Menopausal transition alters female skeletal muscle transcriptome

Objectives Although skeletal muscle is a target of hormonal regulation, the muscle transcriptome, including messenger-RNA (mRNA), long non-coding RNAs (lncRNAs), and microRNAs (miRNAs) has not previously been studied across the menopausal transition. Thus, we took a multi-RNA-omics approach to get insight into transcriptome-wide events of menopause. Methods We included baseline and follow-up muscle samples from seven early (EarlyMT) and 17 late perimenopausal (LateMT) women transitioning to early postmenopause during the study. Total RNA was sequenced and differential expression (DE) of the transcriptome was investigated. Gene functions were investigated with pathway analyses and protein level expression with Western Blot. Results We found 30 DE mRNA genes in EarlyMT and 19 in LateMT participating in pathways controlling cell death, growth, and interactions with the external environment. Lack of protein level changes may indicate a specific role of the regulatory RNAs during menopause. 10 DE lncRNA transcripts but no DE lncRNA genes were identified. No DE miRNAs were found. We identified putative regulatory networks likely to be affected by estradiol availability. Changes in gene expression were correlated with changes in body composition variables, indicating that muscularity and adiposity regulators are affected by menopausal transition. We also found correlations between gene expression and physical activity levels. Conclusions The observed DE genes and their regulatory networks offer novel mechanistic insights into factors affecting body composition during and after menopause. Our results imply that physiological deteriorations orchestrated by the muscle transcriptome likely depend on the magnitude of hormonal change and are influenced by physical activity

Role of Menopausal Transition and Physical Activity in Loss of Lean and Muscle Mass: A Follow-Up Study in Middle-Aged Finnish Women

In midlife, women experience hormonal changes due to menopausal transition. A decrease especially in estradiol has been hypothesized to cause loss of muscle mass. This study investigated the effect of menopausal transition on changes in lean and muscle mass, from the total body to the muscle fiber level, among 47–55-year-old women. Data were used from the Estrogenic Regulation of Muscle Apoptosis (ERMA) study, where 234 women were followed from perimenopause to early postmenopause. Hormone levels (estradiol and follicle stimulating hormone), total and regional body composition (dual-energy X-ray absorptiometry (DXA) and computed tomography (CT) scans), physical activity level (self-reported and accelerometer-measured) and muscle fiber properties (muscle biopsy) were assessed at baseline and at early postmenopause. Significant decreases were seen in lean body mass (LBM), lean body mass index (LBMI), appendicular lean mass (ALM), appendicular lean mass index (ALMI), leg lean mass and thigh muscle cross-sectional area (CSA). Menopausal status was a significant predictor for all tested muscle mass variables, while physical activity was an additional significant contributor for LBM, ALM, ALMI, leg lean mass and relative muscle CSA. Menopausal transition was associated with loss of muscle mass at multiple anatomical levels, while physical activity was beneficial for the maintenance of skeletal muscle mass

Total and regional body adiposity increases during menopause—evidence from a follow‐up study

For women, menopausal transition is a time of significant hormonal changes, which may contribute to altered body composition and regional adipose tissue accumula-tion. Excess adiposity, and especially adipose tissue accumulation in the central body region, increases women's risk of cardiovascular and metabolic conditions and affects physical functioning. We investigated the associations between menopausal progres-sion and total and regional body adiposity measured with dual- energy X- ray absorpti-ometry and computed tomography in two longitudinal cohort studies of women aged 47– 55 (n= 230 and 148, mean follow- up times 1.3 ± 0.7 and 3.9 ± 0.2 years, mean baseline BMI 25.5 kg/m2). We also examined associations between menopausal pro -gression and skeletal muscle fiber characteristics, as well as adipose tissue- derived adipokines. Relative increases of 2%– 14% were observed in regional and total body adiposity measures, with a pronounced fat mass increase in the android area (4% and 14% during short- and long- term follow- ups). Muscle fiber oxidative and glycolytic capacities and intracellular adiposity were not affected by menopause, but were dif-ferentially correlated with total and regional body adiposity at different menopausal stages. Menopausal progression and regional adipose tissue masses were positively associated with serum adiponectin and leptin, and negatively associated with resistin levels. Higher diet quality and physical activity level were also inversely associated with several body adiposity measures. Therefore, healthy lifestyle habits before and during menopause might delay the onset of severe metabolic conditions in women

