Intra- and interobserver analysis in the morphological assessment of early stage embryos during an IVF procedure: a multicentre study

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Activité des cellules d'oviducte sur le développement in vitro des embryons bovins

Doctorat en sciences biologiques -- UCL,199

Effect of estrus-associated glycoprotein and tissue inhibitor of metalloproteinase-1 secreted by oviduct cells on in vitro bovine embryo development.

The effect of two glycoproteins (estrus-associated glycoprotein [EGP] and tissue inhibitor of metalloproteinase [TIMP-1]) secreted by bovine oviduct cells on in vitro bovine embryo development was assessed. A first set of experiments was conducted to determine whether the embryotrophic activity of the bovine oviduct-conditioned medium (BOCM) was correlated with the presence of EGP or TIMP-1. EGP and TIMP-1 were detected in BOCM, supporting the development of 22% zygotes to the blastocyst stage, as well as in BOCM yielding a low blastocyst rate (3-4% blastocysts). These glycoproteins do not seem to be necessary for bovine embryo development up to the blastocyst stage in our BOCM. In a second experiment, zygotes were cultured in modified synthetic oviduct fluid (mSOF) supplemented with different concentrations (0.5, 5, 50, and 500 micrograms/ml) of purified bovine EGP. In the third experiment, since purified bovine TIMP-1 was not available, zygotes were cultured in BOCM depleted of TIMP-1 by immunoprecipitation treatment. Adding EGP to mSOF, or removing TIMP-1 from BOCM, did not affect bovine embryo development up to the blastocyst stage, or mean number of cells per blastocyst after 8 days of culture. The results indicate that, under our culture conditions, EGP and TIMP-1 do not play an important role in sustaining bovine embryo development, and do not influence blastocyst quality, assessed in terms of total number of cells per embryo

Effect of high molecular weight factors present in bovine oviduct-conditioned medium on in vitro bovine embryo development.

To investigate the presence of embryotrophic factors in bovine oviduct-conditioned medium (BOCM), the high molecular weight fraction (> 10 KDa) from BOCM was added to 3 chemically defined embryo culture media (TCM199, DMEM/F12 and modified synthetic oviduct fluid [mSOF]). Zygotes were obtained by in vitro maturation and fertilization of oocytes. Conditioning of TCM199 with oviduct cells increased both cleavage to the 5- to 8-cell stage (59 vs 37%) and further development to the blastocyst stage (19 vs 4%). The low molecular weight fraction (< 10 KDa) of BOCM maintained development to the 5- to 8-cell stage but did not allow development to the blastocyst stage. Adding the high molecular weight fraction to the inactive low molecular weight fraction restored bovine embryo development up to the blastocyst stage. This embryotrophic effect of the high molecular weight fraction was not observed when this fraction was added to TCM199 or DMEM/F12 medium. Whereas adding this fraction to mSOF medium significantly (P<0.05) increased embryo development up to the blastocyst stage (36%) in comparison with that of mSOF (15%) or BOCM (14%). These results show that BOCM contains high molecular weight factors promoting embryo development up to the blastocyst stage. Some chemically defined media mask the effect of these embryotrophic factors

In vitro development of bovine embryos in Buffalo rat liver- or bovine oviduct-conditioned medium.

A culture system for bovine embryos was developed using Buffalo rat liver cell (BRL) line-conditioned medium without serum. Zygotes, obtained by in vitro maturation and fertilization of oocytes, were cultured either in unconditioned medium (TCM 199 or DMEM/F12) or in the same medium conditioned by bovine oviduct or BRL cells. No serum was added during conditioning or during embryo culture. The DMEM/F12 medium was superior to TCM 199 for development of bovine embryos to the 5 to 8-cell stage: on average between 50 and 57% of the embryos reached this stage after 2 d of culture in DMEM/F12 or in conditioned medium, while 36% reached this stage in TCM 199. Further development to the blastocyst stage was enhanced by conditioning. The highest percentage of blastocysts was achieved in DMEM/F12 medium conditioned with BRL cells (30%). The yield of blastocysts was similar in TCM 199 and in DMEM/F12 media conditioned with bovine oviduct cells (22 versus 20%), but after conditioning with BRL cells, DMEM/F12 medium yielded a higher percentage of blastocysts than TCM 199 (30 versus 18%). This might be explained by the fact that viability of BRL cells was better in DMEM/F12 medium than in TCM 199 when serum was omitted. Blastocysts produced in BRL-conditioned medium had a higher number of cells than blastocysts obtained in bovine oviduct-conditioned medium, and their transfer to recipients led to pregnancies and birth of calves. In conclusion, culture of bovine embryos in DMEM/F12 medium conditioned with BRL cells without serum led to the development of good-quality blastocysts and is thus a promising method for producing embryos for the study of potential embryotrophic factors. The use of rat liver cell lines guarantees against bovine viruses and allows for better production of embryos

Effects of co-culture and embryo number on the in vitro development of bovine embryos.

