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Knowledge of action of progesterone (P4) on the human preimplantation embryo is lacking. The objective of this study was to determine expression of a mitochondrial P4 receptor (PR-M) in the trophectoderm (TE) and the inner cell mass (ICM) of the human blastocyst and to determine P4-induced gene expression during growth from the cleavage to the blastocyst stage. Previously cryopreserved cleavage stage embryos were treated with P4 (10−6 M) or vehicle until blastocyst development. Cells from the TE and the ICM of dissected euploid embryos underwent RNA-seq analysis, while other embryos were used for analysis of nuclear PR (nPR) and PR-M expression. PR-M expression was confirmed in the TE, the ICM, and a human embryonic stem cell line (HESC). Conversely, nPR expression was absent in the TE and the ICM with low expression in the HESC line. RNA-seq analysis revealed P4 effects greater in the TE with 183 significant pathway changes compared to 27 in the ICM. The TE response included significant upregulation of genes associated with DNA replication, cell cycle phase transition and others, exemplified by a 7.6-fold increase in the cell proliferation gene, F-Box Associated Domain Containing. The majority of ICM pathways were downregulated including chromosome separation, centromere complex assembly and chromatin remodeling at centromere. This study confirms that human blastocysts express PR-M in both the TE and the ICM, but lack expression of nPR. P4-induced gene regulation differs greatly in the two cell fractions with the predominant effect of cell proliferation in the TE and not the ICM. [ABSTRACT FROM AUTHOR]

Introduction: MicroRNAs (miRNAs) are important regulators of many biological functions, including embryo implantation and development. Recently, it has been reported that miRNAs in biofluids are predictive for physiological and pathological processes. Objectives: In this study, we aim to investigate whether the miRNAs secreted by human embryos in culture medium can be used as embryonic biomarkers. Methods: The culture media were prospectively collected from embryos of patients at reproductive medicine center with informed consent. A high-throughput miRNA sequencing method was applied to detect the miRNA profiles in the human embryo culture media. After bioinformatics analysis and screening of differentially expressed miRNAs, quantitative real-time polymerase chain reaction (qRT-PCR) assay was subsequently performed to further confirm the sequencing results with mixed samples. Furthermore, we performed droplet digital PCR (ddPCR) to verify the target miRNAs at single sample level. Receiver operating characteristic (ROC) analyses were performed for differentially expressed miRNAs. Results: Compared with embryos with failed pregnancy, the embryos with successful pregnancy secreted different miRNA profiles into the culture media, which were predicted to be involved in multiple biological processes. Validated by droplet digital polymerase chain reaction (ddPCR), the expression of hsa-miR-26b-5p and hsa-miR-21-5p in the culture media of cleavage embryos with successful pregnancy was significantly lower than that of embryos with failed pregnancy. Moreover, the Receiver Operating Characteristic (ROC) curve analysis indicated that hsa-miR-26b-5p and hsa-miR-21-5p could serve as potential biomarkers for reproductive outcomes. Conclusion: Together, our findings highlight the important predictive potential of miRNAs secreted by human embryos in culture media, which is meaningful for non-invasive embryo selection in assisted reproductive technology.

Abstract Background Morphological features are the most common criteria used to select human embryos for transfer to a receptive uterine cavity. However, such characteristics are not valid for embryos in cellular arrest. Even aneuploid embryos can have normal morphology, and some euploid embryos have aberrant morphology. The aim of this study was to quantify the expression profile of hsa-miR-21-3p, -24-1-5p, -191-5p, and -372-5p in culture media on day 5 of in vitro embryo development, and compare the profiles of two groups of media classified by outcome: successful (n = 25) or unsuccessful (n = 25) implantation pregnancy. Methods Fifty patients were accepted in the Department of Reproductive Biology of a Hospital in México City, based on the Institutional inclusion criteria for in vitro fertilization. Embryos were transferred to the women on day 5 of cultivation, and the culture media were collected. RNA was isolated from each culture medium with TRIzol reagent, and microRNA (miRNA) expression was detected through RT-PCR with specific primers. Expression bands were quantified by reading optical density. Results There was a 5.2-fold greater expression of hsa-miR-191-5p in the pregnancy-related culture media (p ≤ 0.001) and a 1.6-fold greater level of hsa-miR-24-1-5p (p = 0.043) in the media corresponding to non-pregnant women. No significant difference existed between the two groups hsa-miR-21-3p (p = 0.38) or hsa-miR-372-5p (p = 0.41). Conclusions Regarding adequate in vitro embryo development, hsa-miR-191-5p could possibly represent a positive biomarker, while has-miR-24-1-5p may indicate poor prognosis. This former miRNA modulates IGF2BP-1 and IGF2R, associated with the implantation window. On the other hand, hsa-miR-24-1-5p may be related to a poor prognosis of human embryo development.

