BOR Papers in Press. Published on October 15, 2014 as DOI:10.1095/biolreprod.114.124248 1   

 

Dynamic Proteomic Profiles of In Vivo- and In Vitro-Produced Mouse Post-Implantation Extra-Embryonic Tissues and Placentas1 RUNNING TITLE: Proteomic profiles of in vitro produced placentas SUMMARY SENTENCE: Comparative proteomic analysis shows in vitro produced extra-embryonic tissues and placentas are characterized by aberrant genetic information processing, altered energy metabolism, and disorganized cytoskeletons and intracellular transport. KEYWORDS: Assisted reproductive technology, in vitro fertilization, extra-embryonic tissues, placenta, proteome. Linlin Sui,4,5 Lei An,4,5 Kun Tan,5 Zhuqing Wang,5 Shumin Wang,5 Kai Miao,5 Likun Ren,5 Li Tao,5 Shuzhi He,5 Yong Yu,5 Jinzhou Nie,5 Qian Liu,6 Lei Xing,6 Zhonghong Wu,5 Zhuocheng Hou,3,5 and Jianhui Tian2,5 5 Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Sciences and Technology, China Agricultural University, Haidian, Beijing, China 6 BGI Tech Solutions Co., Ltd., Beishan Industrial Zone, Shenzhen, China   1 This work was supported by grants from the National High-Tech R&D Program (Nos. 2011AA100303, 2013AA102506) and the National Key Technology R&D Program (Nos. 2011BAD19B01, 2011BAD19B03, 2011BAD19B04). 2 Correspondence: Jianhui Tian, Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Sciences and Technology, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China. E-mail: [email protected]. 3 Correspondence: Zhuocheng Hou, Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Sciences and Technology, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China. E-mail: [email protected]. 4 These authors contributed equally to this work.   

ABSTRACT As the interface between the mother and the developing fetus, the placenta is believed to play an important role in assisted reproductive technology (ART)-induced aberrant intrauterine and postnatal development. However, the mechanisms underlying aberrant placentation remain unclear, especially during the extra-embryonic tissue development and early stage of placental formation. Using a mouse model, this investigation provides the first comparative proteomic analysis of in vivo (IVO) and in vitro produced (IVP) extra-embryonic tissues and placentas after in vivo fertilization and development, or in vitro fertilization and culture, respectively. We identified 165 and 178 differentially expressed proteins (DEPs) between IVO and IVP extra-embryonic tissues and placentas on Embryonic Day 7.5 (E7.5) and E10.5, respectively. Many DEPs were functionally associated with genetic information processing, such as impaired de novo DNA methylation, as well as post-transcriptional, translational and post-translational dysregulation. These novel findings were further confirmed by global hypomethylation, and a

  Copyright 2014 by The Society for the Study of Reproduction.

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lower level of correlation was found between the transcriptome and proteome in the IVP groups. In addition, numerous DEPs were involved in energy and amino acid metabolism, cytoskeleton organization and transport, and vasculogenesis and angiogenesis. These disturbed processes and pathways are likely to be associated with embryonic intrauterine growth restriction, an enlarged placenta and impaired labyrinth morphogenesis. This study provides a direct and comprehensive reference for the further exploration of the placental mechanisms that underlie ART-induced developmental aberrations. INTRODUCTION Assisted reproductive technologies (ART) are increasingly used to help overcome infertility. It is estimated that approximately 1.5 million ART cycles are performed annually worldwide, resulting in 350,000 live births [1, 2]. ART can lead to a series of health problems, including embryonic loss [3], preterm birth and perinatal mortality [4], low birth weight [5], pre-eclampsia [6], and an increased risk of childhood and adulthood diseases [7, 8]. However, the mechanisms that underlie these problems are still unclear. In previous studies, gene and protein expression patterns of in vivo (IVO) and in vitro produced (IVP) embryos have been profiled in mouse [9-11], bovine [12, 13] and porcine [14-16] models, which have provided a reference for understanding the underlying mechanisms of these adverse outcomes. However, the majority of these studies have focused on the embryos or conceptuses. The placenta, which is critical for fetal development, may also contribute to ART-induced aberrations. As the interface between the mother and the developing fetus, the placenta is not only responsible for the maternal-fetal exchange of water, nutrients and electrolytes [17], but also forms a barrier against the maternal immune system and deleterious environmental conditions [18]. In addition, the production of hormones by the placenta is essential for the maintenance of pregnancy [19]. Abnormal placentation leads to compromised intrauterine fetal development and may also be associated with postnatal aberrations, including the adult onset of diseases [20, 21]. Recent works by Rinaudo’s group showed that in vitro derived placentas displayed impaired levels of transplacental nutrient transport, which result in decreased fetal growth and birth weight [22, 23]. A population study showed that ART-conceived pregnancies had increased placental weight and increased placental weight/birth weight ratios, which are associated with adverse pregnancy outcomes [24]. To date, the underlying mechanisms of ART-induced aberrant placentation remain highly elusive. Limited high-throughput analyses of gene expression patterns of in vitro derived placentas during early pregnancy have been conducted in the mouse [25] and bovine [26]. However, with regards to the crucial role of post-transcriptional and translational regulation in placental development and function [27, 28], proteomic analysis, which focuses on direct functional molecules, would provide a greater understanding of the underlying mechanisms that are responsible for the aberrant development and function of in vitro derived placentas. This study aimed to determine the differential protein profiles between in vivo (IVO) and in vitro produced (IVP) extra-embryonic tissues and placentas, and investigate the early stage of placental development in particular. These profiles will provide a reference for the investigation of potential mechanisms that are responsible for ART-induced aberrant placentation and concomitant abnormal embryonic development. Based on our dynamic comparative analysis, we found that IVP extra-embryonic tissues and placentas were characterized by a series of dysregulated genetic information processing processes, including de novo DNA methylation, post-transcriptional regulation, translational and post-translational regulation. The data also

 

