[doktorsko delo]
Vesna Šalamun (Author), Eda Vrtačnik-Bokal (Mentor), Luca Lovrečić (Co-mentor)

Abstract

Debelost se pri ženskah s sindromom policistični jajčnikov (PCOS) pojavlja med 40 in 80 %. Poveča tveganje za razvoj PCOS in vpliva na reproduktivne značilnosti. Debelost in PCOS sta povezana z anovulacijo, zmanjšano plodnostjo, povečanim tveganjem za splav in s slabšim izidom nosečnosti pri novorojenčkih in materi. Gre za motnjo pri delovanju osi hipotalamus-hipofiza-jajčniki, kar vodi do anovulacij, ostali vzroki pa so v spremenjeni kakovosti jajčnih celic in receptivnosti endometrija. Znano je, da debelost negativno vpliva na spontano zanositev, kar potrjuje podaljšani čas do zanositve, prav tako pa negativno vpliva na izid postopkov zunajtelesne oploditve (ZTO). Znižana stopnja živorojenosti je v negativni povezavi z indeksom telesne mase. Izrazito slaba prognoza je pri ženskah, pri katerih je debelost kombinirana s PCOS. Zmanjšana plodnost pri debelosti je lahko posledica povišane inzulinske rezistence, oksidativnega stresa, lipotoksičnosti ali kroničnega vnetja. Čeprav je hujšanje izborna metoda zdravljenja neplodnosti pri bolnicah z debelostjo in s PCOS, so načini zmanjšanja telesne mase (diete, fizična aktivnost, metformin) v klinični praksi pogosto nezadovoljivi. Novejše raziskave so zato usmerjene v iskanje novih učinkovitih načinov zmanjšanja telesne mase. Na Kliničnem oddelku za endokrinologijo, diabetes in presnovne bolezni Interne klinike UKC Ljubljana so razvili izviren način zmanjšanja telesne mase pri bolnicah z debelostjo in s PCOS s kombiniranim zdravljenjem z metforminom in liraglutidom, ki je analog glukagonu podobnega peptida-1 (GLP-1). Dokazali so pomembno zmanjšanje telesne mase v primerjavi z do sedaj uporabljenim zdravljenjem z metforminom. Dejstvi, da so receptorji za GLP-1 (GLP-1R) anatomsko porazdeljeni po celotnem reproduktivnem sistemu in da so opaženi učinki GLP-1 na predkliničnih modelih in v kliničnih raziskavah v reproduktivnem sistemu, nakazujeta na vlogo GLP-1 v povezavi z reproduktivnim in metabolnim sistemom. Predklinični rezultati kažejo, da ima GLP-1 večinoma spodbujevalni učinek na os hipotalamus-hipofiza. Farmakološko spodbujanje z GLP-1R zavira delovanje gonadotropinov v hiperkaloričnih stanjih metaboličnega neravnovesja. Predvideva se, da imajo GLP-1 in GLP-1 agonisti (GLP-1A) tudi protivnetne in antifibrotične učinke na različna periferna reproduktivna tkiva, kot so jajčniki, endometrij in testisi, ki so spremenjeni pri debelosti, sladkorni bolezni in PCOS. V raziskavi smo želeli ugotoviti, ali zmanjšanje telesne mase s kombinacijo metformina in liraglutida pri preiskovankah z debelostjo in s PCOS vpliva na kakovost jajčnih celic in receptivnost endometrija v času vgnezditvenega okna in posledično na stopnjo oploditve ter zanositve. Klinične rezultate smo preverili in pojasnili z analizo celotnega transkriptoma endometrijskih celic v času predvidenega vgnezditvenega okna in z izražanjem genskih označevalcev patofiziološko pomembnih procesov v kumulusnih celicah (KC). Petnajst preiskovank smo neposredno pred vstopom v postopek ZTO vključili v 12-tedensko zdravljenje z metforminom in liraglutidom (raziskovalna skupina). Petnajst preiskovank ni bilo zdravljenih pred vstopom v postopek ZTO (kontrolna skupina) V raziskovalni skupini je 2/3 preiskovank shujšalo za najmanj 5 % izhodiščne telesne mase. Primerljivi rezultati so bili doseženi tudi z zmanjšanjem obsega pasu in znižanjem mase, volumna in površine visceralnega maščobnega tkiva, izboljšanjem metabolnih ter nekaterih endokrinoloških značilnosti. Primerjava rezultatov ZTO med raziskovalno in kontrolno skupino ni pokazala razlik glede porabljenih odmerkov gonadotropinov in izidov postopkov. Poleg štirih preiskovank iz raziskovalne skupine, ki so zanosile v postopku ZTO, sta spontano zanosili še dve preiskovanki, ena med prejemanjem terapije in ena po neuspelem postopku ZTO. Primerjava endometrijskih transkriptomov raziskovalne in kontrolne skupine je pokazala, da je bilo izražanje 1036 genov statistično značilno različno med skupinama (p ? 0,05); v raziskovalni skupini je bilo 478 genov bolj izraženih in 558 genov manj izraženih. Med njimi so bili taki geni, ki smo jih lahko povezali z delovanjem liraglutida (PCSK2, ACTN3 in NOS2). Z analizo poglavitnih komponent variance vseh transkriptomov smo ugotovili, da se glede na ovulatorni status ločijo na ovulatorno in anovulatorno podskupino. Z namenom izključitve anovulatornih vplivov na endometrij smo izvedli primerjavo transkriptomov endometrija med ovulatornima podskupinama. Ugotovili smo, da sta se ovulatorni podskupini razlikovali; 822 genov je bilo bolj in 408 manj izraženih. Med njimi so bili taki geni, ki smo jih lahko povezali z delovanjem liraglutida (GSK3, TAC1 in CCKBR). Z namenom natančnejšega razumevanja bioloških mehanizmov hujšanja z zdravili smo na podlagi različno izraženih genov izvedli funkcionalne analize transkriptomov. Med ugotovljenimi