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The article discusses the role played by epigenetics in the field of reproductive medicine. The natural times when developmental epigenetic reprogramming takes place in gametes almost matches the time of human assisted reproduction when the gametes and embryos are handled in an in vitro environemnt. Information is also given on genomic imprinting and in vitro culture of embryos.

Background: In order to optimize IVF strategies, particularly with the use of single embryo transfer, good predictive models are required. Here, we develop a model to allow such prediction, and the structure of the models point to more general conclusions about the mode of action of prognostic factors.Methods: Anonymized data from consecutive embryo transfers in five IVF centres in the UK for the 2000-2005 period were extracted and the morphological grade based on common scoring criteria was included. There were 16 096 (12 487 fresh and 3609 frozen) transfers, for 8775 couples, available for analysis. Live birth data were fitted to a model with separate sub-models for embryo and recipient effects [the 'Embryo-Uterus' (EU) model]. All covariates were included, with sub-model selection using Akaike's information criterion.Results: Age, number of embryos created, attempt number, previous history of pregnancy, duration of infertility, day of transfer and tubal diagnosis were all identified as significant prognostic factors, along with embryo grade and growth rate. Frozen transfers were substantially less likely to lead to a live birth with odds ratios of 1/3 to 1/2 compared with fresh transfers, with no evidence of differential loss for any particular patient group. Age acts predominantly through the embryo component with only a weak effect on the uterus. The embryo number, attempt number, previous pregnancies and duration of infertility act predominantly through the uterine environment. Both sub-models show significant heterogeneity between centres.Conclusions: The EU modelling framework has generated a model for predicting outcomes of embryo-transfer procedures, subject to the limitations of routinely collected data. With this large data set, the model allows identification of factors that act specifically on embryo viability or maternal receptivity. Variability in the two components between centres with similar overall outcomes suggests scope for further optimization of IVF treatment. [ABSTRACT FROM AUTHOR]

Two cases are reported of successful pregnancies following long-term semen banking prior to chemotherapy and radiotherapy for malignancy. With the first case, the patient banked semen at the age of 20 years prior to chemotherapy for Hodgkin's disease; 11 years later the thawed semen was used for IVF with intracytoplasmic sperm injection (ICSI), resulting in twins being born following the transfer of frozen-thawed embryos. In the second case, the patient banked semen at the age of 17 years prior to chemotherapy and radiotherapy for acute myeloid leukaemia; 8 years later it was used for ICSI, resulting in triplets being born following the transfer of frozen-thawed embryos. These cases support long-term semen banking for men whose future fertility may be compromised by suppression of spermatogenesis secondary to administration of chemo/radiotherapy treatment. The advent of successful ICSI combined with embryo cryopreservation has increased the chance of thawed cryopreserved semen achieving fertilization. Banking of a single ejaculate prior to commencement of chemotherapy/radiotherapy treatment may preserve potential fertility without compromising the oncology treatment. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor commenting on the August 9 Editorial concerning the ethical aspects of egg sharing and the risks associated with in-vitro fertilization.

Implantation failure is an important impediment to increasing success rates in assisted reproductive technologies. Knowledge of the cascade of morphological and molecular events at implantation remains limited. Cell surface CD44 and hyaluronate (HA) have been reported in the uterus, but a role in intercellular interaction at implantation remains to be evaluated. Mouse embryos were co-cultured with human Ishikawa endometrial epithelial monolayers over 2 days. Attachment was tenuous during the first 24 h, after which it became stable, leading to breaching of the monolayer. The effects of enzymatically reducing the density of HA, or introducing a function-blocking antibody to CD44, were monitored during progression from weak to stable embryonic attachment. Hyaluronidase-mediated removal of surface HA from the epithelial cells enhanced the speed of attachment, while a similar treatment of embryos had no effect. The antibody to CD44 caused retardation of initial attachment. These results suggest that CD44-HA binding could be employed by embryos during initial docking, but the persistence of HA in epithelial cells might be detrimental to later stages of implantation by retarding attainment of stable attachment., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

