Preantral follicle culture and oocyte quality.

The formation of high-quality oocytes depends on complex stage-specific interactions between the germ cell and the somatic compartment involving endocrine, paracrine, and autocrine regulation. Cooperativity in bidirectional signalling and metabolism in response to factors in the microenvironment drive growth, proliferation, cell cycle regulation, spindle formation and the establishment of epigenetic marks in oocytes. This is essential to ensure faithful chromosome segregation and to achieve high oocyte quality, with far-reaching consequences for embryo survival, development and the health of offspring. Oocytes reach developmental capacity throughout early meiotic stages up to full growth and acquisition of nuclear and cytoplasmic maturational competence during folliculogenesis. Improved preantral follicle culture in which ideally intimate contacts between oocyte and somatic cells are retained provides unique opportunities to assess the effects of microenvironment, growth factors, hormones, cryopreservation and environmental exposure on folliculogenesis and oocyte quality. An optimised follicle culture can contribute to the generation of high-quality oocytes for use in fertility preservation in cancer patients, animal breeding and the preservation of endangered species. The past decade has brought about major advances in follicle culture from different species. Recent advances in preantral follicle culture are discussed to assess the effects of environment, adverse exposure, cryopreservation and age on oocyte quality.

Improved cryotolerance and developmental potential of in vitro and in vivo matured mouse oocytes by supplementing with a glutathione donor prior to vitrification.

STUDY QUESTION: Can supplementation of media with a glutathione (GSH) donor, glutathione ethyl ester (GEE), prior to vitrification protect the mouse oocyte from oxidative damage and critical changes in redox homeostasis, and thereby improve cryotolerance? SUMMARY ANSWER: GEE supplementation supported redox regulation, rapid recovery of spindle and chromosome alignment after vitrification/warming and improved preimplantation development of mouse metaphase II (MII) oocytes. WHAT IS KNOWN ALREADY: Cryopreservation may affect mitochondrial functionality, induce oxidative stress, and thereby affect spindle integrity, chromosome segregation and the quality of mammalian oocytes. GEE is a membrane permeable GSH donor that promoted fertilization and early embryonic development of macaque and bovine oocytes after IVM. STUDY DESIGN, SIZE, DURATION: Two experimental groups consisted of (i) denuded mouse germinal vesicle (GV) oocytes that were matured in vitro in the presence or absence of 1 mM GEE (IVM group 1) and (ii) in vivo ovulated (IVO) MII oocytes that were isolated from the ampullae and exposed to 1 mM GEE for 1 h prior to vitrification (IVO group 2). Recovery of oocytes from both groups was followed after CryoTop vitrification/warming for up to 2 h and parthenogenetic activation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Reactive oxygen species (ROS), spindle morphology and chromosome alignment were analyzed by confocal laser scanning microscopy (CLSM) and polarization microscopy in control and GEE-supplemented MII oocytes. The relative overall intra-oocyte GSH content was assessed by analysis of monochlorobimane (MBC)-GSH adduct fluorescence in IVM MII oocytes. The GSH-dependent intra-mitochondrial redox potential (EmGSH) of IVM MII oocytes was determined after microinjection with specific mRNA at the GV stage to express a redox-sensitive probe within mitochondria (mito-Grx1-roGFP2). The absolute negative redox capacity (in millivolts) was determined by analysis of fluorescence of the oxidized versus the reduced form of sensor by CLSM and quantification according to Nernst equation. Proteome analysis was performed by quantitative 2D saturation gel electrophoresis (2D DIGE). Since microinjection and expression of redox sensor mRNA required removal of cumulus cells, and IVM of denuded mouse oocytes in group 1 induces zona hardening, the development to blastocysts was not assessed after IVF but instead after parthenogenetic activation of vitrified/warmed MII oocytes from both experimental groups. MAIN RESULTS AND ROLE OF CHANCE: IVM of denuded mouse oocytes in the presence of 1 mM GEE significantly increased intra-oocyte GSH content. ROS was not increased by CryoTop vitrification but was significantly lower in the IVM GEE group compared to IVM without GEE before vitrification and after recovery from vitrification/warming (P < 0.001). Vitrification alone significantly increased the GSH-dependent intra-mitochondrial redox capacity after warming (EmGSH, P < 0.001) in IVM oocytes, presumably by diffusion/uptake of cytoplasmic GSH into mitochondria. The presence of 1 mM GEE during IVM increased the redox capacity before vitrification and there was no further increase after vitrification/warming. None of the reproducibly detected 1492 spots of 2D DIGE separated proteins were significantly altered by vitrification or GEE supplementation. However, IVM of denuded oocytes significantly affected spindle integrity and chromosome alignment right after warming from vitrification (0 h) in group 1 and spindle integrity in group 2 (P < 0.05). GEE improved recovery in IVM group as numbers of oocytes with unaligned chromosomes and aberrant spindles was not significantly increased compared to unvitrified controls. The supplementation with GEE for 1 h before vitrification also supported more rapid recovery of spindle birefringence. GEE improved significantly development to the 2-cell stage for MII oocytes that were activated directly after vitrification/warming in both experimental groups, and also the blastocyst rate in the IVO GEE-supplemented group compared to the controls (P < 0.05). LARGE SCALE DATA: None LIMITATIONS, REASONS FOR CAUTION: The studies were carried out in a mouse model, in IVM denuded rather than cumulus-enclosed oocytes, and in activated rather than IVF MII oocytes. Whether the increased GSH-dependent intra-mitochondrial redox capacity also improves male pronuclear formation needs to be studied further experimentally. The influence of GEE supplementation requires also further examination and optimization in human oocytes before it can be considered for clinical ART. WIDER IMPLICATIONS OF THE FINDINGS: Although GEE supplementation did not alter the proteome at MII, the GSH donor may support cellular homeostasis and redox regulation and, thus, increase developmental competence. While human MII oocyte vitrification is an established procedure, GEE might be particularly beneficial for oocytes that suffer from oxidative stress and reduced redox capacity (e.g. aged oocytes) or possess low GSH due to a reduced supply of GSH from cumulus. It might also be of relevance for immature human oocytes that develop without cumulus to MII in vitro (e.g. in ICSI cycles) for ART. STUDY FUNDING AND COMPETING INTERESTS: The study has been supported by the German Research Foundation (DFG FOR 1041; EI 199/3-2). There are no conflict of interests.