Alkiobiopsian DNA-eristysmenetelmän vaikutus allele dropout -ilmiöön

Lapsettomuus ja lasten saantiin liittyvät vaikeudet ovat yleistyvä ongelma ympäri maailman. Tehokkain hoitomuoto on koeputkihedelmöitys (IVF). Koeputkihedelmöityshoito mahdollistaa myös alkiodiagnostiikan, jossa perinnöllistä tautia kantavien potilaiden alkiot tutkitaan tarkoituksena vähentää riskiä saada sairas lapsi. Tämän opinnäytetyön tarkoituksena oli tutkia alkiodiagnostiikan merkittävää ongelmaa, allele dropout-ilmiötä (ADO). Allele dropout on ilmiö, jossa vain toinen tutkittavan geenin alleeleista monistuu polymeraasiketjureaktiossa (PCR). Monistumisvirhe voi aiheuttaa pahimmillaan virheellisen diagnoosin. Allele dropoutia esiintyy analyyseissa, joissa lähdemateriaalia on hyvin vähän. Sen syiksi on esitetty esimerkiksi epäonnistunutta solujen hajotusta, DNA:n eheyteen vaikuttavia tekijöitä ja polymeraasiketjureaktion epäoptimaalisia olosuhteita. Menetelmien kehittämisen tavoitteena on vähentää ilmiön esiintyvyyttä ja täten parantaa alkiodiagnostiikan luotettavuutta. Tässä opinnäytetyössä verrattiin vakiintunutta solujen hajottamis- ja DNA:n eristystyötapaa Ovumian tutkimaan muokattuun menetelmään. Tutkittujen menetelmien vaikutusta allele dropoutiin ja PCR:n onnistumiseen arvioitiin. Työssä käytettiin menettelytapoja, jotka ovat osittain salassa pidettäviä. Täten niiden yksityiskohdat on poistettu julkaistavasta versiosta. Tulokseksi saatiin tilastollisesti merkitsevästi alempi allele dropout käytettäessä Ovumian metodia standardimenetelmään verrattuna. Työssä tutkittu muokattu menetelmä voi toimia jatkokehityksen kohteena esimerkiksi tuotekehitykselle.Infertility and other problems concerning having children are a common problem around the world. The most efficient treatment method is in vitro fertilization (IVF). IVF treatments also make preimplantation genetic diagnosis (PGD) possible, where embryos of patients carrying a hereditary disease are analysed with the aim of decreasing the risk of having an affected child. The purpose of this study was to examine a major PGD related problem, a phenomenon called allele dropout (ADO). Allele dropout is manifested when only one of the two alleles in the locus being investigated amplifies during polymerase chain reaction. A mistake or misinterpretation in the amplification process can result in faulty diagnosis. Allele dropout occurs in analyses, where a limited number of source material is present. The factors causing allele dropout are suggested to be for example failed cell lysis, factors concerning DNA integrity and suboptimal conditions in PCR. The purpose of developing current methods is to decrease the incidence of allele dropout and thereby increasing the reliability of preimplantation diagnostics. In this study, a new protocol for cell lysis and DNA extraction was compared with an established method. Incidence of allele dropout and the success rate of PCR were measured. Some of the methods used are regarded as confidential and therefore some details are excluded from the published version. During laboratory work, a statistically significant decrease in allele dropout rate was observed with the new protocol. The method used can act as a starting point for protocol development

Associations of the menopausal transition with body composition : examining the influence of hormonal changes, muscle RNA signaling and lifestyle habits