This proceedings report presents the outcomes from an international Expert Meeting to establish a consensus on the recommended technical and operational requirements for air quality within modern assisted reproduction technology (ART) laboratories. Topics considered included design and construction of the facility, as well as its heating, ventilation and air conditioning system; control of particulates, micro-organisms (bacteria, fungi and viruses) and volatile organic compounds (VOCs) within critical areas; safe cleaning practices; operational practices to optimize air quality while minimizing physicochemical risks to gametes and embryos (temperature control versus air flow); and appropriate infection-control practices that minimize exposure to VOC. More than 50 consensus points were established under the general headings of assessing site suitability, basic design criteria for new construction, and laboratory commissioning and ongoing VOC management. These consensus points should be considered as aspirational benchmarks for existing ART laboratories, and as guidelines for the construction of new ART laboratories., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available., (Copyright © 2015. Published by Elsevier B.V.)

Approximately one-third of IVF cases in the UK are attributed to male factor infertility and in the majority of cases the origin of male infertility is unknown. The integrity of sperm DNA is important both for the success of assisted reproduction and the implications for the off-spring. One type of DNA damage that has not been investigated with respect to fertility outcomes is the adduct N7-methyldeoxyguanosine (N7-MedG), a biomarker for exposure to alkylating agents. A prospective cohort of couples attending for IVF had their N7-MedG levels in sperm measured using an immunoslot blot technique to examine whether sperm N7-MedG levels are associated with male factor infertility, semen quality measures or assisted reproduction outcomes. Sufficient DNA for analysis was obtained from 67/97 couples and N7-MedG was detected in 94% of sperm samples analysed. Men diagnosed with male factor infertility had significantly higher mean levels of N7-MedG in their sperm DNA (P=0.03). Logistic regression analysis showed that N7-MedG levels were significantly negatively associated with the proportion of oocytes successfully fertilised irrespective of the method of fertilisation used (IVF or intra-cytoplasmic sperm injection; ICSI, P<0.001). Therefore exposure to DNA alkylating agents is significantly associated with male infertility and the proportion of oocytes fertilised during assisted reproduction. Reducing such exposure may improve male fertility but further work is required to determine the relative importance of exogenous and endogenous sources of exposure., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: It is estimated that there is at least a 2-fold rise in the incidence of monozygotic twinning after assisted reproductive technology compared with natural conception. This can result in adverse pregnancy outcomes., Methods: We searched MEDLINE, EMBASE and SCISEARCH for studies that estimated the risk of monozygotic twinning and its association with any particular assisted reproductive technique. Monozygotic twinning was defined by ultrasound or Weinberg criteria. A meta-analysis of the proportion of monozygotic twins was performed using both fixed and random effects models., Results: The search revealed 37 publications reporting on the incidence of monozygotic twins after assisted reproductive techniques. Twenty-seven studies met the inclusion criteria and were included in the meta-analysis. The summary incidence of monozygotic twins after assisted conception was 0.9% (0.8-0.9%). The incidence of monozygotic twins in natural conception is 0.4%. Blastocyst transfer and intracytoplasmic sperm injection are associated with 4.25 and 2.25 times higher risk of monozygotic twins., Conclusions: The risk of monozygotic twins in assisted conception is 2.25 times higher than the natural conceptions. Larger studies reporting on monozygotic twinning following single-embryo transfer or after post-natal confirmation of zygosity with DNA analysis are warranted before definitive conclusions can be drawn and guidelines produced. In order to provide adequate pre-conceptional counselling, it is important to monitor the incidence of monozygotic twins in both natural and assisted conceptions. We suggest building a national multiple pregnancy database based on accurate diagnosis of zygosity.

