Human in vitro oocyte maturation is not associated with increased imprinting error rates at LIT1, SNRPN, PEG3 and GTL2

Abstract

STUDY QUESTION

Does in vitro maturation (IVM) of cumulus-enclosed germinal vesicle (GV) stage oocytes retrieved from small antral follicles in minimally stimulated cycles without an ovulatory hCG dose induce imprinting errors at LIT1, SNRPN, PEG3 and GTL2 in human oocytes?

SUMMARY ANSWER

There is no significant increase in imprinting mutations at LIT1, SNRPN, PEG3 and GTL2 after IVM of cumulus-enclosed GV oocytes from small antral follicles in minimally stimulated cycles without hCG priming.

WHAT IS KNOWN ALREADY

Animal models have generally demonstrated correct methylation imprint establishment for in vitro grown and matured oocytes. For human IVM, well-designed studies allowing conclusions on imprint establishment are currently not available.

STUDY DESIGN, SIZE, DURATION

Immature oocyte–cumulus complexes from 2 to 9 mm follicles were retrieved in polycystic ovary syndrome (PCOS) subjects in minimally stimulated cycles without hCG priming and matured in vitro. In vivo grown oocytes were retrieved after conventional ovarian stimulation for IVF/ICSI or after ovulation induction. Imprinting error rates at three maternally methylated (LIT1, SNRPN and PEG3) and one paternally methylated (GTL2) imprinted genes were compared in 71 in vitro and 38 in vivo matured oocytes.

PARTICIPANTS/MATERIALS, SETTING, METHODS

The limiting dilution bisulfite sequencing technique was applied, allowing increased sensitivity based on multiplex PCR for the imprinted genes and the inclusion of non-imprinted marker genes for cumulus cell DNA contamination.

MAIN RESULTS AND THE ROLE OF CHANCE

In vitro as well as in vivo matured oocytes showed only a few abnormal alleles, consistent with epimutations. The abnormalities were more frequent in immature than in mature oocytes for both groups, although no significant difference was reached. There was no statistically significant increase in imprinting errors in IVM oocytes.

LIMITATIONS, REASONS FOR CAUTION

This single cell methylation analysis was restricted to a number of well-selected imprinted genes. Genome-wide methylation analysis of single human oocytes is currently not possible.

WIDER IMPLICATIONS OF THE FINDINGS

IVM is a patient-friendly alternative to conventional ovarian stimulation in PCOS patients and is associated with reduced gonadotrophin costs and avoidance of OHSS. The results of this study show for the first time that optimized human IVM procedures have no significant effects on the establishment of maternal DNA methylation patterns at LIT1, SNRPN, PEG3 and GTL2.

STUDY FUNDING/COMPETING INTEREST(S)

This study was supported by research funds from Agentschap voor Innovatie door Wetenschap en Technologie (IWT-TBM 110680), Wetenschappelijk Fonds Willy Gepts (WFWG 2011) and German Research Foundation (HA 1374/12-2). There are no competing interests.

IVM, oocyte, genomic imprinting, DNA methylation