysed using FACS Diva software (BD). Gating was set as in Fig. 3A and B. The percentages of mitochondrial apoptotic SOL- and NIL-DMSCs were compared.
FITC Annexin V assay
The assay was performed using FITC Annexin V kit (Trevigen, Inc., USA) as previously described (Kusuma et al., 2017). Gating was set as in Fig. 4A and B. Based on the expression of Annexin V and propidium iodide, cells were classified into four quadrants: Q1: necrosis (Annexin V−/Propidium Iodide+), Q2: late apoptosis (Annexin V+/Propidium Iodide+), Q3: viable/healthy (Annexin V−/Propidium Iodide−), or Q4: early apoptosis (Annexin V+/Propidium Iodide−). The percentage of SOL- and NIL-DMSCs (n = 8 and n = 7, respectively) in each quadrant was compared.
Oxidative stress assays.
ALDEFLUOR aldehyde dehydrogenase detection assay
The assay was performed using ALDEFLUOR Kit (STEMCELL Technologies, Canada) as previously described (Kusuma et al., 2017). Gating was set as in Fig. 5A and B. The percentages of SOL- and NIL-DMSCs (n = 8 and n = 7, respectively) that positively expressed aldehyde dehydrogenase (ALDH) (ALDHbr cells) were compared.
TBARS (TCA method) lipid peroxidation assay
The assay was performed according to the manufacturer’s instructions (Cayman Chemical, USA) to quantify the lipid peroxidation marker, malondialdehyde (MDA), produced by SOL- and NIL-DMSCs. MDA reacted with thiobarbituric acid (TBA) generated thiobarbituric acid reactive substances (TBARS), for which the absorbance was measurable at 535 nm. This assay was performed on 2 × 106 DMSCs homogenised in 50 µl PBS (Gibco, USA). The absorbance of each sample was measured using a SpectraMax Plus 384 microplate reader and processed using SoftMax Pro 5 software. Samples were tested in duplicate. The lipid peroxidation level of SOL and NIL samples (n = 7 and n = 5, respectively) proportional to the absorbance value was compared.
ELISAs for IL-6 and IL-8 detection
Serum-starvation and cell lysate collection.
SOL- and NIL-DMSCs (n = 8 and n = 7, respectively, 70% confluent) were serum starved for 48 h in α-MEM supplemented with 1% FCS to normalise the starting point of cytokine production. Complete removal of FCS from the growth medium (α-MEM complete) was not feasible since DMSCs detached when being cultured in this condition, as shown through decreased cell indices in xCELLigence real-time proliferation assay (Supplementary Fig. S7). DMSCs cultured in growth medium supplemented with 1% FCS maintained viability and proliferation comparable to cells cultured in the standard growth medium supplemented with 10% FCS (Supplementary Fig. S7). Therefore, 1% FCS-supplemented medium was used as the serum-starved medium for DMSCs in this study. The serum-starved medium volume was also reduced to 75% from the standard volume to concentrate the secreted cytokine. After 48 h, the medium was collected, then centrifuged at 500g for 5 min and 2000g for 10 min to remove cell debris. DMSCs were collected after dissociation with TrypLE (Life Technologies, USA). Cells were lysed in radioimmunoprecipitation assay buffer (RIPA buffer, Life Technologies, USA) supplemented with 0.002% AEBSF (MP Biomedicals, USA), then centrifuged at 14 000g, 4°C for 15 min. Supernatants (cell lysates) were collected. The protein concentration of the cell lysates was then measured using Pierce BCA method using the kit from Sigma Aldrich, USA.
ELISAs
ELISA kits (Crux Biolab, Australia) were utilised according to the manufacturer’s instruction to quantify the concentration of IL-6 and IL-8 secreted by SOL- and NIL-DMSCs (n = 8 and n = 7, respectively). IL-6 and IL-8 concentrations were normalised to the protein concentration before being compared.
Western blot for p38 mitogen-activated protein kinase detection
Western blot was performed to determine the level of p38 mitogen-activated protein kinase (MAPK) protein (Cell Signaling, USA) in SOL- and NIL-DMSCs (n = 5 per group). Briefly, the cell lysate containing 30 μg of protein from each sample was electrophoresed on a 12% acrylamide SDS PAGE gel (Bio-Rad) and transferred to PVDF membranes. The membrane was blocked with 5% w/v bovine serum albumin (BSA) in TBST for 1 h and incubated overnight at 4°C rabbit anti-p38 primary antibodies. The membrane was washed three times for 10 min each with TBST prior to being incubated anti-rabbit horseradish peroxidase-conjugated IgG for 60 min at room temperature. The membrane signal was detected with ECL prime (GE Healthcare) and the membranes were visualised using a Luminescent Analyzer LAS4000 System (Fujifilm). The signal intensity of each band was calculated in arbitrary units and normalised to the loading control (the house-keeping protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)).
Mass spectrometry-based lipid analyses
Sample preparation.
SOL- and NIL-DMSCs at P5 (n = 7 per group) were serum starved for 48 h. Prior to cell lysis, cells were resuspended in 400 µl PBS (Gibco, USA) and only 50 µl of this cell suspension was lysed and homogenised in RIPA buffer (Life Technologies, USA). Cell lysates were used to measure the protein concentration of the samples using Pierce BCA method (Sigma Aldrich, USA). Cells resuspended in PBS was aliquoted to microtubes at a volume that would give the equivalent of 12 µg protein/sample. Each sample was then spiked in with 5 µl SPLASH® LIPIDOMIX® Mass Spec Standard (Avanti Polar Lipids, Inc., USA) Lipid components of these mixtures were extracted using Folch’s lipid extraction protocol (Folch et al., 1957).
