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. 2023 Sep 29;40(12):2913–2923. doi: 10.1007/s10815-023-02951-4

Characterization of the long noncoding RNA transcriptome in human preimplantation embryo development

Le Zhang 1,#, Hailong Sun 1,#, Xiujuan Chen 1,✉
PMCID: PMC10656396  PMID: 37770818

Abstract

Purpose

Infertility remains a human health burden globally. Only a fraction of embryos produced via assisted reproductive technologies (ARTs) develop to the blastocyst stage in vitro. lncRNA abundance changes significantly during human early embryonic development, indicating vital regulatory roles of lncRNAs in this process. The aim of this study is to obtain insights into the transcriptional basis of developmental events.

Methods

scRNA-seq data and SUPeR-seq data were used to investigate the lncRNA profiles of human preimplantation embryos. The top 50 highly expressed unique and shared lncRNAs in each stage of preimplantation development were identified. Comparative analysis of the two datasets was used to verify the consistent expression patterns of the lncRNAs. Differentially expressed lncRNAs were identified and subjected to functional enrichment analysis.

Results

The lncRNA profiles of human preimplantation embryos in the E-MTAB-3929 dataset were similar to those in the GSE71318 dataset. The ratios of overlap among the top 50 highly expressed lncRNAs between two pairs of stages (2-cell stage vs. 4-cell stage and 8-cell stage vs. morula) were aberrantly low compared with those between other stages. Each stage of preimplantation development exhibited unique and shared lncRNAs among the top 50 highly expressed lncRNAs. Among the between-group comparisons, the 2-cell stage vs. 4-cell stage showed the highest number of differentially expressed lncRNAs. Functional enrichment analysis revealed that differentially expressed lncRNAs and their associated super enhancers and RNA binding proteins (RBPs) are closely involved in regulating embryonic development. These lncRNAs could function as important cell markers for distinguishing fetal germ cells.

Conclusions

Our study paves the way for understanding the regulation of developmental events, which might be beneficial for improved reproductive outcomes.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10815-023-02951-4.

Keywords: LncRNAs, Human embryo, Embryonic development, LncRNA expression, Functional enrichment

Introduction

Infertility is a disease in which a couple fails to achieve pregnancy after 12 months with regular and unprotected sexual intercourse [1]. Infertility is a global human health burden. It has been estimated that more than 186 million people suffer from infertility worldwide [2]. A survey reported an increase in the age-standardized prevalence rate of infertility from 1990 to 2017 in 195 countries and territories of 0.370% per year for women and 0.291% per year for men [3]. In the Western world, one in every seven couples suffers from infertility, while in developing countries, this number is higher, at one in every four couples including reproductive-aged women [2].

Preimplantation embryo development refers to the set of embryonic processes that occur before transfer to the uterus. Preimplantation embryo development begins with oocyte fertilization by the sperm to form the zygote. The observation of two-pronuclear (2PN) oocytes indicates normal fertilization [4]. Then, zygotes undergo several divisions, from 1-cell to 2-cell to 4-cell to 8-cell [5]. Subsequently, the 16–32 cells compact into a morula [5]. Cell differentiation of the morula results in the formation of trophectoderm cells (TE) and the inner cell mass (ICM) of the blastocyst [6]. However, only 40% of human embryos can develop to the blastocyst stage in vitro [7]. The lower rates of development to blastocysts correspond to the loss of preimplantation embryos, which is one of the major reasons for relatively low live birth rates [8]. Long noncoding RNAs (lncRNAs) are a class of noncoding transcripts that are longer than 200 nucleotides in length [9]. LncRNAs can regulate gene expression at the epigenetic level, transcriptional level, post-transcriptional level and translational level through interacting with DNA, RNA and proteins [10]. Specifically, lncRNAs regulate chromatin remodeling [11], activation and interference of transcription [12], mRNA stabilization [13], modulation of the translational factors [14], and so on. Current evidence indicates that lncRNA abundance changes significantly during human early embryonic development, suggesting its vital regulatory roles in this process [15]. Therefore, investigating the lncRNA expression of the human preimplantation embryo will not only pave the way for understanding the regulation of developmental events but also uncover the genetic causes of embryo loss. The aim of this study is to provide a new direction for improved reproductive outcomes.