To determine whether glutamine consumption is associated with embryo quality and aneuploidy, a retrospective study was conducted in an in vitro fertilization center. Spent embryo culture media from patients undergoing assisted reproduction treatment and preimplantation genetic testing (PGT) were obtained on day 3 of in vitro culture. Embryo quality was assessed for cell number and fragmentation rate. PGT for aneuploidy was performed using whole genome amplification and DNA sequencing. Glutamine levels in spent embryo culture media were analyzed by gas chromatography–mass spectrometry. The results demonstrated that glutamine was a primary contributor to the classification of the good-quality and poor-quality embryos based on the orthogonal partial least-squares discriminant analysis model. Glutamine consumption in the poor-quality embryos was significantly higher than that in the good-quality embryos (P < 0.05). A significant increase in glutamine consumption was observed from aneuploid embryos compared with that from euploid embryos (P < 0.01). The Pearson correlation coefficients between embryo quality and glutamine consumption, and between aneuploidy and glutamine consumption, were 0.430 and 0.757, respectively. The area under the ROC curve was 0.938 (95% CI: 0.902–0.975) for identifying aneuploidy. Animal experiments demonstrate that increased glutamine consumption may be a compensatory mechanism to mitigate oxidative stress. Our data suggest that glutamine consumption is associated with embryo quality and aneuploidy. Glutamine may serve as a molecular indicator for embryo assessment and aneuploidy testing. [ABSTRACT FROM AUTHOR]

Study Question: Do embryos from sibling oocytes assigned to distinct single-step media culture systems demonstrate differences in early embryo development, morphokinectics or aneuploidy rates?Summary Answer: Embryo quality, morphokinetic parameters and aneuploidy rates from trophectoderm biopsy were similar between sibling embryos cultured in distinct media systems from the time of gamete isolation.What Is Known Already: Studies on the effect of commercially available embryo culture media systems have demonstrated inconsistent impact on human embryonic development, morphokinetics, aneuploidy rates and clinical outcomes. In addition, these studies have been primarily randomized at the level of the embryo or the patient to culture media.Study Design, Size, Duration: Prospective sibling oocyte cohort derived from 200 subjects undergoing IVF at a tertiary academic medical center between February 2018 and November 2019.Participants/materials, Setting, Methods: Sibling oocytes were allocated to Global® or SAGE® media system based upon laterality of ovary from which they were retrieved. All embryos were cultured in a time-lapse incubator. Blastocysts underwent trophectoderm biopsy for preimplantation genetic testing for aneuploidy using next-generation sequencing.Main Results and the Role Of Chance: One hundred twenty-seven subjects (n = 127) had paired blastocysts for biopsy in each culture media system. There was no difference in top quality blastocyst formation (47.1 ± 31.0 vs 48.1 ± 27.2%; P = 0.87) nor aneuploidy rate (62.3 ± 34.0 vs 56.1 ± 34.4%; P = 0.07) for sibling embryos cultured in Global versus SAGE media system. Embryo morphokinetic parameters including time to each cell division from two cells (t2) to eight cells (t8), time to morula stage (tM), time to blastocele formation (tSB), time to fully formed blastocyst (tB) and time to expansion of the blastocyst (tEB) were similar between paired blastocysts from each culture media system.Limitations, Reasons For Caution: Pregnancy outcomes and offspring health data were not available for analysis.Wider Implications Of the Findings: Commercially available culture media may not have a differential impact on embryo development and blastocyst aneuploidy rate when patient and stimulation-related factors are held constant.Study Funding/competing Interest(s): There was no external funding for this study. C.H. is owner of a consultancy company, IVF Professionals, Chief Scientific Officer at Apricity, Executive Director at TMRW and co-owner and shareholder of Aria Fertility. She has received speaker fees, consulting fees and travel support from Cooper Surgical and Vitrolife. J.B. is an employee and shareholder of vitrolife.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Culture media supplemented with reproductive fluids (RF) have been used in livestock species, improving the efficiency and quality of in vitro produced embryos. However, usefulness in humans is still unknown. In this study, we collected human reproductive fluids (HRFs) ex vivo (from 25 patients undergoing abdominal hysterectomy plus bilateral salpingectomy) and in vivo (from 31 oocyte donors). Afterward, protocols to evaluate their osmolality, pH, total protein concentration, endotoxin level, and sterility were optimized, establishing security ranges for their use as natural additives. In addition, a functional assay was developed with bovine embryos grown in vitro in a medium supplemented with 1% of collected HRFs. Finally, a proof of concept was performed with six patients on post ovulation day 2 to evaluate the full-term viability of embryos grown in media supplemented with autologous uterine fluid, collected under in vivo conditions. Two of the embryos resulted in successful pregnancy and delivery of healthy babies. In conclusion, this study establishes a complete quality control sheet of HRFs as additives for embryo culture media and shows first preliminary data on obtaining healthy offspring derived from embryos grown in media supplemented with HRFs.

Culture media supplemented with reproductive fluids (RF) have been used in livestock species, improving the efficiency and quality of in vitro produced embryos. However, usefulness in humans is still unknown. In this study, we collected human reproductive fluids (HRFs) ex vivo (from 25 patients undergoing abdominal hysterectomy plus bilateral salpingectomy) and in vivo (from 31 oocyte donors). Afterward, protocols to evaluate their osmolality, pH, total protein concentration, endotoxin level, and sterility were optimized, establishing security ranges for their use as natural additives. In addition, a functional assay was developed with bovine embryos grown in vitro in a medium supplemented with 1% of collected HRFs. Finally, a proof of concept was performed with six patients on post ovulation day 2 to evaluate the full-term viability of embryos grown in media supplemented with autologous uterine fluid, collected under in vivo conditions. Two of the embryos resulted in successful pregnancy and delivery of healthy babies. In conclusion, this study establishes a complete quality control sheet of HRFs as additives for embryo culture media and shows first preliminary data on obtaining healthy offspring derived from embryos grown in media supplemented with HRFs. [ABSTRACT FROM AUTHOR]

