{"id":{"repo_id":"helsinki","oai_identifier":"oai:helda.helsinki.fi:10138/593521"},"canonical_url":"https://search.dev.ndltd.org/etd/helsinki/oai:helda.helsinki.fi:10138/593521","repository":{"repo_id":"helsinki","name":"University of Helsinki","base_url":"https://helda.helsinki.fi/server/oai/request"},"display":{"title":"Vaginal Microbiota : Sampling, Clinical Equivalence, and Combined Sequencing","abstract":"Vaginal microbiota is an important determinant of vaginal health, and it has been studied for over 100 years. The novel technologies, especially sequencing, have revealed the unforeseen complexity of vaginal microbiota communities, and revealed associations to various adverse outcomes including pre-term birth, cancer, and acquisition of sexually transmitted diseases. New data about the vaginal microbiota is cumulating rapidly, but more comprehensive and robust study methods are needed to unravel mechanisms and causalities. The vaginal microbiota is traditionally divided into categories based on microscopic findings on lactobacilli, other bacteria, and host cells. These categories have usually been “normal”, “bacterial vaginosis”, “candidiasis”, and for the last 20 years also “aerobic vaginitis”. However, even the most common of these categories, bacterial vaginosis, is still not fully understood and lacks efficient treatment with lasting effect. At the time when the research for this dissertation began, there were no previous studies on the vaginal microbiota being carried out in Finland and only a handful of studies across Europe. First, we performed a pilot study that set out to identify the feasibility, reproducibility, and comparability of seven different sampling strategies in a study cohort of ten women. We observed that several strategies, including self-sampling, were suitable for vaginal microbiota sampling. Next, we collected samples from 50 healthy Finnish women and through questionnaires, gathered extensive background variables from all participants to study the effect of technical, biological, and socioeconomic factors on the vaginal microbiota and identify potential confounding variables in the study. Furthermore, we used light microscopy to visually categorise samples based on the microbial communities observed and compare them to the sequencing results. To our surprise socioeconomic factors of the subject had larger impact on vaginal microbiota in variance partitioning analysis than technical or hormonal factors. Educational level of the subject was the main factor among socioeconomic factors. Light microscopy and sequencing produced coherent results. Thus, this study underlines the importance on background factors as covariates in microbiota research and reassures clinicians to continue using microscopy in clinical work. The rapidly advancing research on vaginal bacterial communities have shadowed the research on vaginal fungal communities as only a handful of publications have assessed them with sequencing. We developed a parallel sequencing method to study vaginal microbiota and mycobiota in a single sequencing run, and since taxonomic profiling with the ever-growing reference databases was getting noisy, we also developed a text-mining based annotation algorithm. We were able to profile bacterial and fungal communities accurately with over 30% cost reduction compared to separate bacterial and fungal amplicon sequencing. We validated the parallel sequencing and our novel annotation algorithm with shotgun metagenomic sequencing. We failed to validate the fungal results with metagenome, as even a very deep metagenomic sequencing is not able to fully cover the fungal communities of vagina.","abstract_html":"Vaginal microbiota is an important determinant of vaginal health, and it has been studied for over 100 years. The novel technologies, especially sequencing, have revealed the unforeseen complexity of vaginal microbiota communities, and revealed associations to various adverse outcomes including pre-term birth, cancer, and acquisition of sexually transmitted diseases. New data about the vaginal microbiota is cumulating rapidly, but more comprehensive and robust study methods are needed to unravel mechanisms and causalities. The vaginal microbiota is traditionally divided into categories based on microscopic findings on lactobacilli, other bacteria, and host cells. These categories have usually been “normal”, “bacterial vaginosis”, “candidiasis”, and for the last 20 years also “aerobic vaginitis”. However, even the most common