Mass spectrometry analyses.
For the lipid species belonging to the lipid class that was not included in the SPLASH® LIPIDOMIX® mixture (Avanti Polar Lipids, Inc., USA), the relative quantification was determined directly using the peak area of the lipid species, with the assumption of total lipid analysed was equal for all samples as it had been normalised based on the protein concentration of the samples.
Statistical analyses
Data distribution was estimated using the graphical assessment of skewness. Parametric data were analysed using unpaired student t-test (two-tailed), while non-parametric data were analysed using Mann–Whitney U test. Statistical analyses were performed using GraphPad Prism 5.01 software. For xCELLigence migration, Annexin V, ALDH, TBARS lipid peroxidation and ELISAs, the data were generated from the combination of two different batches of patient samples. One sample was tested in both batches as a replication control. Prior to data merger, data were normalised using the average value of each group. The statistical significance was set at *P < 0.05, **P < 0.01 and ***P < 0.001.
Results
Influence of patient-to-patient variation
Clinical characteristics of placental donors are summarised in Supplementary Table S1. The data show the clinical characteristics of patients undergoing SOL and NIL were not significantly different (P > 0.05).
SOL- and NIL-DMSCs expressed stem cell-like properties and were of the maternal origin
Primary cells were isolated from the decidua basalis that remained attached to the maternal side of the placenta after delivery. After one passage (P1), the morphology of the adhered primary cells was uniform under the microscope. All primary cells (P1) showed the typical fibroblastic morphology of MSCs (Supplementary Fig. S1).
Flow cytometry analyses showed that ≥95% cells from both patient groups positively expressed MSC-specific surface markers i.e. CD90, CD 166, CD 73, CD 44 and CD 105 and ≤5% of the cells were positive for haematopoietic markers i.e. CD 45, CD 19 and HLA-DR (Supplementary Fig. S2). Primary cells from both SOL and NIL patients underwent adipogenic and osteogenic differentiation, as shown through the formation of lipid droplets (Supplementary Fig. S3) and calcium deposits (Supplementary Fig. S4), respectively. Primary cells formed colonies when seeded at low density, as expected of MSCs (Supplementary Fig. 5).
FISH analyses of primary cells prepared from placentae of male newborns were carried out. Primary cells were of maternal origin, as shown by the presence of two X chromosomes signals and the absence of Y chromosome signals (Supplementary Fig. S6). FISH analyses are not informative on cells from placentae of female newborn. Cell preparations from the placentae of both female and male newborns were carried out with the same methods, and it was assumed that cell preparations from female newborns did not contain female newborn cells, only maternal cells.
In conclusion, since primary cells isolated from term placentae of patients undergoing SOL and NIL were consistent with the minimum criteria set by ISCT for MSCs (Parolini et al., 2008) and were of maternal origin, they were subsequently referred to SOL-DMSCs and NIL-DMSCs, respectively.
SOL-DMSCs showed functional decline and increased cellular damage compared with NIL-DMSCs
Given that stem cell exhaustion is a hallmark of ageing (Lopez-Otin et al., 2013) and MSCs are the predominant stem cell type in the decidua (Abomaray et al., 2016), we expected to see signs of ageing in DMSCs in labour samples (SOL-DMSCs). We measured cell functions that undergo significant deterioration in other studies of MSCs from aged donors or ageing-related diseases.
The complete profile of cell attachment, proliferation and survival of SOL- and NIL-DMSCs is shown in Fig. 1A. SOL-DMSCs had a significantly reduced proliferation (Fig. 1C), fewer surviving cells (Fig. 1D), a higher percentage of mitochondrial apoptotic cells (Fig. 3C) and necrotic cells (Fig. 4C), and higher levels of lipid peroxidation as shown by higher MDA production (Fig. 5D), as well as higher IL-6 production (Fig. 6A) compared with NIL-DMSCs (P < 0.05). Western blot analyses showed SOL-DMSCs exhibited significantly higher protein levels of p38 MAPK compared with NIL-DMSCs (P < 0.05, Fig. 7B).
xCELLigence real-time proliferation assay of DMSCs. (A) The lines represent the averaged cell indices of NIL and SOL-DMSCs (n = 10 per group). The attachment phase was designated as 0–15 h, the proliferation phase was 15–120 h, the survival phase was from 120 to 300 h and the detachment/death phase was 300 h to the completion of the experiment at 400 h. (B) There were no significant differences in the attachment of SOL- and NIL-DMSCs (P > 0.05). SOL-DMSCs showed a statistically significant reduction of (C) proliferation and (D) cell survivability compared with NIL-DMSCs. Data were presented as mean ± SEM. P-value was calculated using unpaired student t-test. *P < 0.05; **P < 0.01. DMSC, decidual mesenchymal stem/stromal cell; SOL, spontaneous onset of labour; NIL, not in labour.
Not all cell functions were affected. There were no statistically significant differences in attachment capacity (Fig. 1B), the percentage of viable/healthy, early apoptotic, late apoptotic and total apoptotic cells (Fig. 4C), the percentage of ALDHbr cells (Fig. 5C), or IL-8 production (Fig. 6B) between SOL- and NIL-DMSCs (P > 0.05).
