Browsing by Author "Voložonoka, Ludmila"

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    Causes and Genomic Approaches to Female Reproductive Failure. Doctoral Thesis
    (Rīga Stradiņš University, 2021) Voložonoka, Ludmila; Miskova, Anna; Kempa, Inga
    Clinical recognition of the genetic causes of female reproductive failure using increasingly advancing genetic technologies to preserve patient safety and move towards personalized treatment application is a major challenge of reproductive medicine in the 21st century. The aim of this thesis was to demonstrate a reliable application of advanced genomic techniques in different stages of female reproductive failure in real-life clinical or research scenarios. Several genetic approaches were exploited – starting from the multifactor genetic testing of preimplantation embryos to select the ones free of inherited monogenic conditions and chromosomal aberrations, then following with the analysis of fetal material in case of early pregnancy loss using array comparative genomic hybridization and short tandem repeat analysis to exclude maternal cell contamination, and finally using next generation sequencing technology to analyze genetic landscape leading to preterm delivery in women with cervical insufficiency. The practical work described here was published as three scientific articles now forming three chapters of this thesis. Array comparative genomic hybridization combined with loci-specific genetic testing techniques allowed for a versatile and reliable analysis of preimplantation embryos to select the ones free of genetic conditions analyzed, and in combination with microsatellite analysis it also allowed to access the chromosomal causes of early pregnancy loss while reducing the misdiagnosis caused by maternal cell contamination. Next generation sequencing application allowed to identify the disruptive variants potentially contributive to the development of non-syndromic cervical insufficiency. Pathway enrichment analysis of variant genes from our cohort revealed an increased variation burden in genes playing roles in tissue mechanical and biomechanical properties. Literature analysis allowed to conclude that number of genes can be reliably attributed to female reproductive failure and an increasing number of genes form a pool of good candidates. In order to develop diagnostic gene panels and facilitate genetic advancement inclusion in the clinical practice of female reproduction, a standardized clinical gene-disease validity assessment of the identified genes has to be performed and best practice guidelines have to be composed.
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    Causes and Genomic Approaches to Female Reproductive Failure. Summary of the Doctoral Thesis
    (Rīga Stradiņš University, 2021) Voložonoka, Ludmila; Miskova, Anna; Kempa, Inga
    Clinical recognition of the genetic causes of female reproductive failure using increasingly advancing genetic technologies to preserve patient safety and move towards personalized treatment application is a major challenge of reproductive medicine in the 21st century. The aim of this thesis was to demonstrate a reliable application of advanced genomic techniques in different stages of female reproductive failure in real-life clinical or research scenarios. Several genetic approaches were exploited – starting from the multifactor genetic testing of preimplantation embryos to select the ones free of inherited monogenic conditions and chromosomal aberrations, then following with the analysis of fetal material in case of early pregnancy loss using array comparative genomic hybridization and short tandem repeat analysis to exclude maternal cell contamination, and finally using next generation sequencing technology to analyze genetic landscape leading to preterm delivery in women with cervical insufficiency. The practical work described here was published as three scientific articles now forming three chapters of this thesis. Array comparative genomic hybridization combined with loci-specific genetic testing techniques allowed for a versatile and reliable analysis of preimplantation embryos to select the ones free of genetic conditions analyzed, and in combination with microsatellite analysis it also allowed to access the chromosomal causes of early pregnancy loss while reducing the misdiagnosis caused by maternal cell contamination. Next generation sequencing application allowed to identify the disruptive variants potentially contributive to the development of non-syndromic cervical insufficiency. Pathway enrichment analysis of variant genes from our cohort revealed an increased variation burden in genes playing roles in tissue mechanical and biomechanical properties. Literature analysis allowed to conclude that number of genes can be reliably attributed to female reproductive failure and an increasing number of genes form a pool of good candidates. In order to develop diagnostic gene panels and facilitate genetic advancement inclusion in the clinical practice of female reproduction, a standardized clinical gene-disease validity assessment of the identified genes has to be performed and best practice guidelines have to be composed.
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    First preimplantation genetic testing case for monogenic disease in Latvia
    (2017-12-22) Perminov, Dmitry; Voložonoka, Ludmila; Korņejeva, Liene; Jokste-Pīmane, Evija; Blumberga, Arita; Krasucka, Sandra; Seimuškina, Nellija; Kovaļova, Irina; Fodina, Violeta; Rīga Stradiņš University
    Huntington’s disease (HD) is fatal neurodegenerative disease caused by a (CAG) triplet repeat expansion in the Huntingtin (HTT) gene. Inheritance pattern of the disease is autosomal dominant and onset depending on triplet repeat count. Transgenerational HD transmission can be avoided by preimplantation genetic diagnosis (PGD). Here, we report the first preimplantation genetic testing case for monogenic disease, in Latvia. The result of our work led to the birth of healthy child with normal HTT alleles in his genome. We describe a PGD strategy and testing algorithm that can be applied to any couple at risk of transmitting monogenic disease.