of these categories, bacterial vaginosis, is still not fully understood and lacks efficient treatment with lasting effect. At the time when the research for this dissertation began, there were no previous studies on the vaginal microbiota being carried out in Finland and only a handful of studies across Europe. First, we performed a pilot study that set out to identify the feasibility, reproducibility, and comparability of seven different sampling strategies in a study cohort of ten women. We observed that several strategies, including self-sampling, were suitable for vaginal microbiota sampling. Next, we collected samples from 50 healthy Finnish women and through questionnaires, gathered extensive background variables from all participants to study the effect of technical, biological, and socioeconomic factors on the vaginal microbiota and identify potential confounding variables in the study. Furthermore, we used light microscopy to visually categorise samples based on the microbial communities observed and compare them to the sequencing results. To our surprise socioeconomic factors of the subject had larger impact on vaginal microbiota in variance partitioning analysis than technical or hormonal factors. Educational level of the subject was the main factor among socioeconomic factors. Light microscopy and sequencing produced coherent results. Thus, this study underlines the importance on background factors as covariates in microbiota research and reassures clinicians to continue using microscopy in clinical work. The rapidly advancing research on vaginal bacterial communities have shadowed the research on vaginal fungal communities as only a handful of publications have assessed them with sequencing. We developed a parallel sequencing method to study vaginal microbiota and mycobiota in a single sequencing run, and since taxonomic profiling with the ever-growing reference databases was getting noisy, we also developed a text-mining based annotation algorithm. We were able to profile bacterial and fungal communities accurately with over 30% cost reduction compared to separate bacterial and fungal amplicon sequencing. We validated the parallel sequencing and our novel annotation algorithm with shotgun metagenomic sequencing. We failed to validate the fungal results with metagenome, as even a very deep metagenomic sequencing is not able to fully cover the fungal communities of vagina.","abstract_has_math":false,"creators":["Virtanen, Seppo"],"institution":"Helsingin yliopisto","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-04-04","date_published":"2025-04-04","updated_at":"2026-08-21T22:21:56Z","subjects":["naistentaudit ja synnytykset"],"languages":["eng"],"rights":["This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.","Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.","Publikationen är skyddad av upphovsrätten. Den får läsas och skrivas ut för personligt bruk. Användning i kommersiellt syfte är förbjuden."],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10138/593521","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"source_record":{"url":"https://helda.helsinki.fi/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Ahelda.helsinki.fi%3A10138%2F593521","prefix":"dim"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Virtanen, Seppo"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2025-03-05T06:47:52Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2025-03-05T06:47:52Z"]},{"key":"dc:date.issued","label":"Date","values":["2025-04-04"]},{"key":"dc:publisher","label":"Institution","values":["Helsingin yliopisto","Helsingfors universitet","University of Helsinki"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["naistentaudit ja synnytykset"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.","Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.","Publikationen är skyddad av upphovsrätten. Den får läsas och skrivas ut för personligt bruk. Användning i kommersiellt syfte är förbjuden."