Non-invasive cell-free DNA-based approach for the diagnosis of clinical miscarriage: A retrospective study.

OBJECTIVE: To evaluate cell-free DNA (cfDNA) testing as a non-invasive approach to detecting aneuploidies in clinical miscarriages. DESIGN: A retrospective cohort study of women with pregnancy loss. SETTING: Hospitals and genetic analysis laboratories. POPULATION OR SAMPLE: Pregnancy losses in the period 2021-2022. METHODS: Results derived from non-invasive cfDNA testing (Veriseq NIPT Solution V2) of maternal blood and invasive analysis of products of conception (POC) (Ion ReproSeq) compared in 120 women who suffered a miscarriage. MAIN OUTCOME MEASURES: Concordance rate results, cfDNA testing performance, non-informative rate (NIR) and fetal fraction (FF). RESULTS: We found no significant differences in the NIR between invasive (iPOC) and non-invasive (niPOC) analysis of POC (10.0% [12/120] versus 16.7% [20/120]). Of 120 samples, 90 provided an informative result in iPOC and niPOC groups (75%). cfDNA analysis correctly identified 74/87 (85.1%) samples (excluding triploidies). Sensitivity and specificity were 79.4% and 100%, respectively; all discordant cases were female. A binomial logistic model suggested fetal sex as the only variable influencing the concordance rate (P = 0.035). A Y-chromosome-based FF estimate allowed the optimal reclassification of cfDNA of non-informative male fetuses and a more accurate evaluation of cfDNA testing performance. The difference between the two FF estimates (native algorithm and Y-chromosome-based) suggests that female non-concordant cases may represent non-informative cases. CONCLUSIONS: Cell-free DNA-based testing provides a non-invasive approach to determining the genetic cause of clinical miscarriage.

Inserting Cre recombinase into the Prolactin 8a2 gene for decidua-specific recombination in mice.

An estimated 75% of unsuccessful pregnancies are due to implantation failure. Investigating the causes of implantation failure is difficult as decidualization and embryo implantation is a dynamic process. Here, we describe a new decidua-specific iCre recombinase mouse strain. Utilizing CRISPR/Cas9-based genome editing, a mouse strain was developed that expresses iCre recombinase under the control of the endogenous prolactin family 8, subfamily a, member 2 (Prl8a2) promoter. iCre recombinase activity was examined by crossing with mTmG/+ or Sun1-GFP reporter alleles. iCre activity initiated reporter expression at gestational day 5.5 in the primary decidual zone and continued into mid-gestation (gestational day 9.5), with expression highly concentrated in the anti-mesometrial region. No reporter expression was observed in the ovary, oviduct, pituitary, or skeletal muscle, supporting the tissue specificity of the Prl8a2iCre in the primary decidual zone. This novel iCre line will be a valuable tool for in vivo genetic manipulation and lineage tracing to investigate functions of genetic networks and cellular dynamics associated with decidualization and infertility.

Cell-free fetal DNA testing performance and fetal fraction estimation are not affected in ART-conceived pregnancies.

