Extended in vitro culture of human embryos demonstrates the complex nature of diagnosing chromosomal mosaicism from a single trophectoderm biopsy.

STUDY QUESTION: What is the accuracy of preimplantation genetic testing for aneuploidies (PGT-A) when considering human peri-implantation outcomes in vitro? STUDY ANSWER: The probability of accurately diagnosing an embryo as abnormal was 100%, while the proportion of euploid embryos classified as clinically suitable was 61.9%, yet if structural and mosaic abnormalities were not considered accuracy increased to 100%, with a 0% false positive and false negative rate. WHAT IS ALREADY KNOWN: Embryo aneuploidy is associated with implantation failure and early pregnancy loss. However, a proportion of blastocysts are mosaic, containing chromosomally distinct cell populations. Diagnosing chromosomal mosaicism remains a significant challenge for PGT-A. Although mosaic embryos may lead to healthy live births, they are also associated with poorer clinical outcomes. Moreover, the direct effects of mosaicism on early pregnancy remain unknown. Recently, developed in vitro systems allow extended embryo culture for up to 14 days providing a unique opportunity for modelling chromosomal instability during human peri-implantation development. STUDY DESIGN, SIZE, DURATION: A total of 80 embryos were cultured to either 8 (n = 7) or 12 days post-fertilisation (dpf; n = 73). Of these, 54 were PGT-A blastocysts, donated to research following an abnormal (n = 37) or mosaic (n = 17) diagnosis. The remaining 26 were supernumerary blastocysts, obtained from standard assisted reproductive technology (ART) cycles. These embryos underwent trophectoderm (TE) biopsy prior to extended culture. PARTICIPANTS/MATERIALS, SETTING, METHODS: We applied established culture protocols to generate embryo outgrowths. Outgrowth viability was assessed based on careful morphological evaluation. Nine outgrowths were further separated into two or more portions corresponding to inner cell mass (ICM) and TE-derived lineages. A total of 45 embryos were selected for next generation sequencing (NGS) at 8 or 12 dpf. We correlated TE biopsy profiles to both culture outcomes and the chromosomal status of the embryos during later development. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 73 embryos cultured to 12 dpf, 51% remained viable, while 49% detached between 8 and 12 dpf. Viable, Day 12 outgrowths were predominately generated from euploid blastocysts and those diagnosed with trisomies, duplications or mosaic aberrations. Conversely, monosomies, deletions and more complex chromosomal constitutions significantly impaired in vitro development to 12 dpf (10% vs. 77%, P < 0.0001). When compared to the original biopsy, we determined 100% concordance for uniform numerical aneuploidies, both in whole outgrowths and in the ICM and TE-derived outgrowth portions. However, uniform structural variants were not always confirmed later in development. Moreover, a high proportion of embryos originally diagnosed as mosaic remained viable at 12 dpf (58%). Of these, 71% were euploid, with normal profiles observed in both ICM and TE-derived lineages. Based on our validation data, we determine a 0% false negative and 18.5% false positive error rate when diagnosing mosaicism. Overall, our findings demonstrate a diagnostic accuracy of 80% in the context of PGT-A. Nevertheless, if structural and mosaic abnormalities are not considered, accuracy increases to 100%, with a 0% false positive and false negative rate. LIMITATIONS REASONS FOR CAUTION: The inherent limitations of extended in vitro culture, particularly when modelling critical developmental milestones, warrant careful interpretation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings echo current prenatal testing data and support the high clinical predictive value of PGT-A for diagnosing uniform numerical aneuploidies, as well as euploid chromosomal constitutions. However, distinguishing technical bias from biological variability will remain a challenge, inherently limiting the accuracy of a single TE biopsy for diagnosing mosaicism. STUDY FUNDING, COMPETING INTEREST(S): This research is funded by the Ghent University Special Research Fund (BOF01D08114) awarded to M.P., the Research Foundation-Flanders (FWO.KAN.0005.01) research grant awarded to B.H. and De Snoo-van't Hoogerhuijs Stichting awarded to S.M.C.d.S.L. We thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Chromosomal mosaicism in human blastocysts: the ultimate challenge of preimplantation genetic testing?