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indicated that IVP extra-embryonic tissues and placentas had abnormal energy metabolism, cytoskeletal organization, transport, and vasculogenesis. Together with our recent investigation of the proteomics of IVO and IVP post-implantation embryos [11], we propose that the exposure of pre-implantation embryos to in vitro manipulation and culture may not only notably change the protein profile of post-implantation embryos, but also alter the protein profile of post-implantation extra-embryonic tissues and placentas. These changes in protein expression patterns could contribute to the aberrant development and function of IVP placentas. To our knowledge, this is the first comparative analysis of proteomic profiles between IVP and IVO extra-embryonic tissues and placenta, and provides a reference for the further exploration of placental mechanisms that underlie ART-induced aberrations. MATERIALS AND METHODS Animal Preparation F1 female mice (ICR, 5-6 weeks) and F1 male mice (ICR, 8-9 weeks) were fed ad libitum and housed in a room with a controlled light cycle (12L:12D). All studies adhered to procedures that are consistent with the China Agricultural University Guide for the Care and Use of Laboratory Animals. Females were superovulated by an intraperitoneal (i.p.) injection of 5 IU equine chorionic gonadotropin (eCG), followed 48 h later by an i.p. injection of 5 IU human chorionic gonadotropin (hCG). In the IVO groups, F1 females were natural mated with F1 males after the hCG injection. Experimental Design This study mainly focused on the potential mechanisms responsible for the ART-induced aberrant placentation and concomitant abnormal embryonic development. A well-established experimental design [25, 29] was adopted to test the effect of in vitro fertilization and culture on the proteome of extra-embryonic tissues and placentas during the post-implantation period. As illustrated in Figure 1, all female mice were superovulated and divided into two segments at random. After either in vivo fertilization and development (IVO groups as control) or in vitro fertilization and culture (IVP groups), blastocysts were collected and transferred to pseudopregnant recipients. At E7.5 and E10.5, recipient females were sacrificed, and conceptuses with normal morphology, which were more likely to establish a successful pregnancy, were collected from the uterus of recipients [11]. At E7.5, extra-embryonic tissues, including the ectoplacental cone and extra-embryonic ectoderm, were separated from the epiblast (defined as the IVO 7.5 and IVP 7.5 groups). At E10.5, placentas were sampled after the removal of the yolk sac and embryos (defined as the IVO 10.5 and IVP 10.5 groups). Due to the small yield of protein in one extra-embryonic tissue or placenta, and the limited source of IVP samples, a single pooling strategy was used in our study as reported by previous similar studies [12, 13, 25, 30]. Karp et al. also established that this pooling strategy is appropriate in experiments with low sample yields, and could reduce biological variance, thus increasing the power to detect changes [31]. Moreover, the use of a single pooling strategy was also dependent on the primary aim of our study, which is mainly focusing on the potential mechanisms responsible for the ART induced aberrant placentation and concomitant abnormal embryonic development. ART-induced disorders always display population characteristics. Therefore, single pooling is a rational strategy to ensure a relatively large sample size, which was critical for our study to ensure that the pooled sample was representative of the population. Additionally, considering the requirement of high stability for individual pools, each pool

 

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underwent three technical replicates, and statistical analysis was performed based on the technical replicates [13]. This methodology was also approved by previous reviews regarding the statistical analysis of high-throughput data [32, 33]. In Vitro Fertilization and Embryo Culture In vitro fertilization was performed according to a standard well-accepted procedure [34]. All technical procedures were performed by skilled technicians under strictly controlled conditions and an optimized environment. At 14 h post-hCG treatment, cumulus-enclosed oocyte complexes (COCs) were recovered from oviducts and cumulus cells were removed by digestion with hyaluronidase (Sigma–Aldrich) for 3–5 min. After rinsing, oocytes were kept in the incubator in 60-µl drops of HTF medium (Sage, Bedminster, NJ, USA). Sperm was collected from the cauda epididymis and capacitated for 1 h in HTF medium at 37°C and 5% CO2. After 4 h in the incubator, zygotes were washed and then transferred to 60-µl drops of KSOM medium (Millipore, Billerica, MA, USA). The zygotes were cultured to the blastocyst stage at 37°C in a 5% CO2 atmosphere. Blastocyst Collection and Embryo Transfer In the IVO groups, embryos at the blastocyst stage were obtained from donor females by flushing the uterus with M2 medium. Pseudopregnant female mice were mated to vasectomized males 3.5 days prior to embryo transfer. Twelve blastocysts from the IVO or IVP group were transferred to a single recipient, with six embryos in each uterine horn. Collection of Post-Implantation Extra-Embryonic Tissues and Placentas The criteria for selecting samples for pooling were based on developmental progress and the corresponding morphology, which have been commonly used in previous related studies [9, 12, 25]. At E7.5 and E10.5, conceptuses that showed typical morphological features according to well-established landmarks [35, 36] were selected for pooling. In detail, at E7.5, a well-developed conceptus at the gastrulation stage was characterized by the sealed-off amniotic cavity and the formation of three distinct cavities (amniotic cavity, exocoelom and ectoplacetal cleft). The epiblast was separated from the extraembryonic ectoderm. At E10.5, a definitive placenta was formed, and the well-developed embryos were characterized by the deepening of the lens pit and the appearance of the physiological umbilical hernia. At E7.5, the conceptuses covered with the decidual mass were gently teased away from the uterus, the decidua in which the conceptus embedded was peeled off, and the parietal yolk sac was opened to expose the visceral yolk sac endoderm layer. The extra-embryonic tissues, including the ectoplacental cone and extra-embryonic ectoderm, were separated from the epiblast using microdissecting watchmaker's forceps under a stereomicroscope. At E10.5, after the removal of the yolk sac and embryos, the placentas were sampled. All sampled extra-embryonic tissues and placentas were collected and dropped immediately into liquid nitrogen for further use. In addition, to avoid cross-contamination between embryonic and extra-embryonic tissues, we controlled the dissection by detecting the expression of markers specific to the extra-embryonic ectoderm (ETS-related transcription factor, Elf5) [37], ectoplacental cone (Achaete-scute like 2, ASCL2, also known as Mash2) [38, 39], and epiblast (fibroblast growth factor 5, Fgf5) at E7.5 [40, 41]. Protein Sample Preparation

 

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Samples were ground into powder in liquid nitrogen using a mortar and pestle, and 1 ml lysis buffer was added. The suspension was sonicated at 200 W for 15 min and then centrifuged at 30,000 × g for 15 min at 4°C. The supernatant was mixed well with 10% trichloroacetic acid (TCA) and chilled acetone (1:5, v/v) and incubated at -20°C overnight. After centrifugation at 30000 × g at 4°C, the precipitate was washed with chilled acetone three times. The pellet was air-dried and dissolved in 1 ml lysis buffer. The suspension was sonicated at 200 W for 15 min and centrifuged at 30,000 × g for 15 min at 4°C. The supernatant was then transferred to another tube. DTT (10 mM) was added and incubated at 56°C for 1 h to reduce disulfide bonds. Subsequently, iodoacetamide (IAM; 55 mM) were added to block cysteines in the darkroom for 45 min. The supernatant was mixed well with chilled acetone (1:5, v/v) for 2 h at -20°C to precipitate proteins. After centrifugation at 30,000 × g at 4°C, the supernatant was discarded and the pellet was air-dried for 5 min, dissolved in 500 µl 0.5 M triethylammonium bicarbonate (TEAB, Applied Biosystems, Milan, Italy) and sonicated at 200 W for 15 min. Finally, samples were centrifuged at 30,000 × g for 15 min at 4°C. The supernatant was transferred to a new tube and quantified using the 2-D Quant Kit (GE Healthcare, Amersham Biosciences, Piscataway, NJ, USA) with BSA as a standard according to the manufacturer's instructions prior to the subsequent protein digestion. Briefly, 100 µg of each protein sample in 0.5 M TEAB was carefully taken for digestion with Trypsin Gold at the protein:trypsin ratio of 20:1 at 37°C for 4 h. Trypsin Gold at the same protein:trypsin ratio of 20:1 was added again for a further 8 h of digestion at 37°C. LC-ESI-MS/MS Analysis by LTQ-Orbitrap CID After protein digestion, each peptide sample was desalted using a Strata X column (Phenomenex, Torrance, CA, USA), vacuum-dried and then resuspended in a 200-μl volume of buffer A (2% ACN, 0.1% FA). After centrifugation at 20,000 × g for 10 min, the supernatant was recovered to obtain a peptide solution with a final concentration of approximately 0.5 µg/µl. Ten microliters of the supernatant was loaded into a Shimadzu LC-20AD nano HPLC by the autosampler onto a 2-cm C18 trap column (inner diameter 200 μm), and the peptides were eluted onto a resolving 10 cm analytical C18 column (inner diameter 75 μm) made in-house. The samples were loaded at 15 μl/min for 4 min, and then the 91-min gradient was run at 400 nl/min starting from 2% to 35% buffer B (98% ACN, 0.1% FA), followed by a 5-min linear gradient to 80%, maintenance with 80% buffer B for 8 min, and finally returning to 2% within 2 min. The peptides were subjected to nanoelectrospray ionization followed by tandem mass spectrometry (MS/MS) in an LTQ Orbitrap Velos (Thermo Fisher Scientific, San Jose, CA, USA) coupled online to the HPLC. Intact peptides were detected in the Orbitrap at a resolution of 60,000. Peptides were selected for MS/MS using the collision-induced dissociation (CID) operating mode with a normalized collision energy setting of 35%, and ion fragments were detected in the LTQ at a resolution of 7,500. A data-dependent procedure that alternated between one MS scan followed by ten MS/MS scans was applied for the ten most abundant precursor ions above a threshold ion count of 5,000 in the MS survey scan, with the following Dynamic Exclusion settings: repeat counts, 2; repeat duration, 30 s; and exclusion duration, 120 s. The electrospray voltage applied was 1.5 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 1 × 104 ions were accumulated in the ion trap for the generation of the CID spectra. For MS scans, the m/z scan range was from 350 to 2,000 Da. Proteomic Analysis