SOL-DMSCs showed higher migration capacity compared with NIL-DMSCs throughout the time course (Fig. 2A) with statistically significant differences observed (P < 0.05) at 16 h (Fig. 2B). We suggest that increased migratory capacity of SOL-DMSCs may be related to the inflammatory role(s) of MSCs during labour (see Discussion section).
Migration of DMSCs. (A) X-Y plot of xCELLigence real-time migration over 24 h. The lines represent the averaged cell indices of NIL-DMSCs (n = 7) and SOL-DMSCs (n = 8). (B) SOL-DMSCs showed a statistically significant higher level of migration compared with NIL-DMSCs (P < 0.05) at 16 h. Data were presented as mean ± SEM. P-value was calculated using Mann–Whitney U test. *P < 0.05.
SOL- and NIL-DMSCs expressed the same lipid species, but in different quantities
SOL- and NIL-DMSCs showed statistically significant differences in lipid peroxidation levels (Fig. 5D) and studies report roles for lipids and lipid metabolism during labour (Herrera and Ortega-Senovilla, 2010; Jo et al., 2017; Moayeri et al., 2017; Birchenall et al., 2019). Therefore, we determined the lipid profiles of SOL- and NIL-DMSCs using mass spectrometry. Using LipidSearch, 570 unique lipid species were identified in both SOL- and NIL-DMSCs based on MS/MS fragmentation patterns. These lipid species represented five lipid categories i.e. phospholipids (74%), sphingolipids (17%), neutral lipids (9%), fatty acyls (0.2%) and other lipids (0.2%), consisting of 17 lipid classes (Table I). Lipid classes phosphatidylethanolamine (PE, 24%), phosphatidylcholine (PC, 23%) and sphingomyelin (SM, 10%) contained the highest numbers of lipid species. The percentages were calculated per total count of lipid species identified. These findings were expected since phospholipids and sphingolipids are the major lipid components of plasma membrane. PC, PE, PS and SM contribute to more than half the mass of lipid in most plasma membranes (Alberts et al., 2002). The remaining classes contained <10% of the total identified lipid species.
Summary of the number of identified lipid in SOL- and NIL-DMSCs.
| Lipid category/class | Count | |
|---|---|---|
| Phospholipid (n = 422) | ||
| CL | Cardiolipin | 31 |
| LPC | Lysophosphatidylcholine | 20 |
| LPE | Lysophosphatidylethanolamine | 9 |
| LPI | Lysophosphatidylinositol | 1 |
| PC | Phosphatidylcholine | 129 |
| PE | Phosphatidylethanolamine | 137 |
| PG | Phosphatidylglycerol | 34 |
| PI | Phosphatidylinositol | 46 |
| PS | Phosphatidylserine | 15 |
| Sphingolipids (n = 94) | ||
| Cer | Ceramide | 25 |
| Hex1Cer | Hexosyl ceramide | 6 |
| Hex2Cer | Dihexosyl ceramide | 2 |
| Hex3Cer | Trihexosyl ceramide | 4 |
| SM | Sphingomyelin | 57 |
| Neutral lipids (n = 52) | ||
| TG | Triglyceride | 52 |
| Fatty acyl and other lipids (n = 2) | ||
| AcCA | Acyl carnitine | 1 |
| Co | Coenzyme Q | 1 |
| Total | 570 | |
| Lipid category/class | Count | |
|---|---|---|
| Phospholipid (n = 422) | ||
| CL | Cardiolipin | 31 |
| LPC | Lysophosphatidylcholine | 20 |
| LPE | Lysophosphatidylethanolamine | 9 |
| LPI | Lysophosphatidylinositol | 1 |
| PC | Phosphatidylcholine | 129 |
| PE | Phosphatidylethanolamine | 137 |
| PG | Phosphatidylglycerol | 34 |
| PI | Phosphatidylinositol | 46 |
| PS | Phosphatidylserine | 15 |
| Sphingolipids (n = 94) | ||
| Cer | Ceramide | 25 |
| Hex1Cer | Hexosyl ceramide | 6 |
| Hex2Cer | Dihexosyl ceramide | 2 |
| Hex3Cer | Trihexosyl ceramide | 4 |
| SM | Sphingomyelin | 57 |
| Neutral lipids (n = 52) | ||
| TG | Triglyceride | 52 |
| Fatty acyl and other lipids (n = 2) | ||
| AcCA | Acyl carnitine | 1 |
| Co | Coenzyme Q | 1 |
| Total | 570 | |
Summary of the number of identified lipid in SOL- and NIL-DMSCs.