Background: IVF is limited by low success rates and an unacceptably high multiple pregnancy rate. These outcomes would be improved significantly if a single embryo of high viability could be replaced in each treatment cycle, but widespread acceptance of such a policy is hindered by the lack of predictive factors for embryo selection. We have conducted a retrospective clinical study of a novel non-invasive method of embryo selection based on the depletion/appearance of amino acids in the culture medium., Methods: Fifty-three cycles of IVF treatment using ICSI were studied. Embryos were cultured for 24 h in 4 microl drops of medium containing a physiological mixture of 18 amino acids. The spent medium was analysed for amino acid content by high performance liquid chromatography., Results: The turnover of three amino acids, Asn, Gly and Leu, was significantly correlated with a clinical pregnancy and live birth. These correlations were independent of known predictors, such as female age, basal levels of FSH, embryo cell number and embryo morphological grade., Conclusions: Non-invasive assay of amino acid turnover has the potential to improve significantly the prospective selection of the most viable embryos, or single embryo, for replacement in an IVF cycle.

Advances in cancer treatment have led to significant improvements in the likelihood of reaching remission and long-term survival for men. Chemo- and radiotherapy-induced infertility are significant treatment side effects. Cryopreservation before the start of treatment enables sperm to be stored, thereby preserving the man's potential fertility. Here, we describe the successful use (with ICSI) of sperm cryopreserved prior to cancer treatment, for a total of 21 years. We believe this to be the longest period of sperm cryopreservation, resulting in a live birth, so far reported in the literature.

Insulin and the insulin-like growth factors, IGF-I and IGF-II, have been reported to exert a mitogenic effect on the preimplantation mammalian embryo. Furthermore, it has been proposed that loss of imprinting of the insulin-like growth factor II receptor gene and the consequent over-production of IGF-II may be involved in the aetiology of the Enlarged Offspring Syndrome, which occurs as an artefact of in vitro embryo production. We have previously shown that apoptosis occurs in the preimplantation bovine embryo and is influenced by in vitro culture conditions. We have therefore sought to establish the effects of insulin, IGF-I and IGF-II on apoptosis and cell proliferation in bovine blastocysts in vitro. Zygotes, obtained by in vitro maturation and fertilization of follicular oocytes, were cultured to blastocysts, with or without exogenous growth factors. Embryos were stained with propidium iodide to label all nuclei and by TUNEL to label apoptotic nuclei and analyzed by epifluorescent and confocal microscopy. IGF-I and IGF-II, but not insulin, were found to increase the proportion of embryos which formed blastocysts. Insulin decreased the incidence of apoptosis without affecting blastocyst cell number. IGF-I acted to decrease apoptosis and increase total cell number and IGF-II increased cell number alone. These data suggest roles for insulin and the IGFs as mitogens and/or apoptotic survival factors during early bovine development. Perturbation of IGF-II regulated growth may be involved in fetal oversize., (Copyright 2002 Wiley-Liss, Inc.)

Formation of a fully differentiated, implantation competent blastocyst requires the expression of a complex repertoire of molecules. However, the events that drive morphogenesis are poorly elucidated in the human embryo. In this work, we describe the amplification of representative cDNAs from morphologically and developmentally normal, individual human embryos at all stages from pronucleate to blastocyst. These cDNAs were probed to reveal the temporal expression pattern of cell adhesion molecules thought to play a key role in murine preimplantation embryo development. We demonstrated constitutive expression of beta actin, beta 1 and alpha 6 integrins, ZO-1 and E-cadherin, as shown previously in mouse embryos. No expression of beta 3, alpha 2, alpha 3 or alpha 7 integrins nor of L or P selectin was detected at any stage of preimplantation development. beta 5 integrin showed a regulated pattern of expression and was not expressed in blastocysts, while desmocollin-2 could only be detected at the blastocyst stage. Expression and localization of beta 1, beta 5 and alpha 6 integrins and ZO-1 and E-cadherin proteins was confirmed in blastocyst stage embryos by immunocytochemistry. We have identified differences in the expression of integrin molecules between mouse and human embryos, and propose a role for alpha v beta 5 and alpha 6 beta 1 integrin dimers in the human embryo at implantation.

