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STUDY QUESTION Are the extracellular vesicles (EVs) secreted by the maternal endometrium uptaken by human embryos and is their miRNA cargo involved in implantation and embryo development? SUMMARY ANSWER Data suggest that EVs secreted by human endometrial epithelial cells are internalized by human blastocysts, and transport miRNAs to modulate biological processes related to implantation events and early embryo development. WHAT IS KNOWN ALREADY Successful implantation is dependent on coordination between maternal endometrium and embryo, and EVs role in the required cell-to-cell crosstalk has recently been established. In this regard, our group previously showed that protein cargo of EVs secreted by primary human endometrial epithelial cells (pHEECs) is implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development. However, little is known about the regulation of these biological processes through EVs secreted by the endometrium at a transcriptomic level. STUDY DESIGN, SIZE, DURATION A prospective descriptive study was performed. Endometrial biopsies were collected from healthy oocyte donors with confirmed fertility on the day of oocyte retrieval, 36 h after the LH surge. pHEECs were isolated from endometrial biopsies (n   =   8 in each pool) and cultured in vitro. Subsequently, conditioned medium was collected and EVs were isolated and characterized. Uptake of EVs by human blastocysts and miRNA cargo of these EVs (n   =   3 pools) was analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS EVs were isolated from the conditioned culture media using ultracentrifugation, and characterization was performed using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. EVs were fluorescently labeled with Bodipy-TR ceramide, and their uptake by human blastocysts was analyzed using confocal microscopy. Analysis of the miRNA cargo of EVs was performed using miRNA sequencing, target genes of the most expressed miRNA were annotated, and functional enrichment analysis was performed. MAIN RESULTS AND THE ROLE OF CHANCE EVs measured 100–300 nm in diameter, a concentration of 1.78 × 1011 ± 4.12 × 1010 (SD) particles/ml and expressed intraluminal protein markers Heat shock protein 70 (HSP70) and Tumor Susceptibility Gene 101 (TSG101), in addition to CD9 and CD81 transmembrane proteins. Human blastocysts efficiently internalized fluorescent EVs within 1–2 h, and more pronounced internalization was observed in the hatched pole of the embryos. miRNA-seq analysis featured 149 annotated miRNAs, of which 37 were deemed most relevant. The latter had 6592 reported gene targets, that in turn, have functional implications in several processes related to embryo development, oxygen metabolism, cell cycle, cell differentiation, apoptosis, metabolism, cellular organization, and gene expression. Among the relevant miRNAs contained in these EVs, we highlight hsa-miR-92a-3p, hsa-let-7b-5p, hsa-miR-30a-5p, hsa-miR-24-3p, hsa-miR-21-5p, and hsa-let-7a-5p as master regulators of the biological processes. LIMITATIONS, REASONS FOR CAUTION This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment. WIDER IMPLICATIONS OF THE FINDINGS This study defines potential biomarkers of endometrial receptivity and embryo competence that could be useful diagnostic and therapeutic targets for implantation success, as well as open insight further investigations to elucidate the molecular mechanisms implicated in a successful implantation. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Spanish Ministry of Education through FPU awarded to M.S.-B. (FPU18/03735), the Health Institute Carlos III awarded to E.J.-B. (FI19/00110) and awarded to H.F. by the Miguel Servet Program 'Fondo Social Europeo «El FSE invierte en tu futuro»' (CP20/00120), and Generalitat Valenciana through VALi+d Programme awarded to M.C.C.-G. (ACIF/2019/139). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Study Question: What is the role of transcriptional-enhanced associate (TEA) domain family member 4 (TEAD4) in trophectoderm (TE) differentiation during human embryo preimplantation development in comparison to mouse?Summary Answer: TEAD4 regulates TE lineage differentiation in the human preimplantation embryo acting upstream of caudal-type homeobox protein 2 (CDX2), but in contrast to the mouse in a GATA-binding protein 3 (GATA3)-independent manner.What Is Known Already: Tead4 is one of the earliest transcription factors expressed during mouse embryo preimplantation development and is required for the expression of TE-associated genes. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination, have shown that Tead4-targeted embryos have compromised development and expression of the TE-specific Cdx2 and Gata3 is downregulated. Cdx2 and Gata3 act in parallel pathways downstream of Tead4 to induce successful TE differentiation. Downstream loss of Cdx2 expression, compromises TE differentiation and subsequent blastocoel formation and leads to the ectopic expression of inner cell mass (ICM) genes, including POU Class 5 homeobox 1 (Pou5f1) and SRY-box transcription factor (Sox2). Cdx2 is a more potent regulator of TE fate in mouse as loss of Cdx2 expression induces more severe phenotypes compared with loss of Gata3 expression. The role of TEAD4 and its downstream effectors during human preimplantation embryo development has not been investigated yet.Study Design, Size, Duration: The clustered regularly interspaced short palindromic repeats-clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (CRISPR-Cas9) system was first introduced in pronuclei (PN)-stage mouse zygotes aiming to identify a guide RNA (gRNA), yielding high editing efficiency and effective disruption of the Tead4 locus. Three guides were tested (gRNA1-3), each time targeting a distinct region of Exon 2 of Tead4. The effects of targeting on developmental capacity were studied in Tead4-targeted embryos (n = 164-summarized data from gRNA1-3) and were compared with two control groups; sham-injected embryos (n = 26) and non-injected media-control embryos (n = 51). The editing efficiency was determined by next-generation sequencing (NGS). In total, n = 55 (summarized data from gRNA1-3) targeted mouse embryos were analysed by NGS. Immunofluorescence analysis to confirm successful targeting by gRNA1 was performed in Tead4-targeted embryos, and non-injected media-control embryos. The downregulation of secondary TE-associated markers Cdx2 and Gata3 was used as an indirect confirmation of successful Tead4-targeting (previously shown to be expressed downstream of Tead4). Additional groups of gRNA1 Tead4-targeted (n = 45) and media control (n = 36) embryos were cultured for an extended period of 8.5 days, to further assess the developmental capacity of the Tead4-targeted group to develop beyond implantation stages. Following the mouse investigation, human metaphase-II (MII) oocytes obtained by IVM were microinjected with gRNA-Cas9 during ICSI (n = 74) to target TEAD4 or used as media-control (n = 33). The editing efficiency was successfully assessed in n = 25 TEAD4-targeted human embryos. Finally, immunofluorescence analysis for TEAD4, CDX2, GATA3 and the ICM marker SOX2 was performed in TEAD4-targeted (n = 10) and non-injected media-control embryos (n = 29).Participants/materials, Setting, Methods: A ribonucleoprotein complex consisting of a gRNA-Cas9 mixture, designed to target Exon 2 of Tead4/TEAD4, was microinjected in mouse PN stage zygotes or human IVM MII oocytes along with sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. In mouse, an additional group of Tead4-targeted and media-control embryos was cultured in vitro for an extended period of 8.5 days. Embryonic development and morphology were assessed daily, during culture in vitro of mouse and human embryos and was followed by a detailed scoring at late blastocyst stage. Targeting efficiency following gRNA-Cas9 introduction was assessed via immunostaining and NGS analysis.Main Results and the Role Of Chance: NGS analysis of the Tead4-targeted locus revealed very high editing efficiencies for all three guides, with 100% of the mouse embryos (55 out of 55) carrying genetic modifications resulting from CRISPR-Cas9 genome editing. More specifically, 65.22% (15 out 23) of the PN zygotes microinjected with gRNA1-Cas9, which exhibited the highest efficiency, carried exclusively mutated alleles. The developmental capacity of targeted embryos was significantly reduced (data from gRNA1), as 44.17% of the embryos arrested at the morula stage (2.5 days post coitum), coincident with the initiation of TE lineage differentiation, compared with 8.51% in control and 12.50% in sham control groups. High-quality blastocyst formation rates (Grade 3) were 8.97% in the gRNA1-targeted group, compared with 87.23% in the media-control and 87.50% in the sham group. Immunofluorescence analysis in targeted embryos confirmed downregulation of Tead4, Cdx2, and Gata3 expression, which resulted from successful targeting of the Tead4 locus. Tead4-targeted mouse embryos stained positive for the ICM markers Pou5f1 and Sox2, indicating that expression of ICM lineage markers is not affected. Tead4-targeted embryos were able to cavitate and form a blastocoel without being able to hatch. Extended embryo culture following zona pellucida removal, revealed that the targeted embryos can attach and form egg-cylinder-like structures in the absence of trophoblast giant cells. In human embryos, Exon 2 of TEAD4 was successfully targeted by CRISPR-Cas9 (n = 74). In total, 25 embryos from various developmental stages were analysed by NGS and 96.00% (24 out of 25) of the embryos carried genetic modifications because of gRNA-Cas9 editing. In the subgroup of the 24 edited embryos, 17 (70.83%) carried only mutant alleles and 11 out of these 17 (64.70%) carried exclusively frameshift mutations. Six out of 11 embryos reached the blastocyst stage. In contrast to mice, human-targeted embryos formed blastocysts at a rate (25.00%) that did not differ significantly from the control group (23.81%). However, blastocyst morphology and TE quality were significantly compromised following TEAD4-targeting, showing grade C TE scores, with TE containing very few cells. Immunofluorescence analysis of TEAD4-targeted embryos (n = 10) confirmed successful editing by the complete absence of TEAD4 and its downstream TE marker CDX2, but the embryos generated retained expression of GATA3, which is in contrast to what we have observed and has previously been reported in mouse. In this regard, our results indicate that GATA3 acts in parallel with TEAD4/CDX2 towards TE differentiation in human.Large Scale Data: N/A.Limitations, Reasons For Caution: CRISPR-Cas9 germline genome editing, in some cases, induces mosaic genotypes. These genotypes are a result of inefficient and delayed editing, and complicate the phenotypic analysis and developmental assessment of the injected embryos. We cannot exclude the possibility that the observed differences between mouse and human are the result of variable effects triggered by the culture conditions, which were however similar for both mouse and human embryos in this study. Furthermore, this study utilized human oocytes obtained by IVM, which may not fully recapitulate the developmental behaviour of in vivo matured oocytes.Wider Implications Of the Findings: Elucidation of the evolutionary conservation of molecular mechanisms that regulate the differentiation and formation of the trophoblast lineage can give us fundamental insights into early implantation failure, which accounts for ∼15% of human conceptions.Study Funding/competing Interest(s): The research was funded by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01) and Ghent University (BOF.BAS.2018.0018.01). G.C. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 11L8822N). A.B. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 1298722 N). We further thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Is the innate immunity system active in early human embryo development?Summary Answer: The pattern recognition receptors and innate immunity Toll-like receptor (TLR) genes are widely expressed in preimplantation human embryos and the pathway appears to be active in response to TLR ligands.What Is Known Already: Early human embryos are highly sensitive to their local environment, however relatively little is known about how embryos detect and respond to specific environmental cues. While the maternal immune response is known to be key to the establishment of pregnancy at implantation, the ability of human embryos to detect and signal the presence of pathogens is unknown.Study Design, Size, Duration: Expression of TLR family and related genes in human embryos was assessed by analysis of published transcriptome data (n = 40). Day 5 (D-5) human embryos (n = 25) were cultured in the presence of known TLR ligands and gene expression and cytokine production measured compared to controls.Participants/materials, Setting, Methods: Human embryos surplus to treatment requirements were donated with informed consent from several ART centres. Embryos were cultured to Day 6 (D-6) in the presence of the TLR3 and TLR5 ligands Poly (I: C) and flagellin, with gene expression measured by quantitative PCR and cytokine release into medium measured using cytometric bead arrays.Main Results and the Role Of Chance: TLR and related genes, including downstream signalling molecules, were expressed variably at all human embryo developmental stages. Results showed the strongest expression in the blastocyst for TLRs 9 and 5, and throughout development for TLRs 9, 5, 2, 6 and 7. Stimulation of Day 5 blastocysts with TLR3 and TLR5 ligands Poly (I: C) and flagellin produced changes in mRNA expression levels of TLR genes, including the hyaluronan-mediated motility receptor (HMMR), TLR5, TLR7, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and monocyte chemoattractant Protein-1 (MCP-1) (P < 0.05, P < 0.001 compared to unstimulated controls), and release into culture medium of cytokines and chemokines, notably IL8 (P = 0.00005 and 0.01277 for flagellin and Poly (I: C), respectively).Limitations, Reasons For Caution: This was a descriptive and experimental study which suggests that the TLR system is active in human embryos and capable of function, but does not confirm any particular role. Although we identified embryonic transcripts for a range of TLR genes, the expression patterns were not always consistent across published studies and expression levels of some genes were low, leaving open the possibility that these were expressed from the maternal rather than embryonic genome.Wider Implications Of the Findings: This is the first report of the