| Lipid category/class | Count | |
|---|---|---|
| Phospholipid (n = 422) | ||
| CL | Cardiolipin | 31 |
| LPC | Lysophosphatidylcholine | 20 |
| LPE | Lysophosphatidylethanolamine | 9 |
| LPI | Lysophosphatidylinositol | 1 |
| PC | Phosphatidylcholine | 129 |
| PE | Phosphatidylethanolamine | 137 |
| PG | Phosphatidylglycerol | 34 |
| PI | Phosphatidylinositol | 46 |
| PS | Phosphatidylserine | 15 |
| Sphingolipids (n = 94) | ||
| Cer | Ceramide | 25 |
| Hex1Cer | Hexosyl ceramide | 6 |
| Hex2Cer | Dihexosyl ceramide | 2 |
| Hex3Cer | Trihexosyl ceramide | 4 |
| SM | Sphingomyelin | 57 |
| Neutral lipids (n = 52) | ||
| TG | Triglyceride | 52 |
| Fatty acyl and other lipids (n = 2) | ||
| AcCA | Acyl carnitine | 1 |
| Co | Coenzyme Q | 1 |
| Total | 570 | |
| Lipid category/class | Count | |
|---|---|---|
| Phospholipid (n = 422) | ||
| CL | Cardiolipin | 31 |
| LPC | Lysophosphatidylcholine | 20 |
| LPE | Lysophosphatidylethanolamine | 9 |
| LPI | Lysophosphatidylinositol | 1 |
| PC | Phosphatidylcholine | 129 |
| PE | Phosphatidylethanolamine | 137 |
| PG | Phosphatidylglycerol | 34 |
| PI | Phosphatidylinositol | 46 |
| PS | Phosphatidylserine | 15 |
| Sphingolipids (n = 94) | ||
| Cer | Ceramide | 25 |
| Hex1Cer | Hexosyl ceramide | 6 |
| Hex2Cer | Dihexosyl ceramide | 2 |
| Hex3Cer | Trihexosyl ceramide | 4 |
| SM | Sphingomyelin | 57 |
| Neutral lipids (n = 52) | ||
| TG | Triglyceride | 52 |
| Fatty acyl and other lipids (n = 2) | ||
| AcCA | Acyl carnitine | 1 |
| Co | Coenzyme Q | 1 |
| Total | 570 | |
SOL- and NIL-DMSCs showed significant differences in the lipid quantities of various specific lipid species (P < 0.05, Fig. 8), specifically in the category of: (i) phospholipids i.e. class PE (13%), phosphatidylglycerol (PG, 18%), phosphatidylinositol (PI, 22%), and phosphatidylserine (PS, 33%), (ii) sphingolipids i.e. class ceramide (Cer, 12%) and SM (4%), (iii) neutral lipids, i.e. class triglyceride (TG, 6%) and (iv) fatty acyls, i.e. class acyl carnitine (AcCa). The percentages were calculated per total count of lipid species identified in particular lipid class. Some exceptions were observed in the following lipid classes: cardiolipin (CL), lysophosphatidylcholine (LPC), lysophosphatidylinositol (LPI), lysophosphatidylethanolamine (LPE) from the phospholipid category, hexosyl ceramide (Hex1Cer), dihexosyl ceramide (Hex2Cer), trihexosyl ceramide (Hex3Cer) from the sphingolipid category and coenzyme Q (Co). SOL- and NIL-DMSCs did not show any significant differences in lipid quantities for all lipid species in aforementioned classes (P > 0.05).
Generally, compared with NIL-DMSCs, SOL-DMSCs had lower lipid quantities in almost all identified lipid species, except for PE(32:1_20:4) and PE(32:1_22:5), which showed the opposite trend. The complete lists of all identified lipid species with the individual P-value analyses are presented in Supplementary Tables S2–S13.
Discussion
Ageing SOL-DMSCs may promote placental detachment during labour
During parturition, the DMSC niche undergoes substantial transformation to prepare for pregnancy and labour (Norwitz et al., 2015; Kusuma et al., 2016b). This transformation may act as a stress stimulus for DMSCs, triggering their ageing. SOL-DMSCs showed a significantly lower level of proliferation (Fig. 1A and C) and fewer viable cells (Figs 1D and 4C), as well as higher percentage of mitochondrial apoptotic (Fig. 3C) and necrotic cells (Fig. 4C) compared with NIL-DMSCs (P < 0.05). However, there were no statistically significant differences in the percentage of early, late and total apoptotic cells between SOL- and NIL-DMSCs (P > 0.05), although there was a consistent trend towards a higher percentage of the apoptotic cells in SOL-DMSCs.
Mitochondrial apoptosis assay of DMSCs. Representative gating adjustment for (A) SOL-DMSCs and (B) NIL-DMSCs (n = 10 per group). Healthy cells retained JC-1 dye in cytosol and mitochondrial matrix, emitting the green (JC-1 monomeric, detected as the fluorescence intensity on the X-axis) and red fluorescence (JC-1 aggregate, detected as the fluorescence intensity on the Y-axis). Apoptotic cells retained JC-1 dye in cytosol, emitting green fluorescence only (JC-1 monomeric). (C) SOL-DMSCs showed a statistically significant higher percentage of mitochondrial apoptotic cells compared with NIL-DMSCs (P < 0.05). Data were presented as mean ± SEM. P-value was calculated using unpaired student t-test. *P < 0.05.
Annexin V profile of DMSCs. Representative gating adjustment of (A) SOL-DMSCs (n = 8) and (B) NIL-DMSCs (n = 7). Q1: necrosis (Annexin V−/Propidium Iodide+), Q2: late apoptosis (Annexin V+/Propidium Iodide+), Q3: viable/healthy (Annexin V−/Propidium Iodide−), or Q4: early apoptosis (Annexin V+/Propidium Iodide−). X-axis: the fluorescence intensity for Annexin V expression. Y-axis: the fluorescence intensity for propidium iodide expression. (C) SOL-DMSCs had a significantly higher percentage of necrotic cells compared with NIL-DMSCs (P < 0.05). There were no significant differences in the percentage of apoptotic cells (Q2, Q4, Q2 + Q4) and healthy/viable cells (Q3) between SOL- and NIL-DMSCs (P > 0.05). Data were presented as mean ± SEM. P-value was calculated using Mann–Whitney U test. *P < 0.05.