Background: Morphological features are the most common criteria used to select human embryos for transfer to a receptive uterine cavity. However, such characteristics are not valid for embryos in cellular arrest. Even aneuploid embryos can have normal morphology, and some euploid embryos have aberrant morphology. The aim of this study was to quantify the expression profile of hsa-miR-21-3p, -24-1-5p, -191-5p, and -372-5p in culture media on day 5 of in vitro embryo development, and compare the profiles of two groups of media classified by outcome: successful (n = 25) or unsuccessful (n = 25) implantation pregnancy. Methods: Fifty patients were accepted in the Department of Reproductive Biology of a Hospital in México City, based on the Institutional inclusion criteria for in vitro fertilization. Embryos were transferred to the women on day 5 of cultivation, and the culture media were collected. RNA was isolated from each culture medium with TRIzol reagent, and microRNA (miRNA) expression was detected through RT-PCR with specific primers. Expression bands were quantified by reading optical density. Results: There was a 5.2-fold greater expression of hsa-miR-191-5p in the pregnancy-related culture media (p ≤ 0.001) and a 1.6-fold greater level of hsa-miR-24-1-5p (p = 0.043) in the media corresponding to non-pregnant women. No significant difference existed between the two groups hsa-miR-21-3p (p = 0.38) or hsa-miR-372-5p (p = 0.41). Conclusions: Regarding adequate in vitro embryo development, hsa-miR-191-5p could possibly represent a positive biomarker, while has-miR-24-1-5p may indicate poor prognosis. This former miRNA modulates IGF2BP-1 and IGF2R, associated with the implantation window. On the other hand, hsa-miR-24-1-5p may be related to a poor prognosis of human embryo development. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Does the duration of embryo exposure to hyaluronic acid (HA) enriched medium improve the rate of live birth events (LBEs)? SUMMARY ANSWER The use of embryo transfer (ET) medium rich in HA improves LBE (a singleton or twin live birth) regardless of the duration of exposure evaluated in this study, but does not alter gestation or birthweight (BW). WHAT IS KNOWN ALREADY HA-enriched medium is routinely used for ET in ART to facilitate implantation, despite inconclusive evidence on safety and efficacy. STUDY DESIGN, SIZE, DURATION A cohort study was performed evaluating clinical treatment outcomes before and after HA-enriched ET medium was introduced into routine clinical practice. In total, 3391 fresh ET procedures were performed using low HA and HA-rich medium in women undergoing publicly funded IVF/ICSI treatment cycles between May 2011 and April 2015 were included in this cohort study. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 1018 ET performed using low HA medium were compared with 1198, and 1175 ET following exposure to HA-rich medium for 2–4 h (long HA exposure) or for 10–30 min (short HA exposure), respectively. A multiple logistic regression analysis was used to compare clinical outcomes including BW, gestational age and sex ratios between groups, whilst adjusting for patient age, previous attempt, incubator type and the number of embryos transferred. MAIN RESULTS AND THE ROLE OF CHANCE The use of HA-rich medium for ET was positively and significantly associated with improved clinical pregnancy rate and LBE, for both exposure durations: long HA (odds ratio (OR) = 1.21, 95% CI: 0.99–1.48), short HA (OR = 1.32, 95% CI: 1.02–1.72) and pooled OR = 1.26, 95% CI: 1.03–1.54, relative to the use of low HA medium. A comparative analysis of the risks of early pregnancy loss following long HA exposure (OR = 0.76, 95% CI: 0.54–1.06), short HA exposure (OR = 0.84, 95% CI: 0.54–1.30) and late miscarriage (OR = 0.88, 95% CI: 0.51–1.53) (OR = 1.41, 95% CI 0.72–2.77), were lower and not statistically significant. Similarly, ordinary regression analysis of the differences in BW at both HA exposures; pooled OR = −0.9 (−117.1 to 115.3), and adjusted BW between both HA cohorts; pooled OR = −13.8 (−106.1 to 78.6) did not show any differences. However, a difference in gestational age (pooled OR −0.3 (−3.4 to 2.9)) and sex ratio (pooled OR 1.43 (0.95–2.15)) were observed but these were not statistically significant relative to low HA medium. LIMITATIONS, REASONS FOR CAUTION The strength of a randomized treatment allocation was not available in this evaluation study, therefore effects of unmeasured or unknown confounding variables cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS The result of this large cohort study strengthens the case for using HA-rich medium routinely at transfer, while adding the important clinical information that duration of exposure may not be critical. The composition and effects of commercial IVF culture media on success rate and safety remains a major controversy despite increasing calls for transparency and evidence-based practice in ART. Nonetheless, the lack of differences in BW and gestational age observed in this study were reassuring. However, an appraisal of clinical outcomes and appropriate research investigations are required for the continuous evaluation of efficacy and safety of HA. STUDY FUNDING/COMPETING INTEREST(S) T.A. is funded by a Clinical Doctoral Research Fellowship (CDRF) grant (reference: ICA-CDRF-2015-01-068) from the National Institute for Health Research (NIHR). The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health. The authors declare no conflict of interest. [ABSTRACT FROM AUTHOR]