Naisten biologisen vanhenemisen erityispiirre ovat vaihdevuodet, jotka lähes kaikki naiset käyvät läpi keski-iässä. Vaihdevuosiin liittyvät hormonimuutokset on yhdistetty aiemmin epäsuotuisiin muutoksiin kehonkoostumuksessa ja suorituskyvyssä sekä kasvaneeseen riskiin sairastua aineenvaihduntasairauksiin. Aikaisempaa pitkittäisaineistoa vaihdevuosien siirtymävaiheesta on kuitenkin olemassa vain vähän. Tässä tutkimuksessa selvitettiin pitkittäisaineistossa vaihdevuosien siirtymävaiheen ja menopaussin läpikäymisen vaikutuksia kehon rasvoittumiseen, aineenvaihduntaterveyden indikaattoreille, sekä lihaskudoksen määrälle, transkriptomille ja lihassolujen ominaisuuksille. Lisäksi selvitettiin miten tietyt elintapatekijät olivat yhteydessä edellä mainittuihin muuttujiin. Tutkimuksessa käytettiin Estrogeeni, vaihdevuodet ja toimintakyky (ERMA, n=234)- ja Estrogeeni, mikro-RNA:t ja metabolisten toimintahäiriöiden riski (EsmiRs, n=149)-tutkimusten aineistoja. Molempien tutkimusten alussa naiset iältään 47–55 vuotta olivat joko pre- tai perimenopausaalisia ja tutkimusten lopussa postmenopausaalisia. Naisten verinäytteistä määritettiin veren hormoni- ja adipokiinitasot. Kehonkoostumusta ja rasvanjakautumista mitattiin kaksienergiaisella röntgenabsorptiometrialla, kvantitatiivisella tietokonetomografialla ja antropometrialla. Lihasbiopsioista määritettiin lihaskudoksen tarkempia ominaisuuksia ja transkriptomi. Fyysistä aktiivisuutta, ruokavalion laatua ja hormonivalmisteiden käyttöä tutkittiin kyselylomakkeilla ja aktiivisuusmittarilla. Vaihdevuosien siirtymävaiheen läpikäyminen lisäsi koko kehon ja erityisesti keskivartalon rasvamassaa, sekä vähensi lihasmassaa. Veren leptiinin ja adiponektiinin pitoisuudet nousivat ja resistiinin pitoisuus laski. Vaihdevuosien siirtymävaiheen aikana 49:n lähetti-RNA-molekyylin ilmentymistasot muuttuivat lihaksessa. Korkeampi fyysisen aktiivisuuden määrä ja ruokavalion laatu sekä sukupuolihormonivalmisteiden käyttö olivat yhteydessä matalampaan rasvamassaan, suotuisampaan rasvanjakautumiseen ja korkeampaan lihasmassaan. Vaihdevuodet muokkaavat kehonkoostumusta terveydelle epäsuotuisampaan suuntaan, mutta muutokseen ja sen vaikutuksiin aineenvaihduntaterveydelle voi vaikuttaa terveellisillä elintavoilla. Lihaskudoksessa ilmentymistasoiltaan muuttuneet geenit voivat auttaa ymmärtämään vaihdevuosien aikana havaittujen lihas- ja kehonkoostumuksen muutosten mekanismeja.Women’s hormonal aging is characterized by the cessation of the menstrual cycle during menopause. Menopause-related hormonal changes have previously been associated with increased body adiposity, decreased skeletal muscle mass and function, and decreased metabolic health. However, longitudinal data on the topic remains scarce. The aim of this study was to investigate the longitudinal associations of the menopausal transition with body composition, metabolic health indicators, and skeletal muscle tissue cellular properties and transcriptome. A further aim was to investigate whether physical activity, diet quality, and the use of exogenous hormones were associated with these same variables during mid-life. The data used in this thesis is from the Estrogenic Regulation of Muscle Apoptosis (ERMA) (n=234) and Estrogen, MicroRNAs and the Risk of Metabolic Dysfunction (EsmiRs) (n=149) studies. All the participating women (aged 47-55) were either pre- or perimenopausal at baseline, and postmenopausal at follow-up. Hormone and adipokine levels were measured from blood samples. Body composition and fat distribution were measured using dual-energy X-ray absorptiometry, quantitative computed tomography, and anthropometry. Muscle biopsies of m. vastus lateralis were used to determine muscle tissue properties and transcriptome. Physical activity, diet quality, and the use of exogenous hormones were examined using accelerometers and questionnaires. Menopause was associated with increased total body and waist adiposity and decreased lean and muscle mass. Systemic leptin and adiponectin levels increased, while resistin levels decreased. During the menopausal transition the expression level of 49 protein-coding genes, which take part in important cellular signaling pathways, changed. A higher physical activity level, higher diet quality, and the use of exogenous hormones were associated with less adiposity, gynoid-type fat distribution, and higher lean and muscle mass. The menopausal transition is associated with unfavorable changes in body composition, but these changes and their effects on metabolic health may be alleviated by healthier lifestyle habits. Observed changes in skeletal muscle gene expression may help to understand the mechanistic details of muscle tissue and total body health regulation during menopause