Associations between parturition and reduced MSC proliferation level are only reported in studies of parturition-related diseases (e.g. preeclampsia) (Wang et al., 2012), while associations between parturition and apoptosis are only described in studies of DSCs (Gu et al., 1994). Our study is the first to associate natural parturition with apoptotic or necrotic MSCs.
We postulate that ageing of SOL-DMSCs facilitates placental detachment following labour. The regeneration of connective tissue largely depends on MSCs (Robert, 1980). Given the tenacious attachment between the placenta and decidua, efficient placental detachment may rely on progressive weakening of the decidual tissue as a result of ageing effects on SOL-DMSCs.
The reduced proliferation of SOL-DMSCs may simply be a function of endometrial remodelling rather than a contributor of parturition. However, the effects in SOL-DMSCs are similar to the general ageing process and it is possible that the energy used for maintaining the foetal-maternal interface during pregnancy is redirected to other labour processes.
Necrosis dominates the cellular damage in SOL-DMSCs
The mitochondrial apoptosis assay measures mitochondrial membrane potential, which does not discriminate between apoptosis or necrosis (Kroemer et al., 1998). The FITC Annexin V results showed that necrosis was the predominant type of cell death in SOL-DMSCs. As parturition approaches, labouring foetal membranes feature membrane cells that undergo senescence and secrete SASPs, leading to the release of damage-associated molecular pattern molecules, which is followed by necrosis of the adjacent decidual cells (Menon et al., 2017). Necrosis, unlike apoptosis, is typically accompanied by increased inflammatory responses (Wyllie et al., 1980), and this includes necrosis in the labouring decidua (Menon et al., 2017). This association between necrosis and increased inflammation is supported by our finding that SOL-DMSCs produced significantly higher levels of IL-6 than NIL-DMSCs (P < 0.05, Fig. 6A). Thus, the necrosis of SOL-DMSCs may contribute to the gradual increase in pro-inflammatory responses within the labouring decidua.
SOL-DMSC may form a migration and inflammation response loop to facilitate labour at term
During labour, increased pro-inflammatory responses are found in the decidua (Gomez-Lopez et al., 2014; Norwitz et al., 2015). SOL-DMSCs displayed a pro-inflammatory phenotype, as shown by significantly higher IL-6 production compared with NIL-DMSCs (Fig. 6A). This was consistent with SOL-DMSCs having more necrotic cells than NIL-DMSCs, as described above.
Alternatively, the pro-inflammatory phenotype of SOL-DMSCs may result from functional progesterone (P4) withdrawal during labour. Labour is a state of increased inflammation and involves the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)-mediated inflammatory cascade (Yan et al., 2002; Lin et al., 2018). P4 inhibits NFκB and its withdrawal activates NFκB, which promotes a pro-inflammatory response (Lin et al., 2018). During labour, P4 withdrawal may activate the NFκB-mediated inflammatory cascade in SOL-DMSCs, thereby enhancing the pro-inflammatory phenotype of SOL-DMSCs.
During labour, the secretion of pro-inflammatory factors by SOL-DMSCs may be essential to increase the recruitment and functions (i.e. pro-inflammatory state activation and migration) of various immune cells, such as neutrophils and macrophages, to the decidua (Osman et al., 2003; Ulivi et al., 2014). These immune cells play a crucial role in the onset of labour as they secrete more pro-inflammatory cytokines (e.g. IL-6, IL-1β, IL-8, TNF-α) to the already inflamed decidua, thereby further amplifying the inflammatory responses, up to a certain degree that is sufficient to initiate labour at term (Hamilton et al., 2012; Gomez-Lopez et al., 2014). Multiplex analysis of the chemokine or cytokine secretion profile of SOL-DMSCs could be performed in the future to thoroughly determine the chemoattractive properties of SOL-DMSCs.
With regard to migration, Tlili et al. (2018) reported that inhibiting cell proliferation resulted in increased cell migration. Similar to this finding, SOL-DMSCs in this study showed reduced proliferation and increased migration compared with NIL-DMSCs (P < 0.05, Fig. 2). Among pro-inflammatory cytokines secreted by SOL-DMSCs, IL-6 is a likely contributor to the increased migration potential of SOL-DMSCs. IL-6 induces MSC migration (Pricola et al., 2009; Rattigan et al., 2010) and a study of spheroids showed that the migration of MSCs in the spheroid models was only induced by IL-6 (Lewis et al., 2016; Casson et al., 2018). Given that SOL-DMSCs themselves produce pro-inflammatory cytokines (i.e. IL-6), increased migration of SOL-DMSCs may be required to evenly distribute the immunological responses throughout the labouring decidua for complete and smooth delivery of the neonate and placenta.
