[Show abstract][Hide abstract] ABSTRACT: Extensive epigenetic reprogramming occurs during mammalian gametogenesis and preimplantation development. DNA methylation patterns that are laid down during these stages are essential for subsequent normal foetal development. The requirement for more precise assessment of the epigenetic programming of in vitro-derived human preimplantation embryo has become of paramount importance following the identification of epigenetic diseases that are associated with assisted reproduction and/or infertility. Such techniques are also useful and applicable to experimental reproductive biology. In order to expand our knowledge of epigenetic marks, including DNA methylation, during mammalian reproduction and early development, it is necessary to test new and sufficiently sensitive protocols. There are, however, unique challenges to obtain DNA methylation data from the small cell numbers that are present in the preimplantation embryo. In this protocol, we describe the successful application of Pyrosequencing(®) to yield quantitative DNA methylation data over several CpG sites at differentially methylated regions (DMRs) at imprinted loci in single blastocysts, in this case, human blastocysts. Future developments of the protocol will allow DNA methylation analysis of a more extensive panel of genes for each embryo and at the same time, since the protocol allows for the extraction of mRNA from the embryo, the comparison between DNA methylation and gene expression.
No preview · Article · Jun 2015 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: STUDY QUESTION What are the consequences of polycystic ovary syndrome (PCOS) pathology and metformin-pretreatment in vivo in women with PCOS on the metabolism and steroid production of follicular phenotype- and long-term cultured-granulosa cells
Full-text · Article · Aug 2014 · Human Reproduction
[Show abstract][Hide abstract] ABSTRACT: STUDY QUESTION Is it possible to restore ovarian function and natural fertility following the cryopreservation and autotransplantation of
whole ovaries, complete with vascular pedicle, in adult females from a large monovulatory animal model species (i.e. sheep)?
Full-text · Article · Jun 2014 · Human Reproduction
[Show abstract][Hide abstract] ABSTRACT: Mitochondria are responsible for the production of ATP which drives cellular metabolic and biosynthetic processes. This is the first study to quantify mtDNA copy number across all stages of oogenesis in a large monovulatory species, it includes assessment of the activity of mitochondria in GV and MII oocytes through JC1 staining. Primordial to early antral follicles (n=249) were isolated from sheep ovarian cortex following digestion at 37°C for 1 hr and all oocytes were disaggregated from their somatic cells. Germinal vesicle oocytes (n=133) were aspirated from 3-5 mm diameter antral follicles and mature MII oocytes (n=71) were generated following IVM. The mtDNA copy number in each oocyte was quantified using real-time PCR and showed a progressive, but variable increase in the amount of mtDNA in oocytes from primordial follicles (605±205, n=8) to mature MII oocytes (744,633±115,799, n=13; P<0.05). Mitochondrial activity (P>0.05) was not altered during meiotic progression from GV to MII during IVM. The observed increase in mtDNA copy number across oogenesis reflects the changing ATP demands needed to orchestrate cytoskeletal and cytoplasmic reorganisation during oocyte growth and maturation and the need to fuel the resumption of meiosis in mature oocytes following the preovulatory gonadotrophin surge.
