scholarly journals Pre-Implantation Mouse Embryos Cultured In Vitro under Different Oxygen Concentrations Show Altered Ultrastructures

2020 ◽  
Vol 17 (10) ◽  
pp. 3384
Author(s):  
Manuel Belli ◽  
Paolo Rinaudo ◽  
Maria Grazia Palmerini ◽  
Elena Ruggeri ◽  
Sevastiani Antonouli ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Reproductive Technologies ◽  
Mouse Embryos ◽  
Human Embryos ◽  
Fertilization In Vitro ◽  
Vitro Fertilization

Assisted Reproductive Technologies routinely utilize different culture media and oxygen (O2) concentrations to culture human embryos. Overall, embryos cultured under physiological O2 tension (5%) have improved development compared to embryos cultured under atmospheric O2 conditions (20%). The mechanisms responsible for this remain unclear. This study aimed to evaluate the effect of physiologic (5%) or atmospheric O2 (20%) tension on the microscopic ultrastructure of pre-implantation mouse embryos using Transmission Electron Microscopy (TEM). Embryos flushed out of the uterus after natural mating were used as the control. For use as the control, 2-cells, 4-cells, morulae, and blastocysts were flushed out of the uterus after natural fertilization. In vitro fertilization (IVF) was performed using potassium simplex optimized medium (KSOM) under different O2 tensions (5% and 20%) until the blastocyst stage. After collection, embryos were subjected to the standard preparative for light microscopy (LM) and TEM. We found that culture in vitro under 5% and 20% O2 results in an increase of vacuolated shaped mitochondria, cytoplasmic vacuolization and presence of multi-vesicular bodies at every embryonic stage. In addition, blastocysts generated by IVF under 5% and 20% O2 showed a lower content of heterochromatin, an interruption of the trophectodermal and inner cell mass cell membranes, an increased density of residual bodies, and high levels of glycogen granules in the cytoplasm. In conclusion, this study suggests that in vitro culture, particularly under atmospheric O2 tension, causes stage-specific changes in preimplantation embryo ultrastructure. In addition, atmospheric (20%) O2 is associated with increased alterations in embryonic ultrastructure; these changes may explain the reduced embryonic development of embryos cultured with 20% O2.

NON-INVASIVE METHODS FOR STUDYING THE POTENTIAL OF HUMAN EMBRYO FOR IMPLANTATION

2021 ◽  
pp. 29-37
Author(s):  
Василий Николаевич Попов ◽  
Роман Борисович Стукалин ◽  
Валерия Александровна Грибанова
Keyword(s):  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Uterine Cavity ◽  
Married Couples ◽  
Reproductive Technologies ◽  
Preimplantation Embryos ◽  
Preimplantation Genetic Testing ◽  
Vitro Fertilization ◽  
Non Invasive

В статье проводится анализ представленных на сегодня инвазивных и неинвазивных методов исследования преимплантационных эмбрионов. Показана эффективность преимплантационного генетического тестирования эмбрионов до переноса в полость матки. Также рассмотрены альтернативные менее инвазивные варианты изучения жизнеспособности эмбрионов, которые могли бы являться маркерами успешной имплантации. Проблема бесплодного брака с каждым годом становится все более и более значимой. Для части супружеских пар единственной возможностью рождения ребенка становится лечение методами вспомогательных репродуктивных технологий, эффективность которых остается на сегодняшний день не более 50 %. Особенно важным является поиск новых методик, позволяющих повысить результативность процедур экстракорпорального оплодотворения. В этом направлении крайне интересным является изучение неизвазивных методов оценки имплантационного потенциала эмбрионов. В анализе представлены работы по изучению протеома, метаболома и транскриптома эмбриона. Понимание молекулярного состава культуральных сред, в которых происходило развитие эмбриона до пятых суток культивирования, позволит глубже понять физиологию раннего развития, а также установить неивазивные критерии отбора эмбриона с лучшим имплантационным потенциалом и тем самым повысить эффективность проводимых программ вспомогательных репродуктивных технологий The article analyzes the currently presented invasive and non-invasive methods for studying preimplantation embryos. The efficiency of preimplantation genetic testing of embryos before transfer to the uterine cavity has been shown. Also considered are alternative less invasive options for studying the viability of embryos, which could be markers of successful implantation. The problem of sterile marriage is becoming more and more significant every year. For some married couples, the only possibility of having a child is treatment with methods of assisted reproductive technologies, the effectiveness of which remains at most 50% today. It is especially important to search for new techniques to improve the effectiveness of in vitro fertilization procedures. In this direction, it is extremely interesting to study non-invasive methods for assessing the implantation potential of embryos. The analysis presents works on the study of the proteome, metabolome and transcriptome of the embryo. Understanding the molecular composition of the culture media in which the development of the embryo took place until the fifth day of cultivation will allow a deeper understanding of the physiology of early development and also establish non-invasive criteria for the selection of embryos with the best implantation potential and thereby increase the efficiency of the programs of assisted reproductive technologies