STUDY QUESTION: Does ART-based conception influence fetal fraction (FF) estimation and cell-free fetal DNA (cffDNA) testing performance? SUMMARY ANSWER: Mode of conception (ART versus natural) does not impact FF estimation or cffDNA test informativity rates. WHAT IS KNOWN ALREADY: Pregnancies achieved via ART are increasing, and cffDNA testing is displacing traditional prenatal screening methods due to its high sensitivity and specificity and noninvasive nature. However, conflicting data exist on cffDNA testing performance and FF in ART pregnancies compared with natural pregnancies. STUDY DESIGN, SIZE, DURATION: We performed a case-control study that included 21 558 consecutive pregnancies (spontaneous, n = 15 707; ART, n = 5851). ART-conceived pregnancies were stratified into two groups according to oocyte origin. Samples were collected from April 2015 to September 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included women from different centers worldwide. Blood samples were drawn from the 10th week of gestation onward. Massive parallel whole-genome sequencing was used to analyze cffDNA content in blood plasma. Two different types of technologies (single-end and paired-end) were applied because of analysis technology changes made by the sequencing provider over time. FF was determined using different methods depending on the type of technology used. Cases with an FF <2% or with failure in any quality control metrics were classified as noninformative. An analysis of covariance model was selected to identify which qualitative (sequencing methodology, mode of conception, type (i.e. multiplicity) of gestation and age (women >35 or <35 years old)) and quantitative (gestational age, BMI) variables were predictors of FF value. Multinomial logistic regression was used to evaluate whether the mode of conception impacted cffDNA testing performance. MAIN RESULTS AND THE ROLE OF CHANCE: A univariate t-test demonstrated no significant differences in FF values between ART (median FF = 9.2%) and spontaneous pregnancies (median FF = 9.2%). Also, a multivariate analysis showed that the mode of conception, did not strongly impact the percentage of FF. ART-treated women showed a lower incidence of high-risk cffDNA results compared to women who conceived naturally, specifically for trisomy (T)21 (0.7% versus 1.3%, P = 0.001) and T18 (0.1% versus 0.3%, P = 0.001). A multivariate model stratified by type of aneuploidy suggested that these differences were conditioned by oocyte origin, especially for the T21 risk classification (P < 0.0001). False-positive rates (FPRs) were significantly higher in the ART population, mainly for T13 (P = 0.001) and sexual chromosome aneuploidies (SCAs; P < 0.001). A multivariate model suggested that the differences observed in SCAs were caused by sequencing modality rather than by mode of conception. Likewise, ART-treated women who used their own oocytes had a higher probability of a false positive for T13 (P = 0.004). LIMITATIONS, REASONS FOR CAUTION: Our study lacks follow-up data for low- and high-risk cases of both ART-conceived and naturally conceived pregnancies. Therefore, the results comparing FPR in both populations should be interpreted carefully. Also, collecting information about different ART modalities and regarding preimplantation genetic testing for aneuploidy treatments would help draw definite explanations for the trends observed in this study. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study that demonstrates, with a large sample size, that FF is not influenced by mode of conception, demystifying the notion that patients undergoing ART have a higher probability of noninformative cffDNA testing results. Multivariate models stratified by oocyte origin and type of aneuploidy demonstrated that ART-conceived pregnancies do not have a higher probability of classification as a high-risk pregnancy in prenatal testing. This information is especially valuable to clinicians and genetic counselors when informing patients about the risks and limitations of cffDNA testing in ART pregnancies. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by Igenomix Lab S.L.U. All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Should vanishing twin pregnancies be systematically excluded from cell-free fetal DNA testing?

OBJECTIVE: To demonstrate the feasibility of cell-free DNA (cfDNA) testing in vanishing twin (VT) pregnancies in routine clinical practice. METHODS: Our study included 24 874 singleton and 206 VT consecutive pregnancies. Cell-free DNA was analyzed by massively parallel sequencing. Both aneuploidy analysis (chromosomes 13,18, 21, X, and Y) and fetal fraction estimation were performed according to an Illumina algorithm. Contaminant DNA contribution from the demised co-twin was studied in detail. RESULTS: VT pregnancies exhibited a higher prevalence of screen-positive cases (5.8% vs 2.5%), sex discrepancies (10.2% vs 0.05%), and false positive rates (FPR) (2.6% vs 0.3%) than singleton pregnancies. However, their incidence was significantly lower in tests performed after the 14th week (screen-positive cases: 3.1%; sex discrepancies: 7.8%; and FPR: 0.8%). Among the 12 cases in which cfDNA was performed at two time points, fading of contaminating cfDNA was observed in four cases with a sex discrepancy and in one false positive for trisomy 18, resulting in a final correct result. CONCLUSIONS: Our data suggest VT pregnancies could be included in cfDNA testing as long as it is applied after the 14th week of pregnancy. However, future studies to validate our findings are needed before including VT cases in routine clinical practice. Once established, unnecessary invasive procedures could be avoided, mitigating negative emotional impact on future mothers.

