No correlation between pinopode formation and LIF and MMP2 expression in endometrium during implantation window

Implantation depends on two factors — embryo and endometrium. The period of maximal endometrial receptivity is a poorly understood phenomenon. We decided to look at three possible markers of implantation: pinopodes, leukemia inhibitory factor, and matrix metalloproteinase 2 and their correlations. We included in the study 23 idiopathic infertility patients and 21 patients with recurrent spontaneous abortions of unknown etiology. Twenty one fertile patients were also recruited. A biopsy was used for endometrial dating according to the Noyes and Hertig criteria, and assessed for the presence of pinopodes via a scanning electron microscope. Endometria were examined in Real Time-Polymerase Chain Reaction cycles for the mRNA expression of leukemia inhibitory factor (LIF) and matrix metalloproteinase 2 (MMP2). No difference was found in the stage of pinopodes development, nor in the coverage of endometrial surface between the studied groups. The expression level for LIF mRNA was lower in control patients compared to idiopathic infertility and recurrent miscarriage patients. No difference was detected in the expression of MMP2 between all studied groups. No correlation was found between pinopodes development stage and LIF and MMP2 expressions in endometrium. Of the studied factors, LIF and pinopodes show the most promise as potential markers of endometrial receptivity. However, the results achieved suggest that these markers are independent of each other. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 615–621

Ocena poziomu transkrypcji klaudyny-4 w eutopowym endometrium kobiet z niepłodnością pierwotną i minimalną endometriozą

Introduction: Claudin-4 (CLDN4) is a transmembrane protein, responsible for cellular contact and organization. A different expression of claudin 4 in the endometrium, depending on the menstrual cycle and with peak at the aim of the ‘implantation window’, has been observed. CLDN4 is believed to play an important role in embryo implantation. The aim: The aim of the study was to compare the mRNA CLDN4 expression levels in two subgroups of infertile women (idiopathic infertility or minimal endometriosis) and compare them to fertile controls. Method: The study included 36 women with idiopathic infertility and 24 with minimal endometriosis. The control group comprised 26 women. Eutopic endometrium samples were collected with a Pipelle device during the implantation window. Firstly, mRNA was extracted from the endometrium and reverse transcribed into cDNA. Real time PCR was used for the assessment of relative expression levels. Results: The observed transcription level of CLDN4 did not differ statistically between the studied groups, but was significantly higher when compared to controls. Conclusions: Exceedingly high levels of CLDN4 might negatively influence fertility rates.Wstęp: Klaudyna 4 (CLDN4) jest białkiem transbłonowym, odpowiedzialnym m.in. za wzajemny kontakt komórek i ich organizację. W endometrium zaobserwowano zmienne natężenie ekspresji CLDN4, zależne od fazy cyklu, z maksimum w „oknie implantacyjnym”. Zakłada się, że CLDN4 może odgrywać istotną rolę w procesie implantacji zarodka. Cel pracy: Celem pracy było porównanie poziomu mRNA CLDN4 w grupach kobiet niepłodnych z niepłodnością idiopatyczną i endometriozą minimalnego stopnia w odniesieniu do grupy kontrolnej. Metoda: Plan badań uzyskał zgodę komisji bioetycznej. Do badań zakwalifikowano 36 kobiet z niepłodnością idiopatyczną, 24 z kobiety endometriozą minimalnego stopnia. Grupa kontrolna obejmowała 26 kobiet. Materiał badawczy stanowiło eutopowe endometrium, pobrane pipellą w oknie implantacyjnym. Z bioptatów wyizolowano RNA. cDNA uzyskano przy pomocy odwrotnej transkrypcji. Do wyznaczenia względnego poziomu transkrypcji zastosowano metodę real-time PCR. Wyniki: Poziom transkryptu CLDN4 nie różnił się w sposób istotny statystycznie pomiędzy grupami badanymi, ale był istotnie statystycznie wyższy w obu grupach badanych w odniesieniu do grupy kontrolnej. Wniosek: Zbyt wysoki poziom ekspresji CLDN4 może mieć związek z zaburzonym rozrodem

Assessment of the transcription levels for the complement activation control system in eutopic endometrium in women with two or more consecutive miscarriages of unknown etiology.