Full-text · Article · Mar 2013 · Molecular Human Reproduction
[Show abstract][Hide abstract] ABSTRACT: STUDY QUESTION: Can amino acid profiling differentiate between human oocytes with differing competence to mature to metaphase II (MII) in vitro?SUMMARY ANSWEROocytes which remained arrested at the germinal vesicle (GV) stage after 24 h of in vitro maturation (IVM) displayed differences in the depletion/appearance of amino acids compared with oocytes which progressed to MII and patient age, infertile diagnosis and ovarian stimulation regime significantly affected oocyte amino acid turnover during IVM.WHAT IS KNOWN ALREADYAmino acid profiling has been proposed as a technique which can distinguish between human pronucleate zygotes and cleavage stage embryos with the potential to develop to the blastocyst stage and implant to produce a pregnancy and those that arrest. Most recently, the amino acid turnover by individual bovine oocytes has been shown to be predictive of oocyte developmental competence as indicated by the gamete's capacity to undergo fertilization and early cleavage divisions in vitro.STUDY DESIGN, SIZE, DURATIONThe study was conducted between March 2005 and March 2010. A total of 216 oocytes which were at the GV or metaphase I (MI) stages at the time of ICSI were donated by 67 patients.PARTICIPANTS/MATERIALS, SETTINGS, METHODS
The research was conducted in university research laboratories affiliated to a hospital-based infertility clinic. Oocytes were cultured for 24 h and the depletion/appearance of amino acids was measured during the final 6 h of IVM. Amino acid turnover was analysed in relation to oocyte meiotic progression, patient age, disease aetiology and controlled ovarian stimulation regime. MAIN RESULTS AND THE ROLE OF CHANCE: The depletion/appearance of key amino acids was linked to the maturation potential of human oocytes in vitro. Oocytes which arrested at the GV stage (n = 9) depleted significantly more valine and isoleucine than those which progressed to MI (n = 32) or MII (n = 107) (P < 0.05). Glutamate, glutamine, arginine and valine depletion or appearance differed in MII versus degenerating oocytes (n = 20) (P < 0.05). Glutamine, arginine, methionine, phenylalanine, total depletion and total turnover all differed in oocytes from patients aged < 35 years versus patients ≥35 years (P < 0.05). MII oocytes obtained following ovarian stimulation with recombinant FSH depleted more isoleucine (P < 0.05) and more alanine and lysine (P < 0.05) appeared than oocytes from hMG-stimulated cycles. MII oocytes from patients with a polycystic ovary (PCO) morphology (n = 33) depleted more serine (P < 0.05) than oocytes from women with normal ovaries (n = 61). LIMITATIONS, REASONS FOR CAUTION: Immature oocytes collected at the time of ICSI were used as the model for human oocyte maturation. These oocytes have therefore failed to respond to the ovulatory hCG trigger in vivo (they are meiotically incompetent), and have limited capacity to support embryo development in vitro. The lack of cumulus cells and stress of the conditions in vitro may have influenced turnover of amino acids, and owing to the small sample sizes further studies are required to confirm these findings. WIDER IMPLICATIONS OF THE FINDINGS: The findings provide support for the hypothesis that oocyte metabolism reflects oocyte quality. Longitudinal studies are required to link these functional metabolic indices of human oocyte quality with embryo developmental competence. Oocyte amino acid profiling may be a useful tool to quantify the impact of new assisted reproduction technologies (ART) on oocyte quality.STUDY FUNDING/COMPETING INTERESTSThis project was funded by the UK Biology and Biotechnology Research Council (BB/C007395/1) and the Medical Research Council (G 0800250). K.E.H was in receipt of a British Fertility Society/Merck Serono studentship. H.J.L. is a shareholder in Novocellus Ltd, a company which seeks to devise a non-invasive biochemical test of embryo health.
Full-text · Article · Jan 2013 · Human Reproduction
[Show abstract][Hide abstract] ABSTRACT: Fertility preservation by whole ovarian cryopreservation requires successful cryopreservation of both the ovary and its vascular
supply. Previous work has indicated detrimental effects of both perfusion and cryopreservation on the ovarian vasculature.
