Altered Mesenchymal Stem Cells Mechanotransduction from Oxidized Collagen: Morphological and Biophysical Observations.

Extracellular matrix (ECM) provides various mechanical cues that are able to affect the self-renewal and differentiation of mesenchymal stem cells (MSC). Little is known, however, how these cues work in a pathological environment, such as acute oxidative stress. To better understand the behavior of human adipose tissue-derived MSC (ADMSC) in such conditions, we provide morphological and quantitative evidence for significantly altered early steps of mechanotransduction when adhering to oxidized collagen (Col-Oxi). These affect both focal adhesion (FA) formation and YAP/TAZ signaling events. Representative morphological images show that ADMSCs spread better within 2 h of adhesion on native collagen (Col), while they tended to round up on Col-Oxi. It also correlates with the lesser development of the actin cytoskeleton and FA formation, confirmed quantitatively by morphometric analysis using ImageJ. As shown by immunofluorescence analysis, oxidation also affected the ratio of cytosolic-to-nuclear YAP/TAZ activity, concentrating in the nucleus for Col while remaining in the cytosol for Col-Oxi, suggesting abrogated signal transduction. Comparative Atomic Force Microscopy (AFM) studies show that native collagen forms relatively coarse aggregates, much thinner with Col-Oxi, possibly reflecting its altered ability to aggregate. On the other hand, the corresponding Young's moduli were only slightly changed, so viscoelastic properties cannot explain the observed biological differences. However, the roughness of the protein layer decreased dramatically, from RRMS equal to 27.95 ± 5.1 nm for Col to 5.51 ± 0.8 nm for Col-Oxi (p < 0.05), which dictates our conclusion that it is the most altered parameter in oxidation. Thus, it appears to be a predominantly topographic response that affects the mechanotransduction of ADMSCs by oxidized collagen.

Mesenchymal Stem-Cell Remodeling of Adsorbed Type-I Collagen-The Effect of Collagen Oxidation.

This study describes the effect of collagen type I (Col I) oxidation on its physiological remodeling by adipose tissue-derived mesenchymal stem cells (ADMSCs), both mechanical and proteolytic, as an in vitro model for the acute oxidative stress that may occur in vivo upon distinct environmental changes. Morphologically, remodeling was interpreted as the mechanical rearrangement of adsorbed FITC-labelled Col I into a fibril-like pattern. This process was strongly abrogated in cells cultured on oxidized Col I albeit without visible changes in cell morphology. Proteolytic activity was quantified utilizing fluorescence de-quenching (FRET effect). The presence of ADMSCs caused a significant increase in native FITC-Col I fluorescence, which was almost absent in the oxidized samples. Parallel studies in a cell-free system confirmed the enzymatic de-quenching of native FITC-Col I by Clostridial collagenase with statistically significant inhibition occurring in the oxidized samples. Structural changes to the oxidized Col I were further studied by differential scanning calorimetry. In the oxidized samples, an additional endotherm with sustained enthalpy (∆H) was observed at 33.6 °C along with Col I's typical one at 40.5 °C. Collectively, these data support that the remodeling of Col I by ADMSCs is altered upon oxidation due to intrinsic changes to the protein's structure, which represents a novel mechanism for the control of stem cell behavior.

Effect of cryopreservation on the properties of human endometrial stromal cells used in embryo co-culture systems.

PURPOSE: Along with comparative investigation of the decidualization potential and IL-6 secretion by fresh and frozen ESCs, we also aimed to evaluate the effectiveness of co-culture systems based on fresh or frozen ESCs in terms of clinical pregnancy rates. METHODS: Outcome analysis of a total of 215 IVF cycles with co-culture with fresh or frozen ESCs was performed. Endometrial tissue was obtained from 17 healthy donors. Concentrations of secreted prolactin, IGFBP-1, and IL-6 in conditioned media from cultured fresh and frozen ESCs (decidualized or not) were measured using ELISA or ECLIA. RESULTS: Embryo co-culture with frozen ESCs resulted in a much lower pregnancy rate compared to the alternative system using fresh ESCs. Furthermore, cultivated frozen ESCs showed considerably decreased release of prolactin, IGFBP-1, and IL-6 compared to fresh ESCs, indicating that cryopreservation negatively affects their decidualization potential and cytokine production. CONCLUSIONS: Altogether, this data illustrates the need for optimization and improvement of the existing autologous endometrial co-culture systems.

