scholarly journals Test-tube embryos - mouse and human development in vitro to blastocyst stage and beyond

2019 ◽  
Vol 63 (3-4-5) ◽  
pp. 203-215 ◽  
Author(s):  
Niraimathi Govindasamy ◽  
Binyamin Duethorn ◽  
Hatice O. Oezgueldez ◽  
Yung S. Kim ◽  
Ivan Bedzhov
Keyword(s):  
Embryo Culture ◽  
Assisted Reproductive Technologies ◽  
Basic Research ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Test Tube ◽  
Human Embryos ◽  
Culture Methods

Mammalian embryogenesis is intrauterine and depends on support from the maternal environment. Therefore, in order to directly study and manipulate early mouse and human embryos, fine-tuned culture conditions have to be provided to maintain embryo growth in vitro. Over time, the establishment and implementation of embryo culture methods have come a long way, initially enabling the development of few pre-implantation stages, expanding later to support in vitro embryogenesis from fertilization until blastocyst and even ex utero development beyond the implantation stages. Designing culture conditions that enable near physiological development of early embryos without maternal input, especially during the peri- and post-implantation stages, requires overcoming numerous experimental challenges, and it is still far from optimal. Nevertheless, embryo culture methods are an essential cornerstone of both assisted reproductive technologies and basic research, and these methods provide a platform to understand life’s greatest miracle – the development of a new organism.

scholarly journals MODERN APPROACHES TO CULTIVATION AND AUTOANALYSIS OF HUMAN EMBRYO MORPHODYNAMICS IN VIVO

2021 ◽  
pp. 35-43
Author(s):  
O. V. Shurygina ◽  
G. B. Nemkovskiy ◽  
D. Y. Rusakov ◽  
D. S. Gromenko ◽  
M. I. Taxants ◽  
...  
Keyword(s):  
Human Embryo ◽  
Visual Information ◽  
Assisted Reproductive Technologies ◽  
Medical Center ◽  
Time Lapse ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
The Moment

Relevance: Currently, it is extremely important to identify predictors of the development of a competent embryo that determine its implantation potential. In this case, the predictors are predictive parameters that should be assessed together to rank and select human embryos. We introduced the concept of «human embryo morphodynamic profile» to standardize the description of the development of human embryos cultured in vitro. We identified a set of morphokinetic states that are included in the profile and located on the time scale depending on the moment of their registration. All timing cutoffs (points) are given in chronological order relative to the moment of fertilization. The purpose of the study was to implement an information system utilizing artificial intelligence technologies for an automated formation of the morphodynamic profile of a human embryo based on time-lapse photography of the process of human embryo cultivating to the blastocyst stage. Materials and methods: Visual information about the pre-implantation development of human embryos to the blastocyst stage (0 - 6 days from insemination) was collected using an «Embryovisor» incubator for IVF laboratories with a time-lapse (hyperlapse) video fixation system (LLC «WESTTRADE LTD,” Russia). The embryos were cultivated individually in special microwells of WOW dishes (Vitrolife, Sweden). Visual information about cultured human embryos was collected, marked, and prepared at the Laboratory of assisted reproductive technologies (ART) of the Clinical Hospital IDK CJSC “Medical Company IDK” (Group of Companies “Mother and Child,” Samara, Russia) and the medical center “Semya” (Ufa, Russia). The morphodynamic profile was marked using the EmbryoVisor software (customized version). Graphics and markup information was uploaded to the SberCloud cluster. A convolutional neural network for solving the multiclass classification task was implemented on the Christofari supercomputer of the SberCloud cluster. Results: Based on the available database, we have developed a system for forming the morphodynamic profile of a human embryo, taking into account the placement of markers of fixed morphokinetic states. Conclusion: The ability to record major morphodynamic events and assess them allows a more comprehensive approach to evaluating and ranking developing embryos and selecting the most promising embryo for implantation.

scholarly journals Perturbations in imprinted methylation from assisted reproductive technologies but not advanced maternal age in mouse preimplantation embryos

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Audrey J. Kindsfather ◽  
Megan A. Czekalski ◽  
Catherine A. Pressimone ◽  
Margaret P. Erisman ◽  
Mellissa R. W. Mann
Keyword(s):  
Embryo Culture ◽  
Maternal Age ◽  
Assisted Reproductive Technologies ◽  
Advanced Maternal Age ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Clinical Methods ◽  
First Time