To understand the transcriptional basis of developmental events, we analyzed lncRNA expression patterns in two datasets (E-MTAB-3929 and GSE71318) and discovered the top 50 unique and shared highly expressed lncRNAs in each stage of preimplantation development. Then, we investigated differentially expressed lncRNAs between multiple stages of development. Similar trends in lncRNA expression and function were observed in the two different analysis methods. Functional enrichment analysis revealed that lncRNAs and their associated upstream regulators and downstream targets are closely involved in regulating embryonic development.

Materials and methods

Data availability

The gene expression profiling dataset GSE71318 was downloaded from the GEO database (http://www.ncbi.nlm.nih.gov/geo/). This study used oocytes/embryos at oocyte, zygote, 2-cell, 4-cell, 8-cell, morula, early blastocyst, middle blastocyst, late blastocyst, ICM and TE from blastocyst stages. The scRNA-seq dataset MTAB-3929 was obtained from SCPortalen (https://single-cell.riken.jp/SCPortalen_Database/Home/). This study used 1529 cells obtained from 88 human preimplantation embryos at stages ranging from embryonic days 3 to 7.

Identification of differentially expressed lncRNAs

The DESeq2 package was employed to identify differentially expressed lncRNAs between different stages of preimplantation embryo development in the GSE71318 dataset. Differences with a fold change > 2 or < -2 and FDR p value < 0.05 were considered statistically significant. LncRNAs within these threshold values were defined as differentially expressed lncRNAs.

Enrichment of upstream regulators

Differentially expressed lncRNAs of dataset GSE71318 were subjected to enrichment analyses using LncSEA (http://bio.liclab.net/LncSEA/index.php). A bar chart was used to show cell/tissue-type-specific super enhancers that closely regulate the lncRNAs.

Cell marker analysis

Cell marker enrichment analysis was performed through LncSEA, which can identify cell markers for distinguishing different cell types in tissues. Differentially expressed lncRNAs can be regarded as cell markers to identify fetal germ cells.

RNA binding protein partner prediction for lncRNAs

LncSEA was used to investigate RNA binding proteins (RBPs), the downstream targets of differentially expressed lncRNAs. The roles of interactions between lncRNAs and RBPs in human embryonic development will be verified by revealing how these RBPs contribute to pluripotency and differentiation.

Results

SUPeR-seq transcriptome profiling of lncRNAs in human preimplantation embryos

To investigate the lncRNA profiles in human preimplantation embryos, we analyzed the GSE71318 dataset downloaded from GEO, which contained three oocytes, four zygotes, three 2-cell stage embryos, three 4-cell stage embryos, four 8-cell stage embryos, three morulas, two early blastocysts, two middle blastocysts, two late blastocysts, ICM and TE from three blastocysts. The previous study was aimed to trace the expression of circular RNAs in human preimplantation embryos through GSE71318 dataset [16]. In our study, we explored the lncRNAs expression profiles by analyzing the same dataset. The heatmap showed similar lncRNA expression patterns among oocytes, zygotes, and 2-cell stage embryos (Fig. 1a). Significant differences were observed between consecutive stages (2-cell stage vs. 4-cell stage; 4-cell stage vs. 8-cell stage; 8-cell stage vs. morula; morula vs. blastocyst) (Fig. 1a). ICM and TE exhibited no obvious differences in lncRNA expression (Fig. 1a). Then, we analyzed the top 50 highly expressed lncRNAs between consecutive stages (Supplementary Table 1). We discovered that the ratio of the overlapping top 50 highly expressed lncRNAs in the two groups (2-cell stage vs. 4-cell stage; 8-cell stage vs. morula) was aberrantly low compared with that in the other groups (Fig. 1b). Different expression patterns of lncRNAs might indicate different regulatory mechanisms in human preimplantation embryos.

Fig. 1.

