DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids

Abstract

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.

Keywords: In vitro fertilization; blastocyst; developmental biology; epigenetics; imprinting; pig; reproductive secretions; stem cells.

Figure 1.. Schematic representation of three different sperm processing protocols used for in vitro fertilization.

Swim-up-BSA: NaturARTs PIG medium + BSA; Swim-up-Fluid: NaturARTs PIG medium + POF-LF*. Density gradient centrifugation: centrifugation through a discontinuous Percoll: gradient (45% and 90% v/v). *POF-LF: porcine oviductal fluid collected at the late follicular phase of the estrous cycle. Red box represents the portion of the reproductive tract whose conditions we tried to resemble in vitro. IVF results after using these three different sperm processing protocols are included in Table 1. DOI: http://dx.doi.org/10.7554/eLife.23670.003

Figure 2.. Schematic representation of the different steps of the new IVF/EC system.

Swim-up-BSA or Swim-up-Fluid protocols were used for IVF. Previously, oocytes were preincubated in OF-LF for 30 min. Then, each group of putative zygotes were incubated in different media (0–8 hr, 8–48 hr and 48 hr-7days) as indicated in the diagram. O*: ovary with hemorrhagic corpus luteum; O**: early corpus luteum; OF-LF: oviductal fluid-late follicular phase of the estrous cycle; OF-EL: oviductal fluid-early luteal phase of the estrous cycle; UF-EL: uterine fluid-early luteal phase of the estrous cycle. Swim-up-BSA: NaturARTs PIG medium + BSA; Swim-up-Fluid: NaturARTs PIG medium + POF-LF. TALP: culture medium used for IVF. NCSU23: culture medium used for embryo development in vitro supplemented with sodium lactate, pyruvate and non-essential amino acids (NCSU23a) or with glucose and essential and non-essential amino acids (NCSU23b). DOI: http://dx.doi.org/10.7554/eLife.23670.005

Figure 3.. Gene expressed analysis in blastocysts obtained in vivo, by the Natur-IVF system or by C-IVF system.

(A) Heatmap of global gene expression (with log2 fold change >1.5 and adjusted B-H p-value < 0.05). Numbers denote ID of a specific embryo. (B) Principal Component Analysis (PCA) of the RNA-Seq samples: In vivo embryos (IV, red), Natur-IVF (N, green) and C-IVF (C, blue). Numbers denote ID of specific embryos. (C) Venn diagram with DEGs (Figure 3—source data 1). *, #, § denotes DEGs exclusive for C-IVF, Natur-IVF and In vivo, respectively (Figure 3—source data 2). (D) Heat map of gene expression of key genes associated with embryo development/differentiation, epigenetic reprogramming, cell cycle/cell growth, gene expression and imprinting. DOI: http://dx.doi.org/10.7554/eLife.23670.007

Figure 4.. Distribution of methylation levels and general view of the methylation profiles of 9 individual pig blastocysts.

(A) Distribution of methylation percentages across tiles of 150 CpGs on the pig genome for three groups of blastocysts (In-vivo, C-IVF and Natur-IVF). (B) Random browser shot as example of methylation landscape of the nine individual blastocysts analysed (Chr8:37027152–118458156). The two first rows in the picture represent the genes and CpG islands annotated (Ensembl, RRID:SCR_006773 Sus scrofa 10.2) in the pig genome, respectively. Color scale represents methylation levels from red (highest methylation, up to 25%) to blue (lowest methylation-0%). DOI: http://dx.doi.org/10.7554/eLife.23670.010

Figure 5.. DNA-methylation analysis in blastocysts obtained in vivo, by the Natur-IVF system or by C-IVF system.

(A) Principal Component Analysis (PCA) of the DNA methylation samples: In vivo embryos (red), Natur-IVF (green) and C-IVF (blue). Numbers denote ID of specific embryo. (B) Venn diagram of DMRs by pair-wise comparison (adjusted-p <0.05). Number of DMRs with higher (↑) or lower (↓) methylation in each pair-wise comparison are indicated (Figure 5—source data 1). (C) Heatmap of the 417 DMRs between the C-IVF group and the other two groups (In vivo and Natur-IVF). (D) Heatmap of the 324 DMRs between Natur-IVF group and the other two groups (In vivo and C-IVF). (E) Heatmap of the 448 DMRs between the In vivo group and the other two groups (Natur-IVF and C-IVF). For C, D and E (Figure 5—source data 2): Relative methylation measure as the difference in percent of methylation from the median methylation across all samples. DOI: http://dx.doi.org/10.7554/eLife.23670.012

Figure 6.. Top Diseases and Bio Functions linked by Ingenuity Pathways Analysis to DMRs exclusive for each group with low or high methylation.

DOI: http://dx.doi.org/10.7554/eLife.23670.015

Figure 7.. Methylation differences at IGF2R.

(A) Methylation quantitation at IGF2R from the unbiased analysis of genome methylation in SeqMonk with a fixed size of 150 CpG windows. Mean percentages of methylation are shown by the bars for each group. Blue (unmethylated) and red (methylated) dots represent methylation reads. Asterisks indicate that methylation at the indicated region showed significantly different values (p<0.05) in Natur-IVF (*) and In vivo (**) vs C-IVF. TSS: transcription starting site. (B) Detailed view and methylation quantitation of the CpGi at the identified IGF2R DMR. Red rectangles represent, as indicated, CpG islands of the genes. Black boxes indicate the position of the targeted features, whose mean percentages of methylation are shown by the bars for each group. Blue (unmethylated) and red (methylated) dots represent methylation reads. DOI: http://dx.doi.org/10.7554/eLife.23670.016

Figure 8.. Methylation quantitation at NNAT from the unbiased analysis of genome methylation in SeqMonk with a fixed size of 150 CpG windows.

Black boxes indicate the position of the selected 150 CpG windows, whose mean percentages of methylation are shown by the bars for each group. Blue (unmethylated) and red (methylated) dots represent methylation reads. Asterisks indicate that methylation at the indicated region (black box) showed significantly different values (p<0.05) in Natur-IVF (*) and In-vivo (**) vs C-IVF. DOI: http://dx.doi.org/10.7554/eLife.23670.017

Author response image 1.

DOI: http://dx.doi.org/10.7554/eLife.23670.023