The influence of in vitro fertilization and embryo culture on the embryo epigenetic constituents and the possible consequences in the bovine model

2017 ◽  
Vol 8 (4) ◽  
pp. 411-417 ◽  
Author(s):  
M.-A. Sirard
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technologies ◽  
Genetic Model ◽  
Culture Media ◽  
Reproductive Technologies ◽  
The Body ◽  
Model Systems ◽  
Gene Perturbations

Medically assisted reproductive technologies, such as in vitro embryo production, are increasingly being used to palliate infertility. Eggs are produced following a hormonal regimen that stimulates the ovaries to produce a large number of oocytes. Collected oocytes are then fertilized in vitro and allowed to develop in vitro until they are either frozen or transferred to mothers. There are controversial reports on the adverse impacts of these technologies on early embryos and their potential long-term effects. Using newly developed technological platforms that enable global gene expression and global DNA methylation profiling, we evaluated gene perturbations caused by such artificial procedures. We know that cells in the early embryo produce all cells in the body and are able to respond to their in vitro environment. However, it is not known whether gene perturbations are part of a normal response to the environment or are due to distress and will have long-term impacts. While the mouse is an established genetic model used for quality control of culture media in clinics, the bovine is a large mono-ovulating mammal with similar embryonic kinetics as humans during the studied developmental window. These model systems are critical to understand the effects of assisted reproduction without the confounding impact of infertility and without the limitations imposed by the scarcity of donated human samples and ethical issues. The data presented in this review come mostly from our own experimentation, publications, and collaborations. Together they demonstrate that the in vitro environment has a significant impact on embryos at the transcriptomic level and at the DNA methylation level.

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.

scholarly journals Reproductive fluids, used for the in vitro production of pig embryos, result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM.

2020 ◽  
Author(s):  
Evelynne Paris-Oller ◽  
Sergio Navarro-Serna ◽  
Cristina Soriano-Úbeda ◽  
Jordana Sena Lopes ◽  
Carmen Matas ◽  
...  
Keyword(s):  
Reproductive Performance ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
In Vitro Production ◽  
Aberrant Expression ◽  
Low Efficiency ◽  
The Impact

Abstract Background: In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of a modified IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimally invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits.Results: The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and modified (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group.Conclusions: The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.

scholarly journals DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sebastian Canovas ◽  
Elena Ivanova ◽  
Raquel Romar ◽  
Soledad García-Martínez ◽  
Cristina Soriano-Úbeda ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Rna Seq ◽  
Methylation Analysis ◽  
Number Of Children ◽  
Methylation Patterns

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.

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.

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

scholarly journals Culture Medium and Sex Drive Epigenetic Reprogramming in Preimplantation Bovine Embryos

2021 ◽  
Vol 22 (12) ◽  
pp. 6426
Author(s):  
Sebastian Canovas ◽  
Elena Ivanova ◽  
Meriem Hamdi ◽  
Fernando Perez-Sanz ◽  
Dimitrios Rizos ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technologies ◽  
Developmental Process ◽  
Reproductive Technologies ◽  
Sexually Dimorphic ◽  
Element Analysis ◽  
Global Methylation ◽  
The Impact

Assisted reproductive technologies impact transcriptome and epigenome of embryos and can result in long-term phenotypic consequences. Whole-genome DNA methylation profiles from individual bovine blastocysts in vivo- and in vitro-derived (using three sources of protein: reproductive fluids, blood serum and bovine serum albumin) were generated. The impact of in vitro culture on DNA methylation was analyzed, and sex-specific methylation differences at blastocyst stage were uncovered. In vivo embryos showed the highest levels of methylation (29.5%), close to those produced in vitro with serum, whilst embryos produced in vitro with reproductive fluids or albumin showed less global methylation (25–25.4%). During repetitive element analysis, the serum group was the most affected. DNA methylation differences between in vivo and in vitro groups were more frequent in the first intron than in CpGi in promoters. Moreover, hierarchical cluster analysis showed that sex produced a stronger bias in the results than embryo origin. For each group, distance between male and female embryos varied, with in vivo blastocyst showing a lesser distance. Between the sexually dimorphic methylated tiles, which were biased to X-chromosome, critical factors for reproduction, developmental process, cell proliferation and DNA methylation machinery were included. These results support the idea that blastocysts show sexually-dimorphic DNA methylation patterns, and the known picture about the blastocyst methylome should be reconsidered.

scholarly journals Investigating media that support red wolf (Canis rufus) sperm viability and capacitation in vitro