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10138/593521"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Vaginal microbiota is an important determinant of vaginal health, and it has been studied for over 100 years. The novel technologies, especially sequencing, have revealed the unforeseen complexity of vaginal microbiota communities, and revealed associations to various adverse outcomes including pre-term birth, cancer, and acquisition of sexually transmitted diseases. New data about the vaginal microbiota is cumulating rapidly, but more comprehensive and robust study methods are needed to unravel mechanisms and causalities. The vaginal microbiota is traditionally divided into categories based on microscopic findings on lactobacilli, other bacteria, and host cells. These categories have usually been “normal”, “bacterial vaginosis”, “candidiasis”, and for the last 20 years also “aerobic vaginitis”. However, even the most common of these categories, bacterial vaginosis, is still not fully understood and lacks efficient treatment with lasting effect. At the time when the research for this dissertation began, there were no previous studies on the vaginal microbiota being carried out in Finland and only a handful of studies across Europe. First, we performed a pilot study that set out to identify the feasibility, reproducibility, and comparability of seven different sampling strategies in a study cohort of ten women. We observed that several strategies, including self-sampling, were suitable for vaginal microbiota sampling. Next, we collected samples from 50 healthy Finnish women and through questionnaires, gathered extensive background variables from all participants to study the effect of technical, biological, and socioeconomic factors on the vaginal microbiota and identify potential confounding variables in the study. Furthermore, we used light microscopy to visually categorise samples based on the microbial communities observed and compare them to the sequencing results. To our surprise socioeconomic factors of the subject had larger impact on vaginal microbiota in variance partitioning analysis than technical or hormonal factors. Educational level of the subject was the main factor among socioeconomic factors. Light microscopy and sequencing produced coherent results. Thus, this study underlines the importance on background factors as covariates in microbiota research and reassures clinicians to continue using microscopy in clinical work. The rapidly advancing research on vaginal bacterial communities have shadowed the research on vaginal fungal communities as only a handful of publications have assessed them with sequencing. We developed a parallel sequencing method to study vaginal microbiota and mycobiota in a single sequencing run, and since taxonomic profiling with the ever-growing reference databases was getting noisy, we also developed a text-mining based annotation algorithm. We were able to profile bacterial and fungal communities accurately with over 30% cost reduction compared to separate bacterial and fungal amplicon sequencing. We validated the parallel sequencing and our novel annotation algorithm with shotgun metagenomic sequencing. We failed to validate the fungal results with metagenome, as even a very deep metagenomic sequencing is not able to fully cover the fungal communities of vagina.","Emättimen mikrobisto on tärkeä emättimen terveyden säätelijä, jota on tutkittu yli 100 vuoden ajan. Uudet tekniikat, erityisesti sekvensointi, ovat osoittaneet emätinmikrobiston monimuotoisuuden sekä yhteyksiä useisiin gynekologisiin ongelmiin, kuten ennenaikaiseen synnytykseen, syöpään ja sukupuolitautien tarttuvuuteen. Perinteisesti emättimen mikrobistoa on luokiteltu laktobasillien, muiden bakteerien ja isäntäsolujen mikroskooppisten löydösten perusteella seuraaviin luokkiin ”normaali\", \"bakteerivaginoosi\", \"hiivatulehdus\" ja viimeiset 20 vuotta myös \"aerobinen vaginiitti\". Jopa yleisin näistä, bakteerivaginoosi, on edelleen mikrobiologisesti huonosti ymmärretty, eikä sille ole olemassa pysyvää tehokasta hoitoa. Emätinmikrobiston tutkimukseen ja erityisesti mekanismien ja syysuhteiden selvittämiseen tarvitaankin kattavampia tutkimusmenetelmiä. Tätä väitöskirjatyötä aloitettaessa suomalaisten naisten emätinmikrobistosta ei ollut julkaistu yhtään tutkimusta ja Euroopassakin vain muutamia. Ensimmäisessä osatyössä pystytimme työnkulun emätinnäytteiden uuden sukupolven sekvensointiin (next-generation sequencing, NGS) ja toteutimme pilottitutkimuksen kymmenellä naisella ja seitsemällä näytteenottotavalla löytääksemme parhaan näytteenottostrategian ja testataksemme eri menetelmien toistettavuutta ja vertailukelpoisuutta. Tulokset osoittivat, että emätinmikrobistosta saa toistettavat tulokset usealla eri näytteenottotavalla, mukaan lukien testattavan henkilön itse ottama näyte. Kuten kaikissa tutkimuksissa, myös mikrobiston tutkimuksissa on sekoittavia tekijöitä. Tämän vuoksi keräsimme toisessa osatyössä 50 terveeltä suomalaisnaiselta emätinnäytteet