The occurrence of cell death by apoptosis was examined in blastocyst and preblastocyst stage bovine embryos. Zygotes were obtained by in vitro maturation and in vitro fertilization of oocytes from abattoir derived ovaries. Two-cell to hatched blastocyst stage embryos were stained with propidium iodide to label all nuclei and by terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick end-labelling (TUNEL) to label apoptotic nuclei, and were analysed by epifluorescent and confocal microscopy. Apoptosis was first observed at the 9-16-cell stage of development, decreasing at the morula stage before increasing at the blastocyst stage. Apoptotic dead cell index in day 7 blastocysts was negatively correlated with the total number of cells; the percentage of dead cells ranged from approximately 1 to 10% and occurred predominantly within the inner cell mass. In addition, apoptotic dead cell index was significantly higher (P < 0.05) in blastocysts cultured (from the two-cell stage) in the presence of 10% fetal bovine serum compared with those developed in serum-free medium. Embryos selected for early cleavage at < 29 h after fertilization and cultured together until the blastocyst stage showed a significantly lower rate of apoptosis (P < 0.01) compared with slower cleaving embryos.

We previously demonstrated that exogenous transforming growth factor alpha (TGFalpha) reduces the incidence of apoptosis in mouse blastocysts that develop in vitro but does not result in an increase in cell number or the incidence of development to the blastocyst stage. Thus, TGFalpha may function as a cell survival factor in the preimplantation mouse embryo. To extend these studies, we have now examined the development of TGFalpha-deficient preimplantation embryos in vitro and in vivo in TGFalpha-deficient mothers. We found that in both instances the incidence of apoptosis is dramatically increased in the TGFalpha-deficient blastocysts and that this increase is essentially restricted to the cells of the inner cell mass when the embryos develop in vivo but extends to the trophectoderm cells for embryos that develop in vitro. The absence of endogenous TGFalpha has little effect on the incidence of development to the blastocyst stage and cell number, cell lineage allocation, blastocoel volume, and the timing and incidence of hatching in these blastocysts, when compared to wild-type embryos. These results buttress our previous suggestion that TGFalpha functions as a cell survival factor in the preimplantation mouse embryo.

Mouse blastocysts undergo cell death in the inner cell mass (ICM) as a normal feature of development, but little is known as to how this event is regulated or as to the possible role of survival factors in preimplantation development. The observation that growth factors, which can influence preimplantation development, can act as survival factors in other cell types led us to investigate the effects of culture in vitro, embryo density during culture, and transforming growth factor alpha (TGF alpha) on cell death in the blastocyst. Mouse blastocysts cultured singly from the 2-cell stage in 25 microl of medium KSOM + amino acids showed a approximately 3-fold increase in the incidence of cell death, predominantly in the ICM, relative to blastocysts formed in vivo. Increasing the density of embryo culture to 30 embryos per 25 microl of culture medium accelerated development, increased final blastocyst cell number, and partially (approximately 50%) reduced the increase in cell death induced by culture in vitro. Addition of 0.1 pM TGF alpha to the medium of singly cultured embryos also partially (33%) reduced this increase in cell death without accelerating development or increasing final cell number. Culturing isolated ICMs for 24 h in the presence of 0.1 pM TGF alpha also partially (33%) reduced the increase in cell death. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of whole blastocysts confirmed that cell death as detected by fragmented nuclei was apoptotic, as defined by endonuclease activation. Results of these experiments suggest that endogenously produced growth factors may function as cell survival factors during preimplantation development.

