L’immunologie de la reproduction englobe les principes de l’immunologie générale et les aspects spécifiques de la reproduction et du développement. Les Colony Stimulating Factors (CSFs) sont une illustration de l'application médicale de ce domaine. Dans la famille des CSFs, le Granulocyte-Colony Stimulating Factor (G-CSF) apparaît aujourd'hui comme une thérapie innovante dans divers cas d'échec de la reproduction, bien que ses cibles et ses effets ne soient pas encore clairement établis. Dans ce travail, à travers une revue sur les CSFs dans la reproduction, une étude consacrée aux gènes cibles du G-CSF dans l'endomètre humain, et une étude consacrée aux effets de la supplémentation systémique en G-CSF sur l’implantation embryonnaire murine, nous avons essayé d'approcher certains mécanismes d'action possibles pour cette cytokine. Dans les modèles murins fertiles et pro-abortifs, la supplémentation systémique en G-CSF, ciblant spécifiquement l’endomètre préimplantatoire, modifie les taux d’implantation embryonnaire. Dans l’endomètre humain, certaines dérégulations préimplantatoires de gènes cibles du G-CSF ont également été observées chez les patients infertiles. L'influence du G-CSF sur ces gènes cibles a été également illustrée dans un modèle ex-vivo de culture endométriale. Ces cibles dont l’expression est influencée par le G-CSF sont décrites comme des molécules clés dans le processus implantatoire, intervenant sur l’adhésion embryonnaire, la migration cellulaire, le remodelage des tissus et l'angiogenèse locale. Ces données suggèrent des possibilités de diagnostic préventif et pré-conceptionnel de certains échecs de reproduction, considérés jusqu’à maintenant comme idiopathiques, et de thérapies innovantes orientées, afin d’optimiser la réceptivité du biosenseur endométrial afin de permettre une implantation embryonnaire harmonieuse et une grossesse évolutive. / Reproductive Immunology involves general immunology principles and special aspects of reproduction and development. Colony Stimulating Factors (CSFs) are an illustration of the medical application of this domain. In the CSF family, Granulocyte-Colony Stimulating Factor (G-CSF) appears today as a promising therapy in various cases of reproductive failure although its targets and effects are not clearly established. In this work, through a review on CSFs in reproduction, a study dedicated to human endometrial targets of G-CSF, and a study dedicated to systemic G-CSF supplementation effects on murine embryo implantation, we tried to approach some possible mechanisms of action of this cytokine. In the considered non-abortive and abortion-prone murine models, the timed systemic G-CSF supplementation, targeting specifically the pre implantation endometrium, influenced the embryo implantation process. Some pre conceptual human endometrial dysregulations of G-CSF target genes were also observed in infertile patients. The endometrial influence of G-CSF on these target genes was also illustrated in an ex-vivo model. These molecules under G-CSF influence are described as critically involved in embryo implantation process, by influencing embryo adhesion, cell migration, tissue remodelling and angiogenesis. These data suggest possible pre-conceptual preventive diagnosis of such reproductive failures and future orientated therapies to optimise the endometrial biosensor and the further embryo implantation and ongoing pregnancy.

Infertility affects 15% of reproductive-aged couples. The milieu surrounding the growing embryo is of outmost importance, and should be optimised during in vitro fertilisation (IVF). Many biological processes, such as angiogenesis, coagulation, and immune processes need to be well regulated for a pregnancy to occur and progress normally. Histidine-rich glycoprotein (HRG) is a plasma protein that regulates components of these systems by building complexes with various ligands. A single nucleotide polymorphism (SNP) in HRG, denoted HRG C633T, seem to be of importance for IVF treatment outcomes. The aim of this thesis was to further investigate the proposed human fertility effects of the HRG C633T SNP. According to the findings of this thesis, the HRG C633T genotype is associated with primary recurrent miscarriage. Male HRG C633T genotype is associated with semen characteristics in infertile men, and pregnancy rates following IVF. However, the distribution of the HRG C633T SNP does not differ between infertile and fertile couples. We further examined the role of the region surrounding the HRG C633T SNP for regulation of endometrial angiogenesis and human embryo development. The region affects primary endometrial endothelial cell migration, proliferation and tube-formation in vitro but does not appear to affect human embryo development. No effect of the HRG peptide was noted on the secretome of human embryos. However, early embryos secrete proteins into the surrounding culture media and the level of secretion of VEGF-A, IL-6, EMMPRIN and PlGF is greater in embryos of higher developmental stages. In conclusion, the HRG C633T genotype appears to play a role only if infertility is established. The region surrounding HRG C633T SNP is of relevance in vitro for regulation of human endometrial endothelial cell angiogenesis. To predict which embryos to transfer in IVF, we have highlighted a number of proteins of interest for further investigation.