 

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Mass spectra were analyzed using the software MaxQuant (version 1.2.0.18) [42]. MS/MS spectra were searched using the Andromeda search engine [43] against the International Protein Index (IPI) mouse database version 3.84 containing forward and reverse sequences (119,990 entries in total, including forward and reverse sequences). In the main Andromeda search, precursor mass and fragment mass had an initial mass tolerance of 6 ppm and 0.5 Da, respectively. The search included a fixed modification of cysteine carbamidomethylation and variable modifications of methionine oxidation and protein N-terminal acetylation. The minimal peptide length was set to six amino acids, and a maximum of two miscleavages was allowed. The false discovery rate (FDR) was set to 0.01 for peptide and protein identifications. Proteins were considered to be positively identified when at least two peptides were identified and at least one of which was uniquely assignable to the corresponding sequence. In the case of identified peptides that were shared between two proteins, these were combined and reported as a single protein group. Label-free quantitation analysis was performed in MaxQuant. Briefly, total peptide signals were determined in the mass to charge, elution time and intensity space. For every peptide, corresponding total signals from multiple runs were compared to determine peptide ratios. Pair-wise peptide ratios were only determined when the corresponding peak was detected in both LC-MS runs. For between-sample comparisons, label-free quantification was performed with a minimum of two ratio counts to determine the normalized protein intensities, which were then averaged from the three replicates as the protein abundance for each group. To facilitate data analysis, all protein IDs were mapped to the Ensembl Mus musculus gene IDs or gene symbol using the corresponding tools for data analysis. The significance of protein abundance differences between the IVO and IVP groups was evaluated using a two-sample heteroscedastic t-test (two-tailed), DEPs were considered significant at probability values of P < 0.05. As only one biological replicate represented by a single pool containing from 70 to >300 embryos, which originated from more than 50 donors and multiple independent experimental batches, was available for each time point, our selection of DEPs was based on the statistical analysis of technical replicates, as a previous similar study has reported [13]. This methodology was also approved by previous methodological reviews [32, 33]. Hierarchical clustering was performed using the Cluster 3.1 to assess the similarities of the different replicates. The Database for Annotation, Visualization and Integrated Discovery (DAVID v6.7; http://david.abcc.ncifcrf.gov) was used to annotate biological themes in response to different physiological conditions. The Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) was used to determine the associated pathways. In addition, DEPs were sent to the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING; http://string.embl.de/) to build a protein-protein interaction (PPI) network. Phenotype annotations of DEPs were analyzed based on the Mouse Genome Informatics (MGI, http://www.informatics.jax.org/phenotypes.shtml) Database. The correlation coefficient is frequently used to evaluate the relationship between protein abundance and mRNA expression on a genomic scale [44-46]. To evaluate the relationship between protein abundance and mRNA expression at E7.5 and E10.5, Pearson’s correlation coefficients (PCC) and corresponding P-values between the normalized mRNA (Reads Per Kilobase of exon model per Million mapped reads, RPKM) and protein abundance (Label Free Quantification, LFQ) of each group were calculated using the R software package (http://www.r-project.org). RNA Sequencing (RNA-seq)

 

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Total RNAs were extracted from extra-embryonic tissues and placentas with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Polyadenylated RNAs were isolated using the Oligotex mRNA Midi Kit (Qiagen, Valencia, CA, USA). The RNA-seq libraries were constructed using SOLiD Whole Transcriptome Analysis Kit following the standard protocol (AB, Applied Biosystems) and sequenced on the Applied Biosystems SOLiD platform to generate high-quality single-end reads. The raw reads were aligned to genome sequences, trimming off a nucleotide each from the 5’ and 3’ ends and allowing up to two mismatches. Reads mapped to multiple locations were discarded and only uniquely mapped reads were used for the subsequent analysis. Gene expression levels were measured in RPKM. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) Genomic DNA was isolated from extra-embryonic tissues and placentas using Qiagen DNeasy Kits (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The quality of each DNA sample was analyzed for integrity, purity and concentration on a Nanodrop Spectrophotometer DN-1000 (Nanodrop Technologies, Wilmington, DE, USA). Genomic DNA was then fragmented using a Covarias sonication system (Covarias, Woburn, MA, USA). After sonication, the fragments were denatured to produce single-stranded DNA (ssDNA). Following denaturation, the ssDNA was incubated with monoclonal 5-methyl-cytidine (5mC) antibodies. Magnetic beads conjugated to anti-mouse-IgG were then used to bind the anti-5 mC antibodies, and unbound DNA was removed along with the supernatant. Finally, proteinase K was used to digest the antibodies and the DNA was released and collected for sequencing with the HiSeq 2000 Sequencing System (San Diego, CA, USA). Morphometric and histological analysis, immunostaining E7.5 embryos and extra-embryonic tissues were photographed at E7.5, while the prenatal fetuses and placentas were weighed at E19. E7.5 embryos and extra-embryonic tissues and E19 placentas were isolated quickly and then fixed with 4% paraformaldehyde (Sigma–Aldrich). Samples were dehydrated with an ethanol and toluene series (Beijing Chemical Works) and embedded in paraffin (Leica, Wetzlar, Germany). Serial sections (6-µm thickness) were mounted on gelatin-coated glass slides and stained with hematoxylin and eosin (H&E). For immunohistochemistry analysis, antibodies specific to laminin affinity-isolated antigen-specific antibody (Sigma), were used in 6-µm thick paraffin embedded sections. A Histostain-SP Kit (Zhongshan Golden Bridge Biotechnology) was used to visualize the antigen. Western Blotting Extra-embryonic tissues (n = 24 for each group) and placentas (n = 14 for each group) were collected, homogenized and stored in 2 × Laemmli buffer (Bio-Rad, Hercules, CA) with protease inhibitors. Prior to analysis, the samples were thawed and subsequently heated to 100°C for 10 min. The proteins were separated on 10% or 12% acrylamide gels containing 0.1% SDS and then transferred onto hydrophobic PVDF membranes (Amersham, Piscataway, NJ, USA). The membranes were blocked with 5% non-fat dried milk in TBS containing 0.05% (v/v) Tween-20 for 1 h at room temperature and incubated with a diluted antibody overnight at 4°C, followed by three (20-minute) washes in TBS containing 0.05% (v/v) Tween-20. A peroxidase-conjugated secondary antibody (Zhongshan Golden Bridge Biotechnology, Beijing, China) was added for 1 h, and protein bands were then detected using an ECL-plus system (Amersham, Piscataway, NJ, USA).

