Vertical transmission of microbiomes into embryo culture media and its association with assisted reproductive outcomes.

RESEARCH QUESTION: Can microbes vertically transmit from semen and follicular fluid to embryo culture media during assisted reproductive technology (ART) treatment? DESIGN: Spent embryo culture media (SECM), seminal fluid and follicular fluid samples were collected from 61 couples with infertility undergoing ART treatment at the Prince of Wales Hospital, Hong Kong SAR, China. Metagenomic analysis was conducted using 16s rRNA sequencing to identify the source of microbes in SECM, correlation between the semen microbiome and male infertility, and correlation between the follicular fluid microbiome and female infertility. RESULTS: Microbial vertical transmission into SECM was reported in 82.5% of cases, and semen was the main source of contamination in conventional IVF cases. The increased abundances of Staphylococcus spp. and Streptococcus anginosus in semen had negative impacts on total motility and sperm count, respectively (P < 0.001). Significant increases in abundance of the genera Prophyromonas, Neisseria and Facklamia were observed in follicular fluid in women with anovulation, uterine factor infertility and unexplained infertility, respectively (P < 0.01). No significant correlation was found between the bacteria identified in all sample types and ART outcomes, including fertilization rate, embryo development, number of available embryos, and clinical pregnancy rate. CONCLUSION: Embryo culture media can be contaminated during ART treatment, not only by seminal microbes but also by follicular fluid and other sources of microbes. Strong correlations were found between specific microbial taxa in semen and sperm quality, and between the follicular fluid microbiome and the aetiology of female infertility. However, no significant association was found between the microbiomes of SECM, semen and follicular fluid and ART outcomes.

Prediction model for day 3 embryo implantation potential based on metabolites in spent embryo culture medium.

BACKGROUND: Metabolites in spent embryo culture medium correlate with the embryo's viability. However, there is no widely accepted method using metabolite dada to predict successful implantation. We sought to combine metabolomic profiling of spent embryo culture medium and clinical variables to create an implantation prediction model as an adjunct to morphological screening of day 3 embryos. METHODS: This investigation was a prospective, nested case-control study. Forty-two day 3 embryos from 34 patients were transferred, and the spent embryo culture medium was collected. Twenty-two embryos implanted successfully, and the others failed. Metabolites in the medium relevant to implantation were detected and measured by Liquid Chromatography-Mass Spectrometry. Clinical signatures relevant to embryo implantation were subjected to univariate analysis to select candidates for a prediction model. Multivariate logistical regression of the clinical and metabolomic candidates was used to construct a prediction model for embryo implantation potential. RESULTS: The levels of 13 metabolites were significantly different between the successful and failed groups, among which five were most relevant and interpretable selected by Least Absolute Shrinkage and Selection Operator regression analysis. None of the clinical variables significantly affected day 3 embryo implantation. The most relevant and interpretable set of metabolites was used to construct a prediction model for day 3 embryo implantation potential with an accuracy of 0.88. CONCLUSIONS: Day 3 embryos'implantation potential could be noninvasively predicted by the spent embryo culture medium's metabolites measured by LC-MS. This approach may become a useful adjunct to morphological evaluation of day 3 embryos.

Re-denudation of residual cumulus cells on day 3 increases the accuracy of cell-free DNA detection in spent embryo culture medium.

PURPOSE: To evaluate the effect of two-step denudation on maternal contamination, ploidy concordance between spent embryo culture medium (SCM) and trophectoderm, blastocyst formation, and clinical outcome. METHODS: Sibling embryos of the same couple were re-denuded (treatment) and not re-denuded (control) on day 3, and trophectoderm biopsy and SCM collection were performed on day 5/6. Sex chromosomes of 20 pairs of SCM and biopsy samples were analyzed to determine the reduction in maternal contamination. Blastocyst formation, implantation, and ongoing pregnancy rates were analyzed by recruiting 565 cleavage embryos on day 3. A total of 113 SCM samples and their corresponding trophectoderm results were collected for ploidy concordance analysis. RESULTS: The detection rate of XX between the treatment and control groups was significant (12/20 (60.0%) versus 19/20 (95.0%), p = 0.02). Concordance of sex chromosomes between the two groups was significant (17/20 (85.0%) versus 8/19 (42.1%), p = 0.003). There were no significant differences in blastocyst formation rate, implantation rate, and ongoing pregnancy rate between the two groups. Among the 113 pairs of SCM and its corresponding trophectoderm, 37 cases (33.33%) were completely concordant, 39 cases (35.14%) were partially concordant, and 35 cases (31.53%) were discordant. CONCLUSION: Our results suggest that re-denudation on day 3 reduces the influence of maternal contamination and improves the accuracy of cfDNA detection. Moreover, the procedure had no significant effect on blastocyst formation, implantation, and ongoing pregnancy rates. In addition, the ploidy concordance approached 70% compared with biopsy, which is acceptable but still not ideal.