Embryo implantation depends on two primary factors: the quality of the embryo and endometrial receptivity. Small RNAs have been shown to be potent epigenetic regulators influencing cell proliferation, differentiation, and communication even in the context of early embryonic development. However, previous reports are limited to miRNAs and lack sensitivity. Here, we describe a platform for non-invasive small RNA biomarker discovery and validation from embryo-conditioned culture media (ECCM). We hypothesize that small non-coding RNAs (sncRNAs) are secreted by the embryo into the ECCM and test the limit of detection for profiling sncRNA by deep sequencing and quantitative PCR. In the first set of experiments, we evaluated sequencing sensitivity by comparing sncRNA profiles from pools of 10, 5, 3, and single ECCM drops. Next, we performed a similar test for TaqMan qPCR sensitivity by measuring select sncRNAs in 5, 3 and single drop ECCM pools. Finally, we compared the expression of an sncRNA panel by qPCR in single ECCM vs no-embryo control media. We report the first comprehensive sequencing of sncRNAs in ECCM with a sequencing sensitivity of 3 single embryo drops, capturing ~150 miRNAs and an abundance of tRNA-derived small RNAs (tsRNAs). We then profiled 15 sncRNAs by qPCR and determined that the assay maintains sensitivity in single ECCM drops. Finally, we found significant differences in these sncRNA expression between control and ECCM drops. Improving embryo selection is crucial for reducing time to pregnancy. Here we describe a sensitive technique for biomarker discovery by sequencing and qPCR validation in ECCM, demonstrating that the majority of sncRNAs are embryo derived. We also report an abundance of tsRNAs which suggests these sncRNAs may have functions in endometrial-maternal communication beyond the microRNAs which have been described previously. Abbreviations: PGT-A: Preimplantation genetic testing for aneuploidies; ECCM: Embryo-conditioned culture media; sncRNAs: Small non-coding RNAs; miRNAs: microRNAs; EVs: Extracellular vesicles; PCA: Principal component analysis. [ABSTRACT FROM AUTHOR]

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Objective To evaluate the effect of supplementing single-step embryo culture medium with insulin on human embryo development. Design Comparative study. Setting Two private centers. Patient(s) The study involved a sibling oocyte split of 5,142 retrieved oocytes from 360 patients. Intervention(s) Sibling oocytes split after intracytoplasmic sperm injection for culture from day 0 through day 5 or 6 in insulin-supplemented or control medium. Women were split to receive their embryos from insulin-supplemented or control medium. Main Outcome Measure(s) Clinical pregnancy rate. Result(s) There were significantly higher rates of clinical, ongoing, and twin pregnancies in the insulin-supplemented arm than in the control arm. On day 3, embryo quality and compaction were higher in insulin-supplemented medium. On day 5, insulin supplementation showed higher rates of blastocyst formation, quality, and cryopreservation. Conclusion(s) Insulin supplementation of single-step embryo culture medium from day 0 through day 5 or 6 improved clinical pregnancy rate and human embryo development. However, these findings need further confirmation through a multicenter randomized controlled trial that may include other patient populations and different culture media. [ABSTRACT FROM AUTHOR]

Study Question: In comparison to in vivo development, how do different conditions of in vitro culture ('one step' versus 'sequential medium') impact DNA methylation and hydroxymethylation in preimplantation embryos?Summary Answer: Using rabbit as a model, we show that DNA methylation and hydroxymethylation are both affected by in vitro culture of preimplantation embryos and the effect observed depends on the culture medium used.What Is Known Already: Correct regulation of DNA methylation is essential for embryonic development and DNA hydroxymethylation appears more and more to be a key player. Modifications of the environment of early embryos are known to have long term effects on adult phenotypes and health; these probably rely on epigenetic alterations.Study Design Size, Duration: The study design we used is both cross sectional (control versus treatment) and longitudinal (time-course). Each individual in vivo experiment used embryos flushed from the donor at the 2-, 4-, 8-, 16- or morula stage. Each stage was analyzed in at least two independent experiments. Each individual in vitro experiment used embryos flushed from donors at the 1-cell stage (19 h post-coïtum) which were then cultured in parallel in the two tested media until the 2-, 4-, 8- 16-cell or morula stages. Each stage was analyzed in at least three independent experiments. In both the in vivo and in vitro experiments, 4-cell stage embryos were always included as an internal reference.Participants/materials, Setting, Methods: Immunofluorescence with antibodies specific for 5-methylcytosine (5meC) and 5-hydroxymethylcytosine (5hmeC) was used to quantify DNA methylation and hydroxymethylation levels in preimplantation embryos. We assessed the expression of DNA methyltransferases (DNMT), of ten eleven translocation (TET) dioxigenases and of two endogenous retroviral sequences (ERV) using RT-qPCR, since the expression of endogenous retroviral sequences is known to be regulated by DNA methylation. Three repeats were first done for all stages; then three additional repetitions were performed for those stages showing differences or tendencies toward differences between the different conditions in the first round of quantification.Main Results and the Role Of Chance: The kinetics of DNA methylation and hydroxymethylation were modified in in vitro cultured embryos, and the observed differences depended on the type of medium used. These differences were statistically significant. In addition, the expression of TET1 and TET2 was significantly reduced in post-embryonic genome activation (EGA) embryos after in vitro culture in both tested conditions. Finally, the expression of two retroviral sequences was analyzed and found to be significantly affected by in vitro culture.Limitations Reasons For Caution: Our study remains mostly descriptive as no direct link can be established between the epigenetic changes observed and the expression changes in both effectors and targets of the studied epigenetic modifications. The results we obtained suggest that gene expression could be affected on a large scale, but this remains to be confirmed.Wider Implications Of the Findings: Our results are in agreement with the literature, showing that DNA methylation is sensitive to in vitro culture. As we observed an effect of both tested culture conditions on the tested epigenetic marks and on gene expression, we cannot conclude which medium is potentially closest to in vivo conditions. However, as the observed effects are different, additional studies may provide more information and potential recommendations for the use of culture media in assisted reproductive technology.Study Funding/competing Interests: This work was supported by an 'AMP diagnostic prénatal et diagnostic génétique' 2012 grant from the French Agence de la Biomédecine. This study was performed within the framework of ANR LABEX 'REVIVE' (ANR-10-LABX-73). Authors are members of RGB-Net (TD 1101) and Epiconcept (FA 1201) COST actions. The authors declare that there is no competing interest. [ABSTRACT FROM AUTHOR]