Fig. 1

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SUPeR-seq transcriptome profiling of lncRNAs in human preimplantation embryos. a Heatmap showing the lncRNA expression profiles in human preimplantation embryos. b Venn diagrams representing the expression profiles of the top 50 highly expressed lncRNAs

Top 50 unique and shared highly expressed lncRNAs in each stage of preimplantation development

An UpSet plot was used to investigate the top 50 unique and shared highly expressed lncRNAs in each stage of preimplantation development. The plot shows the number of unique and shared lncRNAs between the eleven groups (Fig. 2a; Supplementary Table 2). Furthermore, we analyzed the top 50 shared highly expressed lncRNAs in embryo development. Some lncRNAs exhibited universal expression patterns, while the expression of other lncRNAs displayed developmental stage-specific patterns (Fig. 2b, c). Seven maternal lncRNAs had the highest expression in oocytes and then were downregulated sharply after the 4-cell stage (FGF14-AS2, RP11-252C15.1, RP11-143A12.3, UBL7-AS1, FLJ20021, SPANXA2-OT1, MAFG-AS1) (Fig. 2b). In addition, stage-specifically expressed lncRNAs were identified in every stage (3 in zygotes; 4 in 2-cell stage; 5 in 4-cell stage; 4 in 8-cell stage; 3 in morula; 4 in early blastocyst; 1 in middle blastocyst; 2 in late blastocyst, 4 in ICM and 3 in TE) (Fig. 2b). The specific expression patterns of these lncRNAs might reflect thqeir intrinsic and vital functions in specific developmental stages and their potential for use as biomarkers. Nine lncRNAs (SNHG16, DANCR, EPB41L4A-AS1, LRRC75A-AS1, PITPNA-AS1, SNHG17, SNHG8, SNHG5, GAS5) were highly expressed in all stages, which indicated their fundamental roles in the regulation of development (Fig. 2c).

Fig. 2.

Fig. 2

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Analysis of the top 50 unique and shared highly expressed lncRNAs in preimplantation embryos. a UpSet plot depicting the number of unique and shared top 50 highly expressed lncRNAs in different stages of preimplantation embryos. b Top 50 highly expressed lncRNAs overlapping in all stages. c Top 50 highly expressed lncRNAs specifically expressed in each stage

Comparative analysis of scRNA-seq data and SUPeR-seq data

To verify the results presented above, we examined lncRNA expression from the 8-cell stage to the time before implantation, as collected in a single-cell RNA-seq dataset (E-MTAB-3929). This dataset contains sequence data for 1529 cells obtained from 88 human preimplantation embryos at stages ranging from embryonic days 3 to 7. The original study revealed the establishment of trophectoderm, epiblast, and primitive endoderm lineages and X chromosome dynamics [17]. In our study, a heatmap was used to show the expression profiles of the lncRNAs. There were obvious differences in the enrichment of lncRNAs from embryonic days 3 to 7 (Fig. 3a). Importantly, the lncRNA expression pattern in dataset E-MTAB-3929 was similar to that in the GSE71318 dataset, as shown in the blue box (Supplementary Fig. S1). The ratio of overlap between the top 50 highly expressed lncRNAs in the 8-cell stage vs. morula was lower than those between other pairs of consecutive stages (Fig. 3b; Supplementary Table 3). This finding was consistent with previous results (Fig. 1b). A Venn diagram of the top 50 highly expressed lncRNAs in E3-E7 was constructed. As shown in Fig. 3c, there were 22 overlapping lncRNAs, 12 lncRNAs specifically expressed in E3, 4 lncRNAs specifically expressed in E4, 4 lncRNAs specifically expressed in E5, 1 lncRNA specifically expressed in E6, and 4 lncRNAs specifically expressed in E7 (Supplementary Table 4). There were 8 lncRNAs universally expressed in both datasets, including SNHG16, DANCR, EPB41L4A-AS1, LRRC75A-AS1, SNHG17, SNHG8, SNHG5, and GAS5 (Fig. 3d). Comparative analysis of the two datasets showed consistent expression patterns of lncRNAs in human preimplantation embryos.

Fig. 3.

