The transcriptome of a human polar body accurately reflects its sibling oocyte

Abstract

Improved methods are needed to reliably and accurately evaluate oocyte quality prior to fertilization and transfer into the woman of human embryos created through in vitro fertilization (IVF). All oocytes that are retrieved and matured in culture are exposed to sperm with little in the way of evaluating the oocyte quality. Furthermore, embryos created through IVF are currently evaluated for developmental potential by morphology, a criterion lacking in quantitation and accuracy. With the recent successes in oocyte vitrification and storage, clear metrics are needed to determine oocyte quality prior to fertilizing. The first polar body (PB) is extruded from the oocyte before fertilization and can be biopsied without damaging the oocyte. Here, we tested the hypothesis that the PB transcriptome is representative of that of the oocyte. Polar body biopsy was performed on metaphase II (MII) oocytes followed by single-cell transcriptome analysis of the oocyte and its sibling PB. Over 12,700 unique mRNAs and miRNAs from the oocyte samples were compared with the 5,431 mRNAs recovered from the sibling PBs (5,256 shared mRNAs or 97%, including miRNAs). The results show that human PBs reflect the oocyte transcript profile and suggests that mRNA detection and quantification through high-throughput quantitative PCR could result in the first molecular diagnostic for gene expression in MII oocytes. This could allow for both oocyte ranking and embryo preferences in IVF applications.

FIGURE 1.

a, reverse transcription and second strand cDNA synthesis using the WTA2 kit. Each WTA2 primer has a pseudo-random nonamer (orange) that is designed to preferentially bind to mRNA sequences (green) over rRNA. After genomic DNA digestion, the first strand of cDNA is synthesized (blue). Following an RNase H step, a second round of cDNA synthesis occurs using the same WTA2 primers with the pseudo-random nonamers. The target library was then subjected to two rounds of 15 cycles of PCR amplification using just the WTA2 primer sequence (red). The WTA2 kit produced 30 ng/μl of cDNA fragments of mRNA between 100 and 300 bases long. b, final library construction and sequencing primer. See “Materials and Methods” for the library preparation procedure. The standard Illumina sequencing primer (striped primer) was not used because every sequenced cluster would have started with the same exact sequence, causing the sequencing reaction to fail. A custom sequencing primer was used, which consisted of the WTA2 primer with the six most 3′ bases of the Illumina sequencing primer. 42 bp were sequenced (hatched), consisting of 9 bp of the pseudo-random nonamer and 33 bp of the unknown mRNA sequence. The first 9 bp and the final base were removed, leaving 32-bp sequences to map against the human genome.

FIGURE 2.

a, all four oocytes samples show a high degree of overlap with each other. The total number of genes for each sample is shown in parentheses, and the total number of all genes in all four samples is equal to 12,708 genes. 66.7% of all genes were detected in at least three of the four oocyte samples, and 50.0% of all genes were detected in all four oocyte samples. b, the larger circle represents the total number of genes in each of the four oocyte samples, and the smaller circle shows the overlap of the four sibling polar bodies. The percentage was calculated by taking the total number of genes shared between the sibling oocyte and polar body and dividing by the total number of genes in the polar body. c, the overlaps of genes transcribed in sibling oocyte and PB samples among the four independent comparisons are represented as described in a. In total, there were 4,973 genes found between all the overlap data sets and 279 genes that were sampled in all eight samples. Of the 4,973 overlap gene set, ∼46% were detected in at least two of the overlaps.

FIGURE 3.

a, the most abundant genes in oocytes are compared with the most abundant genes in polar bodies. Each list is compared in increments of 50 genes testing for significant overlap in each section of 50. Iterations of 50 genes that are labeled in red have significant overlap between each list with a p value <0.05, and the fraction of genes shared between the two lists is on the y axis. The first iteration of 50 had an overlap of nearly 80% (39 of 50), and as each iteration adds to the total length of the shared list, the fraction of shared genes between the two lists approaches 100%. b, the individual p value for each iteration of 50 is shown. The green line is the significance cut-off of p = 0.05.

FIGURE 4.

The 6,355 genes that were sampled in all four oocyte samples were compared with the polar body samples as well as previously published microarray studies of oocytes. Comparing the natural log of the geometric mean of the TMM normalized counts of the oocyte samples (lane 1) and the polar body samples (lane 2); genes that are most abundant in the oocytes samples (dark blue) are more likely to be sampled in the polar body samples and are also found at similar expression levels. Select genes are shown on the left, including genes that have been shown to be highly expressed in oocytes (black text) (10) as well as genes significantly up-regulated in cumulus cells (green text) that were found (16). There is a high variation between the individual polar bodies (lane 3), but genes expressed at a high level in oocytes are more likely to be detected in polar bodies (black) than not (white). There is a very strong correlation between genes that are significantly up-regulated (lane 4) or detected (lane 5) in oocytes with microarray studies and the genes that were most abundant in oocytes and polar bodies in this study. The inverse correlation is found with genes that were significantly up-regulated in the reference compared with this data set.