mehanizmi, ki najpomembneje določajo in vmeščajo pridobljene biološke poti v patofiziologijo hujšanja z zdravili, so mehanizem aktivacije GLP-1R, vnetje, oksidativni stres, presnova glukoze in poraba energije ter apoptoza. S primerjanjem transkriptomov raziskovalne in kontrolne skupine smo izdelali gensko omrežje, ki je posledica vpliva hujšanja z zdravili na endometrij. Središčni gen omrežja je YWHAG, liraglutid pa deluje predvidoma preko gena AKT. Najpomembnejše gensko omrežje, ki smo ga pridobili s primerjavo ovulatorne raziskovalne in kontrolne podskupine, je bilo omrežje s središčnim genom SF3A2. Liraglutid je predvidoma deloval na nivoju genov PGRMC2 in MAPK. Analizirali smo izražanje genov KCzrelih jajčnih celic, ki smo jih pridobili iz kompleksa kumulus – jajčna celica. Izmed osmih izbranih genskih biooznačevalcev, ki so pomembni pri razvoju kakovostnih jajčnih celic, sta se dva pomembno ločevala, ko smo primarjali raziskovalne in kontrolne KC. Izražanje inzulinskega in progesteronskega receptorja je bilo v raziskovalni skupini pomembno višje kot v kontrolni. Ugotovili smo, da je hujšanje z zdravili vplivalo na antropometrične značilnosti preiskovank in izboljšalo metabolno ter androgeno funkcijo. Dokazali smo, da je hujšanje z liraglutidom in metforminom pri preiskovankah z debelostjo in s PCOS spremenilo izražanje genov endometrija v času vgnezditvenega okna. Prav tako smo dokazali, da je hujšanje z liraglutidom in metforminom pri preiskovankah z debelostjo in PCOS vplivalo na različno izražanje določenih genskih biooznačevalcev, pomembnih za razvoj jajčne celice. Kljub temu, da se s hujšanjem z zdravili niso izboljšali klinični rezultati postopkov ZTO, so se izboljšali antropometrični, metabolni in endokrinološki parametri, kar potrjuje upravičenost uvedbe tovrstnega hujšanja pri neplodnih bolnicah z debelostjo in s PCOS. V prihodosti bi bilo treba raziskati, kako tako hujšanje vpliva na zmožnost spontanih zanositev.

Keywords

reprodukcija;endokrinologija;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL MF - Faculty of Medicine
Publisher: [V. Šalamun]
UDC: 618.177(043.3)
COBISS: 122075651 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Transcriptome analysis of cumulus and endometrial cells after treatment with metformin and liraglutide in infertile patients with obesity and polycystic syndrome
Secondary abstract: Obesity occurs in 40 to 80% of women with polycystic ovary syndrome (PCOS). It increases the risk of developing PCOS and affects reproductive characteristics. Obesity and PCOS are associated with anovulation, reduced fertility, an increased risk of miscarriage, and poorer pregnancy outcomes in both the neonate and the mother. The causes originate at the level of the hypothalamic-pituitary-ovarian axis, which leads to anovulation. Other causes are altered oocyte quality and altered endometrial receptivity. It is known that obesity, in addition to its negative impact on spontaneous pregnancy, manifested in a prolonged time to conceive, also has a negative impact on the outcome of in vitro fertilization (IVF) procedures. The live birth rate, which is negatively related to the body mass index, decreases. The prognosis is extremely poor in women in whom obesity is combined with PCOS. Reduced fertility in obesity may be a result of increased insulin resistance, oxidative stress, lipotoxicity, and chronic inflammation. Although weight loss is the method of choice for infertility treatment in patients with PCOS and obesity the methods of weight loss (diet, physical activity, metformin) are often unsatisfactory in the clinical practice. Recent research is focused on finding new effective ways to lose weight. The Department of Endocrinology, Diabetes and Metabolic Diseases of the University Medical Centre Ljubljana’s Division of Internal Medicine has developed an original method of weight loss in patients with obesity and PCOS using combination therapy with metformin and liraglutide, which is a glucagon-like peptide-1 analogue (GLP-1). They have demonstrated a significant reduction in body weight compared to previously used metformin treatment. The two facts that GLP-1 receptors (GLP-1R) are anatomically distributed throughout the reproductive system and that there are observed effects of GLP-1 in preclinical models and clinical studies at the level of the reproductive system suggest a role for GLP-1 in the relationship between reproductive and metabolic systems. Preclinical results suggest that GLP-1 has a predominantly stimulatory effect on the hypothalamic-pituitary axis and that pharmacological stimulation with GLP-1R inhibits the action of gonadotropins in hypercaloric states of metabolic imbalance. It has been suggested that GLP-1 and GLP-1 agonists (GLP-1A) also have anti-inflammatory and antifibrotic effects on various peripheral reproductive tissues, such as the ovaries, endometrium, and testes, which are altered in obesity, diabetes, and PCOS. The study sought to determine