It is generally accepted that culturing embryos in groups or with somatic cells improves both the yield and quality of the blastocysts obtained. The aims of this study were 1) to compare the yield and quality of the embryos obtained after culture in several number conditions and in several culture systems and 2) to assess the effect of co-culture started at various stages of embryo development. Under cell-free culture conditions (modified synthetic oviduct fluid [mSOF] supplemented with 10% fetal calf serum [FCS] 48 h post insemination, the rate of Day 10 blastocysts was lower when embryos were cultured in small groups (1 to 6 per drop) than in large groups (4 versus 23% ; P < 0.01). There was no group effect when embryos were co-cultured either with Buffalo rat liver (BRL) cells in TCM 199, or in a culture system allowing the progressive development of cumulus cells in mSOF, even if co-culture started at 66 or 114 h post insemination. However, embryos cultured singly had lower cell numbers than embryos cultured in large groups when co-culture started at 114 h post insemination. This suggests that 1) somatic cells improve the development of singly cultured bovine embryos up to the blastocyst stage after the 9-16 cell stage; 2) co-culture affects blastocyst cell number of singly cultured embryos by acting roughly between the 5-8 and the 9-16 cell stage; and 3) cooperation between embryos could replace the effect of co-culture either on the yield of blastocysts or on blastocyst cell number. Blastocysts appeared significantly earlier in co-culture with cumulus cells in mSOF than in co-culture with BRL cells in TCM 199 (detection of the blastocysts: 7.3 +/- 0.1 d post insemination with cumulus cells versus 8.1 +/- 0.1 d with BRL cells; P < 0.001) and had a significant higher number of cells (143 +/- 9 versus 85 +/- 11; P < 0.001). This system thus seems suitable for the culture of small numbers of embryos resulting from in vitro maturation and fertilization of oocytes from individual donor cows

Characterization of bovine oviduct epithelial cell monolayers cultured under serum-free conditions.

We have developed a culture system for early bovine embryos in serum-free media conditioned by oviduct cell monolayers. A gentle mechanical procedure for oviduct cell isolation has been applied for this purpose avoiding the use of proteolytic enzymes. The aim of the present study was to identify the cell types present in the monolayers and to examine their fate in primary culture in serum-free or in serum-containing media by means of electronmicroscopical, immunocytochemical, and biochemical analyses. The cell dissociation procedure yielded two cell populations: ciliary cells and secretory cells that gradually dedifferentiate during culture. These cells formed a confluent monolayer after 6 d of culture in Tissue Culture Medium 199 medium supplemented with 10% fetal calf serum. Confluent cells displayed a typical epithelial cell morphology as assessed by phase contrast and electron microscopy and all the cells contained cytokeratin filaments as determined by immunocytochemistry. The overall histoarchitecture of the monolayer was preserved after washing and further culture for 7 d in serum-free medium. However, some degenerative signs indicate that the serum-free culture should not be extended for more than 7 d. Confluent oviduct cells also maintained their metabolic and protein secretory activity when deprived of serum. Total protein content in the culture supernatant linearly increased as a function of time and numerous peaks were detected after separation of proteins by high performance ion exchange chromatography. Protein elution patterns were reproducible and most of the proteins present in the culture medium were neosynthesized as determined by the incorporation of radiolabeled amino acids into nondialyzable proteins

CSS 9453 Qualification des salles propres et monitoring des processus aseptiques dans les banques de matériel corporel humain, les structures intermédiaires et les établissements de production