Fig. 3

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Comparative analysis of scRNA-seq data and SUPeR-seq data. a LncRNA expression profiles of preimplantation embryos from two datasets, GSE71318 and E-MTAB-3929. b Venn diagrams of the top 50 highly expressed lncRNAs in dataset E-MTAB-3929. c Venn diagram illustrating shared and distinct lncRNA expression in preimplantation embryos ranging from embryonic days 3 to 7. d Venn diagram of the common and exclusively expressed lncRNAs among the top 50 highly expressed lncRNAs that are expressed in all stages in the two datasets GSE71318 and E-MTAB-3929

Differential lncRNA expression profiles between consecutive stages of embryo development

Next, we focused on differentially expressed lncRNAs (fold change > 2 or < -2; FDR p value < 0.05). A total of 56 differentially expressed lncRNAs were identified in the comparison of two consecutive stages. Two of these differentially expressed lncRNAs were downregulated in the oocyte vs. zygote group, 45 were upregulated and 7 were downregulated in the 2-cell stage vs. 4-cell stage group, 1 was upregulated and 3 were downregulated in the 4-cell stage vs. 8-cell stage group, 1 was upregulated and 2 were downregulated in the 8-cell stage vs. morula group, and 1 was downregulated in the morula vs. early blastocyst group (Fig. 4a; Supplementary Table 5). Notably, compared with other pairs of stages, the number of differentially expressed lncRNAs in the 2-cell stage vs. 4-cell stage group was highest. Next, we analyzed the expression of these differentially expressed lncRNAs (Fig. 4b). The 4- to 8-cell stage of human preimplantation embryos represents the major period of zygotic gene activation (ZGA) in the maternal-to-zygotic transition (MZT) [18]. We found 9 downregulated differentially expressed lncRNAs whose expression levels were decreased sharply after the 4-cell stage and 47 downregulated differentially expressed lncRNAs whose expression levels increased during the major wave of the MZT after the 4-cell stage (Fig. 4b). By comparing the differentially expressed lncRNAs and the top 50 highly expressed lncRNAs, we found that 32 out of 50 highly expressed lncRNAs were differentially expressed in the 4-cell stage, 30 out of 50 highly expressed lncRNAs were differentially expressed in the 8-cell stage, and 24 out of 50 highly expressed lncRNAs were differentially expressed in morula (Fig. 4c). These data indicated that the two methods produced generally similar results, to a certain extent.

Fig. 4.

Fig. 4

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Differential lncRNA expression profiles between two stages of embryo development. a Identification of differentially expressed lncRNAs between two stages of preimplantation embryos. b Expression of differentially expressed lncRNAs between consecutive stages of embryos. c The number of differentially expressed lncRNAs among the top 50 highly expressed lncRNAs

Functional analysis of differentially expressed lncRNAs

Functional enrichment analysis of the all differentially expressed lncRNAs was performed through LncSEA (http://bio.liclab.net/LncSEA/index.php), which identified lncRNA sets associated with upstream regulators and downstream targets. Through super enhancer enrichment analysis, we discovered that these differentially expressed lncRNAs were regulated by cell/tissue type-specific super enhancers, such as sample_02_124_Stem_cell_Embryonic-stem-cells_Naive and sample_00_012_Stem_cell_H1-hESC (Fig. 5a). To identify important cell markers for distinguishing different cell types in tissues, we conducted cell marker enrichment analysis. These differentially expressed lncRNAs could be regarded as mitotic fetal germ cell, meiotic prophase fetal germ cell, mitotic arrest phase fetal germ cell and oogenesis phase fetal germ cell markers (Fig. 5b). These results indicated that differentially expressed lncRNAs are closely involved in regulating embryonic development. Considering the above findings, we investigated RBPs contributing to the pluripotency and differentiation of these lncRNAs. We found 20 differentiation-related RBPs and 7 pluripotency-related RBPs among the top 100 RBPs (Fig. 5c). A large fraction of differentiation-related RBPs increased in abundance from the 4-cell stage, including DDX6 [19], XRN2 [20], RBM15 [21], KQI [22] and PTBP1 [23, 24] (Fig. 5d). Pluripotency-related RBPs decreased in abundance after the 8-cell stage, including DDX6 [25], DGCR8 [26], TIAL1 [27], HNRNPU [28], LIN28A [29], SOX2 [30], and NANOG [31] (Fig. 5e). These data reinforced the importance of lncRNA-mediated RBPs in stem cell identity.