Liquid biopsy: state of reproductive medicine and beyond.

Liquid biopsy is the process of sampling and analyzing body fluids, which enables non-invasive monitoring of complex biological systems in vivo. Liquid biopsy has myriad applications in health and disease as a wide variety of components, ranging from circulating cells to cell-free nucleic acid molecules, can be analyzed. Here, we review different components of liquid biopsy, survey state-of-the-art, non-invasive methods for detecting those components, demonstrate their clinical applications and discuss ethical considerations. Furthermore, we emphasize the importance of artificial intelligence in analyzing liquid biopsy data with the aim of developing ethically-responsible non-invasive technologies that can enhance individualized healthcare. While previous reviews have mainly focused on cancer, this review primarily highlights applications of liquid biopsy in reproductive medicine.

The biological characteristics of long cell-free DNA in spent embryos culture medium as noninvasive biomarker in in-vitro embryo selection.

An improved understanding of the cfDNA fragmentomics has proved it as a promising biomarker in clinical applications. However, biological characteristics of cfDNA in spent embryos culture medium (SECM) remain unsolved obstacles before the application in non-invasive in-vitro embryo selection. In this study, we developed a Tn5 transposase and ligase integrated dual-library construction sequencing strategy (TDual-Seq) and revealed the fragmentomic profile of cfDNA of all sizes in early embryonic development. The detected ratio of long cfDNA (>500 bp) was improved from 4.23 % by traditional NGS to 12.80 % by TDual-Seq. End motif analysis showed long cfDNA molecules have a more dominance of fragmentation intracellularly in apoptotic cells with higher predominance of G-end, while shorter cfDNA undergo fragmentation process both intracellularly and extracellularly. Moreover, the mutational pattern of cfDNA and the correlated GO biological process were well differentiated in cleavage and blastocyst embryos. Finally, we developed a multiparametric index (TQI) that employs the fragmentomic profiles of cfDNA, and achieved an area under the ROC curve of 0.927 in screening top quality embryos. TDual-Seq strategy has facilitated characterizing the fragmentomic profile of cfDNA of all sizes in SECM, which are served as a class of non-invasive biomarkers in the evaluation of embryo quality in in-vitro fertilization. And this improved strategy has opened up potential clinical utilities of long cfDNA analysis.

miR­519d­3p released by human blastocysts negatively regulates endometrial epithelial cell adhesion by targeting HIF1α.

Successful embryo implantation requires a competent embryo, a receptive endometrium and synchronized communication between them. The selection of embryos with the highest implantation potential remains a challenge in the field of assisted reproductive technology. Moreover, little is known about the precise molecular mechanisms underlying embryo­endometrium crosstalk. MicroRNAs (miRNAs/miRs) have been detected in the spent embryo culture medium (SCM); however, their functions at the preimplantation stage remain unclear. In the present study, human SCM samples were collected during in vitro fertilization/intracytoplasmic sperm injection­embryo transfer and divided into implanted and not­implanted groups according to the clinical pregnancy outcomes. Total RNA was extracted and six miRNAs (miR­372­3p, miR­373­3p, miR­516b­5p, miR­517a­3p, miR­519d­3p and miR­520a­3p) were selected for reverse transcription­quantitative PCR (RT­qPCR) analysis. The results revealed that miR­372­3p and miR­519d­3p were markedly increased in SCM from blastocysts that failed to implant compared with in blastocysts that implanted. The receiver operating characteristic curve analysis revealed that miR­519d­3p was superior to miR­372­3p in predicting pregnancy outcomes. In vitro miRNA uptake and cell adhesion assays were performed to determine whether miR­519d­3p could be taken up by endometrial epithelial cells and to examine the biological roles of miR­519d­3p after internalization. Potential targets of miR­519d­3p were verified using a dual­luciferase reporter system. The results demonstrated that miR­519d­3p was taken up by human endometrial epithelial cells and that it may inhibit embryo adhesion by targeting HIF1α. Using RT­qPCR, western blot analysis and flow cytometry assay, HIF1α was shown to inhibit the biosynthesis of fucosyltransferase 7 and sialyl­Lewis X (sLex), a cell­surface oligosaccharide that serves an important role in embryonic apposition and adhesion. In addition, a mouse model was established and the results suggested that miR­519d­3p overexpression hampered embryo implantation in vivo. Taken together, miRNAs in SCM may serve as novel biomarkers for embryo quality. Furthermore, miR­519d­3p was shown to mediate embryo­endometrium crosstalk and to negatively regulate embryo implantation by targeting HIF1α/FUT7/sLex pathway.