STUDY QUESTION: To what extent does a trophectoderm (TE) biopsy reliably reflect the chromosomal constitution of the inner cell mass (ICM) in human blastocysts? SUMMARY ANSWER: Concordance between TE and ICM was established in 62.1% of the embryos analysed. WHAT IS KNOWN ALREADY: Next generation sequencing (NGS) platforms have recently been optimised for preimplantation genetic testing for aneuploidies (PGT-A). However, higher sensitivity has led to an increase in reports of chromosomal mosaicism within a single TE biopsy. This has raised substantial controversy surrounding the prevalence of mosaicism in human blastocysts and the clinical implications of heterogeneity between the TE and ICM. STUDY DESIGN, SIZE, DURATION: To define the distribution and rate of mosaicism in human blastocysts, we assessed chromosomal profiles of the ICM and multiple TE portions obtained from the same embryo. We evaluated donated embryos with an unknown chromosomal profile (n = 34), as well as PGT-A blastocysts, previously diagnosed as abnormal or mosaic (n = 24). Our intra-embryo comparison included a total of 232 samples, obtained from 58 embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS: Four embryo samples, including the ICM and three distinct TE portions, were acquired from good quality blastocysts by micromanipulation. Whole genome amplification (WGA), followed by NGS was performed on all embryo segments. Profiles were compared between samples from the same embryo, while the results from pretested blastocysts were further correlated to the original report. The embryos investigated in our untested group were obtained from good prognosis patients (n = 25), with maternal age ranging from 23 to 39 years. For the pretested embryo group, maternal age ranged from 23 to 40 years (n = 18). MAIN RESULTS AND THE ROLE OF CHANCE: We uncover chromosomal mosaicism, involving both numerical and structural aberrations, in up to 37.9% of the blastocysts analysed. Within the untested group, the overall concordance between the ICM and all TE portions was 55.9%. A normal ICM was detected in 20.6% of blastocysts for which at least one TE portion showed a chromosomal aberration. Conversely, 17.6% of embryos presented with mosaic or uniform abnormalities within the ICM, while showing normal or mosaic TE profiles. For the pretested blastocysts, the overall concordance between the ICM and all TE samples was 70.8%. However, 50% of embryos previously diagnosed with mosaicism did not confirm the original diagnosis. Notably, 31.3% of embryos with a mosaic aberration reported in the original TE biopsy, revealed a euploid profile in the ICM and all three TE samples. Taken together, concordance between the ICM and all TE portions was established in 62.1% of blastocysts, across both embryo groups. Finally, we could not observe a significant effect of age on embryo mosaicism (P = 0.101 untested group; P = 0.7309 pretested group). Similarly, ICM and TE quality were not found to affect the occurrence of chromosomal mosaicism (P = 0.718 and P = 0.462 untested group; P = 1.000 and P = 0.2885 pretested group). LARGE SCALE DATA: All data that support the findings of this study are available online in Vivar (http://cmgg.be/vivar) upon request. LIMITATIONS, REASONS FOR CAUTION: Evaluating biological variation in some instances remains challenging. The technological limitations of sampling mitotic errors that lead to mosaicism, as well as WGA artefacts, warrant careful interpretation. WIDER IMPLICATIONS OF THE FINDINGS: Our results highlight the complex nature of genetic (in)stability during early ontogenesis and indicate that blastocysts harbour a higher rate of chromosomal mosaicism than may have been anticipated. Moreover, our findings reveal an overall high diagnostic sensitivity and relatively low specificity in the context of PGT-A. This suggests that a considerable proportion of embryos are potentially being classified as clinically unsuitable. Ultimately, more precise quantification will benefit the clinical management of embryo mosaicism. STUDY FUNDING/COMPETING INTEREST(S): M.P. is supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF01D08114). J.T. and L.D. are supported by the agency for innovation through science (131673, 141441). B.H. and this research are supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF15/GOA/011). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Human oocyte calcium analysis predicts the response to assisted oocyte activation in patients experiencing fertilization failure after ICSI.

