Funct Integr Genomics DOI 10.1007/s10142-017-0555-y ORIGINAL ARTICLE Transcriptome profile of the human placenta Marta Majewska1 & Aleksandra Lipka2 & Lukasz Paukszto 3 & Jan Pawel Jastrzebski3 & Kamil Myszczynski3 & Marek Gowkielewicz 2 & Marcin Jozwik2 & Mariusz Krzysztof Majewski1 Received: 18 November 2016 /Revised: 9 February 2017 /Accepted: 16 February 2017 # The Author(s) 2017. This article is published with open access at Springerlink.com Abstract The human placenta is a particular organ that insep- (6497) were identified, and among them 30 were novel. To arably binds the mother and the fetus. The proper develop- gain a better understanding of the biological implications, the ment and survival of the conceptus relies on the essential assembled transcripts were annotated with gene ontology interplay between maternal and fetal factors involved in coop- (GO) terms. Single nucleotide variants were predicted for eration within the placenta. In our study, high-throughput se- the transcripts assigned to each analyzed GO category. Our quencing (RNA-seq) was applied to analyze the global tran- results may be useful for establishing a general pattern of the scriptome of the human placenta during uncomplicated preg- gene expression in the human placenta. Characterizing placen- nancies. The RNA-seq was utilized to identify the global pat- tal transcriptome, which is crucial for a pregnancy’soutcome, tern of the gene expression in placentas (N = 4) from women can serve as a basis for identifying the mechanisms underlying in single and twin pregnancies. During analyses, we obtained physiological pregnancy, as well as may be useful for an early 228,044 transcripts. More than 91% of them were multi-exon, detection of the genomic defects. and among them 134 were potentially unknown protein cod- ing genes. Expression levels (FPKM) were estimated for Keywords RNA-seq . Human . Placenta . Transcriptome 38,948 transcriptional active regions, and more than 3000 of genes were expressed with FPKM >20 in each sample. Additionally, all unannotated transcripts with estimated FPKM values were localized on the human genome. Highly Introduction covered splice junctions unannotated in the human genome Next-generation sequencing (NGS) applied to the tran- Electronic supplementary material The online version of this article scriptome (RNA-Seq) is a technology that uncovers broad (doi:10.1007/s10142-017-0555-y) contains supplementary material, amounts of novel information about the transcript (Lerch which is available to authorized users. et al. 2012). RNA-Seq is a powerful tool suitable for a wide range of large-scale tissue-specific profiling, which includes * Marta Majewska analyses of transcriptome simultaneously for mutation, struc- [email protected] tural alteration, and expression, even the reconstruction of the entire transcriptome in the absence of a reference genome (Hui 1 Department of Human Physiology, Faculty of Medical Sciences, 2014). Despite the need for advanced bioinformatic and time- University of Warmia and Mazury in Olsztyn, Warszawska Str 30, consuming data analysis, the RNA-seq still provides a strong 10-082 Olsztyn, Poland alternative to the use of microarrays in gene expression studies 2 Department of Gynecology and Obstetrics, Faculty of Medical (Grada and Weinbrecht 2013). The discovery of specific genes Sciences, University of Warmia and Mazury in Olsztyn, Niepodleglosci Str 44, 10-045 Olsztyn, Poland crucial for placental development and function can help in understanding and identifying the mechanisms underlying 3 Department of Plant Physiology, Genetics and Biotechnology, Faculty of Biology and Biotechnology, University of Warmia and both normal and pathological pregnancies. Detailing the pla- Mazury in Olsztyn, Oczapowskiego Str 1A, cental tissue transcriptome could provide a valuable resource 10-719 Olsztyn-Kortowo, Poland for genomic studies related to placental disease. Funct Integr Genomics Particularly in the context of established basis, the comple- The frequency of multiple gestations has increased over the tion of the human genome sequencing project and the acces- last few decades, mainly due to the use of assisted reproduc- sibility of the reference human genome enabled the applica- tive technologies with the increased maternal age at childbear- tion of genomic knowledge in clinical practice. The current ing. Twin pregnancies are at higher perinatal risk for both progress of NGS technology is vertiginous, and genomic med- mothers and fetuses than singleton pregnancies (Chauhan icine driven by NGS may improve the diagnosis, prognosis, et al. 2010;Hubinontetal.2015). and therapy of many to-date incurable diseases (Ley et al. The aim of the present study was to identify the tran- 2008;Dietz2010). scriptome