expression and activity of a number of components of the innate immunity TLR system in human embryos. Understanding the role of TLRs during preimplantation human development may be important to reveal immunological mechanisms and potential clinical markers of embryo quality and pregnancy initiation during natural conception and in ART.Study Funding/competing Interest(s): This work was funded by the Ministry of Higher Education, The State of Libya, the UK Medical Research Council, and the NIHR Local Comprehensive Research Network and NIHR Manchester Clinical Research Facility and the European Union's Horizon 2020 Research and Innovation Programmes under the Marie Skłodowska-Curie Grant Agreement No. 812660 (DohART-NET). In accordance with H2020 rules, no new human embryos were sacrificed for research activities performed from the EU funding, which concerned only in silico analyses of recorded time-lapse and transcriptomics datasets. None of the authors has any conflict of interest to declare.Trial Registration Number: n/a. [ABSTRACT FROM AUTHOR]

Study Question: What is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model?Summary Answer: POU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation.What Is Known Already: Clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage.Study Design, Size, Duration: The efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n = 135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n = 23). Four control groups were included: non-injected media-control zygotes (n = 43)/oocytes (n = 48); sham-injected zygotes (n = 45)/oocytes (n = 47); Cas9-protein injected zygotes (n = 23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n = 27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n = 37), media control zygotes (n = 19), and sham-injected zygotes (n = 15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n = 29) and media control zygotes (n = 30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain-B6CBA (n = 52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n = 101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n = 33). Immunofluorescence analysis was performed in human CRISPR-injected (n = 10) and media control (n = 9) human embryos.Participants/materials, Setting, Methods: A gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4 days (mouse) or 6.5 days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing.Main Results and the Role Of Chance: Genomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n = 101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts.Limitations, Reasons For Caution: One of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes.Wider Implications Of the Findings: Comparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis.Study Funding/competing Interest(s): The research was funded by the Ghent University Hospital and Ghent University and supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01). The authors declare no competing interests.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Does a new system-the chip-sensing embryo respiration monitoring system (CERMs)-enable evaluation of embryo viability for potential application in a clinical IVF setting?Summary Answer: The system enabled the oxygen consumption rate of spheroids, bovine embryos and frozen-thawed human embryos to be measured, and this rate corresponded to the developmental potential of embryos.What Is Already Known: To date, no reliable and clinically suitable objective evaluation methods for embryos are available, which circumvent the differences in inter-observer subjective view. Existing systems such as the scanning electrochemical microscopy (SECM) technique, which enables the measurement of oxygen consumption rate in embryos, need improvement in usability before they can be applied to a clinical setting.Study Design, Size, Duration: This is a prospective original research study. The feasibility of measuring the oxygen consumption rate was assessed using CERMs for 9 spheroids, 9 bovine embryos and 30 redundant frozen-thawed human embryos. The endpoints for the study were whether CERMs could detect a dissolved oxygen gradient with high sensitivity, had comparable accuracy to the SECM measuring system with improved usability, and could predict the development of an embryo to a blastocyst by measuring the oxygen consumption rate. The relationship between the oxygen consumption rate and standard morphological evaluation was also examined.Participants/materials, Setting, Methods: We developed a new CERMs, which enables the oxygen consumption rate to be measured automatically using an electrochemical method. The device was initially used for measuring a dissolved oxygen concentration gradient in order to calculate oxygen consumption rate using nine spheroids. Next, we evaluated data correlation between the CERMs and the SECM measuring systems using nine bovine embryos. Finally, the oxygen