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ABSTRACT: Follicle-Stimulating Hormone (FSH) at a wide range of doses is routinely added to culture media during in vitro maturation (IVM) of oocytes, but the effects on oocyte health are unclear. The suggestion that superovulation may cause aneuploidy and fetal abnormalities prompted us to study the potential role of FSH in the genesis of chromosomal abnormalities during meiosis I. Mouse cumulus-oocyte complexes (COCs) isolated from the antral follicles of unprimed, sexually immature B6CBF1 mice were cultured in increasing concentrations of FSH. Following culture, matured oocytes were isolated, spread, stained with DAPI, and the numbers of chromosomes counted. Significantly increased aneuploidy, arising during the first meiotic division, was observed in metaphase II oocytes matured in higher concentrations of FSH (> or =20 ng/ml). The effect of FSH on spindle morphology and chromosome alignment during metaphase I was then explored using immunocytochemistry and three-dimensional reconstruction of confocal sections. High FSH had no effect on gross spindle morphology but did alter chromosome congression during prometaphase and metaphase, with the spread of chromosomes across the spindle at this time being significantly greater in oocytes cultured in 2000 ng/ml compared with 2 ng/ml FSH. Analysis of three-dimensional reconstructions of spindles in oocytes matured in 2000 ng/ml FSH shows that chromosomes are more scattered and farther apart than they are following maturation in 2 ng/ml FSH. These results demonstrate that exposure to high levels of FSH during IVM can accelerate nuclear maturation and induce chromosomal abnormalities and highlights the importance of the judicious use of FSH during IVM.
Biology of Reproduction 01/2005; 72(1):107-18. · 4.01 Impact Factor
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ABSTRACT: Successful in vitro maturation (IVM) of oocytes obtained from medium-sized antral follicles could avoid the need for superovulation for in vitro fertilization. The wide range of doses of FSH used in IVM prompted us to study the effect of varying concentrations of FSH on the dynamics of nutrient uptake and production by individual maturing mouse cumulus-oocyte complexes (COCs). COCs isolated from the antral follicles of unprimed, prepubertal B6CBF(1) mice were cultured individually in increasing concentrations of FSH (0-2000 ng/ml). Following culture, pyruvate, glucose, and lactate uptake or production by individual complexes were noninvasively assessed and compared with the stage of nuclear maturation of the enclosed oocyte. FSH significantly increased oocyte maturation and produced a two- to threefold increase in glucose uptake and lactate production by COCs in which the enclosed oocyte completed maturation. In these COCs, pyruvate was taken up under control conditions but was produced in progressively higher quantities in increasing concentrations of FSH. In COCs where the oocyte failed to complete maturation, pyruvate was taken up (rather than produced) and glucose uptake and lactate production were lower and unaffected by the presence or absence of FSH. This suggests that there is dialogue between cumulus cells and the maturing oocyte that influences FSH responsiveness and substrate metabolism of the whole COC. Finally, inhibition of FSH-stimulated glucose uptake by the PI3-kinase inhibitor LY294002 and the finding of GLUT4 protein in granulosa cells suggest that FSH increases glucose uptake by PI3-kinase-mediated translocation of GLUT4 to the granulosa cell membrane.
Biology of Reproduction 08/2004; 71(1):199-209. · 4.01 Impact Factor
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ABSTRACT: The systemic endocrine environment during the later stages of follicle development has a crucial role in co-ordinating follicular and oocyte maturation before ovulation. Polycystic ovary syndrome (PCOS) is associated with abnormal circulating hormones, abnormal peri-follicular vascularity and significant abnormalities of granulosa cell function. After induction of ovulation, fertilization rates in vivo in women with PCOS are normal, but there is an increased risk of early pregnancy loss, particularly in obese patients. After in-vitro maturation of oocytes or following ovulation induction for IVF, oocyte and embryo quality in vitro are not obviously impaired in PCOS. In some reports however, specific endocrine abnormalities, such as hyperinsulinaemia/insulin resistance, have been noted to be associated with reduced fertilization rates and abnormal early embryonic development.
Reproductive biomedicine online 04/2003; 6(2):181-4. · 2.04 Impact Factor
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ABSTRACT: The clinical use of oocytes matured in vitro for IVF is increasing, but little is known about the effect of culture conditions on oocyte maturation.
Denuded immature oocytes identified following superovulation prior to ICSI were individually matured in one of two commercial media: tissue culture medium (TCM) 199 or modified Eagle medium with Earle's modified salts (MEME). During maturation, depletion of pyruvate and accumulation of lactate in the culture medium were non-invasively measured.
Maturing oocytes took up pyruvate (20-30 pmol/oocyte/h) and produced lactate (2-10 pmol/oocyte/h). Oocytes matured faster in MEME, with significantly more oocytes reaching metaphase II by 24 h after oocyte retrieval compared with TCM 199 (P = 0.03). The oocytes that matured more quickly in MEME had significantly lower lactate production than oocytes that matured more slowly (P = 0.02). In TCM 199, pyruvate uptake by rapidly maturing oocytes was lower than by slowly maturing oocytes (P = 0.05). During the second incubation, from 24 to 48 h post-oocyte retrieval, pyruvate uptake in MEME was 30% lower than in TCM 199 (P = 0.007). Pyruvate uptake and lactate production differed depending on the stage of nuclear maturation: pyruvate uptake and lactate production were greater during germinal vesicle breakdown than during polar body extrusion in MEME (P < 0.05).
We have shown that: (i). pyruvate is a major energy source during oocyte maturation; (ii). the composition of the culture medium can affect the rate of maturation; and (iii). the culture medium and stage of nuclear maturation can affect pyruvate uptake and lactate production.
Human Reproduction 11/2002; 17(11):2950-6. · 4.47 Impact Factor
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ABSTRACT: The advent of human in vitro fertilization (IVF) over 30 years ago has made the oocyte and preimplantation embryo uniquely accessible. This accessibility has given rise to new micromanipulation techniques, such as intracytoplasmic sperm injection for treatment of male infertility, as well as embryo biopsy for preimplantation diagnosis of both genetic disease and aneuploidy, a major cause of early embryo demise and miscarriage. In the UK, average pregnancy rates after IVF and embryo transfer are < 25%, even after transfer of several embryos. Unfortunately, a third of these pregnancies involve multiple gestations. Research is currently focusing on methods to improve IVF success rates while reducing twin and triplet pregnancies and their associated increased morbidity and mortality. One approach is to develop screening methods to identify the most viable embryos, so that transfer of fewer healthy embryos will result in a higher proportion of singleton pregnancies. Screening methods include optimizing culture conditions for prolonged culture and selection of viable blastocysts for transfer, or embryo biopsy and aneuploidy screening. Assisted reproduction is also increasingly important in other branches of medicine: survival rates for cancer sufferers are improving continually and there is now a significant need for approaches to preserve fertility after sterilizing chemo-and radiotherapy treatment. Techniques for cryopreserving male and female gametes or gonadal tissue are being developed, although systems to grow and mature these gametes are in their infancy. Finally, there are also concerns regarding the safety of these new assisted reproductive technologies.
Reproduction (Cambridge, England) 03/2002; 123(2):171-83. · 3.09 Impact Factor
