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?

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.

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.

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, pre-clinical 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.

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 human 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.

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.

Preovulatory Aging In Vivo and In Vitro Affects Maturation Rates, Abundance of Selected Proteins, Histone Methylation Pattern and Spindle Integrity in Murine Oocytes.

Delayed ovulation and delayed fertilization can lead to reduced developmental competence of the oocyte. In contrast to the consequences of postovulatory aging of the oocyte, hardly anything is known about the molecular processes occurring during oocyte maturation if ovulation is delayed (preovulatory aging). We investigated several aspects of oocyte maturation in two models of preovulatory aging: an in vitro follicle culture and an in vivo mouse model in which ovulation was postponed using the GnRH antagonist cetrorelix. Both models showed significantly reduced oocyte maturation rates after aging. Furthermore, in vitro preovulatory aging deregulated the protein abundance of the maternal effect genes Smarca4 and Nlrp5, decreased the levels of histone H3K9 trimethylation and caused major deterioration of chromosome alignment and spindle conformation. Protein abundance of YBX2, an important regulator of mRNA stability, storage and recruitment in the oocyte, was not affected by in vitro aging. In contrast, in vivo preovulatory aging led to reduction in Ybx2 transcript and YBX2 protein abundance. Taken together, preovulatory aging seems to affect various processes in the oocyte, which could explain the low maturation rates and the previously described failures in fertilization and embryonic development.

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.

Bisphenol A Effects on Mammalian Oogenesis and Epigenetic Integrity of Oocytes: A Case Study Exploring Risks of Endocrine Disrupting Chemicals.

Bisphenol A (BPA), originally developed as a synthetic oestrogen, is nowadays extensively used in the production of polymeric plastics. Under harsh conditions, these plastics may release BPA, which then can leach into the environment. Detectable concentrations of BPA have been measured in most analysed samples of human serum, plasma, or urine, as well as in follicular fluid, foetal serum, and amniotic fluid. Here we summarize the evidence about adverse BPA effects on the genetic and epigenetic integrity of mammalian oocytes. We conclude that increasing evidence supports the notion that low BPA concentrations adversely affect the epigenome of mammalian female germ cells, with functional consequences on gene expression, chromosome dynamics in meiosis, and oocyte development. Specific time windows, during which profound chromatin remodelling occurs and maternal imprints are established or protected, appear particularly vulnerable to epigenetic deregulation by BPA. Transgenerational effects have been also observed in the offspring of BPA-treated rodents, although the epigenetic mechanisms of inheritance still need to be clarified. The relevance of these findings for human health protection still needs to be fully assessed, but they warrant further investigation in both experimental models and humans.

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.

Pre- and postovulatory aging of murine oocytes affect the transcript level and poly(A) tail length of maternal effect genes.

Maternal effect genes code for oocyte proteins that are important for early embryogenesis. Transcription in oocytes does not take place from the onset of meiotic progression until zygotic genome activation. During this period, protein levels are regulated posttranscriptionally, for example by poly(A) tail length. Posttranscriptional regulation may be impaired in preovulatory and postovulatory aged oocytes, caused by delayed ovulation or delayed fertilization, respectively, and may lead to developmental defects. We investigated transcript levels and poly(A) tail length of ten maternal effect genes in in vivo- and in vitro- (follicle culture) grown oocytes after pre- and postovulatory aging. Quantitative RT-PCR was performed using random hexamer-primed cDNA to determine total transcript levels and oligo(dT)16-primed cDNA to analyze poly(A) tail length. Transcript levels of in vivo preovulatory-aged oocytes remained stable except for decreases in Brg1 and Tet3. Most genes investigated showed a tendency towards increased poly(A) content. Polyadenylation of in vitro preovulatory-aged oocytes was also increased, along with transcript level declines of Trim28, Nlrp2, Nlrp14 and Zar1. In contrast to preovulatory aging, postovulatory aging of in vivo- and in vitro-grown oocytes led to a shortening of poly(A) tails. Postovulatory aging of in vivo-grown oocytes resulted in deadenylation of Nlrp5 after 12 h, and deadenylation of 4 further genes (Tet3, Trim28, Dnmt1, Oct4) after 24 h. Similarly, transcripts of in vitro-grown oocytes were deadenylated after 12 h of postovulatory aging (Tet3, Trim28, Zfp57, Dnmt1, Nlrp5, Zar1). This impact of aging on poly(A) tail length may affect the timed translation of maternal effect gene transcripts and thereby contribute to developmental defects.

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.

Chronic exposure to a low concentration of bisphenol A during follicle culture affects the epigenetic status of germinal vesicles and metaphase II oocytes.

OBJECTIVE: To determine whether exposure to low concentrations of the endocrine disrupting chemical bisphenol A (BPA) during follicle culture and oocyte growth alters the methylation status of differentially methylated regions (DMRs) of imprinted genes and histone posttranslational modification patterns in mammalian oocytes. DESIGN: Comparative and control study. SETTING: Experimental laboratory. ANIMAL(S): C57/Bl6JxCBA/Ca mice. INTERVENTION(S): Exposure of oocytes to 3 nM or 300 nM BPA during follicle culture from preantral to antral stage. MAIN OUTCOME MEASURE(S): Methylation status of DMRs of maternally imprinted (Snrpn, Igf2r, and Mest) and paternally imprinted gene(s) (H19) in mouse germinal vesicle oocytes; trimethylation of histone H3K9, acetylation of histone H4K12, and distance between centromeres of sister chromatids in metaphase II oocytes. RESULT(S): Exposure to 3 nM BPA was associated with slightly accelerated follicle development, statistically significant increases in allele methylation errors in DMRs of maternally imprinted genes, and statistically significant decreases in histone H3K9 trimethylation and interkinetochore distance. CONCLUSION(S): The disturbances in oocyte genomic imprinting and modification of posttranslational histone and centromere architecture provide the first link between low BPA exposures and induction of epigenetic changes that may contribute to chromosome congression failures and meiotic errors, and to altered gene expression that might affect health of the offspring.

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.