consumption rates of 30 human embryos, which were frozen-thawed on 2nd day after fertilization, were measured by CERMs at 6, 24, 48, 72 and 96 h after thawing with standard morphological evaluation. Furthermore, the developed blastocysts were scored using the blastocyst quality score (BQS), and the correlation with oxygen consumption rate was also assessed.Main Results and the Role Of Chance: The device enabled the oxygen consumption rate of an embryo to be measured automatically within a minute. The oxygen concentration gradient profile showed excellent linearity in a distance-dependent change. A close correlation in the oxygen consumption rates of bovine embryos was observed between the SECM measuring system and CERMs, with a determination coefficient of 0.8203 (P = 0.0008). Oxygen consumption rates of human embryos that have reached the blastocyst stage were significantly higher than those of arrested embryos at 48, 72 and 96 h after thawing (P = 0.039, 0.004 and 0.049, respectively). Thus, in vitro development of frozen-thawed human embryos to the blastocyst stage would be predicted at 48 h after thawing (day 4) by measuring the oxygen consumption using CERMs. Although a positive linear relationship between BQS and the oxygen consumption rate was observed [the determination coefficient was R(2) = 0.6537 (P = 0.008)], two blastocysts exhibited low oxygen consumption rates considering their relatively high BQS. This suggests that morphology and metabolism in human embryos might not correlate consistently.Limitations, Reasons For Caution: Transfer of the embryo and pregnancy evaluation was not performed. Thus, a correlation between oxygen consumption and the in vivo viability of embryos remains unknown. Clinical trials, including embryo transfer, would be desirable to determine a threshold value to elect clinically relevant, quality embryos for transfer. We utilized frozen-thawed human embryos in this study. The effect of these manipulations on the respiratory activity of the embryo is also unknown.Wider Implications Of the Findings: Selection of quality embryos, especially in a single embryo transfer cycle, by CERMs may have an impact on obtaining better clinical outcomes, albeit with clinical trials being required. Furthermore, the early determination of quality embryos by CERMs may enable the omission of long-term in vitro embryo culture to the blastocyst stage. CERMs is scalable technology that can be integrated into incubators and/or other embryo evaluation systems, such as the time-lapse systems, due to its chip-based architecture. Thus, CERMS would enable automatic measurement of oxygen consumption, under 5% CO2, in the near future, in order to reduce oxidative stress from exposure to atmospheric air.Study Funding/competing Interests: This study was supported by grants from the Health and Labor Sciences Research Grant (H24-Hisaichiiki-Shitei-016). The authors have no conflicts of interest.Trial Registration Number: Not applicable. [ABSTRACT FROM AUTHOR]

Background: Little is known about the extent and timing of selection against the embryos that are carriers of unbalanced translocations.Methods: Fluorescence in-situ hybridization (FISH) with probes for chromosomes 13, 14 and 18 was performed, mostly on day 3, on 69 human embryos which were then allowed to develop further in culture to day 5, from five carriers of Robertsonian translocation (RT) t(13;14).Results: Twelve normal/balanced blastocysts were replaced in seven consecutive cycles (day 5). Three cycles resulted in clinical pregnancies. The proportion of blastocysts displaying a normal/balanced karyotype was 56%, while only the 20% of blocked embryos were normal/balanced (chi(2): P < 0.05). All the embryos analysed on day 5, except one, displayed mosaicism. The percentages of diploid cells for chromosomes 13 and 14 were significantly lower than for chromosome 18 (chromosome 13: 49.0 +/- 28.0; chromosome 14: 53.0 +/- 31.8; chromosome 18: 75.7 +/- 20.4; Mann-Whitney test: P < 0.01). The embryos displaying vertical line 62% of diploid cells for at least two of the three chromosomes analysed, more frequently reached the blastocyst stage (blocked embryos: blastocysts chromosome 13: 43.1 +/- 30.3, 64.9 +/- 29.0; chromosome 18: 64.9 +/- 29.0, 83.0 +/- 12.9; Mann-Whitney test: P < 0.01).Conclusions: Normal/balanced embryos developed better but the proportion of abnormal blastocysts was still high. Preimplantation genetic diagnosis is recommended to select normal/balanced embryos from RT t(13;14) carriers. [ABSTRACT FROM AUTHOR]