MicroRNA-30d deficiency during preconception affects endometrial receptivity by decreasing implantation rates and impairing fetal growth.

BACKGROUND: Maternal-embryonic crosstalk between the endometrium and the preimplantation embryo is required for normal pregnancy. Our previous results demonstrated that maternal microRNAs secreted into the endometrial fluid, specifically miR-30d, act as a transcriptomic regulator of the preimplantation embryo by the maternal intrauterine environment. OBJECTIVE: To investigate the reproductive and fetal effects of murine miR-30d deficiency at the maternal-embryonic interface according to the origin of its maternal or embryonic default. STUDY DESIGN: A miR-30d knockout murine model was used as the animal model to investigate the impact of maternal and/or embryonic origin of miR-30d deficiency on embryonic implantation and fetal development. Wild-type and miR-30d knockout pseudopregnant mice were used to study the effect of miR-30d deficiency on the receptivity markers by means of real-time quantitative polymerase chain reaction, immunofluorescence, and western blotting. We assessed receptivity markers and implantation rates in 6 different transfer conditions in which embryos obtained from wild-type, knockout, and knockout embryos pretreated with a miR-30d analog were transferred into either wild-type or knockout pseudopregnant females. The impact of miR-30d deficiency on fetal development was evaluated by analyzing the implantation sites and resorbing sites under physiological conditions at days 5, 6, 8, and 12 of pregnancy. Fetal growth was evaluated by analyzing fetuses and placentas at days 12 and 16 of pregnancy. RESULTS: Maternal miR-30d deficiency induced a significant downregulation of endometrial receptivity markers. In wild-type recipients, miR-30d knockout embryos had poorer implantation rates than wild-type embryos (48.86 ± 14.33% vs 75.00 ± 10.47%, respectively, P = .0061). In miR-30d knockout recipients, the lowest implantation rate was observed when knockout embryos were transferred compared to wild-type embryos (26.04 ± 7.15% and 49.71 ± 8.59%, respectively, P = .0059). A positive correlation (r = 0.9978) was observed for maternal leukemia inhibitor factor expression with implantation rates. Further, the course of gestation was compromised in miR-30d knockout mothers, which had smaller implantation sites, greater rates of resorption, and fetuses with smaller crown-rump length and fetal/placental weight ratio. CONCLUSION: Our results demonstrate that maternal and/or embryonic miR-30d deficiency impairs embryonic implantation and fetal development in the animal model. This finding adds a novel miRNA dimension to the understanding of pregnancy and fetal growth restriction in humans.

Chromosome Y as a marker for sex discrepancies in patients with organ transplants: a case report.

BACKGROUND: Organ transplantations cause discrepancy in results from cell-free DNA (cfDNA) testing, but scientific literature is scarce. CASE: A 33-year old gravida underwent cfDNA testing, which showed high levels of Y chromosome (ChrY) in the maternal bloodstream. The ChrY pattern was comparable to an adult male reference. As a result, cfDNA testing was only informative for autosomes. Routine 20-week ultrasound scan showed no structural alterations and the presence of female external genitalia. Post-clinical research revealed that the patient received a bone marrow transplant from a male donor several years before. Fluorescence in situ hybridization showed that 100% of nuclei analysed from the patient's lymphocytes presented a ChrY. CONCLUSION: This case demonstrates ChrY can be used as a marker to avoid sex discrepancies in certain patients with organ transplants.

Inherited unbalanced reciprocal translocation with 3q duplication and 5p deletion in a foetus revealed by cell-free foetal DNA (cffDNA) testing: a case report.