landscape of the human placenta during uncompli- Reproductive success is deeply linked with precise cated single and twin pregnancies to establish the pattern of maternal-embryo communication, which results in correct pla- normal placental gene expression for further comprehensive cental functioning. The placenta is a primary organ essential analyses. for maintaining the proper development of the human fetus during pregnancy. Any disorder in this unique and delicate molecular crosstalk will have implications resulting in restrict- ed progress in successful and proper embryo growth. Materials and methods Disturbances, if more severe, may lead to pregnancy failure, or even influence the developmental potential of offspring to Collection of placental samples and ethics statement adulthood (Fazeli and Pewsey 2008; Fan et al. 2016). Besides its hormonal and nutritive functions, the placenta also serves All clinical samples were collected at the Clinical Ward for as a barrier against the maternal immune system, the gas ex- Gynecology, Obstetrics and Oncological Gynecology at the change compartment, as well as the fetal waste disposal organ. Regional Specialist Hospital in Olsztyn following informed The nutritional status of the mother and infant from con- written consent from mothers. The experimental protocol ception until early childhood, as well as maternal prenatal was approved by the Bioethics Committee of the Warmia- distress and compromise affect the early programming of the Mazury Medical Chamber (OIL.164/15/Bioet) in Olsztyn, immune function, which might, in consequence, influence the Poland. neuro-development of the embryo (Marques et al. 2013). Moreover, hormones produced by the placenta adjust ma- ternal physiology to couple the body changes during the Placental tissue collection pregnancy’sprogress(Crossetal.2003; Watson and Cross 2005). Additionally, significant differences in efficiency and Placentas (N = 4) were collected from healthy women after growth, depending on the sex of the fetus, have been observed uncomplicated pregnancy from term pregnancy (Table 1), (Forsén et al. 1999; Eriksson et al. 2010). Differences in pla- who underwent a scheduled Cesarean section before the onset cental gene expression may also be correlated with the rate of of labor. Samples of placental tissues were collected immedi- growth and body length or weight as observed in males at ately after delivery; then, small pieces of placentas were fro- birth, in comparison with females with equivalent placental zen in liquid nitrogen. Preserved tissues were stored in the size (Misra et al. 2009). Laboratory of Molecular Diagnostics at −70 °C until further The properly functioning placenta depends on the finely analyses. tuned regulation of relevant genes that affect its structural integrity, and the proper growth and development of its struc- tural components. Thus, any disturbances in gene expression Total RNA extraction and purification may represent a major molecular mechanism underlying path- ological pregnancies (Mikheev et al. 2008). RNA was isolated from the placental tissues using the A profound penetration of the spatial and temporal changes Qiagen RNeasy Kit according to the manufacturer’srec- of the placental transcriptome during development and pathol- ommendations. The RNase-Free DNase Set (Qiagen) was ogy of the pregnancy should be considered essential in iden- used to obtain high-quality RNA with DNA-ase digestion. tifying the origins of diseases such as preeclampsia (PE) and RNA integrity (purity and concentration) was evaluated intrauterine growth restriction (IUGR) that cause stillbirth or using the Tecan InfiniteM200 (Tecan Group AG). The preterm birth. The consequences may extend into adult life, RNA integrity number (RIN) was measured on a with a metabolic syndrome being a dramatic example. The Bioanalyzer 2100 (Agilent). Only samples with the RIN advanced RNA-seq is an effective tool for analysis of the >8 were subjected to further analysis. Fifteen microliters placental transcriptome to elucidate possible molecular mech- of total RNA extracted from each sample was delivered to anisms leading to these diseased states, and to identify candi- OpenExome (Poland) in order to prepare a cDNA library date biomarkers (Cox et al. 2015). and perform RNA-seq. Funct Integr Genomics Table 1 Characteristics of collected placental samples used Sample Maternal age Gestational Sex of fetus Multiplicity of Birth in RNA-seq code (years) age (weeks) gestation weight (g) Hs_p3 34 41 ♂ Single 3900 Hs_p9 32 36 ♂ Twin 2870 Hs_p12 33 37 ♀ Twin 2150 Hs_p14 36 39 ♀ Single 2720 cDNA library construction and transcriptome sequencing Transcriptional active regions Double-stranded cDNA libraries were prepared from to- Clean