ja laajan taustakyselyn tulevien tutkimusten vertailuryhmäksi sekä tutkiaksemme teknisten ja taustatekijöiden vaikutusta emätinmikrobistoon. Lisäksi vertasimme sekvensointituloksia näytteiden mikroskooppiseen luokitteluun. Yllätykseksemme koehenkilön sosioekonomisilla tekijöillä oli suurempi vaikutus emätinmikrobistoon kuin teknisillä tai hormonaalisilla tekijöillä. Sosioekonomisista taustatekijöistä merkittävimmäksi osoittautui koulutustaso. Valomikroskopiatutkimus ja sekvensointi tuottivat yhteneviä tuloksia bakteeriston koostumuksesta. Tulokset korostavat taustatekijöiden tärkeyttä mikrobistotutkimuksen yhteismuuttujina ja puoltavat mikroskoopin käyttöä jatkossakin kliinisessä työssä. Aktiivinen emättimen bakteeriyhteisöjen tutkimus on jättänyt varjoonsa emättimen sieniyhteisöjen tutkimuksen ja vain kourallinen julkaisuja on tutkinut niitä sekvensoinnilla. Kolmannessa osatyössä kehitimme rinnakkaissekvensointimenetelmän tutkiaksemme emättimen bakteereja ja sieniä samassa sekvensointiajossa. Kehitimme myös tekstinlouhintaan perustuvan tunnistusalgoritmin sekvenssien taksonomiseen tunnistukseen. Yhdistelmäsekvensoinilla saatiin profiloitua bakteeri- ja sieniyhteisöt tarkasti yli 30 % kustannussäästöllä verrattuna erilliseen bakteeri- ja sienisekvensointiin. Rinnakkaissekvensoinnin ja tunnistusalgoritmin suorituskyky bakteerien osalta vahvistettiin metagenomisekvensoinnilla. Sienituloksia ei saatu validoitua, koska edes erittäin syvä metagenominen sekvensointi ei nykyisillä tietokannoilla tunnista kaikkia emättimen sieniyhteisöjä."]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Vaginal Microbiota : Sampling, Clinical Equivalence, and Combined Sequencing"]}]}],"canonical_facts":{"dc:creator":["Virtanen, Seppo"],"dc:date.accessioned":["2025-03-05T06:47:52Z"],"dc:date.available":["2025-03-05T06:47:52Z"],"dc:date.issued":["2025-04-04"],"dc:description.abstract":["Vaginal microbiota is an important determinant of vaginal health, and it has been studied for over 100 years. The novel technologies, especially sequencing, have revealed the unforeseen complexity of vaginal microbiota communities, and revealed associations to various adverse outcomes including pre-term birth, cancer, and acquisition of sexually transmitted diseases. New data about the vaginal microbiota is cumulating rapidly, but more comprehensive and robust study methods are needed to unravel mechanisms and causalities. The vaginal microbiota is traditionally divided into categories based on microscopic findings on lactobacilli, other bacteria, and host cells. These categories have usually been “normal”, “bacterial vaginosis”, “candidiasis”, and for the last 20 years also “aerobic vaginitis”. However, even the most common of these categories, bacterial vaginosis, is still not fully understood and lacks efficient treatment with lasting effect. At the time when the research for this dissertation began, there were no previous studies on the vaginal microbiota being carried out in Finland and only a handful of studies across Europe. First, we performed a pilot study that set out to identify the feasibility, reproducibility, and comparability of seven different sampling strategies in a study cohort of ten women. We observed that several strategies, including self-sampling, were suitable for vaginal microbiota sampling. Next, we collected samples from 50 healthy Finnish women and through questionnaires, gathered extensive background variables from all participants to study the effect of technical, biological, and socioeconomic factors on the vaginal microbiota and identify potential confounding variables in the study. Furthermore, we used light microscopy to visually categorise samples based on the microbial communities observed and compare them to the sequencing results. To our surprise socioeconomic factors of the subject had larger impact on vaginal microbiota in variance partitioning analysis than technical or hormonal factors. Educational level of the subject was the main factor among socioeconomic factors. Light microscopy and sequencing produced coherent results. Thus, this study underlines the importance on background factors as covariates in microbiota research and reassures clinicians to continue using microscopy in clinical work. The rapidly advancing research on vaginal bacterial communities have shadowed the research on vaginal fungal communities as only a handful of publications have assessed them with sequencing. We developed a parallel sequencing method to study vaginal microbiota