<p>Uvod: Infertilitet pogađa 10-15% parova reproduktivnog doba. Vanetesna oplodnja (VTO) je najefikasniji vid tret-mana infertiliteta, ali uprkos značajnom napretku stopa uspeha VTO u proseku iznosi oko 30% po ciklusu. Glavnim razlogom neuspeha smatra se neadekvatan kvalitet embriona, dok se pretpostavlja da u 10-20% slučajeva razlog neuspeha leži u neadekvatnoj receptivnosti uterusa. Na osnovu inicijalnih istraživanja histeroskopija, koja predstvalja zlatni standard u dijagnostici i tretmanu patologije kavuma uterusa, se često izvodi u svakodnevnoj kliničkoj praksi kako bi se povećala uspe&scaron;nost VTO. Uprkos &scaron;irokoj primeni i dalje ne postoji dovoljno kvalitetnih dokaza o realnoj ulozi histeroskopije na ishod VTO kako kod patolo&scaron;kih stanja kavuma tako i rutinski, pre prvog ili rekurentnog poku&scaron;aja VTO. Cilj disertacije bio je da se utvrdi uticaj sprovođenja histeroskopije na ishod VTO, ustanovi učestalost prethodno neprepoznate patologije kavuma uterusa, kao i da se ispitaju stavovi pacijenata o primeni rutinske histeroskopije pred VTO. Materijal i metode: Istraživanje je sprovedeno u Kliničkom centru Vojvodine, u formi prospektivne studije u dve sukcesivne etape od 01.01.2015. do 01.04.2017. U prvoj etapi poređen je ishod VTO kod pacijentkinja kojima pred postupak VTO nije sprovedena histeroskopija (Grupa A), pacijentkinja kod kojih je dobijen uredan nalaz histeroskopije pred postupak VTO (Grupa B) i pacijentkinja gde je pred postupak VTO dobijen patolo&scaron;ki nalaz kavuma na histeroskopiji koji je u istom aktu tertian (Grupa C). Druga etapa istraživanja predstavljala je randomiziranu kontrolisanu studiju (RCT &ndash; randomised controlled trial). Nakon verifikacije urednog ultrazvučnog nalaza pred prvi postupak VTO, pacijentkinje su randomizirane u Grupu A2 kojima pred postupak VTO nije sprovedena histeroskopija i Grupu B2 kojima je pred postupak VTO sprovedena rutinska histeroskopija. Statistička analiza sprovedena je upotrebom odgovarajućeg softvera (JMP Ver. 9). Poređeni su podaci o osnovnim karakteristikama pacijenata, toka i ishoda ciklusa VTO. Primarni parametar ishoda bila je stopa kliničke trudnoće po embriotransferu. Pored analize ishoda primarno konstruisanih grupa, urađena je analiza i naknadno konstruisanih subgrupa, kao i predikcioni model uspeha VTO baziran na logističkoj regresiji. Rezultati: Studija je uključila 253 pacijentkinje (52 pacijentkinja iz Grupe A, 50 iz Grupe B, 50 iz Grupe C, 51 iz Grupe A2 i 50 iz Grupe B2). Nije postojala statistički značajna razlika u karakteristikama pacijentkinja, parametrima ovarijalne rezerve, broju dobijenih jajnih ćelija ni drugim parametrima toka postupka VTO među posmatranim grupama. U prvoj etapi istraživanja dobijena je statistički značajno (p=0,013) veća stopa kliničkih trudnoća kod pacijentkinja kojima je pred postupak VTO sprovedena histeroskopija - 50 % za Grupu B i 42% za grupu C u odnosu na 30,77% kod pacijentkinja bez histeroskopije (Grupa A), bez statistički značajne razlike među histeroskopskim grupama. U drugoj etapi istraživanja stopa kliničkih trudnoća prilikom upotrebe rutinske histeroskopije pred prvu VTO (Grupa B2) iznosila je 46% naspram 31,37% kod pacijentkinja bez histeroskopije pred prvu VTO (Grupa A2), iako uočena razlika nije dostigla statističku značajnost (p =0,089), uz relativan rizik (RR) za ostvarivanje kliničke trudnoće nakon primene histeoskopije uiznosio od 1,47 (95% CI 0,88-2,43) (p=0,13). Analizon subgrupa kod 100 pacijentkinja sa rutinski sprovedenom histeroskopijom pred VTO i 103 pacijentkinje bez histeroskopije pred VTO, dobijena je statistički značajnao veća stopa kliničkih trudnoća (48% naspram 31,07%, istim redom), uz RR od 1,54 (95% CI 1,08-2,20) (p=0,013), kao i stopa tekućih trudnoća od RR 1,49 (CI 1,01-2,19) (p= 0,039). Analiza ukupnog uticaja izvođenja histeroskopije pred VTO dobila je statistički značanjno veću stopu kliničkih trudnoća po ET za grupu histeroskopije uz RR 1,48 (CI 1,06-2,07) (p=0,017). Histeroskopijom je nakon urednog ultrazvučnog nalaza ustanovljeno postojanje patolo&scaron;kog nalaza kod 34,65% pacijenata i to 22,7% major patologije i 11,88% minor patologije kavuma. Nije postojala statistički značajna razlika u uspehu VTO u odnosu na sam nalaz histeroskopije. 