A continuous flow, perifusion culture system for 8- to 16-cell bovine embryos derived from in vitro culture

Despite hundreds of scientific papers published, no system has resulted in in vitro embryos comparable to those produced in vivo. We hypothesised that assembling the most pertinent elements of IVF studies into one system would result in a highly efficient in vitro culture system. Here we report the in vitro production of bovine embryos using a culture system with strict environmental conditions that produces very-good-quality embryos at high rates. This system consists of a sequential culture system with media composition based on recent reports that characterise the bovine female reproductive tract (Hugentobler et al. 2007 Mol. Reprod. Dev. 74, 445–454; Hugentobler et al. 2007 Theriogenology 68, 538–548; Hugentobler et al. 2008 Mol. Reprod. Dev. 75, 496–503). This system uses a 3-step culture media to prevent toxicity resulting from ammonium accumulation and nutrient depletion and also to adjust the component concentrations to support embryo needs at different developmental stages. Fatty acid-free BSA is used as the protein source and the culture is in droplets under high-quality paraffin oil at 38.5°C under 6.8% CO2, 5% O2 and 88.2% N2. Numerous other aspects were investigated to limit embryo stresses (Lane et al. 2008 Reprod. Fertil. Dev. 20, 23–32) during manipulations, including the use of mini-incubators and very-high-purity gas combined with stringent laboratory practices. In the first year using this new embryo production system, 2839 oocytes were fertilized, resulting in a transferable blastocyst rate of 51%. Of the 1448 embryos produced, 779 were transferred fresh at our facility with pregnancy rates of 55 and 49% at 28 and 60 days, respectively. Pregnancy rates were directly related to the quality of the embryos transferred as 61% of grade 1 embryos transferred induced a pregnancy at Day 28, compared with 41% of grade 2 embryos. Pregnancy induction is not the only indication of good embryo quality. As is well-documented, in vitro-produced bovine embryos do not tolerate slow freezing, so vitrification was applied to surmount this intolerance. However, this is difficult to apply to industry because direct transfer of vitrified embryos is challenging. We hypothesised that the improvement of embryo culture would result in embryos that could tolerate slow freezing. Grade 1 blastocysts (n = 229) were frozen in 1.6M ethylene glycol and 0.1 M sucrose using standard slow freezing procedures. A very high proportion (91%) of frozen–thawed in vitro-produced embryos re-expanded after 24 h of culture with a good quality inner cell mass. Subsequently, 45 grade 1 blastocysts were frozen and transferred, giving pregnancy rates of 58% at Day 60. In conclusion, combining good-quality culture media and conditions resulted in the production of in vitro embryos that were very efficient at inducing pregnancies and tolerating slow freezing, which makes it now possible to consider direct transfer of frozen in vitro-produced bovine embryos.

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Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos

Theriogenology ◽

10.1016/s0093-691x(01)00471-x ◽

2001 ◽

Vol 55 (5) ◽

pp. 1117-1126 ◽

Cited By ~ 85

Author(s):

A. Gutiérrez-Adán ◽

P. Lonergan ◽

D. Rizos ◽

F.A. Ward ◽

M.P. Boland ◽

...

Keyword(s):

Sex Ratio ◽

In Vitro Culture ◽

Culture System ◽

Bovine Embryos ◽

Blastocyst Development ◽

In Vitro Culture System ◽

Kinetics Of

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In-vitro culture system for mesenchymal progenitor cells derived from waste human ovarian follicular fluid

Reproductive BioMedicine Online ◽

10.1016/j.rbmo.2014.06.006 ◽

2014 ◽

Vol 29 (4) ◽

pp. 457-469 ◽

Cited By ~ 14

Author(s):

Federica Riva ◽

Claudia Omes ◽

Roberto Bassani ◽

Rossella E Nappi ◽

Giuliano Mazzini ◽

...

Keyword(s):

In Vitro Culture ◽

Progenitor Cells ◽

Follicular Fluid ◽

Culture System ◽

Mesenchymal Progenitor Cells ◽

In Vitro Culture System ◽

Ovarian Follicular Fluid

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P–218 Analysis of the occurrence of microbial contamination in IVF culture system and the effect of microorganisms on embryo development and clinical outcomes

Human Reproduction ◽

10.1093/humrep/deab130.217 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

F Du ◽

R Li ◽

Q Zhang ◽

W Wang

Keyword(s):