A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys.

STUDY QUESTION: What clinical practices, patient management strategies and experimental methods are currently being used to preserve and restore the fertility of prepubertal boys and adolescent males? SUMMARY ANSWER: Based on a review of the clinical literature and research evidence for sperm freezing and testicular tissue cryopreservation, and after consideration of the relevant ethical and legal challenges, an algorithm for the cryopreservation of sperm and testicular tissue is proposed for prepubertal boys and adolescent males at high risk of fertility loss. WHAT IS KNOWN ALREADY: A known late effect of the chemotherapy agents and radiation exposure regimes used to treat childhood cancers and other non-malignant conditions in males is the damage and/or loss of the proliferating spermatogonial stem cells in the testis. Cryopreservation of spermatozoa is the first line treatment for fertility preservation in adolescent males. Where sperm retrieval is impossible, such as in prepubertal boys, or it is unfeasible in adolescents prior to the onset of ablative therapies, alternative experimental treatments such as testicular tissue cryopreservation and the harvesting and banking of isolated spermatogonial stem cells can now be proposed as viable means of preserving fertility. STUDY DESIGN, SIZE, DURATION: Advances in clinical treatments, patient management strategies and the research methods used to preserve sperm and testicular tissue for prepubertal boys and adolescents were reviewed. A snapshot of the up-take of testis cryopreservation as a means to preserve the fertility of young males prior to December 2012 was provided using a questionnaire. PARTICIPANTS/MATERIALS, SETTING, METHODS: A comprehensive literature review was conducted. In addition, survey results of testis freezing practices in young patients were collated from 24 European centres and Israeli University Hospitals. MAIN RESULTS AND THE ROLE OF CHANCE: There is increasing evidence of the use of testicular tissue cryopreservation as a means to preserve the fertility of pre- and peri-pubertal boys of up to 16 year-old. The survey results indicate that of the 14 respondents, half of the centres were actively offering testis tissue cryobanking as a means of safeguarding the future fertility of boys and adolescents as more than 260 young patients (age range less than 1 year old to 16 years of age), had already undergone testicular tissue retrieval and storage for fertility preservation. The remaining centres were considering the implementation of a tissue-based fertility preservation programme for boys undergoing oncological treatments. LIMITATIONS, REASONS FOR CAUTION: The data collected were limited by the scope of the questionnaire, the geographical range of the survey area, and the small number of respondents. WIDER IMPLICATIONS OF THE FINDINGS: The clinical and research questions identified and the ethical and legal issues raised are highly relevant to the multi-disciplinary teams developing treatment strategies to preserve the fertility of prepubertal and adolescent boys who have a high risk of fertility loss due to ablative interventions, trauma or genetic pre-disposition.

Slow-freezing versus vitrification for human ovarian tissue cryopreservation.

PURPOSE: Ovarian tissue can be cryopreserved prior to chemotherapy using either the slow-freezing or the vitrification method; however, the data on the equality of the procedures are still conflicting. In this study, a comparison of the cryo-damage of human ovarian tissue induced by either vitrification or slow-freezing was performed. METHODS: Ovarian tissue from 23 pre-menopausal patients was cryopreserved with either slow-freezing or vitrification. After thawing/warming, the tissue was histologically and immunohistochemically analyzed and cultured in vitro. During tissue culture the estradiol release was assessed. RESULTS: No significant difference was found in the proportion of high-quality follicles after thawing/warming in the slow-freezing and vitrification group, respectively (72.7 versus 66.7 %, p = 0.733). Estradiol secretion by the ovarian tissue was similar between groups during 18 days in vitro culture (area-under-the-curve 5,411 versus 13,102, p = 0.11). Addition of Sphingosine-1-Phosphate or Activin A to the culture medium did not alter estradiol release in both groups. The proportion of Activated Caspase-3 or 'Proliferating-Cell-Nuclear-Antigen' positive follicles at the end of the culture period was similar between slow-freezing and vitrification. CONCLUSION(S): Slow-freezing and vitrification result in similar morphological integrity after cryopreservation, a similar estradiol release in culture, and similar rates of follicular proliferation and apoptosis after culture.