98,67% pacijenata podržalo je rutinsku upotrebu histeroskopije pred prvi postupak VTO, dok je 83% pacijenata podržavlo rutinsku upotrebu histeroskopije pred svaki postupak VTO. U finalnom predikcionom modelu se uz AUC od 0,748 jedino postojanje visokokvalitetnog embriona uz odnos &scaron;ansi (OR) 7,91 (95% CI 1,80-56,06; p=0,0047), transfer blastociste uz OR 3,80 (95% CI 1,90-7,98; p=0,0001) i izvođenje histeroskopije pred VTO uz OR 2,13 (95% CI 1,14-4,08, p=0,0169) pokazalo statistički značajnim prediktorima trudnoće. Diskusija: Studija je dobila pozitivan uticaj histeroskopije na ishod postupka VTO, iskazan pre svega povećanjem stope kliničkih trudnoća nakon sprovođenja histeroskopije (bilo da je na histeroskopiji nađen uredan ili patolo&scaron;ki nalaz). Dodatna prednost histeroskopije predstavljala je i i detekcija prethodno nepropoznate patologije kavuma. Umeren efekat na ukupno pobolj&scaron;anje stope kliničkih trudnoća prilikom rutinskog sprovođenja histeroskopije pred prvu VTO, koji je statističku značajnost dostigao tek analizom subgrupa u skladu je sa nalazima novijih dobro dizajniranih studija koji donekle limitiraju nekritičku upotrebu histeroskopije. Biolo&scaron;ko obja&scaron;njenje potencijalnog pozitivnog uticaja histeroskopije najverovatnije leži u detekciji i tretmanu prethodno nepropoznate patologije kavuma, olak&scaron;avanju procedure embriotransfera, kao i humoralnim i molekularnim promenama koje nastaju u endometrijumu kao posledica odgovarajuće histeroskopske traume a koji su u dosa&scaron;anjim istraživanjima apostrofirani kao faktori koji mogu povećati receptivnost uterusa. Zaključak: Histeroskopija je efikasna, bezbedna i visoko prihvatljiva procedura koja dovodi do povećanja uspeha VTO u standardnim kliničkim indikacijama (prethodnog neuspelog postupka VTO i sumnje na patolo&scaron;ki nalaz kavuma uterusa) bilo da se na samoj histeroskopiji nađe uredan ili patolo&scaron;ki nalaz. Rutinska primena histeroskopije pred prvi postupak VTO se na osnovu rezultata studije ne može smatrati apsolutno opravdanom usled statistički nedovoljno značajnog povećanja stope kliničke trudnoće. Uzev&scaron;i u obzir visoku prihvatljivost od strane pacijenata i najverovatniji pozitivan efekat na stopu trudnoće primena rutinske histeroskopije pred prvu VTO bila bi opravdana ukoliko se implementira koncept ambulantne histeroskopije.</p> / <p>Introduction: Infertility affects 10-15% of all couples. In vitro fertilisation (IVF) is the most effective method of infertility treatment, but despite a significant improvement, success rate of IVF is still around 30% per cycle. The main reason for the IVF failure is inadequate embryo quality, but in 10-20% of cases the cause of IVF failure lies in impaired uterine receptivity. Based on earlier studies hysteroscopy, gold standard in the diagnosis and treatment of uterine cavity pathology, is often performed to increase IVF success. Despite its wide use, there is lack of high quality evidence regarding real contribution of hysteroscopy on IVF outcome in situations of uterine cavity pathology or routinely prior to first IVF or after recurrent implantation failure. The aim of this dissertation was to determine the influence of performing hysteroscopy on IVF outcome, as well as the incidence of previously unrecognized uterine pathology, and to examine patient&#39;s attitudes about performing routine hysteroscopy prior to IVF. Material and methods: The research was conducted in a prospective manner in two successive stages at Clinical Center of Vojvodina from 01.01.2015. until 01.04.2017. During first stage of the study IVF outcome was compared between patients who did not have a hysteroscopy prior to IVF (group A), patients with normal hysteroscopic finding prior to the IVF (Group B) and patients with abnormal hysteroscopic findings prior to IVF which was treated at the same time (Group C). The second stage of the study was a randomized controlled trial (RCT). After verification of normal ultrasound findings prior to the first IVF, patients were randomized to group A2 in who me hysteroscopy was not performed and group B2 who had routine hysteroscopy prior to first IVF. Statistical analysis was carried out using the appropriate statistical software (JMP Ver. 9). Patient characteristics, course and outcome of IVF cycle were compared between groups. The primary outcome was clinical