Otsides nõela heinakuhjast – endometrioosi biomarkerid

Endometrioosi diagnoosimiseks puuduvad siiani mitteinvasiivsed ja minimaalselt invasiivsed biomarkerid ning haiguse diagnoos põhineb peamiselt laparoskoopilisel operatsioonil ja endometrioosikollete histoloogilisel uuringul. Kuigi endometrioosi biomarkereid on vereplasmast ja -seerumist, uriinist, menstruaalverest, emakaõõne aspiraadist ja ka endomeetriumi koest aktiivselt otsitud, on leitud markerite usaldusväärsus endometrioosi diagnoosimisel jäänud siiski tagasihoidlikuks. Ülevaates on tehtud kokkuvõte endometrioosi biomarkerite otsingute hetkeseisust ja leitud markerite kasutatavusest kliinilises praktikas. Eesti Arst 2017; 96(6):335–34

Allelic estrogen receptor 1 (ESR1) gene variants predict the outcome of ovarian stimulation in in vitro fertilization

The outcome of in vitro fertilization (IVF) depends substantially on the effectiveness of controlled ovarian hyperstimulation (COH) induced by administration of follicle-stimulating hormone (FSH). In COH, endogenously produced estrogens extend the action of FSH in stimulating folliculogenesis. We determined the associations between genetic variations in estrogen receptor ESR1 and ESR2 genes and etiology of female infertility, and analysed the influence of these variations on COH outcome—the quantity and quality of oocytes retrieved. ESR1 PvuII T/C (rs2234693) and XbaI A/G (rs9340799) single-nucleotide polymorphisms (SNPs) and (TA)n microsatellite polymorphism, as well as ESR2 RsaI G/A (rs1256049) SNP and (CA)nmicrosatellite polymorphism were genotyped in 159 IVF patients. The ovarian response to FSH was diminished in patients with endometriosis when compared to tubal factor infertility. ESR1 PvuII and XbaI as well as ESR2 RsaI SNPs were associated with the microsatellite length of the respective genes. Shorter ESR1 (TA)n was linked with a higher risk for unexplained infertility, whereas longer ESR1 (TA)n associated with PvuII*C allele were predictive of a better COH, but not clinical pregnancy outcome in an age-independent manner. These data suggest the variations in ESR1 gene, in addition to the age of a woman, may predict the COH outcome in IVF

Differentially-Expressed miRNAs in Ectopic Stromal Cells Contribute to Endometriosis Development : The Plausible Role of miR-139-5p and miR-375

microRNA (miRNA) expression level alterations between endometrial tissue and endometriotic lesions indicate their involvement in endometriosis pathogenesis. However, as both endometrium and endometriotic lesions consist of different cell types in various proportions, it is not clear which cells contribute to variability in miRNA levels and the overall knowledge about cell-type specific miRNA expression in ectopic cells is scarce. Therefore, we utilized fluorescence-activated cell sorting to isolate endometrial stromal cells from paired endometrial and endometrioma biopsies and combined it with high-throughput sequencing to determine miRNA alterations in endometriotic stroma. The analysis revealed 149 abnormally expressed miRNAs in endometriotic lesions, including extensive upregulation of miR-139-5p and downregulation of miR-375 compared to eutopic cells. miRNA transfection experiments in the endometrial stromal cell line ST-T1b showed that the overexpression of miR-139-5p resulted in the downregulation of homeobox A9 (HOXA9) and HOXA10 expression, whereas the endothelin 1 (EDN1) gene was regulated by miR-375. The results of this study provide further insights into the complex molecular mechanisms involved in endometriosis pathogenesis and demonstrate the necessity for cell-type-specific analysis of ectopic tissues to understand the interactions between different cell populations in disease onset and progression.Peer reviewe

Chemosensitivity and chemoresistance in endometriosis - differences for ectopic versus eutopic cells