 

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RESULTS Morphometric and Phenotypic Analysis In the present study, IVP embryos had a comparable ability to achieve implantation as IVO embryos with an implantation of 78.60% compared to 80.14%, respectively. However, IVP embryos showed a significantly lower survival rate during the early-gestational stage (E7.5, 46.80% vs. 37.75%, P < 0.05), and this difference extended into the prenatal period. The percentage of morphologically aberrant conceptuses (including embryonic and extra-embryonic tissues) in the IVP group was approximately 10% higher than that in the IVO group at E7.5. The percentage of morphologically aberrant conceptuses (including embryonic and extra-embryonic tissues) in the IVP group was 62.5%, which was approximately 10% higher than observed in the IVO group at E7.5. At E7.5, the amniotic cavity is divided into three distinct sections, the amniotic, exocoelom and ectoplacetal cleft. In the current investigation, well-developed extra-embryonic tissues were distinctly divided into the ectoplacental cone and extra-embryonic ectoderm (Figure 2A, a and b). In the IVP group, an increase in delayed embryonic and extra-embryonic development was observed (Figure 2A, c-f), as well as disorganized or degenerating extra-embryonic tissues (Figure 2A, g and h). These observations were similar to our previous morphological and histological analyses [11]. At E10.5, we found that IVP embryos and placentas showed a greater variability in terms of size compared to the IVO samples (Figure 2B). Previous investigations have shown that processes and manipulations associated with IVP can lead to decreased fetal weight from the mid- to late-gestational periods [22, 23]. Our recently published study also indicated that this deficiency could have originated during the post-implantation period [11]. Here, we found that this trend extends to the prenatal stage. Moreover, the IVP group showed enlarged placentas at the prenatal stage, which produced an increased placental/fetus ratio (Figure 3A). At the prenatal stage, we observed that an increased number of IVP placentas showed aberrant morphology when the relative areas of the labyrinth and spongiotrophoblast layers were quantitatively analyzed in cross sections. Compared with the IVO placentas, the development of both areas were abnormal in IVP placentas (Figure 3B). In particular, the labyrinth layer, which is highly associated with placental vasculogenesis, was impaired in IVP placentas. Moreover, the degree of vasculogenesis as labeled with laminin showed an evident deficiency in IVP placentas from the mid- to late-gestational stage (Figure 3C and D). General Proteomic Profiling of IVO and IVP Extra-Embryonic Tissues and Placentas In the IVO7.5, IVP7.5, IVO10.5, and IVP10.5 groups, 374, 294, 77, and 76 morphologically normal extra-embryonic tissues or placentas were collected and sampled, respectively. Using these samples, we positively identified 953, 969, 897 and 881 proteins in the IVO7.5, IVP7.5, IVO10.5, and IVP10.5 groups, respectively, according to the above-described criteria. Based on the overall similarity of gene expression patterns, hierarchical clustering was performed on the different treatments and technical replicates. The results showed the proteomes of IVP extra-embryonic tissues and placentas were strikingly distinct from those of the IVO groups at both E7.5 and E10.5. Data from each technical replicate from each condition were tightly clustered in the same branch, which confirmed the reliability of our detection system (Figure 4A). The precision of quantitation between the technical replicates was also evaluated by PCC [47]. We determined an average PCC of 0.9977 for the protein level and 0.9734 for the

 

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peptide level (Supplemental Table S1 and Figure S1; Supplemental Data are available online at www.biolreprod.org); the correlation values between technical replicates of the peptide and protein level should be satisfying according to the criteria provided by Waanders (0.87 and 0.98 for the peptide and protein level, respectively) [48]. The well-controlled LC-MS/MS detection system also reflected the good quality of chromatographs (Supplemental Figure S2) and MS/MS spectra (Supplemental Figure S3). Validation of the proteomic data was performed using Western blotting (Supplemental Figure S4). Among the proteins that were significantly altered in the IVP groups, the results of Western blotting displayed similar alterations with fold changes according to LC-MS/MS analysis. When the proteomes were compared between the IVO and IVP groups, 165 proteins and 178 proteins were differentially expressed (P < 0.05) at E7.5 (Supplemental Table S2) and E10.5 (Supplemental Table S3). Among the DEPs observed at E7.5, 46 proteins were upregulated and 119 proteins were downregulated in IVP extra-embryonic tissues. At E10.5, 41 proteins were upregulated and 137 proteins were downregulated in IVP groups (Figure 4B). Among these DEPs, 38 were dysregulated at both time points, which accounted for 23.0% and 21.3% of DEPs from E7.5 and E10.5, respectively. These DEPs were mainly associated with the regulation of gene and protein expression, transport, cytoskeleton organization and immune modulation (Figure 4C). Functional Proteomic Profiling of IVO and IVP Extra-Embryonic Tissues and Placentas The DAVID tool was used to show the Gene Ontology (GO) classification of DEPs between IVP and IVO extra-embryonic tissues from E7.5 (Figure 5A) and placentas from E10.5 (Figure 5B), for which 20 and 26 subcategory terms were respectively identified. Among these subcategories, “primary metabolic processes”, “cellular metabolic processes” and “macromolecular metabolic process” were the three most representative processes; these were involved in glucose metabolism, lipid metabolism and amino acid metabolism. Several other biological processes involved in intracellular localization or transport were also dysregulated in IVP extra-embryonic tissues and placentas, including “cellular localization”, “organelle organization”, “regulation of cellular component organization”, “cellular macromolecular complex subunit organization” and “transport”. A KEGG pathway analysis was performed on the DEPs from E7.5 and E10.5. As shown in Table 1, several pathways associated with genetic information processing (including “Spliceosome”, “Ribosome” and “Ubiquitin mediated proteolysis and proteasome), as well as metabolism pathways (such as “Citrate cycle”, “Glycolysis and Gluconeogenesis”, “Glyoxylate and dicarboxylate metabolism” and “Pentose phosphate pathway”) were identified. Unexpectedly, many pathways involved in human diseases were also represented, including cardiovascular (such as “Dilated cardiomyopathy” and “Arrhythmogenic right ventricular cardiomyopathy”) and neurodegenerative diseases (such as “Parkinson's disease” and “Huntington's disease”). Detailed analysis showed that the DEPs in these disease pathways were mainly involved in cytoskeleton organization and mitochondrial function. Using DEPs as seed nodes, a PPI network was constructed (Figure 6). DEPs were mainly enriched in the terms “post-transcriptional regulation”, “cytoskeleton organization and transport” and “translation regulation” at both E7.5 and E10.5. The two time points also showed time point-specific enrichment in terms such as “Hematopoiesis”, “Aminoacyl-tRNA Biosynthesis” and “DNA replication and cell cycle” at E7.5, and “glucose and energy metabolism” and “Proteasome” at E10.5.