Abstract Background Over the last several decades, the average age of first-time mothers has risen steadily. With increasing maternal age comes a decrease in fertility, which in turn has led to an increase in the use of assisted reproductive technologies by these women. Assisted reproductive technologies (ARTs), including superovulation and embryo culture, have been shown separately to alter imprinted DNA methylation maintenance in blastocysts. However, there has been little investigation on the effects of advanced maternal age, with or without ARTs, on genomic imprinting. We hypothesized that ARTs and advanced maternal age, separately and together, alter imprinted methylation in mouse preimplantation embryos. For this study, we examined imprinted methylation at three genes, Snrpn, Kcnq1ot1, and H19, which in humans are linked to ART-associated methylation errors that lead to imprinting disorders. Results Our data showed that imprinted methylation acquisition in oocytes was unaffected by increasing maternal age. Furthermore, imprinted methylation was normally acquired when advanced maternal age was combined with superovulation. Analysis of blastocyst-stage embryos revealed that imprinted methylation maintenance was also not affected by increasing maternal age. In a comparison of ARTs, we observed that the frequency of blastocysts with imprinted methylation loss was similar between the superovulation only and the embryo culture only groups, while the combination of superovulation and embryo culture resulted in a higher frequency of mouse blastocysts with maternal imprinted methylation perturbations than superovulation alone. Finally, the combination of increasing maternal age with ARTs had no additional effect on the frequency of imprinted methylation errors. Conclusion Collectively, increasing maternal age with or without superovulation had no effect of imprinted methylation acquisition at Snrpn, Kcnq1ot1, and H19 in oocytes. Furthermore, during preimplantation development, while ARTs generated perturbations in imprinted methylation maintenance in blastocysts, advanced maternal age did not increase the burden of imprinted methylation errors at Snrpn, Kcnq1ot1, and H19 when combined with ARTs. These results provide cautious optimism that advanced maternal age is not a contributing factor to imprinted methylation errors in embryos produced in the clinic. Furthermore, our data on the effects of ARTs strengthen the need to advance clinical methods to reduce imprinted methylation errors in in vitro-produced embryos.

Characterization of isolated bovine preantral follicles based on morphology, diameter and cell number

Zygote ◽  
2020 ◽  
Vol 28 (2) ◽  
pp. 154-159
Author(s):  
Juliana I. Candelaria ◽  
Anna C. Denicol
Keyword(s):  
Granulosa Cells ◽  
Developmental Stages ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Culture Conditions ◽  
Preantral Follicles ◽  
Secondary Stage

SummaryPreantral follicles are a potential reservoir of oocytes to be used in assisted reproductive technologies. With the increasing interest in developing techniques to grow preantral follicles in vitro, and as the bovine emerges as an appropriate model species to understand human folliculogenesis, the establishment of an accurate classification of developmental stages is needed. Classification of bovine preantral follicles has been mostly based on histological analysis and estimation models, which may not translate well to correctly characterize preantral follicles isolated from the ovary. In this study, we classified bovine preantral follicles by morphology upon isolation, determined diameter and number of granulosa cells by direct counting, and compared our results with previous studies reporting bovine preantral follicle classification. Follicles were isolated via homogenization of ovary tissue and classified into primary, early secondary and secondary stage based on morphology and number of layers of granulosa cells. Diameter was individually measured and Hoechst 33342 was used as a nuclear stain to count granulosa cells. We found that follicles classified by morphology into primary, early secondary, and secondary had different mean diameter and cell number (P < 0.01); cell number and diameter were positively correlated, as were cell density and cell number in each developmental stage (P < 0.01). Results obtained here were mostly in agreement with previous classifications based on histological sections and on isolated follicles, with some discrepancies. The present data add accuracy to classification of bovine preantral follicles that is critical to optimize culture conditions to produce developmentally competent oocytes.