Résumé Objectif L’objectif de cette étude était de comparer le développement des embryons cultivés dans deux milieux de culture globale : Fert/Sage One Step ® (Origio) et Continuous Single Culture ® (CSC) (Irvine Scientific). Méthodes Une étude prospective autocontrôlée a été réalisée sur des ovocytes mis en fécondation in vitro (FIV) ou micro-injectés (ICSI) dans notre centre de février à juin 2015. Après la mise en fécondation, pour chaque patiente, une moitié de la cohorte a été cultivée dans le milieu Fert/Sage One Step ® (groupe SAGE) et l’autre moitié dans le milieu CSC ® (groupe CSC). Les taux de fécondation et le développement embryonnaire ont été suivis de j1 à j5–j6 si nécessaire. Les embryons présentant < 20 % de fragmentation avec 4 blastomères à j2 ou 8 à j3 étaient qualifiés de « top qualité ». Ceux présentant < 20 % de fragmentation avec 3 à 5 blastomères à j2, ou 6 à 10 blastomères à j3 étaient qualifiés de « bonne qualité ». Les blastocystes de grade B3, B4, B5 avec une masse cellulaire interne de grade A ou B et un trophectoderme de grade A ou B étaient dits de « bonne qualité ». Les embryons utiles étaient définis comme ceux ayant été transférés ou congelés. Résultats Soixante-deux tentatives de FIV et 133 d’ICSI, soit 2059 ovocytes, ont été analysées. Les taux de fécondation n’étaient pas différents entre les 2 groupes, respectivement SAGE vs CSC (FIV : 73,4 % vs 68,3 % [ p = 0,49] ; ICSI : 58,9 % vs 63,8 % [ p = 0,12]). Aucune différence n’a été retrouvée dans le développement embryonnaire, respectivement SAGE vs CSC, à j2 (top embryons j2 FIV : 34,4 % vs 33 % [ p = 0,98] ; ICSI : 42,4 % vs 44,9 % [ p = 0,37] ; et bonne qualité j2 FIV : 44 % vs 50,2 % [ p = 0,07] ; ICSI : 64 % vs 71 % [ p = 0,35]) ; ni à j3 (top embryons j3 FIV : 19,4 % vs 21,3 % [ p = 0,61] ; ICSI : 28,7 % vs 27,4 % [ p = 0,54] ; et bonne qualité j3 FIV : 40,4 % vs 50,2 % [ p = 0,91] ; ICSI : 51 % vs 47,6 % [ p = 0,47]). Les taux de blastoformation n’étaient pas différents, respectivement SAGE vs CSC (FIV : 39,9 % vs 41,5 % [ p = 0,63] avec 42,9 % vs 42,2 % blastocystes de bonne qualité [ p = 0,70] ; ICSI : 41,1 % vs 37,8 % [ p = 0,18] avec 32,9 % vs 40,8 % blastocystes de bonne qualité [ p = 0,13]). Aucune différence n’a été observée concernant le taux d’embryons utiles dans les 2 groupes SAGE et CSC (FIV : 52,8 % et 55,2 % [ p = 0,83] ; ICSI : 62,4 % et 61,7 % [ p = 0,70]). Conclusion La culture embryonnaire dans le milieu global Fert/Sage One Step ® vs le milieu global Continuous Single Culture ® ne montre pas de différence dans le développement embryonnaire, ni dans le taux d’embryons utiles, transférés ou congelés. Objective The aim of this study was to compare embryo development cultured in two single-step media commercially available: Fert/Sage One Step ® (Origio) and Continuous Single Culture ® (CSC) (Irvine Scientific). Methods A prospective auto-controlled study of sibling oocytes from women undergoing conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) was performed in our center from February to June 2015. After fertilization, for every patient, half of oocytes were cultured in the single-step Fert/Sage One Step ® (serie SAGE) and the other half in the single-step CSC ® (serie CSC). Fertilization and embryo morphology rates were assessed by day 1 to day 5–6 if needed. Embryo presenting < 20% of fragmentation and 4 cells at day 2, 8 cells at day 3 were qualified as “top quality”. Embryo with < 20% of fragmentation and 3–5 cells at day 2, 6–10 cells at day 3 were qualified as “good quality”. Blastocyst B3, B4, B5 with A or B inner cell mass and A or B trophectoderm were qualified as “good quality”. Transferred or frozen embryos were qualified as useful embryos. Results Sixty-two attempts of IVF and 133 of ICSI were analyzed, corresponding to 2059 inseminated or micro-injected oocytes. Fertilization rate were not different between the 2 series, respectively SAGE vs CSC (IVF: 73.4% vs 68.3% [ P = 0.49]; ICSI: 58.9% vs 63.8% [ P = 0.12]). No difference was found for embryo morphology, respectively SAGE vs CSC, at day 2 (top quality embryo at day 2 IVF: 34.4% vs 33% [ P = 0.98]; ICSI: 42.4% vs 44.9% [ P = 0.37]; and good quality embryo at day 2 IVF: 44% vs 50.2% [ P = 0.07]; ICSI: 64% vs 71% [ P = 0.35]); no difference at day 3 (top quality embryo at day 3 IVF: 19.4% vs 21.3% [ P = 0.61]; ICSI: 28.7% vs 27.4% [ P = 0.54]; and good quality embryo at day 3 IVF: 40.4% vs 50.2% [ P = 0.91]; ICSI: 51% vs 47.6% [ P = 0.47]). Blastocyst development rate were not different, respectively SAGE vs CSC (IVF: 39.9% vs 41.5% [ P = 0.63] with 42.9% vs 42.2% of good quality blastocyst [ P = 0.70]; ICSI: 41.1% vs 37.8% [ P = 0.18] with 32.9% vs 40.8% of good quality blastocyst [ P = 0.13]). No difference was found in the useful embryo rate in the 2 series SAGE vs CSC (IVF: 52.8% vs 55.2% [ P = 0.83]; ICSI: 62.4% vs 61.7% [ P = 0.70]). Conclusion Embryo development and rate of useful embryos, transferred or frozen, were not different according to the embryo culture in single-step media Fert/Sage One Step ® vs single-step Continuous Single Culture ® . [ABSTRACT FROM AUTHOR]