pregnancy rate (CPR) per embryotransfer. In addition to analyzing the IVF outcomes in primarily defined groups, subgroup analysis was also performed, as well as IVF success pre-diction model based on logistic regression. Results: The study included 253 patients (52 patients in Group A, 50 in Group B, 50 in Group C, 51 in Group A2 and 50 in Group B2). There was no statistically significant difference in patient characteristics, ovarian reserve parameters, number of retrieved oocytes, or other relevant parameters of IVF course between the observed groups. In the first stage of the study there was statistically significant (p = 0.013) higher clinical pregnancy rate in patients who had a hysteroscopy before IVF - 50% for Group B and 42% for group C versus 30,77 % in patients without hysteroscopy before IVF (Group A), without statistically significant difference between hysteroscopic groups. In the second stage of the study, routine hysteroscopy prior to first IVF (Group B2) led to clinical pregnancy rate 46% versus 31.37% in patients without hysteroscopy prior to first IVF (Group A2), although without statistical significance (p = 0.089. Relative risk (RR) for achieving clinical pregnancy after performing hysteroscopy was 1.47 (95% CI 0.88-2.43) (p = 0.13). Subgroup analysis of 100 patients with routinely performed hysteroscopy before IVF and 103 patients without hysteroscopy prior to the IVF showed statistically significant higher rates of clinical pregnancies (48% versus 31.07%, in the same order), with RR of 1.54 (95% CI 1.08-2.20), (p = 0.013), and for ongoing pregnancies RR was 1.49 (95% CI 1.01-2.19) (p = 0.039). Overall effect of performing hysteroscopy prior to IVF resulted in a statistically significant increase in the clinical pregnancy with RR 1.48 (95% CI 1.06-2.07) (p = 0.017). After normal ultrasound finding hysteroscopy revealed 34.65% of pathological finding, 22.7% of major and 11.88% of minor pathology of the cavity). There was no statistically significant difference in IVF outcome based on hysteroscopy findings. 98.67% of patients supported the routine use of hysteroscopy before the first IVF procedure, while 83% of patients supported the routine use of the hysteroscopy before every IVF procedure. In the final prediction model, with the AUC of 0.748, only the presence of high quality embryos with odds ratio (OR) 7,91 (95% CI 1,80-56,06; p=0,0047), blastocyst transfer with OR 3,80 (95% CI 1,90-7,98; p=0,0001) and performing hysteroscopy prior to IVF with OR 2,13 (95% CI 1,14-4,08, p=0,0169) proved to be statistically significant predictors of pregnancy. Discussion: The study shoved a positive influence of hysteroscopy on the IVF outcome by increasing clinical pregnancy rate after performing hysteroscopy (whether hysteroscopy revealed normal or pathological finding). Additional benefit of hysteroscopy was detection of previously unrecognized uterine pathology. A moderate effect on the overall improvement in clinical pregnancy rate with use of routine hysteroscopy, which reached statistical significance only by subgroup analysis, is in line with findings of recent well designed studies that somewhat limit the noncritical use of hysteroscopy. A biological explanation of the potential positive effect of hysteroscopy is most likely due to detection and treatment of the previously unrecognized uterine pathology, facilitating embryotransfer procedure, as well as the humoral and molecular changes that occur in the endometrium as a consequence of the hysteroscopic trauma. Those changes were hypothesized as factors that can increase uterine receptivity by numerous research. Conclusion: Hysteroscopy is an effective, safe and highly acceptable procedure that increases IVF success when performed for accepted clinical indications (previous IVF failures, pathological findings of uterine cavity), whether hysteroscopy reveals normal or pathological finding. The routine use of hysteroscopy prior to first IVF based on this study can not be considered justified since increase in clinical pregnancy rate did not reach statistical significance. Given the high acceptance of this concept by the patients and moderate but probable positive effect on IVF outcome, implementation of routine hysteroscopy prior to first VTO would be justified only in office hysteroscopy setting.</p>