and mycobiota in a single sequencing run, and since taxonomic profiling with the ever-growing reference databases was getting noisy, we also developed a text-mining based annotation algorithm. We were able to profile bacterial and fungal communities accurately with over 30% cost reduction compared to separate bacterial and fungal amplicon sequencing. We validated the parallel sequencing and our novel annotation algorithm with shotgun metagenomic sequencing. We failed to validate the fungal results with metagenome, as even a very deep metagenomic sequencing is not able to fully cover the fungal communities of vagina.","Emättimen mikrobisto on tärkeä emättimen terveyden säätelijä, jota on tutkittu yli 100 vuoden ajan. Uudet tekniikat, erityisesti sekvensointi, ovat osoittaneet emätinmikrobiston monimuotoisuuden sekä yhteyksiä useisiin gynekologisiin ongelmiin, kuten ennenaikaiseen synnytykseen, syöpään ja sukupuolitautien tarttuvuuteen. Perinteisesti emättimen mikrobistoa on luokiteltu laktobasillien, muiden bakteerien ja isäntäsolujen mikroskooppisten löydösten perusteella seuraaviin luokkiin ”normaali\", \"bakteerivaginoosi\", \"hiivatulehdus\" ja viimeiset 20 vuotta myös \"aerobinen vaginiitti\". Jopa yleisin näistä, bakteerivaginoosi, on edelleen mikrobiologisesti huonosti ymmärretty, eikä sille ole olemassa pysyvää tehokasta hoitoa. Emätinmikrobiston tutkimukseen ja erityisesti mekanismien ja syysuhteiden selvittämiseen tarvitaankin kattavampia tutkimusmenetelmiä. Tätä väitöskirjatyötä aloitettaessa suomalaisten naisten emätinmikrobistosta ei ollut julkaistu yhtään tutkimusta ja Euroopassakin vain muutamia. Ensimmäisessä osatyössä pystytimme työnkulun emätinnäytteiden uuden sukupolven sekvensointiin (next-generation sequencing, NGS) ja toteutimme pilottitutkimuksen kymmenellä naisella ja seitsemällä näytteenottotavalla löytääksemme parhaan näytteenottostrategian ja testataksemme eri menetelmien toistettavuutta ja vertailukelpoisuutta. Tulokset osoittivat, että emätinmikrobistosta saa toistettavat tulokset usealla eri näytteenottotavalla, mukaan lukien testattavan henkilön itse ottama näyte. Kuten kaikissa tutkimuksissa, myös mikrobiston tutkimuksissa on sekoittavia tekijöitä. Tämän vuoksi keräsimme toisessa osatyössä 50 terveeltä suomalaisnaiselta emätinnäytteet ja laajan taustakyselyn tulevien tutkimusten vertailuryhmäksi sekä tutkiaksemme teknisten ja taustatekijöiden vaikutusta emätinmikrobistoon. Lisäksi vertasimme sekvensointituloksia näytteiden mikroskooppiseen luokitteluun. Yllätykseksemme koehenkilön sosioekonomisilla tekijöillä oli suurempi vaikutus emätinmikrobistoon kuin teknisillä tai hormonaalisilla tekijöillä. Sosioekonomisista taustatekijöistä merkittävimmäksi osoittautui koulutustaso. Valomikroskopiatutkimus ja sekvensointi tuottivat yhteneviä tuloksia bakteeriston koostumuksesta. Tulokset korostavat taustatekijöiden tärkeyttä mikrobistotutkimuksen yhteismuuttujina ja puoltavat mikroskoopin käyttöä jatkossakin kliinisessä työssä. Aktiivinen emättimen bakteeriyhteisöjen tutkimus on jättänyt varjoonsa emättimen sieniyhteisöjen tutkimuksen ja vain kourallinen julkaisuja on tutkinut niitä sekvensoinnilla. Kolmannessa osatyössä kehitimme rinnakkaissekvensointimenetelmän tutkiaksemme emättimen bakteereja ja sieniä samassa sekvensointiajossa. Kehitimme myös tekstinlouhintaan perustuvan tunnistusalgoritmin sekvenssien taksonomiseen tunnistukseen. Yhdistelmäsekvensoinilla saatiin profiloitua bakteeri- ja sieniyhteisöt tarkasti yli 30 % kustannussäästöllä verrattuna erilliseen bakteeri- ja sienisekvensointiin. Rinnakkaissekvensoinnin ja tunnistusalgoritmin suorituskyky bakteerien osalta vahvistettiin metagenomisekvensoinnilla. Sienituloksia ei saatu validoitua, koska edes erittäin syvä metagenominen sekvensointi ei nykyisillä tietokannoilla tunnista kaikkia emättimen sieniyhteisöjä."],"dc:format.mimetype":["application/pdf"],"dc:identifier.uri":["http://hdl.handle.net/10138/593521"],"dc:language.iso":["eng"],"dc:publisher":["Helsingin yliopisto","Helsingfors universitet","University of Helsinki"],"dc:rights":["This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.","Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.","Publikationen är skyddad av upphovsrätten. Den får läsas och skrivas ut för personligt bruk. Användning i kommersiellt syfte är förbjuden."],"dc:subject":["naistentaudit ja synnytykset"],"dc:title":["Vaginal Microbiota : Sampling, Clinical Equivalence, and Combined Sequencing"]},"updated_at":"2026-08-21T22:21:56Z"}