Dicarbonyl stress and glyoxalases in ovarian function.

The ovary is the main regulator of female fertility. Changes in maternal health and physiology can disrupt intraovarian homoeostasis thereby compromising oocyte competence and fertility. Research has only recently devoted attention to the involvement of dicarbonyl stress in ovarian function. On this basis, the present review focuses on clinical and experimental research supporting the role of dicarbonyl overload and AGEs (advanced glycation end-products) as key contributors to perturbations of the ovarian microenvironment leading to lower fertility. Particular emphasis has been given to oocyte susceptibility to methylglyoxal, a powerful glycating agent, whose levels are known to increase during aging and metabolic disorders. According to the literature, the ovary and the oocyte itself can rely on the glyoxalase system to counteract the possible dicarbonyl overload such as that which may occur in reproductive-age women and patients with PCOS (polycystic ovarian syndrome) or diabetes. Overall, although biochemical methods for proper evaluation of dicarbonyl stress in oocytes and the ovarian microenvironment need to be established, AGEs can be proposed as predictive markers and/or therapeutic targets in new strategies for improving reproductive counselling and infertility therapies.

Endoplasmic reticulum-derived multilamellar bodies in oocytes of mouse follicle cultures under oxidized low-density lipoprotein treatment.

OBJECTIVE: Multilamellar bodies associated with an organized endoplasmic reticulum (ER) arise in various somatic cell types, and a subtype called multivesicular bodies is described in oocytes. Both entities, so far undetermined in significance, may occur in oocytes of follicles under oxidative stress. In preovulatory follicles, oxidative stress appears to be caused by oxidized low-density lipoprotein (ox-LDL). METHOD: Cultures of preantral mouse follicles were treated with 100 µg/ml ox-LDL or normal LDL (n-LDL) for 12-48 h or for 12 days during antral follicle growth followed by in vitro ovulation and harvest of cumulus oophorus complexes (COCs) with metaphase II (MII) oocytes on day 13. Preantral follicles, COCs, or MII oocytes were immunostained with anti-tubulin antibody or stained with actin-binding phalloidin for confocal microscopy. Ultrathin sections were prepared for electron microscopy. RESULTS: Preantral follicles exposed to n-LDL or ox-LDL developed normally, and MII oocytes in COCs possessed normal spindles with well-aligned chromosomes. In contrast, treated cumulus cells underwent apoptosis. Only the ox-LDL-treated preantral follicle oocytes showed ER-derived multilamellar bodies (EMBs) of type I, consisting of rough ER membranes for the envelope. The MII oocytes of COCs showed type II EMBs consisting of smooth/vesicular ER and were more prominent after ox-LDL than after n-LDL exposure. Degenerating mitochondria were prominent in oocytes of the ox-LDL group and judged as a sign of oxidative stress. CONCLUSION: Oxidative stress presumably induces damage of proteins and organelles in the oocytes. The EMBs might sequester the damaged structures for oocyte survival. Thus, EMBs could represent a novel form of autophagy.

Vitrification at the pre-antral stage transiently alters inner mitochondrial membrane potential but proteome of in vitro grown and matured mouse oocytes appears unaffected.