143 TRANSCRIPTIONAL PROFILING BY HIGH-THROUGHPUT SEQUENCING OF PORCINE PRE- AND PERI-IMPLANTATION EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 184 ◽  
Author(s):  
S. C. Isom ◽  
J. R. Stevens ◽  
R. Li ◽  
L. D. Spate ◽  
W. G. Spollen ◽  
...  
Keyword(s):  
Somatic Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Assisted Reproductive Technologies ◽  
Inner Cell Mass ◽  
Expression Profiles ◽  
Cell Mass ◽  
Reproductive Technologies ◽  
Regulation Of Transcription

Significant embryo mortality occurs at or around the time of implantation or attachment in virtually all mammalian species studied to date, even in naturally conceived embryos. Embryos resulting from assisted reproductive technologies (ART) are even more susceptible to peri-implantation failure. Herein we describe our effort to characterise the transcriptomes of embryonic disc (ED) and trophoblast (TE) cells from porcine embryos derived from AI, IVF, parthenogenetic oocyte activation (PA) and somatic cell nuclear transfer (NT) on Days 10, 12 and 14 of gestation. The IVF, PA and somatic cell NT embryos were generated using in vitro–matured oocytes, cultured overnight in vitro and then transferred at the 1- to 2-cell stage into appropriately synchronized recipient gilts. On the appropriate collection day, embryos were flushed from the uterus and ED was separated from TE by mechanical dissection. Double-stranded cDNA from the collected samples was sequenced using the GAII platform from Illumina (San Diego, CA, USA). The resulting sequencing reads were aligned to a custom swine transcriptome database (see Isom et al. 2010). A generalized linear model was fit for each of 41 693 genomic regions, for ED and TE samples separately, accounting for embryo type, gestation day and their interaction and using total lane read count as a normalizing offset. Genes with significant embryo type differences (controlling the false discovery rate at 0.10) were subsequently tested for differences between IVF and each of AI, PA and NT. Those genes with significant post hoc differences (either up- or down-regulated compared with IVF) were characterised in terms of gene ontologies and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using a gene set enrichment test. Bone morphogenetic protein signalling was down-regulated (KEGG; P = 0.0099; adjusted to control for FDR at 0.05) in the ED of IVF embryos when compared with AI embryos. In TE cells from IVF embryos, ubiquitin-mediated proteolysis and ErbB signalling (adj P = 0.031 for both pathways) were aberrantly regulated when compared with AI embryos. Of particular interest is the observation that expression of genes involved in chromatin modification (GO:BiologicalProcess; q-value = 0.00005) and epigenetic regulation of transcription (q = 0.00007) was very significantly disrupted in inner cell mass cells from NT embryos compared with IVF embryos. Surprisingly, no such disruption of the epigenetic machinery was observed in the TE cells from NT embryos. In summary, we have used high-throughput sequencing technologies to compare gene expression profiles of various ART embryo types during peri-implantation development. We expect that these data will provide important insight into the root causes of (and possible opportunities for mitigation of) suboptimal development of embryos derived from ART. Funding was received from NIH R01 RR013438 and Food for the 21st Century (RSP) and the Utah Agricultural Experiment Station (UTA00151 and UTA00560 for S. C. Isom and J. R. Stevens, respectively).