audience: researcher, professional, student, popularizationConformément à l’Arrêté Royal (AR) du 28 septembre 2009 fixant les normes de qualité et de sécurité pour le don, le prélèvement, l’obtention, le contrôle, le traitement, le stockage et la distribution de matériel corporel humain (AR, 2009), les banques de matériel corporel humain (MCH), les structures intermédiaires et les établissements de production doivent disposer d’installations adaptées – salles propres – pour le traitement du MCH. Avant la mise en service ou à la suite de travaux, le bon fonctionnement des salles propres doit être démontré grâce à l’exécution de tests de classification et de qualification. Afin de limiter autant que possible le risque de contamination microbiologique, notamment de contamination croisée, pendant la production des MCH, des processus aseptiques doivent être mis en œuvre en fonction du type de MCH et de l’application chez l’être humain. L’efficacité de ces processus aseptiques doit être surveillée entre autre par l’évaluation des particules et du nombre d’unités formant colonies (UFC) constaté dans l’air, sur les surfaces, les consommables, les appareils, les vêtements et/ou les empreintes digitales. Les différents contrôles sont établis sur base de l’analyse de risques. L’avis CSS 8699 relatif aux recommandations en matière de validation et de contrôle de l’environnement au sein des banques de MCH, les structures intermédiaires et les établissements de production date de 2012. À la suite de l’obtention de nouvelles informations, de l’apparition de nouvelles technologies et de l’évolution des exigences, ainsi que la révision de la norme internationale pour la classification et le contrôle des salles propres (NBN EN ISO 14644-1 en 2), le Conseil Supérieur de la Santé (CSS) a décidé de revoir l’avis CSS 8699. Dans le cadre de l’évaluation de l’avis CSS 8699, les problèmes suivants ont été identifiés et ont été pris en compte dans le présent avis : L’avis CSS 8699 se limitait à la classification et au contrôle de l’environnement et n’apportait aucun conseil concernant le contrôle des processus de production aseptiques : o Le matériel source (MCH) est limité et il existe une grande variabilité intrinsèque entre les matériels sources ; o Pour certains types de MCH, le matériel source est contaminé avant ou lors du prélèvement ; o La production des MCH s’effectue souvent à petite échelle ; o Il existe une grande diversité des processus de production aseptiques au sein de différents types de banques de MCH ; o Les processus de production aseptiques comportent souvent de nombreuses opérations manuelles ; o Tous les processus de prélèvement et de production ne peuvent pas être effectués de manière aseptique. Harmonisation avec les concepts de gestion des risques ; La terminologie utilisée dans l’avis CSS 8699 n’était pas toujours univoque ; Une nouvelle version des normes internationales NBN EN ISO14644-1 et 2 a été publiée en 2016 et enrichie de nouvelles informations et techniques dans le cadre de la classification et du contrôle des salles propres à l’aide de compteurs de particules.En 2014, 8,5 % de toutes les réactions indésirables graves recensées en Europe à la suite de la transplantation de MCH étaient imputables à des infections, principalement des contaminations avec des bactéries et des champignons. La prévention des infections consécutives à la transplantation de MCH est un important défi que doivent relever les banques de MCH, les structures intermédiaires et les établissements de production. Il convient sur ce plan de tenir compte de 4 risques majeurs de contamination : Le risque de contamination du MCH durant le prélèvement (type d’environnement où le prélèvement est effectué, durée d’exposition du MCH à l’environnement direct, pathologie sous-jacente chez le donneur, etc.) ; Le risque de contamination du MCH durant le traitement (classe de propreté de la salle blanche où le traitement est effectué, durée d’exposition du MCH à l’environnement direct, nombre de manipulations critiques exécutées, type de traitement, etc.) ; Le risque de contamination croisée avec un autre MCH (efficacité de la procédure de nettoyage et de désinfection appliquée, type de MCH, donneurs infectieux, instruments ou consommables contaminés, etc.) ; Le risque d’infections post-opératoires durant la transplantation ou l’administration du MCH chez le patient (statut immunitaire du patient, forme d’administration, site d’administration, etc.). Conformément aux législations européenne et belge, les banques de MCH, les structures intermédiaires et les établissements de production doivent disposer d’infrastructures adaptées – « salles propres » – pour maîtriser les risques de contamination durant le traitement du MCH. Le CSS recommande de déterminer la qualité de l’air appropriée de ces salles propres à l’aide d’une analyse des risques documentée prenant en compte l’ensemble des risques potentiels de contamination par l’environnement direct et le processus de production et répondant au moins à la classe de propreté minimale telle que définie dans l’AR de 2009. Afin d’assurer la qualification des salles propres, il convient d’élaborer un plan directeur de validation déterminant les tests de qualification à réaliser ou à répéter, les moments où ceux-ci doivent être réalisés ou répétés, ainsi que l’état de fonctionnement dans lequel ils doivent être réalisés ou répétés. Par ailleurs, le CSS préconise la mise sur pied d’un programme de monitoring permettant de déceler les évolutions des tendances de la contamination microbiologique au cours du processus de production aseptique et d’adopter les mesures correctrices en temps opportun. Le programme de monitoring doit identifier, contrôler et suivre les points critiques du processus de production aseptique, identifiés sur la base d’une analyse des risques documentée. Les valeurs d’alertes et de seuils doivent être spécifiées par salle propre contrôlée, par état de fonctionnement et par processus aseptique

Three year results of in vitro production of bovine embryos in serum-poor bovine oviduct conditioned medium. An overview.

This paper presents a synthesis of 3 year results of in vitro production of bovine embryos in medium previously conditioned by bovine oviduct epithelial cells. In Louvain-la-Neuve, Belgium, a total of 18356 oocytes were matured and inseminated in vitro: 13967 (76%) had cleaved at 3 days post-insemination and 3593 (26%) became blastocysts using this culture system. Our data show that conditioned medium can be stored frozen for up to 3 years without significant loss of activity and is resistant to lyophilization. One single batch of conditioned medium was tested within the same period in four different laboratories and yielded variable results: 27 and 37% blastocysts/cleaved embryos in two of them and only 7 and 0% in the two others whereas in each case more than 30% blastocysts were obtained with the local reference co-culture system. In one laboratory, the batch of oil used to overlay the culture drops had a detrimental effect on the blastocyst rate in conditioned medium but not in co-culture