Increased oxidative stress in SOL-DMSCs may be crucial for the timing of parturition
Ageing and parturition are both associated with increased levels of oxidative stress. Gestational tissues, including the decidua, are constantly exposed to high levels of oxidative stress, a consequence of elevated pro-inflammatory responses during labour. The decidua is also exposed to physical changes during delivery e.g. sudden exposure to the O2-rich extra-uterine environment, suppressed utero-placental blood circulation and higher consumption of O2. These all contribute to higher production of reactive oxygen species (ROS) at term (Diaz-Castro et al., 2015; Polettini et al., 2015). The finding that SOL-DMSCs had a significantly higher level of lipid peroxidation compared with NIL-DMSCs (P < 0.05, Fig. 5D) was therefore expected. Increased lipid peroxidation levels in the decidua have been reported in patients with faster timing of parturition (e.g. in cases of spontaneous abortion (Sugino et al., 2000)), suggesting a role in the initiation of parturition. In patients experiencing spontaneous abortion, increased levels of lipid peroxidation were associated with decreased levels of superoxide dismutase (SOD) and increased levels of prostaglandin F2α (PG-F2α) (Sugino et al., 2000). Acceleration of parturition was purported to be due to early induction of PG-F2α synthesis as the response to rapid accumulation of ROS and their end products (i.e. lipid peroxide) in the decidua (Hemler and Lands, 1980). This mechanism may also occur in DMSCs during normal term parturition, but at a rate and degree that is sufficient for parturition to proceed without evoking pathological effects.
Oxidative stress profile of DMSCs. Representative gating adjustment of (A) SOL-DMSCs (n = 8) and (B) NIL-DMSCs (n = 7). X-axis: the fluorescence intensity of ALDH expression. Y-axis: the side scatter profile. P1: ALDHbr cells. (C) There were no significant differences in the percentage of ALDHbr cells between SOL- and NIL-DMSCs (P > 0.05). (D) SOL-DMSCs (n = 7) showed a statistically significant higher production of MDA, which is a measure of potential for lipid peroxidation, compared with NIL-DMSCs (n = 5, P < 0.05). Data were presented as mean ± SEM. P-value was calculated using Mann–Whitney U test. *P < 0.05.
Pro-inflammatory secretion profile of DMSCs. (A) SOL-DMSCs (n = 8) produced significantly higher amount of IL-6 compared with NIL-DMSCs (n = 7, P < 0.05). (B) There were no significant differences in IL-8 production between SOL- and NIL-DMSCs (P > 0.05). IL-6 and IL-8 concentrations were normalised per 1000 µg/ml cell lysate protein. Data were presented as mean ± SEM. P-value was calculated using Mann–Whitney U test. *P < 0.05.
Ageing MSCs characteristically have increased levels of oxidative stress, which is often accompanied by reduced antioxidant defences (Chen et al., 2017). One of the major antioxidant components of DMSCs is the ALDH family of enzymes, which detoxify various aldehydes (Vasiliou and Nebert, 2005). DMSCs that express high levels of ALDH in the cell population are called ALDHbr cells in flow cytometry assays. We previously reported that DMSC populations have a high percentage of ALDHbr cells (Kusuma et al., 2016a). However, in this study, SOL-DMSCs did not show any significant differences in the percentage of ALDHbr cells compared with NIL-DMSCs (P > 0.05; Fig. 5C). This suggests that ALDH is not the major antioxidant system used by DMSCs during labour at term and instead, other antioxidant systems are employed but are yet to be identified.
Potential roles of decidual p38 MAPK activation in parturition
SOL-DMSCs showed significantly higher levels of p38 MAPK protein compared with NIL-DMSCs (P < 0.05, Fig. 7B). In murine deciduae, increased p38 MAPK activation was associated with gradual shortening of telomere length, resulting in progressive, telomere-dependent decidual senescence as normal gestation progressed (Bonney et al., 2016). Bonney et al. (2016) suggested p38 MAPK-dependent decidual senescence may promote parturition through SASP secretion. Alternatively, decidual p38 MAPK activation may promote labour by mediating the activation of decidual leukocyte populations. Li et al. (2015) showed that decidual natural killer T cells were activated through the Toll-like receptor, CD1 and inflammatory signalling following the activation of p38 and ERK pathways.
p38 MAPK protein levels in DMSCs. (A) Western blot of p38 and GAPDH protein levels in SOL-DMSCs and NIL-DMSCs. The full blot of p38 protein levels is presented in Supplementary Fig. S8. (B) Quantitation of the bands in Fig. 7A. SOL-DMSCs showed significantly higher levels of p38 MAPK protein compared with NIL-DMSCs (n = 5 per group, P < 0.05). Values were normalised to the house-keeping protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data were presented as mean ± SEM. P-value was calculated using unpaired student t-test. *P < 0.05.
SOL-DMSCs may be involved in similar p38 MAPK-dependent molecular events as those described above. However, our study is the first to report the association between increased levels of p38 MAPK protein, labour and stem cells (i.e. DMSCs). Studies reporting p38 MAPK levels in labouring decidual cells are limited (Sheller-Miller et al, 2018) and further studies are required to determine the mechanism of action of p38 MAPK in these cells.
SOL-DMSC lipids may supply energy for labour
SOL-DMSCs showed a significant decrease in their lipid quantities compared with NIL-DMSCs (P < 0.05, Fig. 8). Most changes in the lipid quantities of SOL-DMSCs were in the phospholipid and sphingolipid species, which are the major components of plasma membrane. With regard to labour, changes in lipid quantities of SOL-DMSCs may be associated with various signalling pathway changes in the decidual tissue in preparation for labour at term.