scholarly journals A Preclinical Evaluation towards the Clinical Application of Oxygen Consumption Measurement by CERMs by a Mouse Chimera Model

2019 ◽  
Vol 20 (22) ◽  
pp. 5650
Author(s):  
Takashi Kuno ◽  
Masahito Tachibana ◽  
Ayako Fujimine-Sato ◽  
Misaki Fue ◽  
Keiko Higashi ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Human Embryos ◽  
Embryo Morphology ◽  
Vitro Fertilization ◽  
Cell Numbers ◽  
Mitochondrial Dna Copy Number ◽  
Future Fertility

We have developed an automated device for the measurement of oxygen consumption rate (OCR) called Chip-sensing Embryo Respiratory Measurement system (CERMs). To verify the safety and the significance of the OCR measurement by CERMs, we conducted comprehensive tests using a mouse model prior to clinical trials in a human in vitro fertilization (IVF) program. Embryo transfer revealed that the OCR measured by CERMs did not compromise the full-term development of mice or their future fertility, and was positively correlated with adenosine triphosphate (ATP) production and the mitochondrial membrane potential (ΔΨm), thereby indirectly reflecting mitochondrial oxidative phosphorylation (OXPHOS) activity. We demonstrated that the OCR is independent of embryo morphology (the size) and number of mitochondria (mitochondrial DNA copy number). The OCR correlated with the total cell numbers, whereas the inner cell mass (ICM) cell numbers and the fetal developmental rate were not. Thus, the OCR may serve as an indicator of the numbers of trophectoderm (TE) cells, rather than number or quality of ICM cells. However, implantation ability was neither correlated with the OCR, nor the embryo size in this model. This can probably be attributed to the limitation that chimeric embryos contain non-physiological high TE cells counts that are beneficial for implantation. CERMs can be safely employed in clinical IVF owing to it being a safe, highly effective, non-invasive, accurate, and quantitative tool for OCR measurement. Utilization of CERMs for clinical testing of human embryos would provide further insights into the nature of oxidative metabolism and embryonic viability.

Do assisted reproductive technologies and in vitro embryo culture influence the epigenetic control of imprinted genes and transposable elements in children?

2020 ◽  
Author(s):  
J Barberet ◽  
C Binquet ◽  
M Guilleman ◽  
A Doukani ◽  
C Choux ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Embryo Culture ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Randomized Study ◽  
Reproductive Technologies ◽  
Vitro Fertilization ◽  
Epigenetic Control ◽  
Culture Influence

Abstract STUDY QUESTION Do assisted reproductive technologies (ART) and in vitro embryo culture influence the epigenetic control of imprinted genes (IGs) and transposable elements (TEs) in children? SUMMARY ANSWER Significant differences in the DNA methylation of IGs or transposon families were reported between ART and naturally conceived children, but there was no difference between culture media. WHAT IS KNOWN ALREADY There is concern that ART may play a role in increasing the incidence of adverse health outcomes in children, probably through epigenetic mechanisms. It is crucial to assess epigenetic control, especially following non-optimal in vitro culture conditions and to compare epigenetic analyses from ART-conceived and naturally conceived children. STUDY DESIGN, SIZE, DURATION This follow-up study was based on an earlier randomized study comparing in vitro fertilization outcomes following the use of two distinct culture media. We compared the epigenetic profiles of children from the initial randomized study according to the mode of conception [i.e. ART singletons compared with those of a cohort of naturally conceived singleton children (CTL)], the type of embryo culture medium used [global medium (LifeGlobal) and single step medium (Irvine Scientific)] and the mode of in vitro fertilization (i.e. IVF versus ICSI). PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 57 buccal smears were collected from 7- to 8-year-old children. The DNA methylation profiles of four differentially methylated regions (DMRs) of IGs (H19/IGF2: IG-DMR, KCNQ1OT1: TSS-DMR, SNURF: TSS-DMR, and PEG3: TSS-DMR) and two TEs (AluYa5 and LINE-1) were first assessed by pyrosequencing. We further explored IGs and TEs’ methylation changes through methylation array (Human MethylationEPIC BeadChip referred as EPIC array, Illumina). MAIN RESULTS AND THE ROLE OF CHANCE Changes in the IGs’ DNA methylation levels were found in ART children compared to controls. DNA methylation levels of H19/IGF2 DMR were significantly lower in ART children than in CTL children [52% versus 58%, P = 0.003, false discovery rate (FDR) P = 0.018] while a significantly higher methylation rate was observed for the PEG3 DMR (51% versus 48%, P = 0.007, FDR P = 0.021). However, no differences were found between the culture media. After observing these targeted modifications, analyses were performed at wider scale. Again, no differences were detected according to the culture media, but imprinted-related DMRs overlapping promoter region near the genes major for the development (MEG3, BLCAP, and DLX5) were detected between the ART and CTL children. LIMITATIONS, REASONS FOR CAUTION The sample size could seem relatively small, but the high consistency of our results was ensured by the homogeneity of the cohort from the initial randomized study, the standardized laboratory techniques and the robust statistical analyses accounting for multiple testing. WIDER IMPLICATIONS OF THE FINDINGS Although this study did not report DNA methylation differences depending on the culture medium, it sheds light on epigenetic changes that could be observed in some children conceived by ART as compared to CTL children. The clinical relevance of such differences remains largely unknown, and it is still unclear whether such changes are due to some specific ART procedures and/or to parental infertility. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by funding from the Agence Nationale pour la Recherche (‘CARE’-ANR JCJC 2017). The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER Not concerned.