BACKGROUND: Since 2011, screening maternal blood for cell-free foetal DNA (cffDNA) fragments has offered a robust clinical tool to classify pregnancy as low or high-risk for Down, Edwards, and Patau syndromes. With recent advances in molecular biology and improvements in data analysis algorithms, the screening's scope of analysis continues to expand. Indeed, screening now encompassess additional conditions, including aneuploidies for sex chromosomes, microdeletions and microduplications, rare autosomal trisomies, and, more recently, segmental deletions and duplications called copy number variations (CNVs). Yet, the ability to detect CNVs creates a new challenge for cffDNA analysis in couples in which one member carries a structural rearrangement such as a translocation or inversion. CASE PRESENTATION: We report a segmental duplication of the long arm of chromosome 3 and a segmental deletion of the short arm of chromosome 5 detected by cffDNA analysis in a 25-year-old pregnant woman. The blood sample was sequenced on a NextSeq 550 (Illumina) using the VeriSeq NIPT Solution v1 assay. G-band karyotyping in amniotic fluid only detected an abnormality in chromosome 5. Next-generation sequencing in amniocytes confirmed both abnormalities and identified breakpoints in 3q26.32q29 and 5p13.3p15. The foetus died at 21 weeks of gestation due to multiple abnormalities, and later G-band karyotyping in the parents revealed that the father was a carrier of a balanced reciprocal translocation [46,XY,t(3;5)(q26.2;p13)]. Maternal karyotype appeared normal. CONCLUSION: This case provides evidence that extended cffDNA can detect, in addition to aneuploidies for whole chromosomes, large segmental aneuploidies. In some cases, this may indicate the presence of chromosomal rearrangements in a parent. Such abnormalities are outside the scope of standard cffDNA analysis targeting chromosomes 13, 18, 21, X, and Y, potentially leading to undiagnosed congenital conditions.

Endometrial Liquid Biopsy Provides a miRNA Roadmap of the Secretory Phase of the Human Endometrium.

CONTEXT: Endometrial liquid biopsy (ELB) is a minimally invasive alternative for research and diagnosis in endometrial biology. OBJECTIVE: We sought to establish an endometrial micro ribonucleic acid (miRNA) roadmap based on ELB during the secretory phase of the menstrual cycle in both natural and hormonal replacement therapy (HRT) cycles. DESIGN: Human ELB samples (n = 58) were obtained from healthy ovum donors undergoing a natural and an HRT cycle consecutively. miRNA profiles were identified using next-generation sequencing (NGS). For functional analysis, messenger ribonucleic acid targets were chosen among those reported in the endometrial receptivity analysis. RESULTS: The human endometrial secretory phase is characterized by a dynamic miRNA secretion pattern that varies from the prereceptive to the receptive stages. No differences in miRNA profiles were found among natural versus HRT cycles in the same women, reinforcing the similarities in functional and clinical outcomes in natural versus medicated cycles. Bioinformatic analysis revealed 62 validated interactions and 81 predicted interactions of miRNAs differentially expressed in the HRT cycle. Annotation of these genes linked them to 51 different pathways involved in endometrial receptivity. CONCLUSION: This NGS-based study describes the miRNA signature in human ELB during the secretory phase of natural and HRT cycles. A consistent endometrial miRNA signature was observed in the acquisition of endometrial receptivity. Interestingly, no significant differences in miRNA expression were found in natural versus HRT cycles reinforcing the functional clinical similarities between both approaches.

Extracellular Vesicles in Human Reproduction in Health and Disease.