whether weight loss with the combination of metformin and liraglutide in subjects with PCOS and obesity affected oocyte quality and endometrial receptivity during the implantation window and consequently the rate of fertilization and conception. Clinical results were interpreted by endometrial transcriptome analysis at the time of the predicted implantation window and by the expression of genetic markers of pathophysiologically important processes at the cumulus cell (CC) level. In the study group, 2/3 of the subjects lost 5% or more of their baseline body weight. Comparable results were also achieved in waist circumference reduction and in reducing the mass, volume, and surface area of visceral adipose tissue, improving metabolic and some endocrinological characteristics. Comparison of IVF results between the study and control groups showed no differences in gonadotropin doses consumed and procedure outcomes. In addition to the 4 subjects from the study group who became pregnant during the IVF procedure, two other subjects spontaneously conceived, one while undergoing therapy and one after a failed IVF procedure. A comparison of endometrial transcriptome profiles of the study and control groups showed that the expression of 1036 genes was statistically significantly different between the groups (p <0.05); 478 genes were overexpressed and 558 genes were underexpressed in the study group. This included some genes that we were able to link to the action of liraglutide, for example, PCSK2, ACTN3, and NOS2. By analysing the major variance components of all transcriptome profiles, we found that they differ according to their ovulatory status into ovulatory and anovulatory subgroups. In order to exclude anovulatory effects on the endometrium, we compared endometrial transcriptome profiles between ovulatory subgroups. We found that the ovulatory subgroups differed; 822 genes were overexpressed and 408 underexpressed. It included such genes that we were able to link to the action of liraglutide, for example, the gene for GSK3, TAC1, and CCKBR. To better understand the biological mechanisms of weight loss with medication, we performed functional transcriptome analyses based on differentially expressed genes in group comparison. The mechanism of GLP-1R activation, inflammation, oxidative stress, glucose and metabolism homeostasis, and apoptosis are among the identified mechanisms that most importantly determine and place the acquired biological pathways in the pathophysiology of weight loss with medication. By comparing the transcriptome profiles of the study and control groups, we created a genetic network that results from the effect of weight loss with medication on the endometrium. The central gene of the network is YWHAG, and liraglutide is expected to act through the AKT. The most important gene network obtained by comparing the ovulatory study and control subgroups was the network with the central SF3A2 gene. Liraglutide is expected to act at the PGRMC2 and MAPK levels. The expression of CC genes obtained from the cumulus-oocyte complex of mature oocytes was analysed. A group of 2 out of the 8 gene biomarkers important in the development of quality oocytes significantly differed between study and control CCs. Insulin and progesterone receptor expression was significantly higher in the study group compared to the control group. We established that weight loss with medication had a statistically significant effect on the anthropometric characteristics of the subjects and improved metabolic and androgenic function. We confirmed that weight loss with liraglutide and metformin in subjects with obesity and PCOS altered endometrial gene expression during the implantation window. We also confirmed that weight loss with liraglutide and metformin in subjects with PCOS and obesity affects the different expression of certain genetic biomarkers important for oocyte development. Although weight loss with medication did not improve the clinical outcomes of IVF procedures, it improved anthropometric, metabolic, and endocrinological parameters, which confirms the justification for the implementation of weight loss with metformin and liraglutide protocol in infertile patients with obesity and PCOS. In the future, it would be necessary to examine how such weight loss improves the possibility of spontaneous pregnancies.
Secondary keywords: Obesity;Physiopathology;Polycystic ovary syndrome;Therapy;Metformin;Liraglutide;Weight loss;Endometrium;Gene expression;Cumulus cells;Infertility;Fertilization in vitro;Women;Data interpretation, statistical;Debelost;Patofiziologija;Sindrom policističnih jajčnikov;Terapija;Liraglutid;Izguba teže;Endometrij;Gensko izražanje;Kumulusne celice;Neplodnost;In vitro oploditev;Ženske;Statistična interpretacija podatkov;Ginekologija;Terapevtika;Univerzitetna in visokošolska dela;
Type (COBISS): Dissertation
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Medicinska fak.
Pages: 113 f.
ID: 13805853