Research question: Endometriosis is a common gynaecological disease defined by the presence of endometrium-like tissue outside the uterus. This complex disease, often accompanied by severe pain and infertility, causes a significant medical and socioeconomic burden; hence, novel strategies are being sought for the treatment of endometriosis. Here, we set out to explore the cytotoxic effects of a panel of compounds to find toxins with different efficiency in eutopic versus ectopic cells, thus highlighting alterations in the corresponding molecular pathways. Design: The effect on cellular viability of 14 compounds was established in a cohort of paired eutopic and ectopic endometrial stromal cell samples from 11 patients. The biological targets covered by the panel included pro-survival enzymes, cytoskeleton proteins, the proteasome and the cell repair machinery. Results: Protein kinase inhibitors GSK690693, ARC-775 and sorafenib, proteasome inhibitor bortezomib, and microtubuledepolymerizing toxin monomethyl auristatin E were more effective in eutopic cells. In contrast, 10 mu mol/l of the anthracycline toxin doxorubicin caused cellular death in ectopic cells more effectively than in eutopic cells. The large-scale sequencing of mRNA isolated from doxorubicin-treated and control cells indicated different survival strategies in eutopic versus ectopic endometrium. Conclusions: Overall, the results confirm evidence of large-scale metabolic reprogramming in endometriotic cells, which underlies the observed differences in sensitivity towards toxins. The enhanced efficiency of doxorubicin interfering with redox equilibria and/or DNA repair mechanisms pinpoints key players that can be potentially used to selectively target ectopic lesions in endometriosis.Peer reviewe

Loote reesusstaatuse mitteinvasiivne diagnostika – prenataalse diagnostika uus võimalus Eestis

Rasedusaegne reesuskonflikt on üks tähtsamaid loote ja vastsündinu hemolüütilise haiguse tekkepõhjuseid. Reesuskonflikt tekib, kui reesusnegatiivne naine kannab reesuspositiivset loodet. Loote reesusstaatuse määramine, mille kõige täpsemaks meetodiks on invasiivne geneetiline sünnieelne diagnostika (SeD), võimaldab teostada õigeaegset reesuskonflikti profülaktikat ja ravi. Eesti Arst 2005; 84 (12): 853–85

Whole exome sequencing of benign pulmonary metastasizing leiomyoma reveals mutation in the BMP8B gene

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.Peer reviewe

Chromosomal scan of single sperm cells by combining fluorescence-activated cell sorting and next-generation sequencing

PurposeThe purpose of this study was to develop a feasible approach for single sperm isolation and chromosome analysis by next-generation sequencing (NGS).MethodsSingle sperm cells were isolated from semen samples of normozoospermic male and an infertile reciprocal translocation (RcT) carrier with the 46,XY,t(7;13)(p12;q12.1) karyotype using the optimized fluorescence-activated cell sorting (FACS) technique. Genome profiling was performed using NGS.ResultsFollowing whole-genome amplification, NGS,and quality control, the final chromosome analysis was performed on 31 and 6 single cell samples derived from the RcT carrier and normozoospermic male, respectively. All sperm cells from normozoospermic male showed a normal haploid 23-chromosome profile. For the RcT carrier, the sequencing data revealed that 64.5% of sperm cells harbored different variants of chromosome aberrations, involving deletion of 7p or 7q, duplication of 7p, and duplication of 13q, which is concordant with the expected chromosome segregation patterns observed in balanced translocation carriers. In one sample, a duplication of 9q was also detected.ConclusionsWe optimized FACS protocol for simple and efficient isolation of single human sperm cells that subsequently enabled a successful genome-wide chromosome profiling and identification of segmental aneuploidies from these individual cells, following NGS analysis. This approach may be useful for analyzing semen samples of infertile men or chromosomal aberration carriers to facilitate the reproductive risk assessment.Peer reviewe

Aromatase gene (CYP19A1) variants, female infertility and ovarian stimulation outcome: a preliminary report