BACKGROUND: Vitrification is a fast and effective method to cryopreserve ovarian tissue, but it might influence mitochondrial activity and affect gene expression to cause persistent alterations in the proteome of oocytes that grow and mature following cryopreservation. METHODS: In part one of the study, the inner mitochondrial membrane potential (Ψ(mit)) of JC-1 stained oocytes from control and CryoTop vitrified pre-antral follicles was analyzed by confocal microscopy at Day 0, or after culture of follicles for 1 or 12 days. In part two, proteins of in vivo grown germinal vesicle (GV) oocytes were subjected to proteome analysis by SDS polyacrylamide gel electrophoresis, tryptic in-gel digestion of gel slices, and one-dimensional-nano-liquid chromatography of peptides on a multi-dimensional-nano-liquid chromatography system followed by mass spectrometry (LC-MS/MS) and Uniprot Gene Ontology (GO) analysis. In part three, samples containing the protein amount of 40 GV and metaphase II (MII) oocytes, respectively, from control and vitrified pre-antral follicles cultured for 12 or 13 days were subjected to 2D DIGE saturation labeling and separated by isoelectric focusing and SDS gel electrophoresis (2D DIGE), followed by DeCyder(Tm) analysis of spot patterns in three independent biological replicates. Statistical and hierarchical cluster analysis was employed to compare control and vitrified groups. RESULTS: (i) Mitochondrial inner membrane potential differs significantly between control and vitrified GV oocytes at Day 0 and Day 1, but is similar at Day 12 of culture. (ii) LC-MS/MS analysis of SDS gel fractionated protein lysates of 988 mouse GV oocytes revealed identification of 1123 different proteins with a false discovery rate of <1%. GO analysis assigned 811 proteins to the 'biological process' subset. Thirty-five percent of the proteins corresponded to metabolic processes, about 15% to mitochondrion and transport, each, and close to 8% to oxidation-reduction processes. (iii) From the 2D-saturation DIGE analysis 1891 matched spots for GV-stage and 1718 for MII oocyte proteins were detected and the related protein abundances in vitrified and control oocytes were quantified. None of the spots was significantly altered in intensity, and hierarchical cluster analysis as well as histograms of p and q values suggest that vitrification at the pre-antral stage does not significantly alter the proteome of GV or MII oocytes compared with controls. CONCLUSIONS: Vitrification appears to be associated with a significant transient increase in Ψ(mit) in oocyte mitochondria, which disappears when oocyte/cumulus cell apposition is restored upon development to the antral stage. The nano-LC-MS/MS analysis of low numbers of oocytes is useful to obtain information on relevant biological signaling pathways based on protein identifications. For quantitative comparisons, saturation 2D DIGE analysis is superior to LC-MS/MS due to its high sensitivity in cases where the biological material is very limited. Genetic background, age of the female, and/or stimulation protocol appear to influence the proteome pattern. However, the quantitative 2D DIGE approach provides evidence that vitrification does not affect the oocyte proteome after recovery from transient loss of cell-cell interactions, in vitro growth and in vitro maturation under tested conditions. Therefore, transient changes in mitochondrial activity by vitrification do not appear causal to persistent alterations in the mitochondrial or overall oocyte proteome.

Evidence that carbonyl stress by methylglyoxal exposure induces DNA damage and spindle aberrations, affects mitochondrial integrity in mammalian oocytes and contributes to oocyte ageing.

BACKGROUND: Highly reactive carbonyl compounds formed during glycolysis, such as methylglyoxal (MG), can lead to the formation of 'advanced glycation end products' (AGE) and carbonyl stress. Toxic AGEs are suspected to accumulate and play a role in reducing quality and developmental potential of mammalian oocytes of aged females and in PCOS and diabetic patients. Whether and how MG and AGE affect young and aged oocytes at the cellular level is unknown. METHODS: The study consists of three parts. In Part A expression of MG-detoxifying enzymes glyoxalases 1 and 2 was analysed by RT-PCR at different stages of maturation in denuded oocytes (DO), cumulus-enclosed oocytes (CEO) and metaphase (M)II oocytes of the CD-1 mouse to obtain information on stage-specific susceptibility to carbonyl stress. DO and CEO from young and aged females and from stimulated cycles were exposed to MG during maturation in vitro to assess also age-related changes in sensitivity to carbonyl stress induced by MG. Induction of apoptosis by MG on in vitro maturing DO was assessed by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling test. In Part B of the study, DO from large antral follicles of ovaries of adult, young MF-1 mice in late diestrous were exposed to MG to assess direct influences of MG and AGEs formed during continuous exposure to MG on rate and kinetics of maturation to MII, on DNA integrity (by γ-H2AX staining) in the germinal vesicle (GV) stage, and on spindle formation and chromosome alignment (by tubulin and pericentrin immunofluorescence and polarization microscopy), and chromosome segregation (by C-banding) during in vitro maturation. Since MG and AGEs can affect functionality of mitochondria in Part C, mitochondrial distribution and membrane potential was studied using JC-1 probe. Expression of a redox-sensitive mito-Grx1-roGFP2 protein in mitochondria of maturing oocytes by confocal laser scanning microscopy was employed to determine the inner mitochondrial glutathion (GSH)/glutathion disulfide (GSSG)-dependent redox potential. RESULTS: Part A revealed that mRNA for glyoxalases decreases during meiotic maturation. Importantly, cumulus from aged mice in CEO obtained from stimulated cycles does not protect oocytes efficiently from MG-induced meiotic arrest during in vitro maturation. Part B showed that the MG-induced meiotic delay or arrest is associated with significant rises in spindle aberrations, chromosome congression failure and aberrant telophase I in oocytes. MG exposure of meiotically arrested GV-stage oocytes significantly increases the numbers of γ-H2AX spots in the nucleus suggesting increased DNA damage, while MG exposure during maturation affects chromatin condensation and induces chromosome lagging at anaphase I. Moreover, Part C revealed that carbonyl stress by chronic exposure to MG is associated with delays in changes in mitochondrial distribution and altered inner-mitochondrial GSH/GSSG redox potential, which might be particularly relevant for cytoskeletal dynamics as well as processes after fertilization. Sensitivity to a meiotic block by MG appears dependent on the genetic background. CONCLUSIONS: The sensitivity to carbonyl stress by MG appears to increase with maternal age. Since MG-exposure induces DNA damage, meiotic delay, spindle aberrations, anaphase I lagging and epimutation, aged oocytes are particularly at risk for such disturbances in the absence of efficient protection by cumulus. Furthermore, disturbances in mitochondrial distribution and redox regulation may be especially critical for fertilization and developmental competence of oocytes exposed to MG and carbonyl stress before or during maturation, for instance, in aged females, or in PCOS or diabetic patients, in agreement with recent suggestions of correlations between poor follicular and embryonic development, lower pregnancy rate and presence of toxic AGEs in serum, irrespective of age.