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    Management of Pregnancy with Cervical Shortening: Real-Life Clinical Challenges
    (2023-03-26) Kornete, Anna; Voložonoka, Ludmila; Zolovs, Maksims; Rota, Adele; Kempa, Inga; Gailīte, Linda; Rezeberga, Dace; Miskova, Anna; Department of Obstetrics and Gynaecology; Scientific Laboratory of Molecular Genetics; Statistics Unit
    Background and Objectives: Preterm birth is the leading cause of neonatal mortality worldwide and may be responsible for lifelong morbidities in the survivors. Cervical shortening is one of the common pathways to preterm birth associated with its own diagnostic and management challenges. The preventive modalities that have been tested include progesterone supplementation and cervical cerclage and pessaries. The study aimed to assess the management strategies and outcomes in a group of patients with a short cervix during pregnancy or cervical insufficiency. Materials and Methods: Seventy patients from the Riga Maternity Hospital in Riga, Latvia, were included in the prospective longitudinal cohort study between 2017 and 2021. Patients were treated with progesterone, cerclage, and/or pessaries. The signs of intra-amniotic infection/inflammation were assessed, and antibacterial therapy was given when the signs were positive. Results: The rates of PTB were 43.6% (n = 17), 45.5% (n = 5), 61.1% (n = 11), and 50.0% (n = 1) in progesterone only, cerclage, pessary, and cerclage plus pesssary groups, respectively. The progesterone therapy was associated with a reduced preterm birth risk (x2(1) = 6.937, p = 0.008)), whereas positive signs of intra-amniotic infection/inflammation significantly predicted the risk of preterm birth (p = 0.005, OR = 3.82, 95% [CI 1.31–11.11]). Conclusions: A short cervix and bulging membranes, both indicators of intra-amniotic infection/inflammation, are the key risk factors in preterm birth risk predictions. Progesterone supplementation should remain at the forefront of preterm birth prevention. Among patients with a short cervix and especially complex anamnesis, the preterm rates remain high. The successful management of patients with cervical shortening lies between the consensus-based approach for screening, follow-up, and treatment on the one side and personalising medical therapy on the other.
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    Sievietes reproduktīvās mazspējas iemesli un genomiskās pieejas to risināšanai. Promocijas darba kopsavilkums
    (Rīgas Stradiņa universitāte, 2021) Voložonoka, Ludmila; Miskova, Anna; Kempa, Inga
    Sievietes reproduktīvās mazspējas iemesli un genomiskās pieejas to risināšanai Sievietes reproduktīvās mazspējas ģenētisko cēloņu identificēšana, izmantojot mūsdienīgas ģenētiskās tehnoloģijas, vienlaikus saglabājot pacientu drošību un virzoties uz personalizētas ārstēšanas lietošanu, ir reproduktīvās medicīnas izaicinājums 21. gadsimtā. Darba mērķis bija demonstrēt genomisko tehnoloģiju lietojumu dažādos sievietes reproduktīvās mazspējas etapos reālos klīniskajos vai pētījumu apstākļos. Tika lietotas vairākas genomiskās metodoloģijas – pirmsimplantācijas embriju testēšanai, lai atlasītu embrijus bez iedzimtas monogēnas patoloģijas un hromosomālajām aberācijām, salīdzinošā genoma hibridizācija uz mikročipiem un mikrosatelītu analīze augļa hromosomu analīzei pārtraukušās grūtniecības materiālā, nosakot mātes šūnu kontaminācijas klātbūtni paraugā, un, visbeidzot, nākamās paaudzes sekvencēšana dzemdes kakla nepietiekamības izraisītu priekšlaicīgu dzemdību ģenētiskās etioloģijas raksturošanai. Paveiktais praktiskais darbs tika publicēts trijos zinātniskajos rakstos, kas veido trīs darba sadaļas. Salīdzinošā genoma hibridizācija uz mikročipiem apvienojumā ar lokusa ģenētiskās testēšanas metodēm deva iespēju veikt pirmsimplantācijas analīzi, lai atlasītu embrijus bez testētās patoloģijas, savukārt kombinācijā ar mikrosatelītu analīzi ļāva noteikt hromosomālās patoloģijas izraisošas agrīnas grūtniecības pārtraukšanos, vienlaikus mazinot mātes šūnu kontaminācijas izraisītas kļūdainas diagnozes iespēju. Lietojot nākamās paaudzes sekvencēšanu, tika identificēti gēnu varianti, kas potenciāli veicina nesindromiskas dzemdes kakla nepietiekamības attīstību. Gēnu ceļu bagātināšanas analīze atklāja palielinātu gēnu variācijas slogu gēnos, kas nodrošina audu mehānisko un biomehānisko izturību. Literatūras analīze ļauj secināt, ka gēnu skaits ar zināmu ietekmi uz sievietes reproduktīvās mazspējas attīstību nepārtraukti aug un aizvien lielāks gēnu skaits veido labu kandidātu kopu, kas gaida replikācijas pētījumus. Diagnostisko gēnu paneļu klīniskajai izveidei un lietošanai un ģenētisko sasniegumu iekļaušanai sievietes reprodukcijas klīniskajā praksē nepieciešama standartizēta identificēto gēnu klīniskās validitātes novērtēšana un labās prakses vadlīniju izstrāde.