 

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The MGI database showed many phenotypes associated with embryonic and placental development (Supplemental Table S4 and S5). We observed many proteins involved in energy metabolism, such as fatty acid synthase (Fasn) and lactate dehydrogenase A (Ldha), which were annotated with terms such as “embryonic lethality” and “decreased body weight and size”. We also found numerous cytoskeletal proteins, such as talin 1 (Tln1) and tubulin alpha 1A (Tuba1a), were associated with phenotypes such as “abnormal placenta morphology” and “embryonic growth retardation”. At both E7.5 and E10.5, many of these dysregulated proteins, including keratin 8 (Krt8) and vimentin (Vim), were also associated with vasculogenesis and angiogenesis. Related phenotypes included “abnormal placental labyrinth vasculature morphology”, “abnormal angiogenesis” and “abnormal vascular development”. Our results also indicated that IVP post-implantation extra-embryonic tissues and placentas may have suffered from defective hematopoiesis. Furthermore, many DEPs, including scaffold attachment factor B (Safb) and tissue factor pathway inhibitor (Tfpi), were associated with phenotypes such as “abnormal hematopoiesis”, “absent visceral yolk sac blood islands” and “abnormal erythropoiesis”. Correlation between Transcriptome and Proteome, and Global Methylation Profiling During the past 2 years, our research group has profiled the dynamic transcriptomes (RNA-seq) and methylome (methylated DNA immunoprecipitation-seq, MeDIP-seq) of IVO and IVP extra-embryonic tissues and placentas. Using these transcriptomic data, the levels of mRNAs (measured in RPKM) and corresponding proteins (measured by LFQ), were analyzed to obtain a PCC for each group (Supplemental Tables S6 and S7). We used PCC to evaluate the relationship between the mRNA and protein abundances of the IVO and IVP groups at each time point. The PCCs were 0.35 (P = 0.0005), 0.28 (P = 1.34e-5), 0.23 (P = 0.003), and 0.15 (P = 0.05) in the IVO 7.5, IVP 7.5, IVO 10.5 and IVP 10.5 groups, respectively. At both time points, IVP extra-embryonic tissues and placentas showed a lower PCC (Figure 7A), which suggested that the level of correlation between mRNA and protein abundance was lower in the IVP extra-embryonic tissues and placentas than those of the IVO. The downregulation of DNA methyltransferase 3A (Dnmt3a), and its co-factor, histone deacetylase 2 (Hdac2), as well as two important enzymes in the methylation pathway, methylenetetrahydrofolate dehydrogenase (Mthfd1) and S-adenosylhomocysteine hydrolase (Ahcy), led us to hypothesize that IVP extra-embryonic tissues and placentas underwent a compromised process of de novo DNA methylation (Figure 7B). To prove this hypothesis, we performed a hierarchical cluster analysis based on the enrichment of MeDIP reads in the promoter, gene body and 3’UTR regions (Supplemental Table S8). We found that more differentially methylated regions (DMRs) in the IVP extra-embryonic tissues were hypomethylated compared to IVO samples (Figure 7C). In addition, the global hypomethlation was particularly apparent in the gene body region, we found more DMRs in the gene body region showed an IVP-specific deficiency of DNA methylation (indicated by arrow in Figure 7C). DISCUSSION To date, investigations of ART-related aberrations have focused on the contribution and impact of embryos and fetuses, and the possible involvement of the placenta in the development of such aberrations remains largely unknown. To our knowledge, this is the first comparative analysis of proteomic profiles between in vivo- and in vitro-derived extra-embryonic tissues and placentas. Our data showed distinct protein patterns in extra-embryonic tissues and placentas associated with IVO and IVP conditions during early pregnancy. Using functional clustering methods, we

 

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found the DEPs were mainly associated with the regulation of gene and protein expression, energy and amino acid metabolism, cytoskeleton organization and transport, and vasculogenesis, angiogenesis and hematopoiesis. Disturbed Regulation of Gene and Protein Expression at Multiple Levels In the present study, we observed IVP extra-embryonic tissues and placentas were characterized by disturbed genetic information processing, including those involved in the “Spliceosome”, “Ribosome” and “Proteasome”. These novel findings were further confirmed by consistently lower correlation levels between mRNA and proteins in the IVP groups. In previous studies, post-translational, translational and post-translational modifications have been proposed to result in decreased levels of correlation between mRNA and proteins [49, 50]. Our data showed that the lower mRNA-protein correlations were not only reflected on the genomic scale, but also for specific proteins. We noticed that the fold changes of many DEPs were highly variable between the mRNA and protein level. In particular, some proteins were not obviously altered at the mRNA level, but were significantly different at the protein level. It suggested that the fold changes of these proteins were largely attributable to the disturbed post-transcriptional, translational and post-translational processes. Alternative splicing, catalyzed by the spliceosome, is prevalent in higher eukaryotes and plays a major role in the generation of proteomic diversity and gene regulation. Up to 59% of human genes express more than one mRNA by alternative splicing [51]. Previous studies have shown that alternative splicing is an important developmental regulatory mechanism for the placenta [52]. Abnormal variations in splicing have been implicated in diseases such as various forms of cancer and Alzheimer’s disease [53]. In the placenta, alternative splicing has been suggested to be associated with pre-eclampsia (PE), gestational diabetes mellitus (GD) [20, 54, 55], gestational hypertension and fetal intrauterine growth restriction (IUGR) [56]. We found that all of the DEPs associated with spliceosomes at E7.5, including PRP6 pre-mRNA splicing factor 6 (Prpf6) and splicing factor 3b, subunit 2 (Sf3b2), as well as most of the DEPs in this pathway, such as heterogeneous nuclear ribonucleoprotein U (Hnrnpu), Hnrnpc and HnrnpM, were downregulated in the IVP groups. Among the differentially expressed spliceosomal proteins, Sf3a1 was consistently downregulated from E7.5 to E10.5, which suggested that Sf3a1 contributes largely to the aberrant post-transcriptional regulation in IVP extra-embryonic tissues and placentas. The results of the KEGG pathway and PPI network analyses indicated that IVP extra-embryonic and placental tissues also had aberrant translational processes. At E10.5, nearly all ribosomal proteins, including ribosomal proteins S15A (Rps15a), Rps19, Rps6, Rpl4, Rps16 and Rpsa, showed a significant depression in IVP placentas, which indicated the impaired activity or functioning of ribosomes. As the central workhorses responsible for mRNA decoding and protein biosynthesis, dysfunction of the ribosome may lead to aberrant translational activity, which is believed to be associated with the pathology of many diseases [57]. Previous studies have reported the dysregulation of ribosomal proteins in the placenta of patients who suffered from PE, IUGR and spontaneous abortion [58, 59]. In addition, at the two time points studied in this current investigation, many factors that are essential for the translation process, including eukaryotic translation initiation factors (Eif3c, Eif3g, Eif3i, Eif2s3x, Eif3f and Eif3b) and eukaryotic translation elongation factors (Eef1g and Eef1b2), were also dysregulated. A recent study has demonstrated that the translation initiation factor plays an essential role in the function of extravillous trophoblast cells, which are critical for placental implantation [60]. Related

 