Corrigendum to: The ethics of preconception expanded carrier screening in patients seeking assisted reproduction.

[This corrects the article DOI: 10.1093/hropen/hoaa063.].

The ethics of preconception expanded carrier screening in patients seeking assisted reproduction.

Expanded carrier screening (ECS) entails a screening offer for carrier status for multiple recessive disorders simultaneously and allows testing of couples or individuals regardless of ancestry or geographic origin. Although universal ECS-referring to a screening offer for the general population-has generated considerable ethical debate, little attention has been given to the ethics of preconception ECS for patients applying for assisted reproduction using their own gametes. There are several reasons why it is time for a systematic reflection on this practice. Firstly, various European fertility clinics already offer preconception ECS on a routine basis, and others are considering such a screening offer. Professionals involved in assisted reproduction have indicated a need for ethical guidance for ECS. Secondly, it is expected that patients seeking assisted reproduction will be particularly interested in preconception ECS, as they are already undertaking the physical, emotional and economic burdens of such reproduction. Thirdly, an offer of preconception ECS to patients seeking assisted reproduction raises particular ethical questions that do not arise in the context of universal ECS: the professional's involvement in the conception implies that both parental and professional responsibilities should be taken into account. This paper reflects on and provides ethical guidance for a responsible implementation of preconception ECS to patients seeking assisted reproduction using their own gametes by assessing the proportionality of such a screening offer: do the possible benefits clearly outweigh the possible harms and disadvantages? If so, for what kinds of disorders and under what conditions?

Chromosomal and cytoplasmic context determines predisposition to maternal age-related aneuploidy: brief overview and update on MCAK in mammalian oocytes.

It has been known for more than half a century that the risk of conceiving a child with trisomy increases with advanced maternal age. However, the origin of the high susceptibility to nondisjunction of whole chromosomes and precocious separation of sister chromatids, leading to aneuploidy in aged oocytes and embryos derived from them, cannot be traced back to a single disturbance and mechanism. Instead, analysis of recombination patterns of meiotic chromosomes of spread oocytes from embryonal ovary, and of origins and exchange patterns of extra chromosomes in trisomies, as well as morphological and molecular studies of oocytes and somatic cells from young and aged females, show chromosome-specific risk patterns and cellular aberrations related to the chronological age of the female. In addition, analysis of the function of meiotic- and cell-cycle-regulating genes in oogenesis, and the study of the spindle and chromosomal status of maturing oocytes, suggest that several events contribute synergistically to errors in chromosome segregation in aged oocytes in a chromosome-specific fashion. For instance, loss of cohesion may differentially predispose chromosomes with distal or pericentromeric chiasmata to nondisjunction. Studies on expression in young and aged oocytes from human or model organisms, like the mouse, indicate that the presence and functionality/activity of gene products involved in cell-cycle regulation, spindle formation and organelle integrity may be altered in aged oocytes, thus contributing to a high risk of error in chromosome segregation in meiosis I and II. Genes that are often altered in aged mouse oocytes include MCAK (mitotic-centromere-associated protein), a microtubule depolymerase, and AURKB (Aurora kinase B), a protein of the chromosomal passenger complex that has many targets and can also phosphorylate and regulate MCAK localization and activity. Therefore we explored the role of MCAK in maturing mouse oocytes by immunofluorescence, overexpression of a MCAK-EGFP (enhanced green fluorescent protein) fusion protein, knockdown of MCAK by RNAi (RNA interference) and inhibition of AURKB. The observations suggest that MCAK is involved in spindle regulation, chromosome congression and cell-cycle control, and that reductions in mRNA and protein in a context of permissive SAC (spindle assembly checkpoint) predispose to aneuploidy. Failure to recruit MCAK to centromeres and low expression patterns, as well as disturbances in regulation of enzyme localization and activity, e.g. due to alterations in activity of AURKB, may therefore contribute to maternal age-related rises in aneuploidy in mammalian oocytes.

DNA integrity, growth pattern, spindle formation, chromosomal constitution and imprinting patterns of mouse oocytes from vitrified pre-antral follicles.