Extensive evidence suggests that the release of membrane-enclosed compartments, more commonly known as extracellular vesicles (EVs), is a potent newly identified mechanism of cell-to-cell communication both in normal physiology and in pathological conditions. This review presents evidence about the formation and release of different EVs, their definitive markers and cargo content in reproductive physiological processes, and their capacity to convey information between cells through the transfer of functional protein and genetic information to alter phenotype and function of recipient cells associated with reproductive biology. In the male reproductive tract, epididymosomes and prostasomes participate in regulating sperm motility activation, capacitation, and acrosome reaction. In the female reproductive tract, follicular fluid, oviduct/tube, and uterine cavity EVs are considered as vehicles to carry information during oocyte maturation, fertilization, and embryo-maternal crosstalk. EVs via their cargo might be also involved in the triggering, maintenance, and progression of reproductive- and obstetric-related pathologies such as endometriosis, polycystic ovarian syndrome, preeclampsia, gestational diabetes, and erectile dysfunction. In this review, we provide current knowledge on the present and future use of EVs not only as biomarkers, but also as therapeutic targeting agents, mainly as vectors for drug or compound delivery into target cells and tissues.

Daily, seasonal and monthly variations in ozone levels recorded at the Turia river basin in Valencia (Eastern Spain).

INTRODUCTION: The Turia river basin, located in the east of the Iberian Peninsula, drains into the Mediterranean Sea near the city of Valencia (population, 814,208). The predominance of sea-breeze fluxes favours the inland transport of pollutants from the city up the basin where ozone concentrations exceeding the threshold for protection of human health are systematically recorded during the summer months. METHODS: This work analyses the variability in ozone levels by examining their spatial and temporal distribution in a Mediterranean river basin downwind from a city within the period 2005-2008. Orographic determinants and atmospheric fluxes induce strong variations in ozone measurements, even on relatively close locations. CONCLUSIONS: Results show a different behaviour of the monthly means and the daily cycles depending on the season of the year and the measuring environment, with summer/winter ratios ranging from 2.4 in cities to 1.6 inland, and mean values always higher in the interior of the basin. Daily cycles show significant summer/winter differences related to the predominant situations of anticyclonic stability in winter, which limit ventilation, and the predominant breeze circulations in summer. Results also show a "weekend effect" at urban and medium-distance stations. At the most inland station, the weekend/weekday behaviour differs according to the season of the year; weekend ozone levels are higher in spring, autumn and winter, and lower in summer, coinciding with the predominance of local wind cycles that favour air mass penetration inland from the coast.

The use of experimental data and their uncertainty for assessing ozone photochemistry in the Eastern Iberian Peninsula.

Observation-based methods are useful tools to explore the sensitivity of ozone concentrations to precursor controls. With the aim of assessing the ozone precursor sensitivity in two locations: Paterna (suburban) and Villar del Arzobispo (rural) of the Turia river basin in the east of Spain, the photochemical indicator O(3)/NO(y) and the Extent-of-Reaction (EOR) parameter have been calculated from field measurements. In Paterna, the O(3)/NO(y) ratio varied from 0 to 13 with an average value of 5.1 (SD 3.2), whereas the averaged value for the EOR was 0.43 (SD 0.14). In Villar del Arzobispo, the O(3)/NO(y) ratio changed from 5 to 30 with a mean value of 13.6 (SD 4.7) and the EOR gave an averaged value of 0.72 (SD 0.11). The results show two different patterns of ozone production as a function of the location. The suburban area shows a VOC-sensitive regime whereas the rural one shows a transition regime close to NO(x)-sensitive conditions. No seasonal differences in these regimes are observed along the monitoring campaigns. Finally, an analysis of the influence of the measurement quality of NO(y), NO(x) and O(3) on the uncertainty of the O(3)/NO(y) ratio and the EOR was performed showing that the uncertainty of O(3)/NO(y) is not dependent on either its value or the individual values of O(3) and NO(y) but just on the quality of O(3) and NO(y) measurements. The maximum uncertainty is 26% as long as the combined uncertainties of O(3) and NO(y) remain below the 7.5%. The case of the EOR is different and its uncertainty depends on both the value of the EOR parameter and the individual concentration values of NO(y) and NO(x). The uncertainty of the EOR estimation can be very high (>200%) if the combined uncertainties of both NO(y) and NO(x) are high (>7.5%), or especially, if u(NO(y)) and u(NO(x)) differ considerably from each other (>3.5%).