The inheritance, replication and perpetuation of the sperm centriole in the early human embryo are reported. Both normal monospermic and abnormal dispermic embryos (n = 127) were examined by transmission electron microscopy. Centrioles were traced from fertilization to the hatching blastocyst stage. The sperm proximal centriole is introduced into the oocyte at fertilization and remains attached to the expanding spermhead during sperm nuclear decondensation, as it forms the male pronucleus. A sperm aster is initially formed after the centriole duplicates at the pronuclear stage. At syngamy, centrioles occupy a pivotal position on opposite spindle poles, when the first mitotic figure is formed. Bipolar spindles were found in the majority of embryos, while tripolar spindles were seen in four dispermic embryos at syngamy. Two single centrioles were detected at two poles of two tripolar spindles, while two additional centrioles were located on the sides of a bipolar spindle of a dispermic embryo. Sperm tails were detected near spindle poles at syngamy and in later embryos. Typical centrioles showing the characteristic pin-wheel organization of nine triplets of microtubules were evident During centriolar replication, the daughter centriole grows laterally from the parent and gradually acquires pericentriolar material (PCM). The two centrioles are surrounded by a halo of electron-dense PCM, which nucleates microtubules, thus making it a typical centrosome. The usual alignment of diplosomes at right angles to each other was maintained. Centrioles were detected at all stages of embryonic cleavage from the 1-cell through 8-cell stages, right up to the hatching blastocyst stage. They were closely associated with nuclei at interphase, when they were often replicating, and were prominently located at spindle poles during the first four cell cycles. In blasto-cysts, they were detected in trophoblast, embryoblast and endoderm cells respectively. It is evident that the sperm centrosome is the functional active centrosome in the human, while the female is inactive but may contribute some centrosomal material to the zygote centrosome. It is very likely that the paternal centriole is the ancestor of the centrioles in fetal and adult somatic cells. [ABSTRACT FROM PUBLISHER]

Videocinematography and image analysis procedures were utilized to evaluate the effect of conventional and coculture methodologies on morphological parameters in human embryos derived from in-vitro fertilization (IVF). Following 24–30 h of in-vitro development, cocultured embryos had more acceptable morphological features and less fragmentation present than embryos cultured in medium alone. Cocultured embryos were more advanced at the time of replacement when compared with conventionally cultured embryos. Zona pellucida variation (≥20%) also occurred more frequently in cocultured embryos. The morphological characteristic most enhanced after coculture was blastomere expansion. Patients who became pregnant across both culture treatments had a higher proportion of morphologically normal embryos replaced than patients who failed to achieve an ongoing pregnancy. Clinical pregnancy rate for patients following coculture was 49%, which was greater ( < 0.05) than the 29% detected for patients with embryos in the conventional culture group. [ABSTRACT FROM PUBLISHER]

The presence of cell adhesion molecules on human oocytes, early embryos, and pre-hatched blastocysts was examined by indirect immunofluorescence and compared to the distribution found on first trimester villous placenta with the same antibodies. Six integrin subunits (α3, αV, β1, β3, β4, β5) were observed consistently throughout preimplantation development. Evidence was also obtained for the presence of integrin subunits α2, α4, αL, β2, and β7 on a small number of oocytes. A more restricted developmental analysis of E-cadherin, ICAM-1, NCAM, and VCAM-1 demonstrated that these cell adhesion molecules are also present on oocytes and early embryos. L-selectin was detected on oocytes but was not found on 8-cell embryos. The oocyte and early blastomeres have complex surfaces in which the integrin and CAM families are represented. [ABSTRACT FROM PUBLISHER]

A total of 304 human pronuclear zygotes and cleaved embryos from the 2- to 9-cell stages, obtained during invitro fertilization attempts, were photographed and retrospectively analysed after transfer for their morphology and size in relation to their developmental stage, using the Imagenia programme of a Biocom 500 image analyser. Morphometric parameters were calculated from the perimeters, surface measurements, theoretical diameters and circularity factors for the different structures analysed. This report provides the morphometric characteristics of living embryos. For the whole population the mean values were: 157.4 μm for the external zona pellucida diameter, 121.8 μm for the internal zona pellucida diameter, 17.9 μm for the thickness of the zona pellucida and 117.2 μm for the embryo cell mass diameter. The morphometric characteristics of the pronuclear-stage population were significantly different from the cleaved cell stages. If the zona pellucida and cell mass embryo diameters increased slowly from the 2- to 9-cell stages, embryonic external diameters