Physiological, metabolic and transcriptional postnatal phenotypes ofin vitrofertilization (IVF) in the mouse

2017 ◽  
Vol 8 (4) ◽  
pp. 403-410 ◽  
Author(s):  
S. K. Feuer ◽  
P. F. Rinaudo
Keyword(s):  
Assisted Reproductive Technologies ◽  
Biological Significance ◽  
Great Majority ◽  
Reproductive Technologies ◽  
Culture Conditions ◽  
Molecular Evidence ◽  
Metabolic Dysfunction ◽  
Molecular Physiology ◽  
The Life Course

Approximately 1–4% of children today are conceived using assisted reproductive technologies (ARTs), includingin vitrofertilization (IVF). IVF is considered safe and the great majority of these children are healthy, yet there is increasing physiological and molecular evidence from animal models that ART is associated with postnatal metabolic and cardiovascular alterations. Understanding the mechanisms underlying these changes and determining whether they have biological significance is of paramount importance for optimizing the design of culture conditions and improving the health of ART children across the life course. In this review, we examine the evidence of molecular changes present in adult tissues of rodent offspring generated by preimplantation manipulation of gametes and embryos. Although embryo manipulationin vitrocan induce common transcriptional effects in the blastocyst, transcriptional and metabolomic signatures in adult IVF tissues are largely tissue-specific. However, there is pervasive evidence of oxidative stress and metabolic dysfunction, indicating a lasting effect of IVF on molecular physiology.

246 IN VITRO PRODUCTION OF SPRINGBOK (ANTIDORCAS MARSUPIALIS) EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 239 ◽  
Author(s):  
R. Krisher ◽  
A. Auer ◽  
K. Clark ◽  
K. Emsweller ◽  
S. Rogers ◽  
...  
Keyword(s):  
South Africa ◽  
Oocyte Maturation ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Genetic Management ◽  
Blastocyst Development ◽  
Antidorcas Marsupialis

The objective of this experiment was to develop in vitro embryo production (IVP) technologies in springbok (Antidorcas marsupialis), a southern African antelope. Springbok, a fairly common species on game farms in parts of South Africa, may be used as a model species for gamete rescue and IVP techniques to be applied to the conservation of other threatened antelope species. Springbok belong to the family bovidae, subfamily antilopinae, tribe antilopini, which comprises about twenty species in genera Gazella, Antilope, Procapra, Antidorcas, Litocranius, and Ammodorcas. In this tribe alone, there are 4 species or subspecies that are critically endangered, 3 that are endangered, and 10 that are considered vulnerable, demonstrating the need for antelope conservation efforts. In addition, our studies contributed to the South African biological resource bank, so that banked springbok semen and embryos might be used in the future for managed genetic contribution to isolated captive or wild populations via assisted reproductive technologies. Oocytes were recovered (3 replicates) from ovaries obtained at supervised culls for management purposes in South Africa, and cultured in defined Gmat or undefined TCM-199 with FCS maturation medium for 28-30 h (Brad et al. 2004 Reprod. Fertil. Dev. 16, 223). Oocytes were fertilized with frozen-thawed springbok epididymal spermatozoa in modified SOF fertilization medium with caffeine (Herrick et al. 2004 Biol. Reprod. 71, 948–958). Eighteen hours after insemination, a randomly selected subset of the zygotes were fixed to determine fertilization success. The remaining zygotes were cultured in G1/G2 media. On Day 7 of culture, embryos were analyzed for development to the morula or blastocyst stage. A total of 259 selected oocytes were collected from 50 females (5.2 selected oocytes/female on average). There was no difference in the percentage of oocytes normally fertilized (2 pronuclei, PN) between oocytes matured in Gmat (n= 43; 12%) and those matured in TCM-199 (n= 42; 10%). There were significantly (P &lt; 0.05) more oocytes penetrated (e2 PN) when matured in TCM (50%) compared to Gmat (23%). There were no differences in embryonic cleavage or morula/blastocyst development (of total oocytes inseminated) between treatments (Gmat,n= 89, 54%, 9.0%; TCM-199, n= 85, 68%, 9.4%, respectively). In both treatments, the average blastocyst grade was 2.125 using the standard bovine grading system (Curtis, Cattle Embryo Transfer Procedure, 1991). In conclusion, in vitro oocyte maturation, fertilization, and embryo culture to the blastocyst stage is possible in springbok. Importantly, blastocysts can be produced in vitro under semi-defined conditions, demonstrating that oocyte maturation without serum does support developmental competence. This is important for the potential international movement of IVP embryos to be used for genetic management in the conservation of antelope species.