Quantification of lipid species identified in SOL- and NIL-DMSCs. Significant differences in lipid quantities between SOL- and NIL-DMSCs (n = 7 per group, P < 0.05) were found in various (A) phospholipids, (B) sphingolipids, (C) neutral lipids and (D) fatty acyl. Out of 570 lipid species identified in both SOL- and NIL-DMSCs, only lipid species with P < 0.05 between SOL-DMSCs and NIL-DMSCs are shown in these graphs. Peak area was used for relative quantification of particular lipid species (per 100 µl sample) that were not represented in lipid standards (SPLASH® LIPIDOMIX® Mass Spec Standard, Avanti Polar Lipids, Inc., USA). Data were presented as mean ± SEM. P-value deduced from Mann–Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.00.
Among many labour-associated signalling pathways, those that play an important role in the energy supply for the labour process may be the most affected by changes in SOL-DMSC lipid quantities. Various studies have reported major changes in the lipid profile (i.e. quantities) on the gestational tissue samples as parturition approaches (Herrera and Ortega-Senovilla, 2010; Jo et al., 2017; Moayeri et al., 2017; Birchenall et al., 2019). Among these studies, a pilot study by Birchenall et al. (2019) evaluated and compared the metabolite changes in the maternal and foetal plasma following SOL + vaginal delivery at term in humans (with NIL + elective C-section as control group). Significant changes in the metabolite quantities were found between SOL and NIL samples, with most changes were detected in the lipid categories, particularly in class Cer and AcCa (Birchenall et al., 2019). Birchenall et al. (2019) also discussed the high similarity in metabolite changes between labour and strenuous exercise and suggested that the metabolism process of various macromolecules, such as lipids, may change in order to provide the energy required for these high energy consumption activities (i.e. labour and exercise). Birchenall et al. (2019) also suggested that the energy required for labour was very likely to originate from the maternal tissue (e.g. maternal skeletal muscle, which is derived from osteogenic differentiation of MSCs) since the foetus/neonate is not thought to use its skeletal muscle during labour and delivery. Based on these observations, it is possible that the lipid components of SOL-DMSCs may be catabolised through higher lipolytic activities during labour to provide, at least in part, the energy source that fuels the labour process. Interestingly, similar to parturition, major changes in lipid quantities are reported in several studies of stem cell ageing (Ito and Suda, 2014) and MSC ageing (Kilpinen et al., 2013) with particular changes in the glycerophospholipids (PC, PS, PE and PI) profile. These studies support the notion that changes in lipid quantities of SOL-DMSCs may also result from the ageing process during term labour.
Increased IL-6 and p38 MAPK protein levels, as well as altered lipid profiles, are associated with ageing
Two recent reviews (Cox and Redman, 2017; Sultana et al., 2018) highlighted the role of cellular senescence in ageing of the placenta and decidua and noted that IL-6 is a marker for decidual senescence. IL-6 is an important component of SASP and its expression is associated with genotoxic stress in many human cell types. Moreover, a systemic review and meta-analysis showed that serum levels of IL-6 are a predictor of disability and frailty (Soysal et al., 2016). Finally, an extensive systematic search to identify biomarker candidates for a frailty biomarker panel identified 44 candidate biomarkers. IL-6 was a member of the final core panel of four ageing/frailty biomarkers (IL-6, CXCL10, CXC3CL1 and GDF15) (Cardoso et al., 2018).
Numerous studies show p38 MAPK levels are significantly increased in senescent cells. Furthermore, p38 MAPK activation, in response to various stress stimuli, results in cellular senescence (Wang et al., 2002; Iwasa et al., 2003; Harada et al., 2014; Borodkina et al., 2016). Moreover, p38 MAPK activation continued after the withdrawal of the initial stress stimuli, suggesting the potential involvement of p38 MAPK in the irreversible trait of cellular senescence. Many studies also show the association between increased expression of p38 MAPK and oxidative stress-induced cellular senescence in stem cells (Borodkina et al., 2014, 2016) and other types of cells (Dasari et al., 2006; Barascu et al., 2012; Li et al., 2017). The association between p38 MAPK, oxidative stress and senescence (or ageing) was evident in foetal membranes during pregnancy and parturition. P38 MAPK activation is associated with oxidative stress and DNA damage, which eventually leads to cellular senescence and inflammatory responses (i.e. SASP development) in foetal membrane cells (Menon et al., 2013; Polettini et al., 2015; Menon and Papaconstantinou, 2016).
Sultana et al. (2017) highlighted the effects of oxidative stress on cellular lipid profiles, and serum lipid levels, in placental pathologies associated with premature ageing (preeclampsia, foetal growth restriction, preterm birth) have characteristic changes in lipid distribution and content. Trayssac et al. (2018) highlighted sphingolipids as regulators of ageing and senescence in many cell types and organisms. In particular, the sphingolipid ceramide, which we showed was altered in SOL-DMSCs, is a regulator of cell senescence and ageing.
Changes in DMSC function and lipid profile: a cause or consequence of labour?
In this study, significant ageing-related differences in various important functions (i.e. proliferation, IL-6 production, mitochondrial apoptosis, necrosis and lipid peroxidation) and lipid profiles (i.e. quantities) were detected on SOL- and NIL-DMSCs. These suggest the possible association between active, spontaneous labour and changes in the properties and behaviours of stem cells at term parturition. The possibilities are, either these changes trigger molecular pathways to cause SOL at term or are the consequence of labour itself. The study design of this project was not sufficient to discriminate between cause or consequence with regard to the relationship between labour and the functional and molecular changes in SOL-DMSCs.