Progress has been made towards ascertaining the genetic predictors of ovarian stimulation in IVF. Aromatase cytochrome P450, encoded by the CYP19A1 gene, catalyses a key step in ovarian oestrogen biosynthesis. Hence, the aromatase gene is an attractive candidate for genetic studies. This study aimed to examine the genetic influences of CYP19A1 TCT trinucleotide insertion/deletion (Ins/Del) and (TTTA)n microsatellite intronic polymorphisms on ovarian stimulation outcome and aetiology of female infertility. IVF patients (n = 152) underwent ovarian stimulation according to recombinant FSH and gonadotrophin- releasing hormone antagonist protocol. Del/Del homozygous patients with shorter TTTA repeats exhibited decreased ovarian FSH sensitivity in ovarian stimulation, which may reflect variations in aromatase gene expression during early antral follicle development. Accordingly, this study demonstrates correlations between Del allele and shorter (TTTA)n repeat sizes with smaller ovaries (r = −0.70, P = 0.047) and fewer antral follicles (r = 0.21, P = 0.018) on days 3–5 of spontaneous menstrual cycle, respectively. Furthermore, Del variation linked with low-repeat-number (TTTA)n alleles are involved in enhanced genetic susceptibility to unexplained infertility (adjusted OR = 4.33, P = 0.039) and endometriosis (r = −0.88, P = 0.026), which corroborates evidence on the overlapping patient profiles of ovarian dysfunction in both types of female infertility

Mehe geneetiline viljatus

Geneetilise põhjusega viljatus diagnoositakse 10–15%-l viljatutest meestest. Sagedasemateks teadaolevateks geneetilisteks põhjusteks on autosoomsete ja sugukromosoomide arvu, struktuuri ja ümberpaiknemise anomaaliad. Lisaks on tuvastatud suur hulk reproduktiivsüsteemiga seotud geene ja neis esinevaid geenivariatsioone, mille roll meeste viljatuse patogeneesis ei ole üheselt selge. Senised meeste viljatuse ja geenivariatsioonide vahelised uuringud on olulisi seoseid leidnud vaid vähestel juhtudel. Siiski on geneetilised uuringud kiiresti ja pidevalt arenev valdkond. Saadud teadmised peaksid tooma kliinilisse meditsiini uusi geneetilisi markereid, mis võimaldaksid senisest edukamalt diagnoosida meeste geneetilist viljatust. Artiklis on antud lühiülevaade meeste viljatuse geneetilistest põhjustest ning tutvustatud mõningaid võimalikke mehe viljakust mõjutavaid geene ning geeni polümorfi sme. Eesti Arst 2008; 87(9):628−63

High-throughput mRNA sequencing of stromal cells from endometriomas and endometrium

The aetiology of endometriosis is still unclear and to find mechanisms behind the disease development, it is important to study each cell type from endometrium and ectopic lesions independently. The objective of this study was to uncover complete mRNA profiles in uncultured stromal cells from paired samples of endometriomas and eutopic endometrium. High-throughput mRNA sequencing revealed over 1300 dysregulated genes in stromal cells from ectopic lesions, including several novel genes in the context of endometriosis. Functional annotation analysis of differentially expressed genes highlighted pathways related to cell adhesion, extracellular matrix–receptor interaction and complement and coagulation cascade. Most importantly, we found a simultaneous upregulation of complement system components and inhibitors, indicating major imbalances in complement regulation in ectopic stromal cells. We also performed in vitro experiments to evaluate the effect of endometriosis patients’ peritoneal fluid (PF) on complement system gene expression levels, but no significant impact of PF on C3, CD55 and CFH levels was observed. In conclusion, the use of isolated stromal cells enables to determine gene expression levels without the background interference of other cell types. In the future, a new standard design studying all cell types from endometriotic lesions separately should be applied to reveal novel mechanisms behind endometriosis pathogenesis