Morphological assessment is currently the primary technique for selection of viable embryos for uterine transfer during assisted reproductive techniques, however this method has limited predictive power. The objective of this study was to employ NMR based metabolic profiling analysis of spent embryo culture media to identify novel biomarkers of embryo viability and provide insight into the metabolism of a viable embryo. A total of 37 patients undergoing IVF/ICSI treatment were recruited and 58 media samples were collected from embryos that were transferred back to the uterus. 1H NMR spectra were acquired and analyzed resulting in the quantification of 12 metabolites in the media samples. Analysis of metabolite ratios revealed significant differences between those patients with positive ( n = 27) and negative ( n = 31) urinary βhCG results. Some of the most biologically relevant differences include a 17% increase in the formate to glycine ratio and a 22% decrease in the citrate to alanine ratio in the spent embryo media from the positive pregnancy group. Overall, the results indicate that metabolic profiling may provide a means of identifying biomarkers that aid selection of viable embryos. [ABSTRACT FROM AUTHOR]

In this study, a rapid and simple method which combines drop coating deposition and Raman spectroscopy (DCDR) was developed to characterize the dry embryo culture media (ECM) droplet. We demonstrated that Raman spectra obtained from the droplet edge presented useful and characteristic signatures for protein and amino acids assessment. Using a different analytical method, scanning electron microscopy coupled with energy dispersive X-ray analysis, we further confirmed that Na, K, and Cl were mainly detected in the central area of the dry ECM droplet while sulphur, an indicative of the presence of macromolecules such as proteins, was mainly found at the periphery of the droplet. In addition, to reduce sample preparation time, different temperatures for drying the droplets were tested. The results showed that drying temperature at 50°C can effectively reduce the sample preparation time to 6 min (as compared to 50 min for drying at room temperature, ~25°C) without inducing thermal damage to the proteins. This work demonstrated that DCDR has potential for rapid and reliable metabolomic profiling of ECM in clinical applications. [ABSTRACT FROM AUTHOR]

Objective: To compare the dynamics of early development between embryos cultured in single and sequential media. Design: Randomized, comparative study. Setting: Private IVF centre. Patients: A total of 446 metaphase II oocytes from 51 couples who underwent oocyte retrieval procedure for intracytoplasmic sperm injection. Forty-nine resulted in embryo transfer. Intervention: Oocytes were split between single and sequential media produced by the same manufacturer and cultured in a time-lapse incubator. Main outcome measures: Morphokinetic parameters until the embryos reached the 5-cell stage (t5), utilization, clinical pregnancy and implantation rates. Results: Embryos cultured in single media were advanced from the first mitosis cycle and reached 2- to 5-cell stages earlier. There was not any difference between the durations for cell cycle two (cc2 = t3-t2) and s2 (t4-t3). The utilization, clinical pregnancy and implantation rates did not differ between groups. The proportion of cryopreserved day6 embryos to two pronuclei oocytes was significantly higher in sequential than in single media. Conclusions: Morphokinetics of embryo development vary between single and sequential culture media at least until the 5-cell stage. The overall clinical and embryological parameters remain similar regardless of the culture system. [ABSTRACT FROM AUTHOR]

