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Expressional profiling of the endometrium enables the personalised timing of the window of implantation (WOI). This study presents and evaluates a novel analytical pipeline based on a TAC-seq (Targeted Allele Counting by sequencing) method for endometrial dating. The expressional profiles were clustered, and differential expression analysis was performed on the model development group, using 63 endometrial biopsies spanning over proliferative (PE, n = 18), early-secretory (ESE, n = 18), mid-secretory (MSE, n = 17) and late-secretory (LSE, n = 10) endometrial phases of the natural cycle. A quantitative predictor model was trained on the development group and validated on sequenced samples from healthy women, consisting of 52 paired samples taken from ESE and MSE phases and five LSE phase samples from 31 individuals. Finally, the developed test was applied to 44 MSE phase samples from a study group of patients diagnosed with recurrent implantation failure (RIF). In validation samples (n = 57), we detected displaced WOI in 1.8% of the samples from fertile women. In the RIF study group, we detected a significantly higher proportion of the samples with shifted WOI than in the validation set of samples from fertile women, 15.9% and 1.8% (p = 0.012), respectively. The developed model was evaluated with an average cross-validation accuracy of 98.8% and an accuracy of 98.2% in the validation group. The developed beREADY screening model enables sensitive and dynamic detection of selected transcriptome biomarkers, providing a quantitative and accurate prediction of endometrial receptivity status., (© 2023. Springer Nature Limited.)

Background: Permanent progression of paternal age and development of reproductive medicine lead to increase in number of children conceived with assisted reproductive techniques (ART). Although it is uncertain if ARTs have direct influence on offspring health, advanced paternal age, associated comorbidities and reduced fertility possess significant risks of genetic disorders to the offspring. With a broad implementation of a non-invasive prenatal testing (NIPT), more cases of genetic disorders, including sex discordance are revealed. Among biological causes of sex discordance are disorders of sexual development, majority of which are associated with the SRY gene., Case Presentation: We report a case of a non-invasive prenatal testing and ultrasound sex discordance in a 46,XY karyotype female fetus with an SRY pathogenic variant, who was conceived through an intracytoplasmic sperm injection (ICSI) due to severe oligozoospermia of the father. Advanced mean age of ICSI patients is associated with risk of de novo mutations and monogenic disorders in the offspring. Additionally, ICSI patients have higher risk to harbour infertility-predisposing mutations, including mutations in the SRY gene. These familial and de novo genetic factors predispose ICSI-conceived children to congenital malformations and might negatively affect reproductive health of ICSI-patients' offspring., Conclusions: Oligozoospermic patients planning assisted reproduction are warranted to undergo genetic counselling and testing for possible inherited and mosaic mutations, and risk factors for de novo mutations., (© 2022. The Author(s).)

Non-invasive prenatal testing (NIPT) is a powerful screening method for fetal aneuploidy detection, relying on laboratory and computational analysis of cell-free DNA. Although several published computational NIPT analysis tools are available, no prior comprehensive, head-to-head accuracy comparison of the various tools has been published. Here, we compared the outcome accuracies obtained for clinically validated samples with five commonly used computational NIPT aneuploidy analysis tools (WisecondorX, NIPTeR, NIPTmer, RAPIDR, and GIPseq) across various sequencing depths (coverage) and fetal DNA fractions. The sample set included cases of fetal trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), and trisomy 13 (Patau syndrome). We determined that all of the compared tools were considerably affected by lower sequencing depths, such that increasing proportions of undetected trisomy cases (false negatives) were observed as the sequencing depth decreased. We summarised our benchmarking results and highlighted the advantages and disadvantages of each computational NIPT software. To conclude, trisomy detection for lower coverage NIPT samples (e.g. 2.5M reads per sample) is technically possible but can, with some NIPT tools, produce troubling rates of inaccurate trisomy detection, especially in low-FF samples., Competing Interests: The authors have declared that no competing interests exist. GIPseq authors (A.A., B.B. and J.V.) performed all their computational analyses ’blindly’, without having any information about the analysed samples. They also did not participate in the analysis and interpretation of corresponding results.

