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Updated: Jun 8, 2026
Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes
Published on: April 24, 2019
Detection and quantification of mRNA in single human polar bodies: a minimally invasive test of gene expression
Peter C Klatsky1, Gary M Wessel, Sandra A Carson
1Division of Reproductive Endocrinology and Infertility, Women and Infants Hospital, Alpert School of Medicine, Brown University, 101 Dudley Street, Providence, RI 02905, USA. pklatsky@wihri.org
Abstract:
Proteins and mRNA produced in oogenesis support embryonic development until the zygotic transition, 3 days after fertilization. Since polar bodies can be biopsied with little if any harm to the oocyte, we tested the hypothesis that mRNA originating from expression in the meiotic oocyte is present and detectable in a single polar body prior to insemination. Human oocytes were obtained from patients undergoing controlled ovarian hyperstimulation and intracytoplasmic sperm injection. Immature oocytes were cultured overnight and inspected the following day for maturation. Metaphase II oocytes underwent polar body biopsy followed by reverse transcription without RNA isolation. Sibling oocytes were similarly prepared. Complementary DNA from all samples were pre-amplified over 15 cycles for candidate genes using selective primers. Real-time PCR was performed to detect and quantify relative single-cell gene expression. Polar body mRNA was detected in 11 of 12 candidate genes. Transcripts that were present in greater abundance in the oocyte were more likely to be detected in qPCR replicates from single polar bodies. Pre-amplification of cDNA synthesized without RNA isolation can facilitate the quantitative detection of mRNA in single human polar bodies.
Insights
Messenger RNA (mRNA) from the meiotic oocyte is detectable in human polar bodies. This finding supports using polar bodies for non-invasive genetic assessment of oocyte quality.
Area of Science:
- Reproductive Biology
- Molecular Biology
- Genetics
Background:
- Maternal mRNA and proteins are crucial for early embryonic development until the zygotic transition.
- Polar bodies, byproducts of meiosis, can be biopsied with minimal harm to the oocyte.
Purpose of the Study:
- To test if mRNA from the meiotic oocyte is present and detectable in a single polar body before fertilization.
- To evaluate the potential of polar bodies as a source for assessing oocyte genetic material.
Main Methods:
- Human oocytes were collected during controlled ovarian hyperstimulation.
- Metaphase II oocytes underwent polar body biopsy; complementary DNA (cDNA) was synthesized without RNA isolation.
- Pre-amplification of cDNA followed by quantitative real-time PCR (qPCR) was used to detect and quantify candidate gene mRNA.
Main Results:
- mRNA was detected in polar bodies for 11 out of 12 candidate genes tested.
- Higher transcript abundance in the oocyte correlated with increased detection probability in polar body samples.
- Pre-amplification enabled quantitative mRNA detection in single human polar bodies.
Conclusions:
- Single human polar bodies contain detectable levels of maternal mRNA.
- Polar body biopsy is a viable method for accessing oocyte-derived mRNA.
- This technique offers a promising avenue for non-invasive assessment of oocyte developmental potential.