207 Female Reproductive Fluids and Epigenetics

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 114-114
Author(s):  
Sebastian Canovas ◽  
Raquel Romar ◽  
Pilar Coy
Keyword(s):  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Morphological Changes ◽  
Adult Life ◽  
Specific Effect ◽  
Reproductive Technologies ◽  
Early Embryo ◽  
Vitro Fertilization

Abstract Physiological fertilization, and early embryo development, involves dramatic transcriptomic, epigenetic and morphological changes in a short temporal window. During this period gametes and early embryos are surrounded by reproductive fluids (oviductal and uterine), which contain nutrients, growth factors, hormones and extracellular vesicles acting as carriers of DNA, RNA, proteins and other factors with putative roles in intercellular communication. Under in vitro conditions, and in the absence of these fluids, embryos derived from Assisted Reproductive Technologies (ART) reveal transcriptional and epigenetic differences compared with in vivo embryos, which could result in long-term phenotypic consequences in adult life. Therefore, reproductive fluids supplementation in the culture medium offers an alternative to imitate physiological conditions and decrease these consequences. In vitro, oviductal fluid (OF) can modulate capacitation-associated events and sperm-zona pellucida interactions and contribute to the control of polyspermy in pigs. The use of in vitro fertilization media supplemented with reproductive fluids (Natur-IVF) improves embryo quality and blastocysts hatching ability. Moreover, Natur-IVF embryos show expression and methylation patterns closer to in vivo blastocysts. In cows, supplementation of culture media with reproductive fluids, or some isolated factors, improves blastocyst rate and survival after embryo transfer, and reverses the expression of some altered genes. However, considering the complexity of the oviductal and uterine fluids, it seems difficult that the use of just a few factors in isolation can reverse all undesired consequences of the IVP. On the other hand, sex-specific embryonic plasticity, as a consequence of the oviductal regulatory signals, have been proposed. Thus, we have analysed the sex-specific effect of supplementation with reproductive fluids in bovine embryos and data reveal sex-dependent impact in DNA methylation. All these results confirm that developmental programme can be modulated by reproductive fluids and it shows sex-specific effects. This strategy allows the possibility of minimizing undesired in vitro derived consequences.