STUDY QUESTION: Can human oocyte calcium analysis predict fertilization success after assisted oocyte activation (AOA) in patients experiencing fertilization failure after ICSI? SUMMARY ANSWER: ICSI-AOA restores the fertilization rate only in patients displaying abnormal Ca2+ oscillations during human oocyte activation. WHAT IS KNOWN ALREADY: Patients capable of activating mouse oocytes and who showed abnormal Ca2+ profiles after mouse oocyte Ca2+ analysis (M-OCA), have variable responses to ICSI-AOA. It remains unsettled whether human oocyte Ca2+ analysis (H-OCA) would yield an improved accuracy to predict fertilization success after ICSI-AOA. STUDY DESIGN, SIZE, DURATION: Sperm activation potential was first evaluated by MOAT. Subsequently, Ca2+ oscillatory patterns were determined with sperm from patients showing moderate to normal activation potential based on the capacity of human sperm to generate Ca2+ responses upon microinjection in mouse and human oocytes. Altogether, this study includes a total of 255 mouse and 122 human oocytes. M-OCA was performed with 16 different sperm samples before undergoing ICSI-AOA treatment. H-OCA was performed for 11 patients who finally underwent ICSI-AOA treatment. The diagnostic accuracy to predict fertilization success was calculated based on the response to ICSI-AOA. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients experiencing low or total failed fertilization after conventional ICSI were included in the study. All participants showed moderate to high rates of activation after MOAT. Metaphase II (MII) oocytes from B6D2F1 mice were used for M-OCA. Control fertile sperm samples were used to obtain a reference Ca2+ oscillation profile elicited in human oocytes. Donated human oocytes, non-suitable for IVF treatments, were collected and vitrified at MII stage for further analysis by H-OCA. MAIN RESULTS AND THE ROLE OF CHANCE: M-OCA and H-OCA predicted the response to ICSI-AOA in 8 out of 11 (73%) patients. Compared to M-OCA, H-OCA detected the presence of sperm activation deficiencies with greater sensitivity (75 vs 100%, respectively). ICSI-AOA never showed benefit to overcome fertilization failure in patients showing normal capacity to generate Ca2+ oscillations in H-OCA and was likely to be beneficial in cases displaying abnormal H-OCA Ca2+ oscillations patterns. LIMITATIONS, REASONS FOR CAUTION: The scarce availability of human oocytes donated for research purposes is a limiting factor to perform H-OCA. Ca2+ imaging requires specific equipment to monitor fluorescence changes over time. WIDER IMPLICATIONS OF THE FINDINGS: H-OCA is a sensitive test to diagnose gamete-linked fertilization failure. H-OCA allows treatment counseling for couples experiencing ICSI failures to either undergo ICSI-AOA or to participate in gamete donation programs. The present data provide an important template of the Ca2+ signature observed during human fertilization in cases with normal, low and failed fertilization after conventional ICSI. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Flemish fund for scientific research (FWO-Vlaanderen, G060615N). The authors have no conflict of interest to declare.

Comparison of iron uptake in different Helicobacter species.

Comparison of iron uptake of four Helicobacter species (Helicobacter pylori, Helicobacter felis, Helicobacter acinonyx, and Helicobacter mustelae), associated with various degrees of gastritis in their respective host, with five other species which colonize the intestinal tract of various animals (Helicobacter fennelliae, Helicobacter cinaedi, Helicobacter muridarum, Helicobacter bilis, and Helicobacter hepaticus), demonstrated that the iron acquisition system differed according to the ecological niche of the organism. Gastric Helicobacter, except for H. pylori, which used iron from human lactoferrin, were nonsiderophore-producing organisms and were only able to obtain iron from heme and hemoglobin. Nongastric Helicobacter produced siderophores and were able to use for growth a wide range of iron sources (bovine and human lactoferrin and transferrin, heme, hemoglobin).

Regulation of the transcription of genes encoding different virulence factors in Helicobacter pylori by free iron.