Human endometrium, deciuda and placenta have been shown to express factors that inhibit the complement activation cascade - decay-accelerating factor (DAF), membrane cofactor protein (MCP) and the C3 complement component. In the following study we have analyzed the transcripts levels for DAF, MCP and heparin-binding epidermal growth factor-like growth factor (HB-EGF), the C3 complement component and receptor for vascular endothelial growth factor (VEGFR1) as markers of endometrial unbalance between factors activating the complement system in women with consecutive miscarriages. Study enrolled 30 women with at least two consecutive miscarriages, and 19 healthly women, that comprised the control group. RNA was isolated from endometrial samples. Transcripts levels of DAF and MCP was higher in women with consecutive miscarriages compared to controls, 0.78 vs 5.08 (

Ocena wpływu in vitro niskocząsteczkowej heparyny na ekspresję heparanazy i czynników wzrostu wiążących heparynę w endometrium kobiet z zaburzonym rozrodem

Heparin has a beneficial effect in the treatment of recurrent miscarriages and positively affects implantation rates in the IVF procedure in women with reproductive disorders not associated with thrombophilia. Several studies have indicated that heparin, by blocking the enzymatic activity of heparanase, may affect the structure and function of the extracellular matrix (ECM) and related growth factors. Disturbances in the remodeling (ECM) are believed to be the potential cause of implantation disorders and recurrent miscarriages. Objectives: The aim of the study was the evaluation, on an in vitro model, of the effect of low molecular weight heparin (LMWH) on the expression of heparanase (HPSE) and, important for successful implantation and invasion of trophoblast, heparan sulfate (HS) - binding growth factors, i.e. heparin-binding epidermal growth factor-like (HB-EGF), vascular endothelial growth factor (VEGF-A), fibroblast growth factor (FGF2) in the endometrium, during the implantation window in women with recurrent miscarriage. Method: Biopsy samples, obtained from 10 patients with two or more unexplained miscarriages, were used to construct a co-culture of glandular epithelial cells and stroma. Endometrium in vitro model was supplemented with steroid hormones and enoxaparin 5ug/ml. Using the qPCR, we assessed relative levels of the HPSE, HB-EGF, VEGF-A and FGF2 transcripts in glandular epithelium and stroma in cell culture. Using ELISA, we measured concentrations of the mentioned above factors in culture medium. Results: A statistically significant increase in the relative level of HPSE, HB-EGF, VEGF-A, FGF2 transcripts in the cells of the glandular epithelium and stroma (p<0.001), as well as their increased concentration in the medium of cultures treated with steroid hormones (p<0.001) were observed. However, we found no effect of LMWH supplementation on the level of the investigated factors. Conclusions: Our results show that the importance of the HPSE hydrolytic activity in the endometrium, during the implantation window, may have a secondary function, and/or that beneficial effects of LMWH in women with impaired reproduction have no significant, direct connection with the, catalyzed by HPSE, reconstruction of the ECM and with release of heparin-binding growth factors.Wstęp: Heparyna wykazuje korzystne działanie w leczeniu nawracających poronień, a także wpływa na wzrost odsetka implantacji w programach zapłodnień in vitro u kobiet z zaburzeniami rozrodu, nie związanym z trombofilią. Liczne badania wskazują, że heparyna poprzez blokowanie enzymu heparanazy może wywierać wpływ na strukturę i funkcję macierzy zewnątrzkomórkowej (ECM) i związane z nią czynniki wzrostu. Zaburzenia w remodelingu (ECM) są uznawane za potencjalną przyczynę zaburzeń implantacji i nawracających poronień. Celem tego badania jest ocena na modelu in vitro wpływu niskocząsteczkowej heparyny na ekspresję heparanazy (HPSE) oraz istotnych dla procesu implantacji i inwazji trofoblastu czynników związanych z siarczanem heparanu (HS): wiążącego heparynę nabłonkowego czynnika wzrostu (HB-EGF), czynnika wzrostu śródbłonka (VEGF-A), czynnika wzrostu fibroblastów (FGF2) w endometrium, w oknie implantacyjnym niekoncepcyjnego cyklu u kobiet z nawracającymi poronieniami. Metoda: Bioptaty endometrium pozyskane od 10 pacjentek z dwoma lub więcej niewyjaśnionymi utratami ciąż posłużyły do skonstruowania kokultury komórek nabłonka gruczołowego i podścieliska. Model endometrium in vitro suplementowano hormonami steroidowymi i enoxaparyną sodu o stężeniu 5ug/ml. Przy użyciu metody qPCR oceniono względny poziom transkrypcji HPSE, HB-EGF, VEGF-A, FGF2 w nabłonku gruczołowym i podścielisku. Metodą ELISA oceniono stężenie w/w czynników medium hodowlanym. Wyniki: Zaobserwowano istotny statystycznie wzrost poziomu ekspresji HPSE, HB-EGF, EGF-A, FGF2 w komórkach nabłonka gruczołowego i podścieliska oraz ich stężenia w medium w hodowlach poddanych działaniu hormonów steroidowych. Nie znaleziono natomiast wpływu suplementacji LMWH na poziom badanych czynników. Wnioski: Uzyskane wyniki wskazują, że znaczenie aktywności hydrolitycznej heparanazy w endometrium, w oknie implantacyjnym cyklu niekoncepcyjnego może mieć funkcję drugorzędną i że, korzystne działanie heparyny u kobiet z zaburzonym rozrodem nie ma istotnego, bezpośredniego związku z przebudową macierzy zewnątrzkomórkowej, katalizowaną przez heparanazę i uwalnianiem czynników wzrostu wiążących heparynę