BACKGROUND: Cryopreservation of follicles for culture and oocyte growth and maturation in vitro provides an option to increase the number of fertilizable oocytes and restore fertility in cases where transplantation of ovarian tissue poses a risk for malignant cell contamination. Vitrification for cryopreservation is fast and avoids ice crystal formation. However, the influences of exposure to high concentrations of cryoprotectants on follicle development, oocyte growth and maturation, and particularly, on the DNA integrity and methylation imprinting has not been studied systematically. METHODS: Follicle survival and development, DNA damage, oocyte growth patterns, maturation, spindle formation and chromosomal constitution were studied after Cryo-Top vitrification of mouse pre-antral follicles cultured to the antral stage and induced to ovulate in vitro. Methylation of differentially methylated regions (DMRs) of two maternally (Snrpn and Igf2r) and one paternally (H19) imprinted genes was studied by bisulfite pyrosequencing. RESULTS: Vitrification results in partial or total loss of oocyte-granulosa cell apposition and actin-rich transzonal projections, a transient increase in DNA breaks and a delay in follicle development. However, the oocyte growth pattern, maturation, spindle and chromosomal constitution are not significantly different between the vitrified and the control groups. Vitrification is not associated with elevated levels of imprinting mutations (aberrant methylation of the entire DMR), although the distribution of sporadic CpG methylation errors in the Snrpn DMR appears to differ slightly between control and vitrified oocytes. CONCLUSIONS: DNA breaks appear to be rapidly repaired and vitrification of oocytes inside pre-antral follicles by the Cryo-Top method does not appear to increase risks of abnormal imprinting or disturbances in spindle formation and chromosome segregation.

Risks of aneuploidy induction from chemical exposure: Twenty years of collaborative research in Europe from basic science to regulatory implications.

Although Theodor Boveri linked abnormal chromosome numbers and disease more than a century ago, an in-depth understanding of the impact of mitotic and meiotic chromosome segregation errors on cell proliferation and diseases is still lacking. This review reflects on the efforts and results of a large European research network that, from the 1980's until 2004, focused on protection against aneuploidy-inducing factors and tackled the following problems: 1) the origin and consequences of chromosome imbalance in somatic and germ cells; 2) aneuploidy as a result of environmental factors; 3) dose-effect relationships; 4) the need for validated assays to identify aneugenic factors and classify them according to their modes of action; 5) the need for reliable, quantitative data suitable for regulating exposure and preventing aneuploidy induction; 6) the need for mechanistic insight into the consequences of aneuploidy for human health. This activity brought together a consortium of experts from basic science and applied genetic toxicology to prepare the basis for defining guidelines and to encourage regulatory activities for the prevention of induced aneuploidy. Major strengths of the EU research programmes on aneuploidy were having a valuable scientific approach based on well-selected compounds and accurate methods that allow the determination of precise dose-effect relationships, reproducibility and inter-laboratory comparisons. The work was conducted by experienced scientists stimulated by a fascination with the complex scientific issues surrounding aneuploidy; a key strength was asking the right questions at the right time. The strength of the data permitted evaluation at the regulatory level. Finally, the entire enterprise benefited from a solid partnership under the lead of an inspired and stimulating coordinator. The research programme elucidated the major modes of action of aneugens, developed scientifically sound assays to assess aneugens in different tissues, and achieved the international validation of relevant assays with the goal of protecting human populations from aneugenic chemicals. The role of aneuploidy in tumorigenesis will require additional research, and the study of effects of exposure to multiple agents should become a priority. It is hoped that these reflections will stimulate the implementation of aneuploidy testing in national and OECD guidelines.

Trichlorfon effects on mouse oocytes following in vivo exposure.

Trichlorfon (TCF) is a widely used pesticide, which according to some epidemiological and experimental data, is suspected of being aneugenic in human and mouse cells. In particular, in vitro studies in mouse oocytes showed the induction of aneuploidy and polyploidy at the first meiotic division and of severe morphological alterations of the second meiotic spindle. We have tested the hypothesis that an acute treatment of mice with TCF might similarly affect chromosome segregation in maturing oocytes. Superovulated MF-1 mice were intraperitoneally injected with 400mg/kg TCF or orally administered with 600mg/kg TCF either at the time of or 4h after human chorionic gonadotrophin (HCG) injection. Oocytes were harvested 17h after HCG and metaphase II chromosomes were cytogenetically analyzed. No significant increase of aneuploid or polyploid cells was detected at any treatment condition. A significant (p<0.001) decrease of metaphases showing premature chromatid separation or premature anaphase II in all TCF-treated groups with respect to controls suggested that TCF treatment may have delayed the first meiotic division. To evaluate possible effects of the pesticide upon the second meiotic division, a group of females orally treated with 600mg/kg TCF at resumption of meiosis was mated with untreated males and zygotes were collected for cytogenetic analysis. No evidence of aneuploidy induction was obtained, but the frequency of polyploid zygotes was increased fivefold over the control level (p<0.01). Such polyploid embryos might have arisen from fertilization of oocytes that were either meiotically delayed and still in metaphase I at fertilization or progressed through anaphase II without cytokinesis. These findings show that in vivo studies on aneuploidy induction in oocytes may yield results different from those obtained by in vitro experiments and that both kinds of data may be necessary for risk assessment of environmentally relevant exposures.