Background: Fetal immune tolerance is crucial for pregnancy success. We studied the link between preeclampsia, a severe pregnancy disorder with uncertain pathogenesis, and fetal human leukocyte antigen G (HLA-G) and other genes regulating maternal immune responses., Methods: We assessed sex ratios and regulatory HLA-G haplotypes in population cohorts and series of preeclampsia and stillbirth. We studied placental mRNA expression of 136 genes by sequencing and HLA-G and interferon alpha (IFNα) protein expression by immunohistochemistry., Findings: We found underrepresentation of males in preeclamptic births, especially those delivered preterm or small for gestational age. Balancing selection at HLA-G associated with the sex ratio, stillbirth, and preeclampsia. We observed downregulation of HLA-G, its receptors, and many other tolerogenic genes, and marked upregulation of IFNA1 in preeclamptic placentas., Interpretation: These findings indicate that an evolutionary trade-off between immune tolerance and protection against infections at the maternal-fetal interface promotes genetic diversity in fetal HLA-G, thereby affecting survival, preeclampsia, and sex ratio. We highlight IFNA1 as a potential mediator of preeclampsia and a target for therapeutic trials., Funding: Finnish Medical Foundation, Päivikki and Sakari Sohlberg Foundation, Karolinska Institutet Research Foundation, Scandinavia-Japan Sasakawa Foundation, Japan Eye Bank Association, Astellas Foundation for Research on Metabolic Disorders, Japan Society for the Promotion of Science, Knut and Alice Wallenberg Foundation, Swedish Research Council, Medical Society Liv och Hälsa, Sigrid Jusélius Foundation, Helsinki University Hospital and University of Helsinki, Jane and Aatos Erkko Foundation, Academy of Finland, Finska Läkaresällskapet, Novo Nordisk Foundation, Finnish Foundation for Pediatric Research, and Emil Aaltonen Foundation., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: The study aimed to validate a whole-genome sequencing-based NIPT laboratory method and our recently developed NIPTmer aneuploidy detection software with the potential to integrate the pipeline into prenatal clinical care in Estonia., Method: In total, 424 maternal blood samples were included. Analysis pipeline involved cell-free DNA extraction, library preparation and massively parallel sequencing on Illumina platform. Aneuploidies were determined with NIPTmer software, which is based on counting pre-defined per-chromosome sets of unique k-mers from sequencing raw data. SeqFF was implemented to estimate cell-free fetal DNA (cffDNA) fraction., Results: NIPTmer identified correctly all samples of non-mosaic trisomy 21 (T21, 15/15), T18 (9/9), T13 (4/4) and monosomy X (4/4) cases, with the 100% sensitivity. However, one mosaic T18 remained undetected. Six false-positive (FP) results were observed (FP rate of 1.5%, 6/398), including three for T18 (specificity 99.3%) and three for T13 (specificity 99.3%). The level of cffDNA of <4% was estimated in eight samples, including one sample with T13 and T18. Despite low cffDNA level, these two samples were determined as aneuploid., Conclusion: We believe that the developed NIPT method can successfully be used as a universal primary screening test in combination with ultrasound scan for the first trimester fetal examination., (© 2019 John Wiley & Sons, Ltd.)