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studies have also reported the involvement of placental translation inhibition in the pathology of IUGR and PE [28], as well as early pregnancy loss [61]. The ubiquitin-proteasome system (UPS) is a non-lysosomal proteolysis system responsible for the degradation of irrelevant or misfolded intracellular proteins. The proteasome-dependent regulation of protein abundance, as an important post-translational modification method, plays a crucial role in both physiological and pathological processes [62]. The results of KEGG pathway analysis showed a consistent deregulation of ubiquitin-proteasome pathway (UPP). In our study, all DEPs enriched in UPP were downregulated in IVP groups, including ubiquitin-like modifier activating enzymes (Uba1, Uba2 and Uba6), ubiquitination factor E4B (Ube4b), proteasome 26S subunit, non-ATPase, 11 (Psmd11), proteasome 26S subunit, ATPase 2 (Psmc2) and proteasome subunit, alpha type 1 and 7 (Psma1, Psma7), which shows that proteasome activity was deficient in the IVP extra-embryonic and placental tissues. Previous clinical studies have demonstrated the association of altered placental proteasome activity with PE and IUGR [63] It is widely accepted that ART can disrupt epigenetic modifications during embryogenesis and these epimutations could lead to altered patterns of gene expression [10]. However, to date, the underlying mechanisms that lead to epimutations have been unclear. Key players involved in epigenetic modifications, such as Dnmt3a and its co-factor Hdac2 at E7.5, as well as Hdac1 at E10.5, were dysregulated in IVP extra-embryonic and placental tissues. Dnmt3a, which is crucial for de novo DNA methylation in post-implantation embryos and placentas, was found to be deficient in IVP extra-embryonic tissues at E7.5. In addition, two important enzymes in the methylation pathway, Mthfd1 and Ahcy, were also downregulated at E7.5. These data imply that de novo DNA methylation during the post-implantation period in IVP extra-embryonic tissues may have been incomplete. Analysis of the global methylation profile of the IVP samples confirmed this presumption and indicated that a deficiency of these enzymes was likely to lead to impaired de novo DNA methylation. The global hypomethylation observed in IVP samples was similar to that observed by a previous clinical study in which placental tissue and cord blood from in vitro conceptions showed a lower mean methylation at CpG sites [64]. Global DNA hypomethylation in placentas, especially the hypomethylation of imprinted genes, has been suggested to be associated with IUGR [65]. Altered Energy and Amino Acid Metabolism GO and KEGG analyses showed that IVP extra-embryonic tissues and placentas are characterized by dysregulated metabolic pathways, including the TCA cycle pathway, glycolysis and gluconeogenesis. The placenta can perform glycogenic functions before the fetal liver has acquired this capacity [66]. Fetoplacental glucose is derived entirely from the maternal supply [67]. A crucial function of the placenta is to transfer glucose from the maternal circulation to the fetus. Glucose is then directly used as a source of energy by the placenta or the fetus, or can be stored as glycogen in the placenta [68]. Previously, alterations in placental glucose metabolism were shown to be highly associated with fetal IUGR [69]. In the current investigation, metabolic enzymes in the citrate cycle (TCA cycle) pathway, such as ATP citrate lyase (ACLY) and dihydrolipoamide S-succinyltransferase (DLST), displayed consistent changes from E7.5 to E10.5, as indicated by the GO analysis. The KEGG pathway and PPI network analyses also showed that the TCA cycle pathway was disrupted. In addition, the aberrant metabolism and transport of glucose in the placenta has been associated with placental hypertrophy and fetal hypoglycemia [70]. KEGG pathway analysis also showed decreased levels of glycolysis and gluconeogenesis. It

 

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should be noted that all of enriched DEPs, including glucose phosphate isomerase 1(GPI1), aldolase A (ALDOA), PGM2 (phosphoglucomutase 2) and PGK1 (phosphoglycerate kinase 1), were downregulated in IVP placentas. Glycolysis and gluconeogenesis is critical for the differentiation of human placental trophoblast cells to the syncytiotrophoblast [71], which is essential for the establishment of nutrient circulation between the embryo and the mother, as well as the tolerance of the fetus to the maternal immune system [72]. It is of interest that the DEPs were significantly enriched in pathways involved in the pathology of neural diseases. In detail, these proteins were mainly involved in mitochondrial functions. These mitochondria-associated proteins have been well documented to be involved in the placental transport of carbohydrates, lipids and amino acids, as well as steroidogenesis [73] and pathological processes such as PE [74] and fetal IUGR [69]. Amino acids play a central role in cell function and provide a vital source of energy for fetal growth. At both time points, several DEPs involved in glutamine metabolism were downregulated in IVP tissues, which suggested that glutamine metabolism was impaired. Glutamine plays an extremely important role in fetal and placental nutrition [75]. This unique amino acid, which can be actively synthesized and released by the placenta [76], is delivered into the fetal circulation at a higher rate than all other amino acids [77]. Impaired glutamine metabolism in IVP placentas leads to lower levels of glutamine in the fetal circulation, which is associated with IUGR [78]. In addition, the placental metabolism of glutamine may be closely correlated with progesterone biosynthesis [79], which is a crucial hormone for the maintenance of pregnancy. We found that many proteins involved in energy and amino acid metabolism were associated with the phenotypes such as “embryonic lethality during the peri-implantation period”, “embryonic and fetal growth retardation” and “decreased body weight and size”. These phenotypes were highly concordant with our observations of increased embryonic lethality and retardation during the peri-implantation period. Furthermore, we also found that restricted embryonic and fetal growth was related to a decrease in prenatal fetal weight and an increase in placental weight, which may represent compensatory effects [80]. Although having a comparable ability to achieve implantation, IVP embryos showed significantly lower survival rates during the post-implantation period than IVO embryos. In addition, an increased number of conceptuses in the IVP group showed aberrant morphologies, and those abnormal conceptuses were more likely to be eliminated during this stage. Although the growth of IVP embryos/fetuses were consistently depressed from the early- to late-gestational stage, extra-embryonic tissues/ placentas showed a different pattern. In accordance with the findings of a previous study, the development of IVP extra-embryonic tissues were delayed or retarded at the early-gestational stage, but became progressively larger during gestation [22]; ultimately, they became larger than those in the IVO group, possibly to compensate for their earlier deficiency in size. Aberrant Cytoskeleton Organization and Transport In the present study, many cytoskeleton proteins were found to be differentially expressed in extra-embryonic tissues and placentas. Cytoskeletal components are crucial for the correct development and function of the placenta as they are involved in many key processes during placental formation, such as adhesion, transport and angiogenesis. This was reflected by the results of the MGI analysis, in which many cytoskeleton proteins were annotated with phenotype terms such as “abnormal ectoplacental cone morphology”, “impaired placental function”, “abnormal placenta morphology” and “abnormal trophoblast layer morphology”. Well-organized

 