Abstract: Aminograms of tubal and follicular fluids were obtained using fluids collected by aspiratory puncture from six cats. The amino acids were separated and quantified by high-performance liquid chromatography analysis. The serum of the cats was used as control. The three most prevalent amino acids quantified in cat tubal fluid were glycine, glutamic acid, and taurine. Their mean concentrations were 840 μmol/l (μm), 808 μm and 596 μm, respectively. The three most prevalent amino acids quantified in cat follicular fluid were alanine, glutamine, and taurine. Their mean concentrations were 359 μm, 351 μm, and 258 μm, respectively. This result is consistent with aminograms of tubal fluid previously determined in other mammals. As previously observed in other species and humans, glycine was quantitatively the most abundant and most prevalent free amino acid in cat tubal fluid. The total quantity of amino acids in tubal fluid was similar in cats and other species. However, in contrast with other species studied, hypotaurine was not detected in tubal and follicular fluids of female cats. [Copyright &y& Elsevier]

The aim of this study was to determine whether homocysteine (hcy) concentrations in embryo culture media correlate with pregnancy outcome in assisted reproductive technology (ART) cycles. Forty patients who underwent single embryo transfer at the infertility clinic of a tertiary care center were recruited for this case-control study. Spent embryo culture media from all patients were collected after single embryo transfer on day 3 (n = 40). Hcy concentrations in embryo culture media were analyzed by enzyme cycling method. Patients were grouped according to the diagnosis of a clinical pregnancy. Sixteen patients were pregnant while 24 patients failed to achieve conception. Mean Hcy levels in the culture media were significantly different between the groups (p < 0.003), as 4.58 ± 1.31 μmol/l in the non-pregnant group and 3.37 ± 0.92 μmol/l in the pregnant group. Receiver operator curve analysis for determining the diagnostic potential of Hcy for pregnancy revealed an area under the curve of 0.792 (confidence interval: 0.65–0.94;p < 0.05). A cut-off value of 3.53 μmol/l was determined with a sensitivity of 83.3%, and a specificity of 68.8%. Lower hcy levels were associated with a better chance of pregnancy and better embryo grades. Hcy may be introduced as an individual metabolomic profiling marker for embryos. [ABSTRACT FROM AUTHOR]

In this study, we aimed to determine whether human embryos secrete interleukin-1β (IL-1β) into culture media and its correlation with embryo grade and development. Culture media supernatants of 100 embryos obtained from 39 cycles of 38 patients and cultivated individually were collected 2 and 3 days after intracytoplasmic sperm injection (ICSI). IL-1β concentrations of samples were determined with ELISA and compared with embryo grades and blastomere numbers. Embryo grades and the amount of IL-1β they secreted were found not to be correlated (p:0.559). Numbers of blastomeres each embryo had at 2nd and 3rd days were found to be correlated with IL-1β secreted (p:0.00 and p:0.00, respectively). Mean amount of IL-1β secreted by the embryos from ejaculated sperm cycles were found to be significantly higher than that of embryos from TESE cycles (p:0.016). Patient age and etiology of infertility were not correlated with the amount of IL-1β secreted and embryo grade. In conclusion, preimplantation human embryos secrete IL-1β in their media in amounts correlated with their blastomere numbers. [ABSTRACT FROM AUTHOR]

