Detection and quantification of mRNA
in single human polar bodies: a
minimally invasive test of gene
expression during oogenesis
Peter C. Klatsky1,*, Gary M. Wessel2, and Sandra A. Carson1
1Division of Reproductive Endocrinology and Infertility, Women and Infants Hospital, Alpert School of Medicine, Brown University, 101
Dudley Street, Providence, RI 02905, USA2Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence,
RI 02912, USA
*Correspondence address. Tel: +1-415-200-6259; Fax: +1-401-276-7845. E-mail: email@example.com
Submitted on July 20, 2010; resubmitted on September 3, 2010; accepted on September 7, 2010
abstract: Proteins and mRNA produced in oogenesis support embryonic development until the zygotic transition, 3 days after ferti-
lization. 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 over-
night 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.
Key words: gene expression / infertility / oocyte quality / oocyte / meiosis
The mature oocyte has a unique, cytoplasm-dependent ability to incor-
differentiation. These functions require specific changes in the mRNA
populations that have been documented in experiments using pooled
oocytes (Assou et al., 2006; Evsikov and Evsikova, 2009). The clinical
importance of healthyoocyte development is evidenced by the impress-
forming comparative genomic hybridization across 24 chromosomes,
many euploid blastocysts, fail to implant and develop into living
embryos, suggesting potential problems in either gene expression or
endometrial receptivity (Schoolcraft et al., in press).
Oocyte gene expression can be evaluated in messenger RNA,
which represents genetic functionality and the downstream effect of
epigenetic influences mediating oocyte development. The functional
importance of cytoplasmic factors in the oocyte is supported by
observations that oocytes can parthenogenically divide without
sperm and that enucleated oocytes can support full embryonic devel-
opment after somatic cell nuclear transfer (Briggs and King, 1952;
Rascado et al., 2010). Previous studies of oocyte populations at
varying developmental stages have identified rapid changes in mRNA
profiles that occur during oocyte maturation and subsequent fertiliza-
tion, but most data are limited to cohorts of oocytes (Assou et al.,
2006; Gasca et al., 2007). Few investigators have evaluated genetic
expression in individual oocytes, and no investigators have heretofore
evaluated RNA from individual oocytes without destroying the oocyte
(Hartshorn et al., 2005; Kocabas et al., 2006; Kurimoto et al., 2007).
The developmental potential of individual oocytes varies widely,
making it challenging to evaluate successful oogenesis from gene
expression in pooled oocytes.
The ability to detect and compare individual differences in oocyte
gene expression without harming the oocyte may prove helpful to
clinicians caring for patients using ART (Assou et al., 2008). Such a
technique would allow embryologists to test for the presence or
& The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Molecular Human Reproduction, Vol.16, No.12 pp. 938–943, 2010
Advanced Access publication on September 12, 2010doi:10.1093/molehr/gaq077
at Brown University on January 28, 2011
Funding was provided through seed funds from the Brown University
provost’s office, as well as departmental funds from the Center for
Reproduction and Infertility at Women and Infants Hospital of
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at Brown University on January 28, 2011