Escherichia Coli ◽

In Vitro Culture ◽

Clinical Outcomes ◽

Embryo Transfer ◽

Follicular Fluid ◽

Culture System ◽

Microbial Contamination ◽

Culture Medium ◽

Ivf Et

Abstract Study question what is the source, prevalence, and influence of microbial contamination on in vitro fertilization (IVF) and embryo transfer (ET) cycles? Summary answer Microbial contamination mainly occurs on Day 2, most caused by Escherichia coli carried with semen. ICSI could prevent contamination effectively and get good clinical outcomes. What is known already Microbial contamination occurs in IVF-ET system occasionally, which is hard to stop happening. The IVF culture system and laboratory environment, the patients’ follicular fluid and semen are not absolutely sterile, while the antibiotics in culture medium isn’t effective for all microbe types, and the artificial operations may bring in microbes. Generally, microbial contamination leads to degradation of embryos, reduction the number of embryos available, and infection of female reproductive tract, which would increase the cost of patients’ time, money, and bring psychological damages. A better understanding of embryo contamination in IVF culture system is of added value. Study design, size, duration A total of 29583 IVF-ET cycles were enrolled in this prospective observational study, from January 2010 to December 2020, included 70 microbial contamination cycles discovered in Day1-Day3 (D1-D3) of in vitro culture. Follicular fluid and semen saved on oocyte retrieval day, and culture medium contaminated were examined and identified for microorganisms at each contamination cycle. Participants/materials, setting, methods Compared the contamination rate of different insemination methods (IVF/ICSI/IVF+ICSI), different in vitro culture days (D1-D3), and different samples examination (follicular fluid, semen, culture medium) respectively, identified the source of microorganism types, compared the IVF culture outcomes and clinical outcomes between total contamination group (TC group, 42 cases) and partial contamination group (PC group, 28 cases). Main results and the role of chance A total of 70 microbial contamination cases occurred in 29583 oocyte retrieving cycles (0.24%), and it was observed only in IVF embryos but never in ICSI (Intracytoplasmic sperm injection) embryos. 38 contamination cases occurred on D2 with a highest ratio (54.3%) compared to D1 (32.9%) and D3(12.9%); Compared with follicular fluid, semen was the main cause inducing contamination from D1 to D3, and Escherichia coli in semen and culture medium, Enterococcus faecalis in follicular fluid proved to be the most common sources. Compared with TC group, the PC group showed a lower rate of No-available embryos (21.4% vs 81.0%) and a higher rate of blastocyst formation (41.2% vs 28.6%), In addition, the clinical pregnancy rate of PC group was higher than that of TC group in both fresh and frozen-thawed embryo transfer cycles (31.3% vs 16.7%, 38.5% vs 0.0%). Limitations, reasons for caution Further study is still necessary to better understand the sources that induce microbial contamination embryos, and more efficient methods are required to remove the microbes on these contaminated embryos so as better develop and manage a sterile micro-environment for successful embryo growth. Wider implications of the findings: The differential embryonic microbe types associated to different IVF culture and clinical outcomes in patients undergoing IVF-ET might have profound implications for understanding the microbial sources and making a better management of IVF culture system. Trial registration number Not applicable

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Relative expression of mRNAs related to cavitation process in bovine embryos produced in vivo and in vitro

Revista Brasileira de Zootecnia ◽

10.1590/s1516-35982011000100017 ◽

2011 ◽

Vol 40 (1) ◽

pp. 124-128

Author(s):

Sabine Wohlres-Viana ◽

Mariana Cortes Boite ◽

João Henrique Moreira Viana ◽

Marco Antonio Machado ◽

Luiz Sérgio de Almeida Camargo

Keyword(s):

Culture System ◽

Rna Extraction ◽

Endogenous Control ◽

Bovine Embryos ◽

Relative Expression ◽

Gapdh Gene ◽

Expression Of Genes ◽

In Vitro Culture System

The objectives of this work were to identify and to evaluate possible differences on gene expression of aquaporins and Na/K-ATPases transcripts between embryos in vivo and in vitro produced. For each group, 15 blastocysts distributed in three pools were used for RNA extraction followed by amplification and reverse transcription. The resulting cDNAs were submitted to Real-Time PCR, using the GAPDH gene as endogenous control. It was not possible to identify AQP1 transcripts. Relative expression of AQP3 (1.33 ± 0.78) and AQP11 (2.00 ± 1.42) were not different in blastocysts in vitro and in vivo produced. Na/K-ATPase α1 gene (2.25 ± 1.07) was overregulated whereas Na/K-ATPase β2 transcripts 0.40 ± 0.30) did not differ among blastocysts produced in vitro from those produced in vivo. Transcripts for gene AQP1 are not present in bovine blastocysts. In vitro culture system does not alter expression of genes AQP3, AQP11 and Na/K-ATPase β2 genes, however, it affects expression of Na/K-ATPase α1.

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