2020 ◽  
Vol 1 (1) ◽  
pp. 83-92
Author(s):  
Jennifer B Nagashima ◽  
Marcia de Almeida Monteiro Melo Ferraz ◽  
Sarah H Kamen ◽  
Nucharin Songsasen
Keyword(s):  
Tyrosine Phosphorylation ◽  
Sperm Motility ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
Critically Endangered ◽  
Sperm Viability ◽  
Canis Rufus ◽  
Red Wolf

The red wolf is a critically endangered canid, with ~250 and ~20 individuals in the ex situ and reintroduced wild populations, respectively. Assisted reproductive technologies such as sperm cryopreservation and in vitro fertilization therefore represent critically-needed tools to manage these populations. However, the motility of post-thaw red wolf sperm rapidly declines during in vitro incubation, hindering the ability to develop these technologies. In this study, we evaluated the influence of several culture media (a modified canine capacitation medium (mCCM), a modified North Carolina State University-23 medium (mNCSU-23), a synthetic oviductal fluid (SOF), a fertilization Tyrode’s medium base or Fert-TALP (FERT), and a TRIS-based buffer (TRIS)) on the survival and capacitation of red wolf sperm during extended (18 h) incubation at 38.5°C and 5% CO2. Red wolf sperm motility averaged (±s.e.m.) 73.8 ± 7.1% at the time of collection, and was better maintained over 4 h incubation in mCCM (55.0 ± 9.8%) and mNCSU-23 (54.7 ± 10.4), compared to mSOF (43.8 ± 8.3%), FERT (30 ± 10.5), and TRIS (16.4 ± 4.1%) solutions. Patterns of tyrosine phosphorylation signal, as assessed via immunocytochemistry, indicated induction of capacitation between 2 and 4 h in vitro culture. Tyrosine phosphorylation signal was particularly robust in mCCM and mNCSU-23 incubated sperm, although significant acrosome exocytosis was not observed in response to progesterone supplementation after 3 h incubation in any of the media. In sum, results indicate mCCM and mNCSU-23 are promising base media for the in vitro incubation and capacitation of red wolf sperm, for assisted reproduction applications. Lay summary Development of assisted reproductive technologies such as in vitro fertilization and artificial insemination is of high importance to the genetic management of critically endangered species such as the red wolf (Canis rufus). However, these technologies require the ability to maintain sperm viability and function during extended incubation, which has not been successful for the red wolf thus far. In this study, various culture media developed for sperm/egg/embryo culture in large mammalian species were evaluated for their ability to maintain red wolf sperm motility under physiological incubation conditions. Media and conditions previously utilized for domestic dog sperm were found to best support sperm incubation and capacitation (process of becoming competent to fertilize an egg) in the red wolf, representing a key step for future development of assisted reproductive technologies for the species.

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.

scholarly journals Human Cumulus Cells in Long-Term In Vitro Culture Reflect Differential Expression Profile of Genes Responsible for Planned Cell Death and Aging—A Study of New Molecular Markers

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1265
Author(s):  
Błażej Chermuła ◽  
Wiesława Kranc ◽  
Karol Jopek ◽  
Joanna Budna-Tukan ◽  
Greg Hutchings ◽  
...  
Keyword(s):  
Cell Death ◽  
In Vitro Culture ◽  
Expression Analysis ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Oocyte Quality ◽  
Genetic Profile

In the ovarian follicle, maturation of the oocyte increases in the presence of somatic cells called cumulus cells (CCs). These cells form a direct barrier between the oocyte and external environment. Thanks to bidirectional communication, they have a direct impact on the oocyte, its quality and development potential. Understanding the genetic profile of CCs appears to be important in elucidating the physiology of oocytes. Long-term in vitro culture of CCs collected from patients undergoing controlled ovarian stimulation during in vitro fertilization procedure was conducted. Using microarray expression analysis, transcript levels were assessed on day 1, 7, 15, and 30 of culture. Apoptosis and aging of CCs strictly influence oocyte quality and subsequently the outcome of assisted reproductive technologies (ART). Thus, particular attention was paid to the analysis of genes involved in programmed cell death, aging, and apoptosis. Due to the detailed level of expression analysis of each of the 133 analyzed genes, three groups were selected: first with significantly decreased expression during the culture; second with the statistically lowest increase in expression; and third with the highest significant increase in expression. COL3A1, SFRP4, CTGF, HTR2B, VCAM1, TNFRSF11B genes, belonging to the third group, were identified as potential carriers of information on oocyte quality.

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.

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