Estradiol deficiency and skeletal muscle apoptosis : Possible contribution of microRNAs

Background Menopause leads to estradiol (E2) deficiency that is associated with decreases in muscle mass and strength. Here we studied the effect of E2 deficiency on miR-signaling that targets apoptotic pathways. Methods C57BL6 mice were divided into control (normal estrous cycle, n = 8), OVX (E2 deficiency, n = 7) and OVX + E2 groups (E2-pellet, n = 4). Six weeks following the OVX surgery, mice were sacrificed and RNA isolated from gastrocnemius muscles. miR-profiles were studied with Next-generation sequencing (NGS) and candidate miRs verified using qPCR. The target proteins of the miRs were found using in silico analysis and measured at mRNA (qPCR) and protein levels (Western blot). Results Of the apoptosis-linked miRs present, eleven (miRs-92a-3p, 122-5p, 133a-3p, 214-3p, 337-3p, 381-3p, 483-3p, 483-5p, 491-5p, 501-5p and 652-3p) indicated differential expression between OVX and OVX + E2 mice in NGS analysis. In qPCR verification, muscle from OVX mice had lower expression of all eleven miRs compared with OVX + E2 (p < 0.050). Accordingly, OVX had higher expression of cytochrome C and caspases 6 and 9 compared with OVX + E2 at the mRNA level (p < 0.050). At the protein level, OVX also had lower anti-apoptotic BCL-W and greater pro-apoptotic cytochrome C and active caspase 9 compared with OVX + E2 (p < 0.050). Conclusion E2 deficiency down regulated several miRs related to apoptotic pathways thus releasing their targets from miR-mediated suppression, which may lead to increased apoptosis and contribute to reduced skeletal muscle mass. Abbreviations AIFApoptosis inducing factorBCL2B-cell lymphoma-2 regulator proteinBCL-XLB-cell lymphoma-extra-large regulator proteinBCL-WB-cell lymphoma-like protein 2CASPCaspasecytCCytochrome CE2EstradiolFasLFas ligandGAPDHGlyceraldehyde 3-phosphate dehydrogenaseHSPHeat shock proteinmiRmicroRNANGSNext-generation sequencingOVXOvariectomypeerReviewe

Menopausal symptoms and cardiometabolic risk factors in middle-aged women : A cross-sectional and longitudinal study with 4-year follow-up

Objective To study associations of menopausal symptoms with cardiometabolic risk factors. Study design A cross-sectional and longitudinal study of a representative population sample of 1393 women aged 47–55 years with a sub-sample of 298 followed for four years. The numbers of vasomotor, psychological, somatic or pain, and urogenital menopausal symptoms were ascertained at baseline through self-report. Their associations with cardiometabolic risk factors were studied using linear regression and linear mixed-effect models. Models were adjusted for age, menopausal status, body mass index, the use of hormonal preparations, education, smoking, and alcohol consumption. Main outcome measures Cardiometabolic risk factors included total cholesterol, low-density and high-density lipoprotein cholesterol, blood pressure, glucose, triglycerides, total and android fat mass, and physical activity. Results All cholesterol and fat mass measures had modest positive associations with menopausal symptoms. The number of vasomotor symptoms, in particular, was associated with total cholesterol (B = 0.13 mmol/l, 95 % CI [0.07, 0.20]; 0.15 mmol/l [0.02, 0.28]) and low-density lipoprotein cholesterol (0.08 mmol/l [0.03, 0.14]; 0.12 mmol/l [0.01, 0.09]) in cross-sectional and longitudinal analyses, respectively. However, these associations disappeared after adjusting for confounders. The number of symptoms was not associated with blood pressure, glucose, triglycerides, and physical activity. Menopausal symptoms at baseline did not predict the changes in the risk factors during the follow-up. Conclusions Menopausal symptoms may not be independently associated with cardiometabolic risk, and they do not seem to predict the changes in risk factors during the menopausal transition.peerReviewe