Continuous exposure to bisphenol A during in vitro follicular development induces meiotic abnormalities.

Bisphenol A (BPA), a widely used environmental contaminant, may exert weak estrogenic, anti-androgenic and anti-thyroidic activities. BPA is suspected to possess aneugenic properties that may affect somatic cells and mammalian oocytes. Oocyte growth and maturation depend upon a complex bi-directional signaling between the oocyte and its companion somatic cells. Consequently, disturbances in oocyte maturation may originate either from direct effects of BPA at the level of the oocyte or from indirect influences at the follicular level, such as alterations in hormonal homeostasis. This study aimed to analyze the effects of chronic BPA exposure (3 nM to 30 microM) on follicle-enclosed growth and maturation of mouse oocytes in vitro. Oocytes were cultured and their spindle and chromosomes were stained by alpha-tubulin immunofluorescence and ethidium homodimer-2, respectively. Confocal microscopy was utilized for subsequent analysis. Only follicles that were exposed to 30 microM BPA during follicular development showed a slightly reduced granulosa cell proliferation and a lower total estrogen production, but they still developed and formed antral-like cavities. However, 18% of oocytes were unable to resume meiosis after stimulation of oocyte maturation, and 37% arrested after germinal vesicle breakdown, significantly different from controls (p<0.05). Only 45% of the oocytes extruded a first polar body (p < 0.05). 30 microM BPA led also to a significant increase in meiosis I-arrested oocytes with unaligned chromosomes and spindle aberrations. Oocytes that were able to progress beyond meiosis I, frequently arrested at an abnormal telophase I. Additionally, in many oocytes exposed to low chronic BPA that matured to meiosis II chromosomes failed to congress at the spindle equator. In conclusion, mouse follicle culture reveals non-linear dose-dependent effects of BPA on the meiotic spindle in mouse oocytes when exposure was chronic throughout oocyte growth and maturation.

Exposure of mouse oocytes to bisphenol A causes meiotic arrest but not aneuploidy.

Mouse oocytes isolated from large antral follicles were exposed to a wide range of concentrations of bisphenol A (BPA) during maturation in vitro (50 ng/ml to 10 microg/ml BPA in medium). Exposure to high concentrations of BPA (10 microg/ml) affected spindle formation, distribution of pericentriolar material and chromosome alignment on the spindle (termed congression failure), and caused a significant meiotic arrest. However, BPA did not increase hyperploidy at meiosis II at any tested concentration. Some but not all meiosis I arrested oocytes had MAD2-positive foci at centromeres of chromosomes in bivalents, suggesting that they had failed to pass the spindle checkpoint control. In a second set of experiments prepubertal mice were exposed sub-chronically for 7 days to low BPA by daily oral administration, followed by in vitro maturation of the denuded oocytes to metaphase II in the absence of BPA, as this treatment protocol was previously reported to induce chromosome congression failure and therefore suspected to cause aneuploidy in oocytes. The sub-chronic exposure subtly affected spindle morphology and oocyte maturation. However, as with the exposure in vitro, there was no evidence that low BPA doses increased hyperploidy at meiosis II. In conclusion, the data suggest that mouse oocytes from mice respond to BPA-induced disturbances in spindle formation by induction of meiotic arrest. This response might result from an effective checkpoint mechanism preventing the occurrence of chromosome malsegregation and aneuploidy. Low chronic BPA exposure in vivo as such does not appear to pose a risk for induction of errors in chromosome segregation at first meiosis in mouse oocytes. Additional factors besides BPA may have caused the high rate of congression failure and the temporary increase in hyperploidy in mouse metaphase II oocytes reported previously.

Enhanced polarizing microscopy as a new tool in aneuploidy research in oocytes.

Chromosomal non-disjunction in female meiosis gives rise to reduced fertility and trisomy in humans. Human oocytes, especially from aged women, appear especially susceptible to non-disjunction. The oocyte spindle is crucial for high fidelity of chromosome segregation at meiotic divisions, and alterations in spindle morphology are therefore indicators of adverse conditions during oocyte development that may result in meiotic aneuploidy. In the past, oocytes had to be fixed for spindle analysis, precluding direct non-invasive identification of aneugens and adverse maturation conditions that affect spindle integrity and chromosome behaviour. Aneuploidy research for detection of spindle aberrations was therefore mainly focused on in vivo or in vitro exposed, fixed animal oocytes or cytogenetic analysis of spread oocytes. Orientation independent enhanced polarizing microscopy with nearly circularly polarized light and electronically controlled liquid crystal compensator optics is a new tool to study spindle morphology non-invasively in vivo for qualitative as well as quantitative analysis. Image generation by polarization microscopy depends on the intrinsic optical properties of the spindle with its paracrystalline microtubule lattice. When polarized light passes through such a lattice it induces a splitting of the beam and shift in the plane of vibration and retardation of light (termed birefringence and retardance). Studies of animal oocytes and follicle-cell denuded human oocytes fertilized by intracytoplasmic sperm injection for assisted conception have demonstrated the safety and efficacy of enhanced polarization microscopy. The method can be employed in aneuploidy research for non-invasive dose-response studies to detect spindle aberrations, for instance, in combination with cytogenetic analysis. Due to the non-invasive nature of the technique it may be employed in routine analysis of human oocytes to assess risks by lifestyle factors, and occupational and adverse environmental exposures.