Correlation of the expression of heparanase and heparin-binding EGF-like growth factor in the implantation window of nonconceptual cycle endometrium

Although it was suggested that heparanase (HPSE) may affect implantation and pregnancy, so far there have been no wide-ranging studies on the expression of and possible disturbances in the interactions between HPSE, heparan sulfate (HS) and related growth factors, such as heparin-binding EGF-like growth factor (HB-EGF). The aim of this study was to evaluate whether the expression profile of both HPSE and HB-EGF can be associated with impaired reproduction in the endometrial implantation window, in the non-conception cycle. The study group consisted of 32 women with two or more unexplained, consecutive miscarriages, and 61 idiopathic infertility patients, while the control comprised of 22 women with normal reproductive potential. We compared the expression of HB-EGF and HPSE at the transcript (qPCR) and protein (Western Blot) levels in eutopic endometrium. Also assessed were correlations between both factors in the studied groups. In women with consecutive miscarriages we observed lower HPSE relative transcript (p = 0.003) and lower protein (p = 0.002) level compared with the control group. Level of the HB-EGF protein was decreased (p = 0.017). HPSE mRNA level was higher in idiopathic infertility (p = 0.003) compared with women with miscarriages. We found statistically significant correlations in both transcript and protein levels in all groups (p < 0.05). Our results allow the assumption of the existence of a process by which, in normal human endometrium, HB-EGF expression coincides with the synthesis of HPSE. As a result, the HB-EGF molecule can bind to the HS on the cell surface, enhancing its affinity to the receptor. Then, the release of growth factors associated with HS oligomers occurs that is catalyzed by HPSE. We suggest that one of the causes of unexplained miscarriages may result from the impaired expression of HPSE and HB-EGF

Brak różnic w ekspresji konwertazy proproteinowej 6 u kobiet niepłodnych z endometriozą minimalnego stopnia i u kobiet z niepłodnością idiopatyczną

Abstract Objective: Proprotein convertase 6(PC6) is known to be the key enzyme involved in the transformation of many hormones, cytokines and their receptors into their active forms. Experimental in vitro studies have also proven that lack of PC6 in the endometrium prevents decidualisation. Therefore in our study we have aimed at determining whether infertility in some patients might be attributable to decreased expression of PC6. Material and methods: With the use of RealTime PCR we have studied the expression level of PC6 in receptive phase endometria from 36 idiopathic infertile patients, 26 infertile patients with minimal grade endometriosis and compared those results with fertile, age-matched controls. The endometria were collected 7-9 days after ovulation. Results: There were no statistically significant differences regarding the expression of PC6 in endometria from patients with idiopathic infertility, infertile patients with endometriosis and controls. Conclusions: Since there is no detectable difference in PC6 expression, the decreased expression of PC6 is unlikely to cause infertility.Streszczenie Cel pracy: Konwertaza proproteinowa 6 (PC6) jest kluczowym enzymem biorącym udział w przekształceniu wielu prohormonów, cytokin i ich receptorów w aktywne formy. Badania eksperymentalne in vitro dowiodły, iż brak PC6 uniemożliwia przemianę doczesnową w endometrium. Naszym celem była ocena czy u pacjentek niepłodnych czynnikiem wywołującym niepłodność może być zaburzona ekspresja PC6. Materiał i metoda: Stosujac RT-PCR zbadaliśmy poziom ekspresji PC6 w fazie receptywnej endometrium u 36 kobiet z niepłodnością idiopatyczną, 26 pacjentek z endometriozą minimalną oraz porównaliśmy te wyniki z płodnymi pacjentkami dobranymi pod względem wieku. Endometrium zostało pobrane 7-9 dni po owulacji. Wyniki: Nie stwierdziliśmy statystycznie znamiennych różnic w ekspresji PC6 w endometrium z grupy z niełΠodnością idiopatyczną, niepłodnymi pacjentkami z endometriozą a grupą kontrolną. Wnioski: Wydaje się, że zaburzona ekspresja PC6 nie jest przyczyną niepłodności