Thickness of endometrium: predictor of the effectiveness of IVF/ICSI programs (literature review)

GYNECOLOGY ◽  
2018 ◽  
Vol 20 (1) ◽  
pp. 113-116
Author(s):  
L A Bagdasaryan ◽  
I E Korneyeva
Keyword(s):  
Assisted Reproductive Technologies ◽  
Endometrial Thickness ◽  
Molecular Genetic ◽  
Uterine Cavity ◽  
Reproductive Technologies ◽  
Genetic Characteristics ◽  
Vitro Fertilization ◽  
Need To Evaluate ◽  
The Relationship

The aim of the study is to systematically analyze the data available in the modern literature on the relationship between endometrial thickness and the frequency of pregnancy in the program of assisted reproductive technologies (ART). Materials and methods. The review includes data from foreign and domestic articles found in PubMed on this topic. Results. The article presents data on the relationship between the thickness of the endometrium and the frequency of pregnancy in ART programs. The greatest number of studies is devoted to the evaluation of the relationship between the thickness of the endometrium and the frequency of pregnancy on the day of the ovulation trigger. Data are presented on the existence of a correlation between the thickness of the endometrium measured on the day of the ovulation trigger and the frequency of clinical pregnancy, as well as data on the need to evaluate the structure of the endometrium and the state of subendometric blood flow. The importance of multilayered (three-layered) endometrium as a prognostic marker of success in in vitro fertilization/intracytoplasmic sperm injection programs in the ovum is emphasized. The conclusion. The thickness of the endometrium can not be used as an argument for canceling the cycle or abolishing embryo transfer to the uterine cavity. Further studies in this direction are needed with a study of the morphological and molecular genetic characteristics of the endometrium, which in the future will allow us to evaluate the relationship between the thickness of the endometrium and the probability of pregnancy.

Legal restrictions on pre-implantation genetic diagnostics in the framework of in vitro fertilization procedure: foreign experience and Russian perspective

2019 ◽  
Author(s):  
N.A. Altinnik , S.S. Zenin , V.V. Komarova et all
Keyword(s):  
In Vitro Fertilization ◽  
Assisted Reproductive Technologies ◽  
Genetic Diseases ◽  
Genetic Diagnosis ◽  
Reproductive Technologies ◽  
Legal Regulation ◽  
Genetic Diagnostics ◽  
Vitro Fertilization ◽  
Medical Indications

The article discusses the factors that determine the content of the legal limitations of pre-implantation genetic diagnosis in the framework of the in vitro fertilization procedure, taking into account international experience and modern domestic regulatory legal regulation of the field of assisted reproductive technologies. The authors substantiates the conclusion that it is necessary to legislate a list of medical indications for preimplantation genetic diagnosis, as well as the categories of hereditary or other genetic diseases diagnosed in the framework of this procedure.

scholarly journals Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marino Maemura ◽  
Hiroaki Taketsuru ◽  
Yuki Nakajima ◽  
Ruiqi Shao ◽  
Ayaka Kakihara ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Primitive Endoderm ◽  
Cell Stage ◽  
Supportive Environment ◽  
Mouse Zygotes ◽  
Single Blastomeres ◽  
Multicellular Organisms

AbstractIn multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

scholarly journals Prevalence of pathogenic copy number variants among children conceived by donor oocyte

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sandra Monfort ◽  
Carmen Orellana ◽  
Silvestre Oltra ◽  
Mónica Rosello ◽  
Alfonso Caro-Llopis ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Copy Number ◽  
Assisted Reproductive Technologies ◽  
Copy Number Variants ◽  
Reproductive Technologies ◽  
Significant Excess ◽  
Vitro Fertilization ◽  
Donor Oocyte ◽  
Increased Risk

AbstractDevelopment of assisted reproductive technologies to address infertility has favored the birth of many children in the last years. The majority of children born with these treatments are healthy, but some concerns remain on the safety of these medical procedures. We have retrospectively analyzed both the fertilization method and the microarray results in all those children born between 2010 and 2019 with multiple congenital anomalies, developmental delay and/or autistic spectrum disorder (n = 486) referred for array study in our center. This analysis showed a significant excess of pathogenic copy number variants among those patients conceived after in vitro fertilization with donor oocyte with respect to those patients conceived by natural fertilization (p = 0.0001). On the other hand, no significant excess of pathogenic copy number variants was observed among patients born by autologous oocyte in vitro fertilization. Further studies are necessary to confirm these results and in order to identify the factors that may contribute to an increased risk of genomic rearrangements, as well as consider the screening for genomic alterations after oocyte donation in prenatal diagnosis.

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