This study assessed the effects of blood perfusion, alone or in combination with cryopreservation, on functional effects in
the follicle population and ovarian function in vivo following short-term autotransplantation of the tissue after vascular reanastomosis and measured acute changes in endothelial
cell-related gene expression within the ovarian medulla and pedicle. Following autotransplantation for 7 days, primordial,
transitional and primary follicle densities were significantly reduced (P < 0.05) and stromal Ki67 and caspase-3 expression significantly increased (P < 0.05) in cryopreserved but not fresh or perfused whole ovaries. There was evidence of clot formation and fluorescent microsphere
(FMS) extravasation in the medulla of all cryopreserved ovaries, indicating vascular damage. Utilizing a customized RT–PCR
array or conventional RT–PCR, we found that perfusion alone resulted in down-regulation in the expression of caspase 6 and
thrombospondin 1 (THBS1) genes in the medulla. Following additional cryopreservation, endothelial nitric oxide synthase (eNOS), endothelin 1, endothelin receptor A and Bcl-2 expression were significantly (P < 0.05) down-regulated. In the pedicle, both perfusion and cryopreservation caused a (P < 0.05) down-regulation of eNOS and THBS1, and an up-regulation in Bax expression. Perfusion also caused a down-regulation of TNF and up-regulation of endothelin-2 expression (P < 0.05). In conclusion, this study has identified a number of endothelial cell-related genes expressed in the medulla which
are acutely affected by both cryopreservation and perfusion, supporting the hypothesis that both interventions have deleterious
effects on endothelial cell function.
No preview · Article · Nov 2012 · Molecular Human Reproduction
[Show abstract][Hide abstract] ABSTRACT: There is evidence that expression and methylation of the imprinted paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) gene may be affected by assisted reproductive technologies (ARTs) and infertility. In this study, we sought to assess the imprinting status of the MEST gene in a large cohort of in vitro-derived human preimplantation embryos, in order to characterise potentially adverse effects of ART and infertility on this locus in early human development. Embryonic genomic DNA from morula or blastocyst stage embryos was screened for a transcribed AflIII polymorphism in MEST and imprinting analysis was then performed in cDNA libraries derived from these embryos. In 10 heterozygous embryos, MEST expression was monoallelic in seven embryos, predominantly monoallelic in two embryos, and biallelic in one embryo. Screening of cDNA derived from 61 additional human preimplantation embryos, for which DNA for genotyping was unavailable, identified eight embryos with expression originating from both alleles (biallelic or predominantly monoallelic). In some embryos, therefore, the onset of imprinted MEST expression occurs during late preimplantation development. Variability in MEST imprinting was observed in both in vitro fertilization and intracytoplasmic sperm injection-derived embryos. Biallelic or predominantly monoallelic MEST expression was not associated with any one cause of infertility. Characterisation of the main MEST isoforms revealed that isoform 2 was detected in early development and was itself variably imprinted between embryos. To our knowledge, this report constitutes the largest expression study to date of genomic imprinting in human preimplantation embryos and reveals that for some imprinted genes, contrasting imprinting states exist between embryos.European Journal of Human Genetics advance online publication, 4 July 2012; doi:10.1038/ejhg.2012.102.
Full-text · Article · Jul 2012 · European journal of human genetics: EJHG
[Show abstract][Hide abstract] ABSTRACT: The response of Graafian follicles to pre-ovulatory surge levels of FSH and LH in vivo triggers the terminal differentiation of granulosa cells and oocyte maturation. In polyovular species, the LH-driven signalling uses the epidermal growth factor (EGF)-like ligands AREG, EREG and BTC to promote oocyte maturation and cumulus expansion. This experimental series used a physiologically relevant ovine in vitro maturation (IVM) system to evaluate the impact of exposure to pre-ovulatory levels (100 ng/ml) of LH and FSH on ovine cumulus cell expression of EGF-like ligands in vitro. The serum-free sheep IVM system supported high levels (91.4%) of gonadotrophin-induced maturation of cumulus-enclosed oocytes and embryo development to the blastocyst stage (34.5%). Results were equivalent to a serum-based IVM system (85.1% IVM, 25.8% blastocyst rate; P>0.05) but were significantly different (P<0.05) to serum-free medium without gonadotrophins (69.5% IVM; 8.0% blastocyst rate). Ovine BTC was cloned and sequenced. Gonadotrophin-induced AREG, EREG, BTC and EGFR expressions were quantified in cumulus and mural granulosa cells during IVM. A rapid induction of AREG expression was apparent in both cell types within 30 min of gonadotrophin exposure in vitro. LHCGR (LHR) was detected in mural cells and FSHR in both cumulus and mural granulosa cells. The data confirm the involvement of AREG and EGFR during gonadotrophin-induced cumulus expansion, oocyte maturation and the acquisition of developmental competence by sheep oocytes matured in vitro.