We report an RT-PCR-based assay to analyze the spatial pattern of expression of genes in mouse blastocysts. The assay is based on comparing the amount of an amplicon for a specific gene in isolated inner cell mass cells to that in intact blastocysts and using this ratio to calculate the relative amount of transcript per inner cell mass cell or trophectoderm cell. Validation of the assay is demonstrated by documenting that the level of fgf-4 transcripts in inner cell mass cells is approximately 13 times greater than that in trophectoderm cells, and that the level of endo A transcripts in trophectoderm cells is approximately 18 times greater than that in inner cell mass cells; results of others have shown that fgf-4 and endo A expression are enhanced in the inner cell mass cells and trophectoderm cells, respectively. Using this assay, we find that transcripts for both the EGF receptor and TGF-alpha are present in both the inner cell mass and trophectoderm cells and that on a per cell basis, essentially equal amounts of each transcript are present in these two cell types. We also demonstrate by laser-scanning confocal microscopy that TGF-alpha protein is uniformly present in all cells of the blastocyst.

The role of oxidative phosphorylation in blastocoel development in rats was determined by culturing morula stage embryos for 24 h in the presence of three inhibitors of ATP generation: cyanide, antimycin-A and 2,4-dinitrophenol (DNP). Rat morulae could form blastocysts in concentrations of cyanide that are toxic to the embryos of other mammals. Similar results were obtained with antimycin-A and DNP, although DNP reduced the number of blastocysts that formed. A non-invasive ultramicrofluorometric assay was used on single blastocysts and the glycolytic pathway was shown to be stimulated in the presence of these inhibitors. These results suggest that, uniquely among preimplantation embryos studied, the developing rat blastocyst does not have an absolute requirement for oxidative phosphorylation but may be able to compensate by increasing the amount of glucose consumed and metabolized by glycolysis. This pattern of metabolism may be related to the changing maternal environment during development, with blastocoel cavity formation and implantation taking place in increasingly anoxic conditions.

Preimplantation mammalian development culminates in the formation of a fluid-filled cavity, the blastocoele, which is a prerequisite for successful implantation and further development. The blastocoele is enclosed by a single layer of polarised cells, the trophectoderm, which is the first epithelium formed in development. In embryos of the mouse and the rabbit, a basolaterally located Na+/K(+)-ATPase hydrolyses ATP to drive the vectorial transport of ions, which is responsible for the accumulation of blastocoele fluid. Using non-invasive assays of energy substrate consumption and blastocoele fluid accumulation, experiments were carried out on single preimplantation rat embryos, to establish: (1) the roles of the Na+/K(+)-ATPase and exogenous energy substrates, and (2) the relationship between the consumption and metabolism of energy substrates and fluid accumulation, during blastocoele cavity formation in this species. Ouabain 0.5 mM and energy-substrate-free medium both caused an inhibition in the number of embryos forming a blastocoele in culture, and also reduced the rate of fluid accumulation by day 5 blastocysts collapsed in cytochalasin-D and allowed to re-expand. Ouabain also reduced the consumption of glucose (but not pyruvate) and the production of lactate by re-expanding blastocysts. In the absence of the inhibitor, a direct relationship was seen between fluid accumulation and both glucose (but not pyruvate) consumption and lactate production. However, ouabain had no effect on intact, expanded blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)

The consumption of pyruvate and glucose, and the production of lactate, by single preimplantation embryos, was measured using a noninvasive technique. Embryos were cultured in 300-500-nl microdrops, for 8-12 h at a time, from Day 4 to Day 6 after mating, when they developed from the 8-cell stage to expanded blastocyst. Pyruvate was the predominant substrate at the 8-cell/morula stage; glucose uptake exceeded that of pyruvate after the onset of blastocoel formation. Lactate production increased in parallel with glucose consumption. For most stages, approximately 100% of the glucose uptake was accountable for by lactate production and in some cases an additional source of lactate must be postulated. Culture in vitro had little effect on lactate production, although a lower level of metabolism was observed compared with fresh blastocysts. Rat embryos were capable of developing to blastocysts in the absence of glucose, when lactate production was greatly reduced.