Fig. 5.

Fig. 5

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Functional analysis of differentially expressed lncRNAs. a Bar chart showing cell/tissue-type-specific super enhancers that closely regulate lncRNAs. b Cell marker analysis to identify cell-specific lncRNAs for potential use as cell markers. c RNA binding protein (RBP) analysis to identify RBPs of these lncRNAs contributing to the pluripotency and differentiation. d Expression of 20 differentiation-related RBPs among the top 100 RBPs. e Expression of 7 pluripotency-related RBPs among the top 100 RBPs

Discussion

Comparative analysis of scRNA-seq and SUPeR-seq data revealed that the lncRNA expression patterns in dataset E-MTAB-3929 was similar to that in the GSE71318 dataset. The ratio of the top 50 highly expressed lncRNAs overlapping between consecutive stages was consistent in the two datasets. The top 50 shared highly expressed lncRNAs in each stage of preimplantation development might indicate the fundamental roles of them in embryo development. For instance, lncRNA GAS5 and EPB41L4A-AS1 were highly expressed in all stage of preimplantation development as shown in Fig. 2c. GAS5 facilitated human embryonic stem cell self-renewal by upregulating NODAL expression [32]. EPB41L4A-AS1 was reported to induce metabolic reprogramming in trophoblast cells and placenta tissue derived from miscarried pregnancies [33]. These studies support our speculation. The top 50 unique highly expressed lncRNAs in each stage might show the intrinsic and vital functions in specific developmental stages. For example, NEAT1 could bind to Carm1, facilitating paraspeckle formation to regulate preimplantation development by affecting ICM/TE cell fates [34]. NEAT1 KO mice displayed significantly decreased fertility [35]. These findings are consistent with the result as shown in Fig. 2b. The 4- to 8-cell stage of human preimplantation embryos represents the major period of ZGA in the MZT. It was reported that BCAR4 transcript was abundant from the zygote to the 8-cell stage, rarely expressed in the morula and undetectable in blastocysts in bovine [36]. This maternal profile of BCAR4 was also represented in Fig. 4b of our study. BCAR4 accumulated from oocyte maturation and decreased after 4-cell stage and was not activated at the major period of ZGA in the MZT. In contrast, SNHG3 was dramatically increased from 4- to 8-cell stage, which might represent the specific expression in preimplantation embryos during ZGA. In addition, SNHG3 was crucial for mouse embryonic stem cell self-renewal and pluripotency [37]. These studies strongly corroborate the roles of the screened lncRNAs in regulating the development of preimplantation embryos.

lncRNAs with differential expression between multiple stages of development were investigated. Changes between consecutive stages were our particular focus. We observed no differentially expressed lncRNAs in the zygote to 2-cell transition, indicating a relative quiescence of lncRNA transcription. A large number of differentially expressed lncRNAs were observed between the 2-cell stage and 4-cell stage, with 52 differentially expressed genes identified, 45 of which were upregulated. The following consecutive stages had fewer differentially expressed lncRNAs. Importantly, more than half of the top 50 highly expressed lncRNAs in the 4-cell, 8-cell and morula stages were identified as differentially expressed lncRNAs, suggesting similar trends in lncRNA expression and function in the two different analysis methods.

During major ZGA of human preimplantation embryos, a burst of gene activation occurs at 4- to 8-cell stage, which provided RNAs and proteins supporting development of embryos [38, 39]. This evidence also supported our findings that dramatically changed differentially expressed lncRNAs in 4-cell stage compared to 2-cell stage.