Introduction: Benchtop incubators with small individual chambers have been developed in order to improve the stability of embryo culture conditions reducing the environmental stress during the embryo development. These new dry incubators were designed without any air humidification system in order to prevent bacterial proliferation and to enable the use of time-lapse system. However, an elevated evaporation of the culture media could occur in these conditions. The main objective of the study is to analyse the impact of the used incubator type on the embryo culture media osmolality., Materials and Methods: Microdrops of 50μL of culture media were placed in 60mm diameter culture dishes, and quickly covered with either 7 or 8mL of mineral oil in an IVF workstation with laminar airflow. Two series of culture dishes have been randomly placed either in a humidified incubator or in a dry benchtop incubator. The microdrops of each culture dishes were sampled at D0, D1, D2, D3, and D5 respectively to measure the osmolality in triplicate using a cryoscopic osmometer. The mean values of osmolality in each incubator have been compared respectively on D0, D1, D2, D3 and D5 with appropriate statistical tests, and considered statistically significant when P<0.05., Results: The osmolality of the microdrops placed in the dry benchtop incubator differed significantly after the third day of culture, regardless of the level of mineral oil in the culture dishes. Indeed, using Petri dishes covered respectively with 7 or 8mL of mineral oil, osmolality values of samples from the dry incubator were significantly higher than those from the humidified one, at D3 and D5 (D3/7mL: 273±2.1 vs. 268±1.0mOsm/kg; P=0.02; D3/8mL: 282±8.0 vs. 270±0.7mOsm/kg; P=0.04) and D5 (D5/7mL: 283±1.5 vs. 270±3.6mOsm/kg; P=0.004; D5/8mL: 287±5.6 vs. 268±2.3mOsm/kg; P=0.005). Furthermore, the analysis on paired samples showed that the osmolality in the dry benchtop incubator at D5 using 7mL of oil (283±1.5mOsm/kg; P=0.003) and at D3 (273±2.1mOsm/kg; P=0.016) and D5 (287±5.6mOsm/kg; P=0.009) using 8mL of oil was significantly higher than that measured at D0 (265±1.9mOsm/kg)., Conclusion: A significant increase of the embryo culture media osmolality was observed in the dry benchtop incubator with ambient hygrometry in our standard conditions. Adding 1mL of oil was not sufficient to reduce the evaporation of the media. Although maintained at a physiological level, the impact of the osmolality changes on the in vitro embryo development has to be further determined., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Objective: To characterize nuclear and mitochondrial DNA (mtDNA) in spent culture media from normally developing blastocysts to determine whether it could be used for noninvasive genetic assessment.Design: Prospective embryo cohort study.Setting: Academic center and private in vitro fertilization (IVF) clinic.Patient(s): Seventy patients undergoing intracytoplasmic sperm injection (ICSI) and 227 blastocysts.Intervention(s): Culture media assessment, artificial blastocoele fluid collapse and DNA analysis using digital polymerase chain reaction (dPCR), long-range PCR, quantitative PCR (qPCR), and DNA fingerprinting.Main Outcome Measure(s): Presence of nuclear and mtDNA in three different commercial culture media from Vitrolife and Irvine Scientific, spent embryo media assessment at the cleavage and blastocyst stages of development, and analysis of the internal media controls for each patient that had been exposed to identical conditions as embryo media but did not come into contact with embryos.Result(s): Higher levels of nuclear and mtDNA were observed in the culture media that had been exposed to embryos compared with the internal media controls. Nuclear DNA (∼4 copies) and mtDNA (∼600 copies) could be detected in spent media, and the levels increased at the blastocyst stage. No increase in DNA was detected after artificial blastocoele fluid collapse. Mixed sex chromosome DNA was detected. This originated from contamination in the culture media and from maternal (cumulus) cells. Due to the limited amount of template, the presence of embryonic nuclear DNA could not be confirmed by DNA fingerprinting analysis.Conclusion(s): Currently DNA from culture media cannot be used for genetic assessment because embryo-associated structures release DNA into the culture medium and the DNA is of mixed origin. [ABSTRACT FROM AUTHOR]

There are numerous reports on embryo culture media and conditions in the laboratory, as the subject is multifaceted and complex, reflecting the variation in practice. In this scoping review, we attempt to approach the topic of culture media and conditions from the practitioners' perspective aiming to highlight, in a comprehensive fashion, important aspects regarding the options available, introduce points of debate and controversy, while maintaining the viewpoint of the practicing embryologist's concerns., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objective: The aim of this study was to compare embryo development cultured in two single-step media commercially available: Fert/Sage One Step® (Origio) and Continuous Single Culture® (CSC) (Irvine Scientific)., Methods: A prospective auto-controlled study of sibling oocytes from women undergoing conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) was performed in our center from February to June 2015. After fertilization, for every patient, half of oocytes were cultured in the single-step Fert/Sage One Step® (serie SAGE) and the other half in the single-step CSC®(serie CSC). Fertilization and embryo morphology rates were assessed by day 1 to day 5-6 if needed. Embryo presenting<20% of fragmentation and 4 cells at day 2, 8 cells at day 3 were qualified as "top quality". Embryo with<20% of fragmentation and 3-5 cells at day 2, 6-10 cells at day 3 were qualified as "good quality". Blastocyst B3, B4, B5 with A or B inner cell mass and A or B trophectoderm were qualified as "good quality". Transferred or frozen embryos were qualified as useful embryos., Results: Sixty-two attempts of IVF and 133 of ICSI were analyzed, corresponding to 2059 inseminated or micro-injected oocytes. Fertilization rate were not different between the 2 series, respectively SAGE vs CSC (IVF: 73.4% vs 68.3% [P=0.49]; ICSI: 58.9% vs 63.8% [P=0.12]). No difference was found for embryo morphology, respectively SAGE vs CSC, at day 2 (top quality embryo at day 2 IVF: 34.4% vs 33% [P=0.98]; ICSI: 42.4% vs 44.9% [P=0.37]; and good quality embryo at day 2 IVF: 44% vs 50.2% [P=0.07]; ICSI: 64% vs 71% [P=0.35]); no difference at day 3 (top quality embryo at day 3 IVF: 19.4% vs 21.3% [P=0.61]; ICSI: 28.7% vs 27.4% [P=0.54]; and good quality embryo at day 3 IVF: 40.4% vs 50.2% [P=0.91]; ICSI: 51% vs 47.6% [P=0.47]). Blastocyst development rate were not different, respectively SAGE vs CSC (IVF: 39.9% vs 41.5% [P=0.63] with 42.9% vs 42.2% of good quality blastocyst [P=0.70]; ICSI: 41.1% vs 37.8% [P=0.18] with 32.9% vs 40.8% of good quality blastocyst [P=0.13]). No difference was found in the useful embryo rate in the 2 series SAGE vs CSC (IVF: 52.8% vs 55.2% [P=0.83]; ICSI: 62.4% vs 61.7% [P=0.70])., Conclusion: Embryo development and rate of useful embryos, transferred or frozen, were not different according to the embryo culture in single-step media Fert/Sage One Step® vs single-step Continuous Single Culture®., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)