Since free iron possesses a poor solubility under physiologic conditions and thus becomes a limiting nutrient for growth, a shift from high- to low-iron environmental conditions is an important signal for bacteria to coordinate the regulation of gene expression. Here, we studied and compared the level of transcripts corresponding to the vacA (cytotoxin), ureA (urease), cagA (cytotoxin-associated antigen) and fur (ferric uptake regulator) genes of Helicobacter pylori, grown under iron-sufficient and iron-restricted conditions. A significant increase in the accumulation of vacA and fur transcripts was observed under iron-restricted conditions. This up-regulation by low levels of iron seems to be not directly regulated by Fur, and certainly requires other regulatory factors. No statistical difference was defined in the accumulation of cagA and ureA.

Evaluation of rapid molecular methods for detection of clarithromycin resistance in Helicobacter pylori.

Resistance of Helicobacter pylori to clarithromycin is due to point mutations at position A2143 or A2144 of the rrnH 23S rRNA gene, each mutation creating an additional restriction site for BsaI or MboII. A procedure combining PCR and RFLP analysis was evaluated for detection of these mutations using primers specific for the 23S rRNA gene, and BsaI and MboII enzymes. All clarithromycin-resistant isolates (8/8), as defined by the MIC, were found to be resistant by PCR-RFLP. No clarithromycin-sensitive isolates (14/14) gave a positive reaction.

Identification, characterization, and immunogenicity of the lactoferrin-binding protein from Helicobacter pylori.

Iron acquisition plays an important role in bacterial virulence. Different studies have been initiated to define the mechanism by which Helicobacter pylori acquires iron. We had previously demonstrated that human lactoferrin (HLf) supported full growth of the bacteria in media lacking other iron sources. The ability of H. pylori to use HLf as an iron source had been found to be dependent on cell-to-protein contact. Since lactoferrin has been found in significant amounts in human stomach resection specimens from patients with superficial or atrophic gastritis, the iron uptake of H. pylori via a specific HLf receptor may play a major role in the virulence of H. pylori infection. In this study, by using affinity chromatography with biotinylated HLf and streptavidin-agarose, we identified a 70-kDa lactoferrin-binding protein (Lbp) from outer membrane proteins of H. pylori. This Lbp was only present when H. pylori was grown in an iron-starved medium, suggesting that it serves in iron uptake. Direct binding assays with increasing concentrations of biotinylated HLf demonstrated that the lactoferrin interaction with the outer membrane of H. pylori grown in iron-restricted medium was saturable. Competitive binding experiments with bovine and human lactoferrin and with transferrin of horse, bovine, and human origin indicated that this Lbp appeared highly specific for HLf. A number of other studies have focused on the importance of transferrin and lactoferrin receptors in pathogenic bacteria and their specificity with the host species. This observation might explain the very strict human specificity of H. pylori.

Importance in diagnosis of gastritis of detection by PCR of the cagA gene in Helicobacter pylori strains isolated from children.

The cagA gene has been detected by PCR and DNA hybridization in 45 Helicobacter pylori strains isolated from children. For each child, clinical symptoms, endoscopic aspect of the gastric mucosa, and histological gastritis were evaluated. Gene-positive strains were associated with hemorrhagic gastritis in 66.6% of the children, while gene-negative strains were associated with hemorrhagic gastritis in 11.2% of the children (P = 0.0001). In addition, 88.8% of gene-positive strains were associated with severe histological gastritis (scores of 3 and 4), and gene-negative strains were collected from the gastric mucosa with the same type of infiltration of neutrophils and lymphocytes in the lamina propia in 55.5% of the children. These differences were statistically significant (P = 0.017). Gene-positive strains were also isolated more frequently from children with vomiting (P = 0.04), while the absence of clinical signs was not significantly different in cagA gene-positive or -negative patients. All of these observations confirmed the role of this cagA gene as a marker of gastric inflammation in children. The detection of this gene might be helpful to determine the degree of inflammation of the gastric mucosa in the absence of abdominal symptoms. We might better understand the natural history of H. pylori infection if we studied the evolution of gastritis in children with regard to the cagA status of isolated strains.