Non-invasive prenatal testing (NIPT) enables accurate detection of fetal chromosomal trisomies. The majority of publicly available computational methods for sequencing-based NIPT analyses rely on low-coverage whole-genome sequencing (WGS) data and are not applicable for targeted high-coverage sequencing data from cell-free DNA samples. Here, we present a novel computational framework for a targeted high-coverage sequencing-based NIPT analysis. The developed framework uses a hidden Markov model (HMM) in conjunction with a supplemental machine learning model, such as decision tree (DT) or support vector machine (SVM), to detect fetal trisomy and parental origin of additional fetal chromosomes. These models were developed using simulated datasets covering a wide range of biologically relevant scenarios with various chromosomal quantities, parental origins of extra chromosomes, fetal DNA fractions, and sequencing read depths. Developed models were tested on simulated and experimental targeted sequencing datasets. Consequently, we determined the functional feasibility and limitations of each proposed approach and demonstrated that read count-based HMM achieved the best overall classification accuracy of 0.89 for detecting fetal euploidies and trisomies on simulated dataset. Furthermore, we show that by using the DT and SVM on the HMM classification results, it was possible to increase the final trisomy classification accuracy to 0.98 and 0.99, respectively. We demonstrate that read count and allelic ratio-based models can achieve a high accuracy (up to 0.98) for detecting fetal trisomy even if the fetal fraction is as low as 2%. Currently, existing commercial NIPT analysis requires at least 4% of fetal fraction, which can be possibly a challenge in case of early gestational age (<10 weeks) or high maternal body mass index (>35 kg/m2). More accurate detection can be achieved at higher sequencing depth using HMM in conjunction with supplemental models, which significantly improve the trisomy detection especially in borderline scenarios (e.g., very low fetal fraction) and enables to perform NIPT even earlier than 10 weeks of pregnancy., Competing Interests: The authors have declared that no competing interests exist.

Targeted next-generation sequencing (NGS) methods have become essential in medical research and diagnostics. In addition to NGS sensitivity and high-throughput capacity, precise biomolecule counting based on unique molecular identifier (UMI) has potential to increase biomolecule detection accuracy. Although UMIs are widely used in basic research its introduction to clinical assays is still in progress. Here, we present a robust and cost-effective TAC-seq (Targeted Allele Counting by sequencing) method that uses UMIs to estimate the original molecule counts of mRNAs, microRNAs, and cell-free DNA. We applied TAC-seq in three different clinical applications and compared the results with standard NGS. RNA samples extracted from human endometrial biopsies were analyzed using previously described 57 mRNA-based receptivity biomarkers and 49 selected microRNAs at different expression levels. Cell-free DNA aneuploidy testing was based on cell line (47,XX, +21) genomic DNA. TAC-seq mRNA profiling showed identical clustering results to transcriptome RNA sequencing, and microRNA detection demonstrated significant reduction in amplification bias, allowing to determine minor expression changes between different samples that remained undetermined by standard NGS. The mimicking experiment for cell-free DNA fetal aneuploidy analysis showed that TAC-seq can be applied to count highly fragmented DNA, detecting significant ( p  = 7.6 × 10 -4 ) excess of chromosome 21 molecules at 10% fetal fraction level. Based on three proof-of-principle applications we demonstrate that TAC-seq is an accurate and highly potential biomarker profiling method for advanced medical research and diagnostics., Competing Interests: Kaarel Krjutškov, Mariann Koel, Juha Kere, and Andres Salumets declare that they have submitted an international patent application on the TAC-seq method. The other authors declare no competing interests.

Non-invasive prenatal testing (NIPT) is a recent and rapidly evolving method for detecting genetic lesions, such as aneuploidies, of a fetus. However, there is a need for faster and cheaper laboratory and analysis methods to make NIPT more widely accessible. We have developed a novel software package for detection of fetal aneuploidies from next-generation low-coverage whole genome sequencing data. Our tool - NIPTmer - is based on counting pre-defined per-chromosome sets of unique k-mers from raw sequencing data, and applying linear regression model on the counts. Additionally, the filtering process used for k-mer list creation allows one to take into account the genetic variance in a specific sample, thus reducing the source of uncertainty. The processing time of one sample is less than 10 CPU-minutes on a high-end workstation. NIPTmer was validated on a cohort of 583 NIPT samples and it correctly predicted 37 non-mosaic fetal aneuploidies. NIPTmer has the potential to reduce significantly the time and complexity of NIPT post-sequencing analysis compared to mapping-based methods. For non-commercial users the software package is freely available at http://bioinfo.ut.ee/NIPTMer/ .