were higher and zona pellucida thicknesses were lower in odd than even number blastomere embryos. Preliminary results show that in cases where implantation occurs, the embryo has a lower zona pellucida thickness. A comparison of the different embryo cell stages confirmed the existence of an asynchronous division process during early embryo development. Global results show no evidence of morphometric differences between subpopulations of the embryos according to their microscopic grading. Deviations from the normal asynchronous division process, however, appear to be a new parameter to take into account during embryo scoring. Image analysis is not invasive and could be used to predict the criteria needed for more efficient embryo assessment before transfer. [ABSTRACT FROM PUBLISHER]

The uptake of pyruvate by human embryos derived from natural cycles in the first 24 h following fertilization was examined. Since only one egg was obtained and therefore only one embryo transferred to the woman, it was possible to relate pyruvate consumption by a particular embryo to the outcome of that cycle (pregnancy or no pregnancy). The results showed that embryos have a wide range of pyruvate uptake values (2–53 pmol/embryo/h) but that this variation was reduced significantly to an intermediate range of values in those embryos that were able to implant (10–30 pmol/embryo/h). An association was found between embryo morphology and pyruvate consumption. Morphologically good embryos were more likely to implant if they demonstrated an intermediate pyruvate uptake. However, poor embryos did not implant even if they had a pyruvate uptake of 10–30 pmol/embryo/h. No relationship was found between the type of infertility and pyruvate consumption of individual embryos. It is suggested that the ability of an embryo to implant is multifactorial and that both morphology and pyruvate uptake may be factors. [ABSTRACT FROM PUBLISHER]

We have developed a new procedure, called cell recycling, which combines the two powerful techniques of polymerase chain reaction (PCR) and fluorescent in-situ hybridization (FISH) on the same single cell. A fixed cell is used as the DNA template for PCR, prior to the FISH analysis. Using single blastomeres from mouse embryos as a model system, cell recycling procedures detect the single-copy β-haemoglobin gene sequence at an efficiency of 70% as well as sex chromosome constitution at an efficiency of 74% in the same single cell. Cell recycling will increase the success rate of pregnancy following preimplantation diagnosis for a specific gene defect by identifying embryos with chromosomal abnormalities and eliminating them from the transfer procedure. [ABSTRACT FROM PUBLISHER]

A short fluorescence in-situ hybridization (FISH) procedure using fluorochrome and digoxigenin labelled DNA probes was developed for application in human preimplantation embryos in order to analyse the five chromosomes most involved in human aneuploidy (X, Y, 18, 13 and 21). The chromosomes were fluorescent-stained and detected simultaneously in 157 blastomeres from 30 human embryos. Successful FISH analysis was achieved in 93% of the blastomeres. Aberrations for these chromosomes were found in 70% of abnormally developing monospermic embryos. The majority of normally developing monospermic embryos obtained from older patients were also chromosomally abnormal. By analysing all or most of the cells from these embryos, true mosaicism was distinguished from technique failure. Mosaic embryos, polyploid embryos with ploidies as high as 8n, haploid embryos, embryos monosomic for 13/21 and for X, and embryos trisomic for 13/21 and 18, were common in abnormally developing embryos. In contrast, aneuploidy was the main chromosome abnormality found in normally developing monospermic embryos. [ABSTRACT FROM PUBLISHER]

Fluorescent detection of in-situ hybridization (FISH) with a chromosome 18-specific probe (P5041 B.5 D18Z1) has been used to assess the use of this method for preimplantation diagnosis of aneuploidy. Interphase nuclei (n = 802) have been analysed from 59 normally fertilized embryos developing in vitro at the normal rate between days 2 and 7 postinsemination. The efficiency of hybridization in control cells, as assessed by the proportion with two signals in normal female lymphocytes was 88.9% (n = 353) and with three signals in a trisomic (48,XXX+18) fibroblast cell line 74.0% (n = 290). Fifty-four of the human embryos were considered to be diploid on the basis that the majority of nuclei had two signals. Some nuclei in these embryos had one or no signal, especially on day 2, and tetraploid nuclei were also widespread. Among the remaining five embryos, one 5-cell embryo on day 2 had three hybridization signals in 4/5 nuclei and was trisomic for chromosome 18, one 4-cell embryo on day 2 had only one signal in 4/4 nuclei and was monosomic, and the three other embryos were aneuploid mosaics and/or had multi-nucleated blastomeres. Analysis of the incidence of interphase nuclei with more or less than the diploid number of hybridization signals indicates that more than a single nucleus will be necessary for accurate preimplantation diagnosis of aneuploidy. [ABSTRACT FROM AUTHOR]