[Show abstract][Hide abstract] ABSTRACT: Amino acid profiling has been used to distinguish between human embryos of differing developmental competence. We sought to determine whether amino acid profiling could be used to distinguish between metaphase II (MII) bovine oocytes with different developmental capabilities in vitro. Amino acid turnover was assayed during the final 6 h of in vitro maturation prior to oocytes undergoing individual fertilization in vitro. Following insemination, zygotes were immobilized in groups of 16 on the base of a Petri dish using Cell-Tak tissue adhesive to enable the developmental progress of each to be tracked to the blastocyst stage. Spent droplets of in vitro maturation medium were analyzed by high performance liquid chromatography, which revealed glutamine, arginine, and asparagine were depleted in the greatest quantities. Incompetent MII oocytes that failed to cleave by 72 h postfertilization depleted significantly more glutamine from (P = 0.0006) and released more alanine (P = 0.0001) into the medium than oocytes that cleaved. When cutoff values were selected for the turnover of alanine, arginine, glutamine, leucine, and tryptophan and modeled to predict fertilization and cleavage potential, oocytes that did not exceed the cutoff values for ≥2 of these key amino acids were more likely to cleave. The sensitivity, specificity, accuracy, and positive predictive value of this model were 60.5%, 76.8%, 63.5%, and 92.0%, respectively. Significant differences (P ≤ 0.015) in the consumption/production of alanine and glutamine were also observed when comparing uncleaved oocytes with those that produced blastocysts. The data show that noninvasive amino acid profiling can be used to measure oocyte developmental competence.
Preview · Article · Feb 2012 · Biology of Reproduction
[Show abstract][Hide abstract] ABSTRACT: Transient Receptor Potential Melastatin 3 (TRPM3) is a widely expressed calcium-permeable non-selective cation channel that is stimulated by high concentrations of nifedipine or by physiological steroids that include pregnenolone sulphate. Here we sought to identify steroids that inhibit TRPM3. Channel activity was studied using calcium-measurement and patch-clamp techniques. Progesterone (0.01-10μM) suppressed TRPM3 activity evoked by pregnenolone sulphate. Progesterone metabolites and 17β-oestradiol were also inhibitory but the effects were relatively small. Dihydrotestosterone was an inhibitor at concentrations higher than 1μM. Corticosteroids lacked effect. Overlay assays indicated that pregnenolone sulphate, progesterone and dihydrotestosterone bound to TRPM3. In contrast to dihydrotestosterone, progesterone inhibited nifedipine-evoked TRPM3 activity or activity in the absence of an exogenous activator, suggesting a pregnenolone sulphate-independent mechanism of action. Dihydrotestosterone, like a non-steroid look-alike compound, acted as a competitive antagonist at the pregnenolone sulphate binding site. Progesterone inhibited endogenous TRPM3 in vascular smooth muscle cells. Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified. The data further define a chemical framework for competition with pregnenolone sulphate at TRPM3 and expand knowledge of steroid interactions with TRPM3, suggesting direct steroid binding and pregnenolone sulphate-independent inhibition by progesterone.
[Show abstract][Hide abstract] ABSTRACT: Metabolic studies of mammalian embryos started with the development of in vitro culture systems more than 40 years ago. More recently, metabolic studies have begun to shed light on the requirements of growing oocytes/follicles from the earliest stages of folliculogenesis. While growing oocytes preferentially metabolise pyruvate over glucose, the somatic compartment of ovarian follicles is more glycolytic. The metabolic preferences of the oocyte are reflected in the early zygote, which becomes increasingly dependent on glycolytic energy production as development progresses to the blastocyst stage. Furthermore, the intricate metabolic relationship between each oocyte and its somatic surroundings is critical for oocyte growth and developmental competence. Measurements of amino acid turnover in bovine oocytes indicate that glutamine, arginine and leucine are consistently depleted, while alanine is produced, showing similarities with amino acid turnover in preimplantation embryos. Amino acid profiling is a good predictor of embryo quality and might also turn out to be a predictor of oocyte developmental competence. Finally, recent studies have uncovered lipid metabolism in oocytes and early embryos, suggesting that endogenous fatty acids might be used for energy production. Together, metabolic studies have revealed the multiplicity of energetic substrates used by oocytes and early embryos, and suggest that the versatility of the metabolic pathways available for energy production is key for high developmental potential. Metabolic studies of early embryos are now being applied to follicle culture, and the goal of describing the metabolome of the growing oocyte in its follicle is now very attainable.