Background: Benign metastasizing leiomyoma (BML) is an orphan neoplasm commonly characterized by pulmonary metastases consisting of smooth muscle cells. Patients with BML have usually a current or previous uterine leiomyoma, which is therefore suggested to be the most probable source of this tumour. The purpose of this case report was to determine the possible genetic grounds for pulmonary BML., Case Presentation: We present a case report in an asymptomatic 44-year-old female patient, who has developed uterine leiomyoma with subsequent pulmonary BML. Whole exome sequencing (WES) was used to detect somatic mutations in BML lesion. Somatic single nucleotide mutations were identified by comparing the WES data between the pulmonary metastasis and blood sample of the same BML patient. One heterozygous somatic mutation was selected for validation by Sanger sequencing. Clonality of the pulmonary metastasis and uterine leiomyoma was assessed by X-chromosome inactivation assay., Conclusions: We describe a potentially deleterious somatic heterozygous mutation in bone morphogenetic protein 8B (BMP8B) gene (c.1139A > G, Tyr380Cys) that was identified in the pulmonary metastasis and was absent from blood and uterine leiomyoma, and may play a facilitating role in the metastasizing of BML. The clonality assay confirmed a skewed pattern of X-chromosome inactivation, suggesting monoclonal origin of the pulmonary metastases.

Urinary concentration of doxorubicin and its active metabolite doxorubicinol was followed in two groups of rats after hepatic intra-arterial injection of a single dose of 6 mg/kg doxorubicin or the same dose combined with 30 mg/kg Spherex (DSM). Urinary samples were collected during five days following administration. The concomitant DSM administration caused a significant decrease of the cumulative urinary excretion of intact doxorubicin; average five days of cumulative values were 73 and 103 micrograms respectively in the group with and without DSM. This indicates a DSM-induced reduction in systemic drug exposure. The elimination rates of doxorubicin or its active metabolite were not influenced by DSM. The decrease in excretion of the parent drug was not due to an interaction between the drug and DSM as evident by examining the binding of doxorubicin to DSM in vitro. Instead regionally altered drug distribution seems a plausible explanation to the reduction.

To increase the effectiveness of intra-arterial treatment, embolizing agents are used. DSM (Spherex) is a starch microsphere suspension--mean diameter 45 mu--which can be used repeatedly to produce a transient vascular occlusion for about half an hour. It has been used experimentally and clinically in the treatment of liver cancer to increase the efficacy of drugs such as doxorubicin. In the present study the effect of DSM on the distribution of 14C-labelled doxorubicin was investigated in rats with liver tumours. A previously used pharmacological dose of 30 mg/kg DSM was compared to a lower dose of 12.5 mg/kg, especially regarding the intrahepatic distribution of co-injected drug at two and twelve hours after injection. The lower dose was to found to give a more effective tumour targeting as well as less regional toxicity. The results of the present study show that in experimental models lower doses of DSM should be beneficial.

High input levels can cause saturation and "fast pumping" effects in hearing aids. The resulting distortion products and changes in speech amplitude envelope are examined in the time domain. Spectrograms reveal changes in speech elements not apparent with time domain methods.