Extracellular vesicles and high‐density lipoproteins : Exercise and oestrogen‐responsive small RNA carriers

Decreased systemic oestrogen levels (i.e., menopause) affect metabolic health. However, the detailed mechanisms underlying this process remain unclear. Both oestrogens and exercise have been shown to improve metabolic health, which may be partly mediated by circulating microRNA (c-miR) signalling. In recent years, extracellular vesicles (EV) have increased interest in the field of tissue crosstalk. However, in many studies on EV-carried miRs, the co-isolation of high-density lipoprotein (HDL) particles with EVs has not been considered, potentially affecting the results. Here, we demonstrate that EV and HDL particles have distinct small RNA (sRNA) content, including both host and nonhost sRNAs. Exercise caused an acute increase in relative miR abundancy in EVs, whereas in HDL particles, it caused an increase in transfer RNA-derived sRNA. Furthermore, we demonstrate that oestrogen-based hormonal therapy (HT) allows the acute exercise-induced miR-response to occur in both EV and HDL particles in postmenopausal women, while the response was absent in nonusers.peerReviewe

Role of Menopausal Transition and Physical Activity in Loss of Lean and Muscle Mass : A Follow-Up Study in Middle-Aged Finnish Women

In midlife, women experience hormonal changes due to menopausal transition. A decrease especially in estradiol has been hypothesized to cause loss of muscle mass. This study investigated the effect of menopausal transition on changes in lean and muscle mass, from the total body to the muscle fiber level, among 47–55-year-old women. Data were used from the Estrogenic Regulation of Muscle Apoptosis (ERMA) study, where 234 women were followed from perimenopause to early postmenopause. Hormone levels (estradiol and follicle stimulating hormone), total and regional body composition (dual-energy X-ray absorptiometry (DXA) and computed tomography (CT) scans), physical activity level (self-reported and accelerometer-measured) and muscle fiber properties (muscle biopsy) were assessed at baseline and at early postmenopause. Significant decreases were seen in lean body mass (LBM), lean body mass index (LBMI), appendicular lean mass (ALM), appendicular lean mass index (ALMI), leg lean mass and thigh muscle cross-sectional area (CSA). Menopausal status was a significant predictor for all tested muscle mass variables, while physical activity was an additional significant contributor for LBM, ALM, ALMI, leg lean mass and relative muscle CSA. Menopausal transition was associated with loss of muscle mass at multiple anatomical levels, while physical activity was beneficial for the maintenance of skeletal muscle mass.peerReviewe

Associations of resting and peak fat oxidation with sex hormone profile and blood glucose control in middle-aged women

Background and Aims Menopause may reduce fat oxidation. We investigated whether sex hormone profile explains resting fat oxidation (RFO) or peak fat oxidation (PFO) during incremental cycling in middle-aged women. Secondarily, we studied associations of RFO and PFO with glucose regulation. Method and Results We measured RFO and PFO of 42 women (age 52–58 years) with indirect calorimetry. Seven participants were pre- or perimenopausal, 26 were postmenopausal, and nine were postmenopausal hormone therapy users. Serum estradiol (E2), follicle-stimulating hormone, progesterone, and testosterone levels were quantified with immunoassays. Insulin sensitivity (Matsuda index) and glucose tolerance (area under the curve) were determined by glucose tolerance testing. Body composition was assessed with dual-energy X-ray absorptiometry; physical activity with self-report and accelerometry; and diet, with food diaries. Menopausal status or sex hormone levels were not associated with the fat oxidation outcomes. RFO determinants were fat mass (β = 0.44, P = 0.006) and preceding energy intake (β = −0.41, P = 0.019). Cardiorespiratory fitness (β = 0.59, P = 0.002), lean mass (β = 0.49, P = 0.002) and physical activity (self-reported β = 0.37, P = 0.020; accelerometer-measured β = 0.35, P = 0.024) explained PFO. RFO and PFO were not related to insulin sensitivity. Higher RFO was associated with poorer glucose tolerance (β = 0.52, P = 0.002). Conclusion Among studied middle-aged women, sex hormone profile did not explain RFO or PFO, and higher fat oxidation capacity did not indicate better glucose control.peerReviewe