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Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system

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    Rights statement: © The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Original languageEnglish
Pages (from-to)135–142
Number of pages8
JournalMolecular Human Reproduction
Volume24
Issue number3
Early online date30 Jan 2018
DOIs
StatePublished - 1 Mar 2018

Abstract

STUDY QUESTION: Can complete oocyte development be achieved from human ovarian tissue containing primordial/unilaminar follicles and grown in vitro in a multi-step culture to meiotic maturation demonstrated by the formation of polar bodies and a Metaphase II spindle?

SUMMARY ANSWER: Development of human oocytes from primordial/unilaminar stages to resumption of meiosis (Metaphase II) and emission of a polar body was achieved within a serum free multi-step culture system.

WHAT IS KNOWN ALREADY: Complete development of oocytes in vitro has been achieved in mouse, where in vitro grown (IVG) oocytes from primordial follicles have resulted in the production of live offspring. Human oocytes have been grown in vitro from the secondary/multi-laminar stage to obtain fully grown oocytes capable of meiotic maturation. However, there are no reports of a culture system supporting complete growth from the earliest stages of human follicle development through to Metaphase II.

STUDY DESIGN, SIZE, DURATION: Ovarian cortical biopsies were obtained with informed consent from women undergoing elective caesarean section (mean age 30.7 ± 1.7; range 25-39 years, n = 10).

PARTICIPANTS/MATERIALS, SETTING, METHODS: Laboratory setting. Ovarian biopsies were dissected into thin strips, and after removal of growing follicles were cultured in serum free medium for 8 days (step 1). At the end of this period secondary/multi-laminar follicles were dissected from the strips and intact follicles 100-150 μm in diameter were selected for further culture. Isolated follicles were cultured individually in serum free medium in the presence of 100 ng/ml of human recombinant Activin A (step 2). Individual follicles were monitored and after 8 days, Cumulus Oocyte Complexes (COCs) were retrieved by gentle pressure on the cultured follicles. Complexes with complete cumulus and adherent mural granulosa cells were selected and cultured in the presence of Activin A and FSH on membranes for a further 4 days (step 3). At the end of step 3, complexes containing oocytes greater than 100 μm diameter were selected for in vitro maturation in SAGE medium (step 4) then fixed for analysis.

MAIN RESULTS AND THE ROLE OF CHANCE: Pieces of human ovarian cortex cultured in serum free medium for 8 days (step 1) supported early follicle growth and 87 secondary follicles of diameter 120 ± 6 μm (mean ± sem) could be dissected for further culture. After a further 8 days, 54 of the 87 follicles had reached the antral stage of development. COCs were retrieved by gentle pressure from the cultured follicles and those with adherent mural granulosa cells (n = 48) were selected and cultured for a further 4 days (step 3). At the end of step 3, 32 complexes contained oocytes greater than 100 μm diameter were selected for in vitro maturation (step 4). Nine of these complexes contained polar bodies within 24 h and all polar bodies were abnormally large. Confocal immuno-histochemical analysis showed the presence of a Metaphase II spindle confirming that these in vitro grown oocytes had resumed meiosis but their developmental potential is unknown.

LIMITATIONS, REASONS FOR CAUTION: This is a small number of samples but provides proof of concept that complete development of human oocytes can occur in vitro. Further optimisation with morphological evaluation and fertilisation potential of in vitro grown oocytes is required to determine whether they are normal.

WIDER IMPLICATIONS OF THE FINDINGS: The ability to develop human oocytes from the earliest follicular stages in vitro through to maturation and fertilisation would benefit fertility preservation practice.

STUDY FUNDING/COMPETING INTERESTS: Funded by UK Medical Research Council Grants G0901839 and MR/L00299X/1. No competing interests.

    Research areas

  • Ovary, follicle, oocyte, in vitro growth, IVM, meiosis, human

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