No preview · Article · Jan 2012 · The International journal of developmental biology
[Show abstract][Hide abstract] ABSTRACT: Introduction Oocyte metabolism reflects other aspects of the unique biology of this important cell type. The protracted process of mammalian oogenesis exacts a huge metabolic toll on the presumptive gamete. To ensure that the nutritional needs of oocytes are met oogenesis occurs in concert with folliculogenesis. Folliculogenesis is a lengthy process beginning with a primordial oocyte surrounded by a small number of flattened pregranulosa cells and ending with the ovulation of a fully grown, metaphase II oocyte, some weeks or months later. Throughout their development, oocytes and follicle cells are physically and metabolically linked via a complex network of homologous and heterologous gap junctions . Metabolic coupling of oocytes and somatic cells facilitates the transfer of molecules of <1kDa, including ions, amino acids, pyruvate and glucose, molecules such as adenosine triphosphate (ATP) , and other signaling molecules and meiosis-arresting signals from the somatic compartment of the follicle to the oocyte and vice versa to provide the physiological basis for oocyte and follicle development . While the metabolic cooperativity between oocytes and their companion granulosa cells is dynamic, discrete differences exist between the nutritional needs of oocytes and somatic granulosa cells and throughout their development oocytes are exposed to a changing nutritional environment as the follicular cells undergo proliferation, antral cavity formation, differentiation, and ovulation. In turn, oocytes have been shown to regulate apoptosis and cholesterol biosynthesis and metabolism by the follicular cells and so impact on follicular development .E.
[Show abstract][Hide abstract] ABSTRACT: Familial biparental hydatidiform mole (FBHM) is the only known pure maternal-effect recessive inherited disorder in humans. Affected women, although developmentally normal themselves, suffer repeated pregnancy loss because of the development of the conceptus into a complete hydatidiform mole in which extraembryonic trophoblastic tissue develops but the embryo itself suffers early demise. This developmental phenotype results from a genome-wide failure to correctly specify or maintain a maternal epigenotype at imprinted loci. Most cases of FBHM result from mutations of NLRP7, but genetic heterogeneity has been demonstrated. Here, we report biallelic mutations of C6orf221 in three families with FBHM. The previously described biological properties of their respective gene families suggest that NLRP7 and C6orf221 may interact as components of an oocyte complex that is directly or indirectly required for determination of epigenetic status on the oocyte genome.
Full-text · Article · Sep 2011 · The American Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: We report the first quantitative assessment of DNA methylation for any gene in the human preimplantation embryo to reveal that imprints exist at KvDMR1, RB1, SNRPN, and GRB10 in the human blastocyst. For comparison, in two human embryonic stem cell lines, imprints were also observed at KvDMR1, SNRPN, GRB10, and other imprinted loci, whereas RB1 and MEG3 were hypermethylated.
No preview · Article · May 2011 · Fertility and sterility
[Show abstract][Hide abstract] ABSTRACT: Information on the ovarian follicle reserve in the African elephant (Loxodonta africana) is lacking. This study set out to determine the ratios of early preantral follicles and their relative dimensions in the ovaries of 16 African elephant aged 10-34 years. The ovaries were sectioned histologically. Follicles were counted and classified according to expansion of the pre-granulosa cells. Early primary follicles were the most common (75.8%±11.8%), followed by true primary follicles (23.8%±11.8%), whereas primordial follicles were the most rare (<2%). Measurements made on at least 100 early preantral follicles from each animal (n=1464) indicate that growth in oocyte and nuclear diameters started with transition to the true primary stage P<0.01. This, together with the observed ratios between the three types of early preantral follicles suggest that both classical primordial and early primary follicles contribute to the ovarian reserve in the African elephant.