Trichlorfon-induced polyploidy and nondisjunction in mouse oocytes from preantral follicle culture.

Trichlorfon (TCF), an organophosphate insecticide and potent inhibitor of choline esterases, was previously shown to induce first meiotic nondisjunction and spindle aberrations in isolated, follicle cell-denuded mouse oocytes maturing in vitro. To explore dose-response and direct and indirect, potentially synergistic effects of TCF on the somatic cells and the oocyte within a follicle, we presently employed preantral follicle culture. 100 microg/ml TCF added at the time of hormonally stimulated resumption of meiosis of follicle cell-enclosed mouse oocytes, 16 h before in vitro ovulation, induced significant rises in first meiotic nondisjunction in oocytes from preantral follicle culture. Lower concentrations (6 microg/ml TCF) disturbed polar body formation. Nuclear maturation to meiosis II in absence of cytokinesis resulted in significant increases in polyploidy. Oocytes maturing in follicles in the presence of TCF had aberrant second meiotic spindles. Influences of TCF on somatic cell function were evident by reduced follicular mucification in vitro and deceased progesterone production. In contrast to TCF, acetylcholine (0.1-100 microM) increased progesterone production. The observations therefore suggest that TCF influences oocyte maturation and folliculogenesis directly and indirectly. High TCF is aneugenic at first meiotic division in oocytes, irrespective of the presence or absence of follicle cells. At lower concentrations TCF interferes with spindle formation, chromosome congression at meiosis II, and coordination of nuclear and cytoplasmic maturation, posing risks for second meiotic errors. The observations suggest that chronic TCF exposure during maturation in the follicle may predispose oocytes to the formation of chromosomally unbalanced preimplantation embryos after fertilization.

Human germline gene editing: Recommendations of ESHG and ESHRE.

Technological developments in gene editing raise high expectations for clinical applications, first of all for somatic gene editing but in theory also for germline gene editing (GLGE). GLGE is currently not allowed in many countries. This makes clinical applications in these countries impossible now, even if GLGE would become safe and effective. What were the arguments behind this legislation, and are they still convincing? If a technique can help to avoid serious genetic disorders, in a safe and effective way, would this be a reason to reconsider earlier standpoints? The European Society of Human Reproduction and Embryology (ESHRE) and the European Society of Human Genetics (ESHG) together developed a Background document and Recommendations to inform and stimulate ongoing societal debates. After consulting its membership and experts, this final version of the Recommendations was endorsed by the Executive Committee and the Board of the respective Societies in May 2017. Taking account of ethical arguments, we argue that both basic and pre-clinical research regarding GLGE can be justified, with conditions. Furthermore, while clinical GLGE would be totally premature, it might become a responsible intervention in the future, but only after adequate pre-clinical research. Safety of the child and future generations is a major concern. Future discussions must also address priorities among reproductive and potential non-reproductive alternatives, such as PGD and somatic editing, if that would be safe and successful. The prohibition of human germline modification, however, needs renewed discussion among relevant stakeholders, including the general public and legislators.

Responsible innovation in human germline gene editing: Background document to the recommendations of ESHG and ESHRE.

Technological developments in gene editing raise high expectations for clinical applications, including editing of the germline. The European Society of Human Reproduction and Embryology (ESHRE) and the European Society of Human Genetics (ESHG) together developed a Background document and Recommendations to inform and stimulate ongoing societal debates. This document provides the background to the Recommendations. Germline gene editing is currently not allowed in many countries. This makes clinical applications in these countries impossible now, even if germline gene editing would become safe and effective. What were the arguments behind this legislation, and are they still convincing? If a technique could help to avoid serious genetic disorders, in a safe and effective way, would this be a reason to reconsider earlier standpoints? This Background document summarizes the scientific developments and expectations regarding germline gene editing, legal regulations at the European level, and ethics for three different settings (basic research, preclinical research and clinical applications). In ethical terms, we argue that the deontological objections (e.g., gene editing goes against nature) do not seem convincing while consequentialist objections (e.g., safety for the children thus conceived and following generations) require research, not all of which is allowed in the current legal situation in European countries. Development of this Background document and Recommendations reflects the responsibility to help society understand and debate the full range of possible implications of the new technologies, and to contribute to regulations that are adapted to the dynamics of the field while taking account of ethical considerations and societal concerns.