The degradable starch microspheres (DSM) used have a size of 45 microns and are dissolved by amylase in blood. After intraarterial administration of a mixture of DSM and cytostatic drugs the coinjected drugs remain for a longer time in the target tissue/tumor. A transient hypoxia occurs. Systemic exposure of drugs is decreased. Rats with a carcinoma implanted into the liver were given DSM and drugs via the hepatic artery. DSM did not significantly increase the incorporation of 5-fluorouracil (5-FU) into liver tumor RNA. The incorporation of 5-FU into intestinal and bone marrow RNA increased. DSM increased the antitumor effect of doxorubicin, tauromustine, carmustine and RSU-1069 (aziridine 2-nitroimidazole). Side effects, such as liver and gastric necroses and body weight loss, appeared in some rats. The toxic overspill to the stomach seemed to be reduced by giving the DSM in two parts, with all the cytotoxic drug in the first part. The effect on liver and tumor was not decreased by this procedure. DSM alone had no anti-tumor effect. DSM alone decreased liver UDP-glucuronic acid in tumor-free rats, given either by the hepatic artery or, in the double dose, by the portal vein. DSM alone did not increase liver NADPH-cytochrome c reductase activity or serum ASAT (aspartate-aminotransferase) or ALAT (alanine-aminotransferase), indicating that the DSM are inert to the liver, when infused into the tributary vessels.

The effect of the cytostatic nitrosourea drugs TCNU and BCNU on tumor growth was studied in rats with a transplantable colon adenocarcinoma implanted into the liver. The drugs were given as a single dose into the hepatic artery alone or together with degradable starch microspheres (DSM). The effect of the treatment on tumor growth was measured 7 days later. Compared to control animals, TCNU decreased tumor growth. BCNU alone did not significantly decrease tumor growth. The antitumor effect of both drugs was significantly improved by DSM. The addition of DSM to the drugs caused liver damage in a few rats. There were no differences between different treatment groups regarding body weight changes. DSM may protect bone marrow against BCNU toxicity. Injection of DSM alone had no effect either on tumor growth or on survival.

Indomethacin has been reported to decrease pancreatic blood flow. The drug has been used as an analgesic in acute pancreatitis. As decreased blood flow to the pancreas may detrimentally affect the outcome of acute pancreatitis, we investigated the effects of indomethacin on blood flow in the normal porcine pancreas. Regional blood flows, with special reference to the pancreatic flow, were studied with radioactively labelled microspheres in ketamine-anesthetized pigs before and after intravenous administration of indomethacin 2 mg/kg during 10 min. A transient decrease of cardiac output was seen during the infusion. Basal pancreatic blood flow was significantly increased 10 and 30 min after administration of indomethacin. No significant changes were found in small-intestinal or renal blood flow. We conclude that indomethacin does not reduce blood flow in normal porcine pancreas.

Developing a family of frequency response curves for AGC types of hearing instruments using swept pure tones at varying input levels often produces erroneous results. This problem is caused by exceeding the threshold for activating the AGC circuit at some frequencies but not at other frequencies during the pure-tone sweep, thereby producing a different frequency response from that which would be obtained with a complex input signal such as speech-shaped noise. This measurement artifact may be minimized by ensuring that the threshold for activating the AGC circuit is either always exceeded or never exceeded during the development of a frequency response curve. Three input signals are compared for developing a family of frequency responses for an AGC hearing aid: (1) swept pure tone, (2) swept pure tone with bias tone added, and (3) shaped broad-band noise. The shaped broad-band noise appears to be the input signal of choice.

In a model of secondary liver cancer in the rat an evaluation was made of the influence of degradable starch microspheres (Spherex) on the drug retention in tumor and liver tissue. Sodium pertechnetate was used as a drug model substance and was injected into the hepatic artery alone or with degradable starch microspheres (DSM) in a dose of 6 or 12 mg. The distribution of pertechnetate was measured by a gamma-camera equipped with a high resolution collimator. In rats with liver tumor the total elimination of pertechnetate from the liver was delayed when compared to rats without tumor. The tumor concentration of pertechnetate was higher than that of the surrounding liver tissue, irrespective of the presence of DSM. With a DSM dose of 12 mg there was a significantly higher retention of pertechnetate in the tumor during the whole observation period compared to pertechnetate only. The results of this study indicates that DSM can be of value in regional liver chemotherapy to increase liver tumor drug exposure and to reduce systemic toxicity.