Embryo Culture Conditions and the Epigenome

2018 ◽  
Vol 36 (03/04) ◽  
pp. 211-220 ◽  
Author(s):  
Sneha Mani ◽  
Monica Mainigi
Keyword(s):  
Embryo Culture ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
Common Mechanism ◽  
Success Rates ◽  
Blastocyst Development ◽  
Increased Risk ◽  
Modifiable Factors

AbstractAssisted reproductive technologies (ARTs) lead to an increased risk for pregnancy complications, congenital abnormalities, and specific imprinting disorders. Epigenetic dysfunction is thought to be one common mechanism which may be affecting these outcomes. The timing of multiple ART interventions overlaps with developmental time periods that are particularly vulnerable to epigenetic change. In vitro embryo culture is known to impact blastocyst development, in vitro fertilization (IVF) success rates, as well as neonatal outcomes. Embryo culture, in contrast to other procedures involved in ART, is obligatory, and has the highest potential for causing alterations in epigenetic reprograming. In this review, we summarize progress that has been made in exploring the effects of embryo culture, culture media, and oxygen tension on epigenetic regulation in the developing embryo. In humans, it is difficult to isolate the role of embryo culture on epigenetic perturbations. Therefore, additional well-controlled animal studies isolating individual exposures are necessary to minimize the epigenetic effects of modifiable factors utilized during ART. Findings from these studies will likely not only improve IVF success rates but also reduce the risk of adverse perinatal outcomes.

Reproductive Technologies as Instruments of Meaningful Parenting: Ethics in the Age of ARTs

2002 ◽  
Vol 11 (4) ◽  
pp. 401-410
Author(s):  
D. MICAH HESTER
Keyword(s):  
Science Fiction ◽  
Assisted Reproductive Technologies ◽  
Treatment Options ◽  
Reproductive Technologies ◽  
Test Tube ◽  
Unrelated Donor ◽  
Vitro Fertilization ◽  
Biological Relationship ◽  
Genetic Techniques

Since the decade of the 1970s, and particularly since the first successful test-tube baby in 1978, the development and use of assisted reproductive technologies (ARTs) have grown exponentially. Would-be parents—including those in so-called traditional male-female marriages, unmarried adults, postmenopausal women, and same-sex partnerships—who just over 20 years ago had no recourse for their (in)fertility issues can now pursue their desires to have children with at least a partial, if not, total, genetic and/or biological relationship. Ovulation-stimulating medications, artificial insemination using the sperm of a husband or unrelated donor (AIH or AID, respectively), in vitro fertilization with embryo transfer (IVF-ET), intracytoplasmic sperm injection (ICS), and gamete and zygote intrafallopian transfers (GIFT and ZIFT) are but a few of a host of treatment options ranging in complexity, invasiveness, and expense. And on the horizon are genetic techniques such as cloning—which was once considered “pure” science fiction but in 1997 became what some call an inevitability, with the development of mammalian cloning in the form of the now-famous (if not infamous) Dolly the sheep.

scholarly journals Advantages of the outgrowth model for evaluating the implantation competence of blastocysts

2020 ◽  
Vol 47 (2) ◽  
pp. 85-93
Author(s):  
Jihyun Kim ◽  
Jaewang Lee ◽  
Jin Hyun Jun
Keyword(s):  
Maternal Effects ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Animal Experimentation ◽  
In Utero ◽  
Culture Conditions ◽  
Research Model ◽  
Ethical Problems ◽  
Implantation Process

The implantation process is highly complex and difficult to mimic <i>in vitro</i>, and a reliable experimental model of implantation has yet to be established. Many researchers have used embryo transfer (ET) to assess implantation potential; however, ET with pseudopregnant mice requires expert surgical skills and numerous sacrificial animals. To overcome those economic and ethical problems, several researchers have tried to use outgrowth models to evaluate the implantation potential of embryos. Many previous studies, as well as our experiments, have found significant correlations between blastocyst outgrowth <i>in vitro</i> and implantation in utero by ET. This review proposes the blastocyst outgrowth model as a possible alternative to animal experimentation involving ET in utero. In particular, the outgrowth model might be a cost- and time-effective alternative method to ET for evaluating the effectiveness of culture conditions or treatments. An advanced outgrowth model and further culture of outgrowth embryos could provide a subtle research model of peri- and postimplantation development, excluding maternal effects, and thereby could facilitate progress in assisted reproductive technologies. Recently, we found that outgrowth embryos secreted extracellular vesicles containing specific microRNAs. The function of microRNAs from outgrowth embryos should be elucidated in further researches.