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cytoskeletons are necessary for stable cell-cell and cell-matrix adhesion [81]; therefore, the observed changes of cytoskeletal protein expression may lead to placental adhesion anomalies. Defective placental adhesion (DPA) has been shown to be highly associated with the voluntary termination of second-trimester pregnancy [82], as well as placental abruption [83]. In the current investigation, we also found a deficiency of some adhesion-associated molecules in IVP placentas, including integrin beta 1 (Itgb1, also known as fibronectin receptor beta) and reticulon 4 (Rtn4). The deficiency of Itgb1 or Rtn4 could lead to phenotypes such as “abnormal trophoblast layer morphology”, “embryonic growth arrest”, “decreased fetal size” and “embryonic lethality”. These molecules have been reported to arrange adhesion via the interaction with cytoskeletal proteins, including talin [84], radixin [85] and actin [86] which were also found to be dysregulated in IVP placentas. Some of the cytoskeletal DEPs, including actin, radixin [87] and gelsolin [88], are important components of placental syncytiotrophoblast microvilli, which are critical for maternal-fetal transport [89]. Cytoskeletal structures are also believed to be associated with transport regulation in the human placenta [88]. The aberrant expression or distribution of cytoskeletal proteins in placental microvilli membranes are thought to be relevant to the pathogenesis of PE and IUGR [89]. Many of the observed DEPs were functionally specific to transport regulation. These DEPs could be subcategorized into the following types: 1) proteins involved in transporting molecules between the cytoplasm and the nucleus, such as exportin 1 (Xpo1), importin 4 (Ipo4), importin 7 (Ipo7), karyopherin beta 1 (Kpnb1) and transportin 1 (Tnpo1); 2) membrane transport proteins of the solute carrier family responsible for the transport of glucose, monocarboxylate and adenine, such as solute carrier family 16 member 1 (Slc16a1), solute carrier family 2 member 1 (Slc2a1) and solute carrier family 25 member 4 (Slc25a4); and 3) ATPase or ATP synthase involved in cation transport, such as ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 (Atp2a2), ATPase, Na+/K+ transporting, alpha 1 polypeptide (Atp1a1), ATP synthase, H+ transporting mitochondrial F1 complex and beta subunit (Atp5b), These enzymes belong to the family of ATPases or ATP synthases, which are specifically responsible for cation transport via the synthesis or release of energy. The aberrant expression of these transport proteins has been linked to multiple pregnancy complications, including IUGR [90], PE, gestational diabetes mellitus (GDM) [91, 92] and spontaneous abortion [93]. It should be noted that the majority of differentially expressed cytoskeleton and transport proteins showed consistent dysregulation at two time points. This finding suggests that aberrant cytoskeleton organization and transport in the developing IVP placenta may originate from extra-embryonic tissues of the gastrulae. Impaired Vasculogenesis, Angiogenesis and Hematopoiesis Based on the MGI analysis, we found that many of the dysregulated proteins at both E7.5 and E10.5 that were associated with extra-embryonic tissue and placental development, as well as embryonic survival and development, were also involved in vasculogenesis and angiogenesis. These DEPs included keratin 8 (Krt8), pregnancy zone protein (Pzp), filamin alpha (Flna), vimentin (Vim), methionine aminopeptidase 2 (Metap2), integrin beta 1 (Itgb1) and Atp2a2. Normal vasculogenesis and angiogenesis in the placenta are crucial for the exchange of oxygen and nutrients, which is necessary for fetal growth and development. Placental vascular malformations are highly associated with IUGR [94] and embryonic lethality [95]. These findings were confirmed by the morphological analysis of IVP placentas, in which both the labyrinth layer and vasculogenesis were impaired from the mid- to late-gestational periods.

 

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Correspondingly, the results of both our present and recently published studies [11], together with the findings of Bloise et al. [22], indicate that IVP embryos/fetus have consistently decreased weights and depressed growth. More importantly, we found that some of these DEPs, including Vim, Flna, Itgb1 and Atp2a2, were consistently dysregulated during the post-implantation stage. These DEPs may contribute largely to impaired vasculogenesis and angiogenesis in the IVP placenta. The results of both the PPI network analysis and MGI phenotype annotation indicate that IVP post-implantation extra-embryonic tissues and placentas may have defective hematopoiesis. Many DEPs, including hemopexin (Hpx), kininogen 1 (Kng1), fibrinogen alpha chain (Fga), fibrinogen beta chain (Fgb), transferrin (Trf), mutS homolog 2 (Msh2), integrin beta 1 (Itgb1) and ligase I (Lig1) were functionally associated with hematopoiesis. The dysregulation of these proteins is likely to lead to compromised hematopoiesis. The placenta is a hematopoietic organ during both embryonic and fetal development [96] and defective hematopoiesis in the placenta has been linked with embryonic lethality [97]. Trophoblasts have been shown to be critical for the maintenance of the placental hematopoietic microenvironment [97]; therefore, the DEPs associated with “abnormal trophoblast layer morphology” and “abnormal trophoblast giant cells”, such as Krt8, Krt19, Tln1, Itgb1, Sjogren syndrome antigen B (Ssb) and mitogen-activated protein kinase 1 (Mapk1), could contribute largely to impaired hematopoiesis in IVP placentas. Goldie et al. established that vasculogenesis occurs in parallel with hematopoiesis. Endothelial and hematopoietic cells may share a common progenitor, and there is commonality among the regulatory factors that control the differentiation and differentiated function of both cell lineages [98]. Park et al. also demonstrated the role of vasculogenesis in supporting hematopoiesis [99]. Cytoskeletal proteins and associated adhesion molecules or mediators accounted for a large proportion of the angiogenetic and hematopoietic proteins. If the functional clustering and above analysis are taken together, we propose that the impaired cytoskeletal organization not only leads to impaired transport and adhesion, but also results in compromised vasculogenesis and hematopoiesis in IVP placentas. These phenotypes could then play a central role in the abnormal placentation of IVP groups. By comparing the dynamic proteomic profiles of IVO and IVP extra-embryonic tissues and placentas during the post-implantation period, we observed an increase in abnormal development and function in the IVP placenta and extra-embryonic tissues. IVP extra-embryonic tissues and placentas showed a significant increase in aberrant genetic information processing from upstream de novo DNA methylation to downstream post-translational regulation, which together lead to altered protein production. These changes in protein profiles could significantly compromise cellular functions, including mitochondrial energy metabolism, cytoskeletal organization and transport. Consequent cellular aberrations result in compromised placental development and function, which contribute to retarded fetal growth and embryonic lethality (Figure 8). REFERENCES 1. European Society of Human Reproduction and Embryology, ART fact sheet. In; 2013. http://www.eshre.eu/GuidelinesDandDLegal/ARTDfactDsheet.aspx. 2. Zoll M. Facts about Assisted Reproductive Technology. In 2012. http://www.miriamzoll.net/author/fertilityFacts.html 3. Racowsky C. High rates of embryonic loss, yet high incidence of multiple births in human

 