Lidocaine is a well-documented local anaesthetic often used for paracervical block (PCB) in pregnant women. For this reason, the substance was used early on for PCB in connection with transvaginal follicle aspiration. However, the concentration reached in follicular fluid after PCB or local infiltration of the vaginal wall has not been previously determined. Furthermore, if lidocaine reaches the follicle, it seems important to determine if the concentration reached influences fertilization and early cleavage of the embryo and the pregnancy rate. The study included 46 women who had PCB with lidocaine (50 mg) and 46 women who had no PCB in connection with follicle aspiration. The mean concentration in follicular fluid was 0.36 ± 1.1 μg/ml. There was no significant difference in lidocaine concentration between follicles containing oocytes that were fertilized and those that were not. Furthermore, the fertilization and cleavage rates did not differ significantly in women with and without PCB with lidocaine. The pregnancy rate did not differ between the two groups. It thus seems that the concentration of lidocaine found in the follicular fluid after PCB with 50 mg lidocaine does not negatively affect fertilization of the human oocyte or early cleavage of the human embryo. [ABSTRACT FROM PUBLISHER]

Morphometrical procedures were used to quantitatively evaluate human oocytes and embryos in an IVF programme. The metaphase II oocyte was an irregular 3.5 × 10 μm sphere of 1.05 coefficient of form. The ooplasmic volume of 1.4 × 10 μm was reduced by 10% by fertilization. The zona pellucida behaved as a stable and almost spherical envelope of 1.8 × 10 /m volume and 17 μm thickness. Through the first three cleavages, mean blastomere reduces 28.5% volume per division, evolving from an irregular spherical shape with 0.9 coefficient of form to an ellipsoid (0.8) at the 8-cell stage. The coefficient of diversity between sister blastomeres progressively moved from 1.4 to 1.6 during the first two (2) cleavages. The coefficient of diversity also increased at 3-cell (2.2) and 6-cell (2.6) asynchronous divisions. Morphologically abnormal embryos showed some morphometrical differences. Embryos which successfully implanted and progressed to birth showed a higher coefficient of diversity between sister blastomeres. [ABSTRACT FROM PUBLISHER]

The infrastructure of a 4-cell human embryo undergoing development is described. The embryo recovered from the Fallopian tube 130 h after a single episode of intercourse and 70 h after the luteinizing hormone peak in plasma, was partially surrounded by cumulus cells. The blastomeres, of even size, were nucleated and had a similar distribution of cytoplasmic organelles. No signs of abnormality or cellular degeneration were observed. Transmission electron microscopy of serial sections revealed the presence of primitive desmosomes between cells, abundant vesicles within the blebs of the outer sheet of the nuclear membrane, blebbing of the inner sheet of the nuclear membrane and close association between the inner sheet of the nuclear membrane and the intranuclear annulate lamellae. Nucleolar precursors lacking the structural organization of mature nucleoli were also found. Similarities and differences between this and other pretmplantation human embryos reported earlier are analysed. [ABSTRACT FROM PUBLISHER]

The biochemical basis and mechanism of embryo implementation is poorly understood. The human embryo has to penetrate the endometrial basement membrane and the thick decidual wall during implantation, a process which resembles the active spreading of invasive tumour cells where the degradation of type IV collagen (basement membrane collagen) plays an important role. This study reports that human embryos produce type IV collagen-degrading enzyme activity and the secretion of this enzyme increases with time in culture. The type IV collagen-degrading enzyme activity might facilitate the penetration of the embryo through the decidua, thus emphasizing an active role of the embryo in implantation. On the other hand, unfertilized oocytes secrete low, stable amounts of type IV collagen-degrading enzyme activity in vitro. It was also found that follicular granulosa cells secrete high amounts of type IV collagenolytic activity in culture. It has been previously shown that there is a pre-ovulatory peak in type IV collagenolytic activity in follicular fluid, and it can be assumed that the appearance of this enzyme in the follicular fluid is probably connected to follicular rupture and that it is produced by granulosa cells [ABSTRACT FROM PUBLISHER]