To characterize the functions of the differentially expressed lncRNAs, we performed enrichment analysis using LncSEA. First, we found the upstream regulators of differentially expressed lncRNAs, namely, super enhancers. They were enriched in stem-associated specific cells/tissues. These differentially expressed lncRNAs could function as important cell markers for distinguishing different cell types in tissues, such as mitotic fetal germ cells, meiotic prophase fetal germ cells, mitotic arrest phase fetal germ cells and oogenesis phase fetal germ cell markers. RPBs of differentially expressed lncRNAs were identified as contributors to pluripotency and differentiation. The expression of these RPBs was significantly elevated at the biological transitions from 2-cell to 4-cell embryos, which was consistent with the massive wave of differential lncRNA expression during the same period. Based on gene-deficient mice, loss of QKI [40], SRSF1 [41], and DDX3X [42] led to the early embryonic lethal phenotype, which suggested crucial roles of these differentiation related RPBs in early embryonic development. Pluripotency related RPB SOX2 overexpression in 1-cell embryos led to arrest at the 2-cell stage, and overexpressing SOX2 in 2-cell embryos inhibited 15% of zygotically expressed genes [43]. NANOG controlled ZGA and acted as a key transcription factor participating in preimplantation development [44, 45]. These results indicated that differentially expressed lncRNAs and their associated super enhancers and RBPs are closely involved in regulating embryonic development.

The limitation of this study was the lack of experiments for further verification of the regulatory roles of screened lncRNAs in embryonic development. The existing studies associated with lncRNA-mediated preimplantation embryo development are also limited. More efforts are needed to uncover the detailed mechanisms of their interplay. Despite this limitation, our research still provides new insight into the genetic causes of embryo loss.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (TIF 647 KB) (647.5KB, tif)
Supplementary file2 (XLSX 12 KB) (12.2KB, xlsx)
Supplementary file3 (XLSX 10 KB) (10.2KB, xlsx)
Supplementary file4 (XLSX 12 KB) (11.8KB, xlsx)
Supplementary file5 (XLSX 10 KB) (10.2KB, xlsx)
Supplementary file6 (XLSX 11 KB) (11.3KB, xlsx)

Abbreviations

ARTs

Assisted reproductive technologies

RBPs

RNA binding proteins

2PN

Two-pronuclear

TE

Trophectoderm cells

ICM

The inner cell mass

lncRNAs

Long noncoding RNAs

MZT

Maternal-to-zygotic transition

ZGA

Zygotic gene activation

Author contribution

XC and LZ generated the hypothesis and designed the study. LZ and HS interpreted data and prepared figures. LZ and HS prepared the first draft of the manuscript. LZ edited and prepared the final draft of the manuscript. LZ supervised the secured funding. XC supervised the overall research and interpreted results. All authors read and approved the final manuscript.

Funding

This work was supported by the Natural Science Foundation of Inner Mongolia (2020BS08002); Startup Foundation for Advanced Talents of Inner Mongolia; Program for Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region (NJYT22014); and the Doctoral Scientific Research Foundation of the Affiliated Hospital of Inner Mongolia Medical University (NYFY BS 202111).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

All the authors read and approved the final manuscript.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Le Zhang and Hailong Sun contributed equally.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (TIF 647 KB) (647.5KB, tif)
Supplementary file2 (XLSX 12 KB) (12.2KB, xlsx)
Supplementary file3 (XLSX 10 KB) (10.2KB, xlsx)
Supplementary file4 (XLSX 12 KB) (11.8KB, xlsx)
Supplementary file5 (XLSX 10 KB) (10.2KB, xlsx)
Supplementary file6 (XLSX 11 KB) (11.3KB, xlsx)

Data Availability Statement

The gene expression profiling dataset GSE71318 was downloaded from the GEO database (http://www.ncbi.nlm.nih.gov/geo/). This study used oocytes/embryos at oocyte, zygote, 2-cell, 4-cell, 8-cell, morula, early blastocyst, middle blastocyst, late blastocyst, ICM and TE from blastocyst stages. The scRNA-seq dataset MTAB-3929 was obtained from SCPortalen (https://single-cell.riken.jp/SCPortalen_Database/Home/). This study used 1529 cells obtained from 88 human preimplantation embryos at stages ranging from embryonic days 3 to 7.

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Articles from Journal of Assisted Reproduction and Genetics are provided here courtesy of Springer

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