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65. Koukoura O, Sifakis S, Spandidos DA. DNA methylation in the human placenta and fetal growth (review). Mol Med Rep 2012; 5: 883-889. 66. PADYKULA HA, RICHARDSON D. A correlated histochemical and biochemical study of glycogen storage in the rat placenta. Am J Anat 1963; 112: 215-241. 67. Lager S, Powell TL. Regulation of nutrient transport across the placenta. J Pregnancy 2012; 2012: 179827. 68. Toledo MT, Gomes MM. Placental glycogen metabolism changes during walker tumour growth. Placenta 2004; 25: 456-462. 69. Magnusson AL, Powell T, Wennergren M, Jansson T. Glucose metabolism in the human preterm and term placenta of IUGR fetuses. Placenta 2004; 25: 337-346. 70. Hay WJ. Placental-fetal glucose exchange and fetal glucose metabolism. Trans Am Clin Climatol Assoc 2006; 117: 321-339, 339-340. 71. Bax BE, Bloxam DL. Energy metabolism and glycolysis in human placental trophoblast cells during differentiation. Biochim Biophys Acta 1997; 1319: 283-292. 72. Holland OJ, Linscheid C, Hodes HC, Nauser TL, Gilliam M, Stone P, Chamley LW, Petroff MG. Minor histocompatibility antigens are expressed in syncytiotrophoblast and trophoblast debris: implications for maternal alloreactivity to the fetus. Am J Pathol 2012; 180: 256-266. 73. Federico M, Rebeca M, Oscar F, H, Sofia O, S, Erika G,C, Maria T E. The Role of Mitochondria in Syncytiotrophoblast Cells: Bioenergetics and Steroidogenesis. Obstetrics and Gynecology 2012: 397-482. 74. Wang Y, Walsh SW. Placental mitochondria as a source of oxidative stress in pre-eclampsia. Placenta 1998; 19: 581-586. 75. Neu J, Auestad N, DeMarco VG. Glutamine metabolism in the fetus and critically ill low birth weight neonate. Adv Pediatr 2002; 49: 203-226. 76. Self JT, Spencer TE, Johnson GA, Hu J, Bazer FW, Wu G. Glutamine synthesis in the developing porcine placenta. Biol Reprod 2004; 70: 1444-1451. 77. Battaglia FC. Glutamine and glutamate exchange between the fetal liver and the placenta. J Nutr 2000; 130: 974S-977S. 78. Lin G, Liu C, Feng C, Fan Z, Dai Z, Lai C, Li Z, Wu G, Wang J. Metabolomic analysis reveals differences in umbilical vein plasma metabolites between normal and growth-restricted fetal pigs during late gestation. J Nutr 2012; 142: 990-998. 79. Jerzy K, Makarewicz W, Swierczynski J, Bukato BG, Zelewski L. Mitochondrial glutamine and glutamate metabolism in human placenta and its possible link with progesterone biosynthesis. Placenta 1993; 14 Supl 1: 77-86. 80. Tao L. Placental ratio and fetal growth. HK J Paediatr 1998; 3: 29-31. 81. Parsons JT, Horwitz AR, Schwartz MA. Cell adhesion: integrating cytoskeletal dynamics and cellular tension. Nat Rev Mol Cell Biol 2010; 11: 633-643. 82. Morotti M, Podesta S, Musizzano Y, Venturini PL, Bentivoglio G, Fulcheri E, Ferrero S. Defective placental adhesion in voluntary termination of second-trimester pregnancy and risk of recurrence in subsequent pregnancies. J Matern Fetal Neonatal Med 2012; 25: 339-342. 83. Eskes TK. Clotting disorders and placental abruption: homocysteine--a new risk factor. Eur J Obstet Gynecol Reprod Biol 2001; 95: 206-212. 84. Critchley DR. Cytoskeletal proteins talin and vinculin in integrin-mediated adhesion. Biochem Soc Trans 2004; 32: 831-836. 85. Tang P, Cao C, Xu M, Zhang L. Cytoskeletal protein radixin activates integrin alpha(M)beta(2) by binding to its cytoplasmic tail. FEBS Lett 2007; 581: 1103-1108.

 

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IVP extra-embryonic tissues collected at E7.5. a and b) Representative normal IVO sample. c and d) Representative phenotypes of mildly delayed conceptuses, of which extra-embryonic tissues, including the ectoplacental cone and extra-embryonic ectoderm, were not fully developed. e and f) Representative phenotypes of severely delayed conceptuses, which were arrested at the egg-cylinder stage or displayed a degenerated extra-embryonic ectoderm and embryonic ectoderm. g and h) Representative phenotypes of conceptuses that were characterized by morphologically disorganized or degenerated extra-embryonic tissues. B) Morphological comparison of IVO and IVP embryos and placentas at E10.5. IVP samples showed greater variability in size compared to the IVO samples. Bars = 300 μm (A) and 2 mm (B). Figure 3. Phenotypic and morphological comparisons between IVO and IVP placentas at the mid- to late-gestational stage. A) Prenatal fetal weight and placental weight, as well as the ratio of placental/fetal weight, in IVO and IVP groups at E19. B) Representative cross sections of the entire placenta in the IVO and IVP groups. The areas of the labyrinth and spongiotrophoblast layers, as well as the decidua, were measured, and ratios of different layers to the total areas are shown. C and D) Representative images of laminin-labeled placental vasculogenesis in IVO and IVP groups at E13.5 (C) and E19 (D). The boxed regions are shown at a higher magnification on the right. Abbreviations: L, labyrinth layer; S, spongiotrophoblast layer; D, decidua. Bars = 500 μm (B, C and D) and 100 μm (the higher magnification on the right). Figure 4. General profiling of the proteome between the IVO and IVP groups. A) Hierarchical clustering analyses based on DEPs at E7.5 and E10.5. Clustering was performed on treatments and technical replicates at E7.5 and E10.5. Normalized protein abundance (LFQ intensity) is represented by different colors: red indicates high abundance and green denotes low abundance. B) Distribution of DEPs with different fold changes at E7.5 and E10.5. Red and green bars show proteins that were significantly upregulated and downregulated in IVP groups, respectively (P < 0.05). Dark and pale colors correspond to the different fold changes. C) Comparisons of the DEPs between E7.5 and E10.5. The Venn diagram shows DEPs specific to the E7.5 or E10.5 groups or in common with both. The table shows the major functions of DEPs in common with two time points. Figure 5. GO classification. Analysis using DAVID supplied a simplified overview of the GO subcategories based on the major category of "biological process" for DEPs at E7.5 (A) and E10.5 (B), respectively. The number for each subcategory represents the percentage of enriched DEPs in the total identified DEPs. Figure 6. Protein-protein interaction (PPI) network. DEPs at E7.5 (A) and E10.5 (B) showed a tightly interconnected network from a web-based search of the STRING database. Figure 7. Correlation between the transcriptome and proteome with global methylation profiling. A) Correlation between mRNA and protein abundances in IVO and IVP samples at E7.5 and E10.5. Red dots and lines represent IVP samples, while the blue diamonds and lines represent IVO samples. The trend lines depict the best fit as predicted by linear regression. The Y-axis represents the normalized protein abundance (units = LFQ intensity); the X-axis represents the normalized mRNA expression level (units = RPKM). B) Comparisons of the protein abundance

 

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of DNMT3a, Hdac2, Ahcy, and Mthfd1 between the IVP and IVO groups. Open circles represent each data point; lines represent the mean value for the three technical replicates. C) Heat map of differentially methylated regions (DMRs) between IVO and IVP extra-embryonic tissues in the promoter, gene body, and 3’ downstream regions. Normalized methylation levels are represented by different colors: red indicates relative hypermethylation and black denotes relative hypomethylation. Figure 8. Summary of comparative proteomic profiles between IVO and IVP extra-embryonic and placental tissues.

 

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TABLE 1. Identified KEGG pathways for DEPs at E7.5 and E10.5. E7.5 Spliceosome Citrate cycle (TCA cycle) Ubiquitin mediated proteolysis (proteasome) Dilated cardiomyopathy DNA replication Cell cycle Complement and coagulation cascades

 

E10.5 Spliceosome Citrate cycle (TCA cycle) Ubiquitin mediated proteolysis (proteasome) Dilated cardiomyopathy Ribosome Glycolysis/gluconeogenesis Fc gamma R-mediated phagocytosis Glyoxylate and dicarboxylate metabolism Hypertrophic cardiomyopathy (HCM) Regulation of actin cytoskeleton Parkinson disease Huntington disease Pentose phosphate pathway Focal adhesion Renal cell carcinoma Cysteine and methionine metabolism Arrhythmogenic right ventricular cardiomyopathy (ARVC) Adherens junction Cardiac muscle contraction

Dynamic proteomic profiles of in vivo- and in vitro-produced mouse postimplantation extraembryonic tissues and placentas.

As the interface between the mother and the developing fetus, the placenta is believed to play an important role in assisted reproductive technology (...
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