Additional groups of samples could provide data to distinguish between cause or consequence. For example, further studies could include a group of ‘emergency C-section shortly after SOL’ patients as controls. In general, ‘emergency C-section shortly after SOL’ patients are patients who have briefly experienced the labour process before undergoing C-section surgery due to various medical complications. In terms of the length of labour, the sequence of patient groups, from the shortest to the longest, should be patients undergoing (i) NIL + elective C-section, (ii) emergency C-section shortly after SOL and (iii) SOL + vaginal delivery. If the functional and molecular profile of DMSCs from placentae of patients undergoing ‘emergency C-section shortly after SOL’ (ECS-DMSCs) resemble those of NIL-DMSCs, it is likely that the changes detected in SOL-DMSCs were the consequence of labour. In other words, if the molecular and functional changes that are detected in SOL-DMSCs, and not in NIL-DMSCs, are not detected in ECS-DMSCs where the donors have undergone the initial phase of labour only, then it is likely that the changes in SOL-DMSCs are a consequence of the normal labour process. However, if the functional and molecular profile of ECS-DMSCs is similar to those in SOL-DMSCs, then it is likely that these changes are the cause of labour since the changes are already occurring in the initial phase of the labour process (Fig. 9).
The rationale of the cause-consequence analyses. NIL-DMSCs: DMSCs from placentae of not in labour patients undergoing elective C-section delivery. ECS-DMSCs: DMSCs from placentae of patients who undergo a brief labour process before emergency C-section delivery is performed. SOL-DMSCs: DMSCs from placentae of patients undergoing spontaneous onset of labour + vaginal delivery. ‘-’ indicates a control level of DMSC function and lipid profile, while ‘↓↑’ indicates altered levels of DMSC function and lipid profile.
Nevertheless, it is worth noting that the collection process of emergency C-section placentae for these analyses may be logistically challenging because of low numbers of samples with suitable medical conditions and the unpredictability of sample availability in normal working hours. Most emergency C-section deliveries are performed due to the foetal and/or maternal medical problem; therefore, the possibility of an underlying pathological condition must be taken into consideration when using these samples. To avoid confounding pathological factors, the ideal patient is one who plans to have an elective C-section, but in whom labour contractions start earlier than expected resulting in the ‘emergency C-section shortly after SOL’ and an uncomplicated outcome for the mother or foetus.
Alternatively, ageing-related gene knock-out studies may also be useful to determine the causal relationship between labour and the functional and molecular changes in SOL-DMSCs. For example, an experiment can be performed in pregnant deciduata models (e.g. murine models) where ageing-related genes (e.g. MTOR or BCAT1) in SOL-DMSCs are specifically knocked out. The timing of parturition of the animal models and changes in SOL-DMSC functions are then observed. Knock-out of these ageing-associated genes should result in the restored functions of SOL-DMSCs. If restored functions of SOL-DMSCs results in the delayed timing of parturition, functional changes in SOL-DMSCs are likely to be the cause of labour. When the cells do not age, as reflected by the restored cell functions, labour does not occur. In contrast, if the restored functions of SOL-DMSCs do not affect the timing of parturition, the functional changes of SOL-DMSCs are likely to be the consequences of labour. Whether the cells age or not, labour will still proceed normally.
Future research may also involve exposing NIL-DMSCs to oxidative stress or necrosis inducers to provide a better understanding regarding of the mechanistic transition between SOL- and NIL-DMSCs.
Acknowledgements
The author(s) acknowledge the patients who consented to provide their samples, the clinical research midwives at the Royal Women’s Hospital, Sue Duggan and Moira Stewart, for sample collection, Dr Matthew Burton and Dr Eleanor L. Jones (Murdoch Children’s Research Institute) for flow cytometry analyses, Dr Mark Pertile and Dr Melissa Glass (Victorian Clinical Genetics Services) for FISH analyses, as well as Dr Shuai Nie (Mass Spectrometry and Proteomics Facility (MSPF), Bio21 Molecular Science & Biotechnology Institute) for mass spectrometry analyses. This work was approved by the Human Research Ethics Committees of Royal Women’s Hospital (Projects 10/49 and 12/42).
Authors’ roles
All authors contributed to the conception and design of the study. J.C.W. and R.K. performed the experiments for data acquisition. J.C.W., P.F.J., B.K. and H.M.G. analysed and interpreted the data. J.C.W. wrote the manuscript and prepared the figures, while R.K., M.I.K., H.M.G., P.F.J., S.P.B. and B.K. critically revised the manuscript. All authors read and approved the final manuscript.
Funding
This work was funded by the Department of Maternal-Fetal Medicine Pregnancy Research Centre, Royal Women’s Hospital, the Department of Obstetrics and Gynaecology of the University of Melbourne, and an Australia Awards Scholarships from the Department of Foreign Affairs and Trade (Australia) to J.C.W.
Conflict of interest
J.C.W., M.I.K., H.M.G., S.P.B. and B.K. declare that there are no potential conflicts of interest with respect to the research, authorship and/or publication of this article. P.F.J. reports personal fees from Exopharm Limited during the conduct of the study and outside of the submitted work. R.K. was subsequently employed by Exopharm Limited following the completion of the work described in this study.