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    Unravelling the genetic landscape of cervical insufficiency : Insights into connective tissue dysfunction and hormonal pathways
    (2024-09) Voložonoka, Ludmila; Bārdiņa, Līvija; Kornete, Anna; Krūmiņa, Zita; Rots, Dmitrijs; Minkauskienė, Meilė; Rota, Adele; Strelcoviene, Zita; Vilne, Baiba; Kempa, Inga; Miskova, Anna; Gailīte, Linda; Rezeberga, Dace; Rīga Stradiņš University
    BACKGROUND: The intricate molecular pathways and genetic factors that underlie the pathophysiology of cervical insufficiency (CI) remain largely unknown and understudied. METHODS: We sequenced exomes from 114 patients in Latvia and Lithuania, diagnosed with a short cervix, CI, or a history of CI in previous pregnancies. To probe the well-known link between CI and connective tissue dysfunction, we introduced a connective tissue dysfunction assessment questionnaire, incorporating Beighton and Brighton scores. The phenotypic data obtained from the questionnaire was correlated with the number of rare damaging variants identified in genes associated with connective tissue disorders (in silico NGS panel). SKAT, SKAT-O, and burden tests were performed to identify genes associated with CI without a priori hypotheses. Pathway enrichment analysis was conducted using both targeted and genome-wide approaches. RESULTS: No patient could be assigned monogenic connective tissue disorder neither genetically, neither clinically upon clinical geneticist evaluation. Expanding our exploration to a genome-wide perspective, pathway enrichment analysis replicated the significance of extracellular matrix-related pathways as important contributors to CI's development. A genome-wide burden analysis unveiled a statistically significant prevalence of rare damaging variants in genes and pathways associated with steroids (p-adj = 5.37E-06). Rare damaging variants, absent in controls (internal database, n = 588), in the progesterone receptor (PGR) (six patients) and glucocorticoid receptor (NR3C1) (two patients) genes were identified within key functional domains, potentially disrupting the receptors' affinity for DNA or ligands. CONCLUSION: Cervical insufficiency in non-syndromic patients is not attributed to a single connective tissue gene variant in a Mendelian fashion but rather to the cumulative effect of multiple inherited gene variants highlighting the significance of the connective tissue pathway in the multifactorial nature of CI. PGR or NR3C1 variants may contribute to the pathophysiology of CI and/or preterm birth through the impaired progesterone action pathways, opening new perspectives for targeted interventions and enhanced clinical management strategies of this condition.
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    Whole Genome Amplification in Preimplantation Genetic Testing in the Era of Massively Parallel Sequencing
    (2022-05-01) Voložonoka, Ludmila; Miskova, Anna; Gailīte, Linda; Scientific Laboratory of Molecular Genetics; Department of Obstetrics and Gynaecology
    Successful whole genome amplification (WGA) is a cornerstone of contemporary preimplantation genetic testing (PGT). Choosing the most suitable WGA technique for PGT can be particularly challenging because each WGA technique performs differently in combination with different downstream processing and detection methods. The aim of this review is to provide insight into the performance and drawbacks of DOP-PCR, MDA and MALBAC, as well as the hybrid WGA techniques most widely used in PGT. As the field of PGT is moving towards a wide adaptation of comprehensive massively parallel sequencing (MPS)-based approaches, we especially focus our review on MPS parameters and detection opportunities of WGA-amplified material, i.e., mappability of reads, uniformity of coverage and its influence on copy number variation analysis, and genomic coverage and its influence on single nucleotide variation calling. The ability of MDA-based WGA solutions to better cover the targeted genome and the ability of PCR-based solutions to provide better uniformity of coverage are highlighted. While numerous comprehensive PGT solutions exploiting different WGA types and adjusted bioinformatic pipelines to detect copy number and single nucleotide changes are available, the ones exploiting MDA appear more advantageous. The opportunity to fully analyse the targeted genome is influenced by the MPS parameters themselves rather than the solely chosen WGA.