Full-text · Article · Jan 2011 · Animal reproduction science
[Show abstract][Hide abstract] ABSTRACT: Aim: To explore the molecular basis of clinically observed volume reduction in uterine leomyoma, exposed to mifepristone.
Background: Treatment of uterine leiomyomas with a selective progesterone receptor modulator (SPRM) as an alternative to surgery is of
considerable clinical interest. Steroid homone receptors are overexpressed in leiomyoma tumor tissue compared to adjacent
myometrium and involved in the process of leiomyoma growth. Progesterone receptor modulators such as mifepristone are effective
and well tolerated in reducing myoma volume and vaginal bleeding. In a previously reported study we observed a significant
volume reduction in the dominant myoma in response to mifepristone, but with a wide individual variation (median -23%, range:
-81 to + 19%) in response to treatment. Thus, a study was conducted to explore the molecular basis of good response to mifepristone
Material and Methods: Premenopausal women with uterine leiomyoma (n = 12) received treatment with mifepristone 50 mg every other day for 12 weeks.
Among them, eight women were sub grouped as good (N = 4, median −49%, range −64 to −31%) or poor (N = 4, median −22%, range
− to −21%) respnders. At surgery, biopsies were taken from the periphery of the dominant leiomyoma and total RNA was extracted
to study the gene expression by microarray. The result was further analysed by Ingenuity Pathway Analysis (IPA, Ingenuity®
Systems, www.ingenuity.com) to explore the leading molecular pathway mediating the response to mifepristone. The result from
the microarray was confirmed by real time-PCR. Proliferation marker MKI67 and apoptosis marker TP53 were analysed along with
apoptotic index by TUNEL assay. Ethical permission for this study was obtained prior to start of the study.
Results: Twenty one canonical pathways showed significantly different expression (p < 0,05) on comparing between good and poor responders.
The most differently expressed pathway was Metabolism of Xenobiotics by Cytochrome P450 pathway. The second most significant
pathway and the pathway more relevant to uterine leomyoma growth is the glutathione pathway harboring glutathione-s tranferases
(p = 0.0001, ratio 5%). One of the genes was downregulated (GPX2 – 1.7 fold) and 4 genes belonging to this family were upregulated
(GSTM1 + 8.0-fold), GSTM2(+ 1.5 fold), GSTM3(+ 2.3 fold), GSTM5(+ 2.2 fold) among the good responders. Further analysis by
real time PCR showed GSTM1 was not detectable in biopsies from non responders. No correlations were seen for GSTM1, MKI67
or TP53 versus percentual myoma volume reduction. TUNEL analysis showed no difference in the degree of apoptosis between good
or bad responders to mifepristone.
Conclusion: Our findings indicate that glutothione pathway is involved in the action of mifepristone on leiomyoma volume reduction. GSTM1
positive fenotype is of importance for uterine leiomyoma volume reduction in response to mifepristone exposure in vivo. The mechanism behind the difference in growthregulation is still not clear, but could be suggested to interfere with proliferation
or repression co-regulators related to the degree of metabolism of steroids regulated by GSTMs. The finding in the present
study of a tentative prognostic marker for leiomyoma volume reduction during mifepristone treatment is of potential importance
for the clinical management of millions of women suffering from symptoms from uterine leiomyomas.
Full-text · Article · Jan 2011 · Human Reproduction
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND Ovarian tissue cryopreservation, in combination with autotransplantation or long-term culture, has been proposed as a means
of fertility preservation. However follicle density within ovarian cortex has a profound impact on the success of in vivo and in vitro systems designed to support follicle growth and restore fertility. The objective of this study was to investigate the dye
neutral red (NR) as a tool to quantify follicle density in situ, without compromising follicle viability and developmental potential.