71 The Effect of Two Different In Vitro Culture Media and Mice Embryo Groupings on Hatchability After 24 Hours of Culture

2018 ◽  
Vol 30 (1) ◽  
pp. 174
Author(s):  
N. C. Negota ◽  
M. L. Mphaphathi ◽  
L. P. Nethenzheni ◽  
T. L. Rammutla ◽  
N. R. Serota ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
Significant Difference ◽  
Autocrine Signalling ◽  
Blastocyst Hatching

Mammalian blastocysts must hatch out from the zona pellucida before implantation. In vitro embryo culture and grouping of mice blastocysts are conducive options of assisted reproductive technologies (ART) to speed up the hatching rate of mice embryos. The number of embryos per unit volume has the greatest impact on hatching rates due to autocrine signalling. The study aimed to determine the effect of two in vitro culture (IVC) media (TCM-199 and Ham’s F10) and embryo groupings (1, 2, 3, and 4 embryos per 50-µL droplet) after 24 h of culture on hatching rate. Breeds of C57BL/6 (n = 10) and BALB/c (n = 10) were raised until they reached maturity and bred naturally to produce the first filial generation. The photoperiod was 14 h of light followed by 10 h of darkness in the breeding house, and feed and water were provided ad libitum. Female mice were superovulated using eCG and hCG. The first filial generations from 2 breeds were used for the collection of 160 blastocysts and randomly allocated into 2 IVC media (80 embryos for TCM-199 and 80 embryos for Ham’s F10) and again subjected to 4 embryo groupings (1, 2, 3, and 4 embryos per droplet) treatments. Four replicates were done per treatment group. The general linear model of Minitab version 17 (Minitab Inc., State College, PA, USA) was used to analyse the data. The hatching rate of blastocyst stage was significantly higher for TCM-199 (56.9 ± 27.2) compared with Ham’s F10 (50.0 ± 35.1%). The comparison of all embryo groupings, 1 (20.0 ± 40.5), 2 (28.8 ± 29.7), 3 (59.1 ± 38.8), and 4 (43.8 ± 32.4%) per 50-µL droplet showed significant differences, irrespective of IVC medium and breed. In TCM-199, groupings of 1 (20.0 ± 41.0), 2 (30.0 ± 29.9), 3 (63.3 ± 40.3), and 4 (42.5 ± 33.5%) had a significant difference on blastocyst hatching percent. In Ham’s F10, groupings of 1 (20.0 ± 41.0), 2 (27.5 ± 30.2), 3 (55.0 ± 37.9), and 4 (45.0 ± 32.0%) were significantly different on blastocyst hatching rate. However, an increase in hatching rate was observed for the interaction of media and embryo groupings and especially when embryos were increased per droplet in all breeds. In conclusion, the use of TCM-199 and grouping of 3 embryos per 50-µL droplet during culture had the highest hatching rate compared with the use of Ham’s F10.

Implications of Assisted Reproductive Technologies for Pregnancy Outcomes in Mammals

2020 ◽  
Vol 8 (1) ◽  
pp. 395-413 ◽  
Author(s):  
Peter J. Hansen
Keyword(s):  
Assisted Reproductive Technologies ◽  
Genetic Manipulation ◽  
Adult Life ◽  
Reproductive Technologies ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Long Distance ◽  
Vitro Production

Development of assisted reproductive technologies has been driven by the goals of reducing the incidence of infertility, increasing the number of offspring from genetically elite animals, facilitating genetic manipulation, aiding preservation and long-distance movement of germplasm, and generating research material. Superovulation is associated with reduced fertilization rate and alterations in endometrial function. In vitro production of embryos can have a variety of consequences. Most embryos produced in vitro are capable of establishing pregnancy and developing into healthy neonatal animals. However, in vitro production is associated with reduced ability to develop to the blastocyst stage, increased incidence of failure to establish pregnancy, placental dysfunction, and altered fetal development. Changes in the developmental program mean that some consequences of being produced in vitro can extend into adult life. Reduced competence of the embryo produced in vitro to develop to the blastocyst stage is caused largely by disruption of events during oocyte maturation and fertilization. Conditions during embryo culture can affect embryo freezability and competence to establish pregnancy after transfer. Culture conditions, including actions of embryokines, can also affect the postnatal phenotype of the resultant progeny.

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