TGF superfamily and MMP2, MMP9, TIMP1 genes expression in the endometrium of women with impaired reproduction.

During the putative "implantation window", a period of maximal endometrial receptivity that spans 7-9 days after ovulation, a series of changes on the structural and molecular level occur that render the endometrium susceptible to implantation for the human embryo. Many members of the TGFbetas are expressed by human endometrium at different stages of menstrual cycle. Also studies regarding the MMP2 gene expression and activity of MMP2 in the implantation window have shown a higher expression and activity of MMP2 in women with impaired fertility. We have examined by RT-PCR the expression of TGFbeta2 and MMP2, MMP9 and TIMP1 in 28 patients with idiopathic infertility, 16 patients with unexplained recurrent miscarriage and 16 control women were enrolled in this study. Seven to nine days after ovulation endometrial biopsy by Pipelle or hysteroscopy was performed to assess the expression of TGFbeta2 , MMP2, MMP9 and TIMP1. We found that in endometria from women with idiopathic infertility TGFbeta2 expression was 2.8 fold higher than in endometria from control group and 2.1 fold higher in endometrial samples from women with unexplained recurrent miscarriage compared to the control group. The MMP2, MMP9 and TIMP1 expression in endometrial samples revealed no significant differences between the study groups and control group. There was a statistically significant negative correlation between TGFbeta2 and MMP9 expression in endometria from women in control group. The present investigations suggest that dysregulated TGFbeta2, MMP2, MMP9 and TIMP1 expression are associated with infertility and early pregnancy loss. However the exact mechanism of how overexpression of endometrial TGFbetaand MMPs interferes with implantation may be more complex

HSD3B2, HSD17B1, HSD17B2, ESR1, ESR2 and AR expression in infertile women with endometriosis

Objectives: The development of endometriosis is associated with changes in the expression of genes encoding the 3β-hydroxysteroid dehydrogenase type II (HSD3B2) and 17β-hydroxysteroid dehydrogenase type II (HSD17B2), estrogen receptors 1 (ESR1) and 2 (ESR2) and the androgen receptor (AR). However, little is known about the expression of HSD3B2, HSD17B1, HSD17B2, ESR1 ESR2 and AR during the endometrial phases in eutopic endometrium from infertile women with endometriosis. Material and methods: Using RT-qPCR analysis, we assessed the expression of the studied genes in the follicular and luteal phases in eutopic endometrium from fertile women (n = 17) and infertile women (n = 35) with endometriosis. Results: In the mid-follicular eutopic endometrium, we observed a significant increase in HSD3B2 transcript levels in all infertile women with endometriosis (p = 0.003), in infertile women with stage I/II endometriosis (p = 0.008) and in infertile women with stage III/IV endometriosis (p = 0.009) compared to all fertile women. There was a significant increase in ESR1 tran­scripts in all infertile women with endometriosis (p = 0.008) and in infertile women with stage I/II endometriosis (p = 0.019) and in infertile women with stage III/IV endometriosis (p = 0.023) compared to all fertile women. In the mid-luteal eutopic endometrium, we did not observe significant differences in HSD3B2, HSD17B1, HSD17B2, ESR1, ESR2 and AR transcripts between infertile women with endometriosis and fertile women. Conclusions: Observed significant increase in HSD3B2 and ESR1 transcripts in follicular eutopic endometrium from infer­tile women with endometriosis may be related to abnormal biological effect of E2 in endometrium, further affecting the development of human embryos