Cells isolated from human glioblastoma multiforme express progesterone-induced blocking factor (PIBF).

Glioblastoma multiforme (GBM) is the most common and malignant tumor in the central nervous system. One of the contemporary hypotheses postulates that its pathogenesis is associated with the cancer stem cells (CSCs) which originate from mutations in the normal neural stem cells residing in their specific "niches." Simultaneously with its aggressive development the tumor suppresses the local immune system by different secreted and/or cell expressed factors. Progesterone-induced blocking factor (PIBF) is an immunomodulatory protein with known role in the regulation of the immune response in the reproductive system. Expression of PIBF has been described in some tumors as one of the factors suppressing the anti-tumor immunity. The aim of the present study was to check for the expression of PIBF from cells isolated from six GBMs. To characterize the cultured cells and to study the PIBF expression confocal microscopy, flow cytometry, ELISA, and real-time PCR were used. The results obtained showed expression of markers typical for cancer CSCs and secretion of interleukin 6 by the GBM-derived cultured cells. The results convincingly prove that PIBF is intracellularly expressed by the cultured cells from the all six GBM samples, and this fact is confirmed by three different methods-flow cytometry, confocal microscopy, and real-time PCR. This paper reports for the first time the expression of PIBF by GBM-derived cells cultured in vitro and reveals a new aspect of the immunosuppressive mechanism used by GBM in escaping the immune control.

Conditioned medium from adipose tissue-derived mesenchymal stem cells induces CD4+FOXP3+ cells and increases IL-10 secretion.

Mesenchymal stem cells (MSCs) are a new and promising tool for therapy of autoimmune disorders. In recent years their possibility to take part in the modulation of the immune response is discussed. The exact mechanisms for immunoregulation realized by MSCs are not clear yet, but interactions with other immunoregulatory cells may be involved in this process. The investigation of the influence of MSCs on the expression of FoxP3 and cytokine secretion by T helper cells was the aim of this study. T helper cells were isolated from PBMCs by magnetic separation and MSCs were isolated from human adipose tissue, and CD4⁺ T cells were cultured with conditional medium of MSCs. The methods which were used include flow cytometry, ELISA, and Human Proteome profiler kits. The results demonstrated that secretory factors in MSCs conditional medium lead to increased expression of FoxP3 and increased secretion of IL-10 by T helpers. The obtained results give us opportunity to discuss the interaction between two kinds of immunoregulatory cells: MSCs and FoxP3⁺ T helpers. We suppose that this interaction leads to increased number of immunosuppressive helpers which secrete IL-10. MSCs provide some of their immunosuppressive functions acting on T regulatory cells, and we believe that IL-6 secreted by MSCs is involved in this process.

Morphological and Quantitative Evidence for Altered Mesenchymal Stem Cell Remodeling of Collagen in an Oxidative Environment-Peculiar Effect of Epigallocatechin-3-Gallate.

Mesenchymal stem cells (MSCs) are involved in the process of extracellular matrix (ECM) remodeling where collagens play a pivotal role. We recently demonstrated that the remodeling of adsorbed collagen type I might be disordered upon oxidation following its fate in the presence of human adipose-derived MSC (ADMSCs). With the present study we intended to learn more about the effect of polyphenolic antioxidant Epigallocatechin-3-gallate (EGCG), attempting to mimic the conditions of oxidative stress in vivo and its putative prevention by antioxidants. Collagen Type I was isolated from mouse tail tendon (MTC) and labelled with FITC before being oxidized according to Fe2+/H2O2 protocol. FITC-collagen remodeling by ADMSC was assessed morphologically before and after EGCG pretreatment and confirmed via detailed morphometric analysis measuring the anisotropy index (AI) and fluorescence intensity (FI) in selected regions of interest (ROI), namely: outside the cells, over the cells, and central (nuclear/perinuclear) region, whereas the pericellular proteolytic activity was measured by de-quenching fluorescent collagen probes (FRET effect). Here we provide morphological evidence that MTC undergoes significant reorganization by the adhering ADMSC and is accompanied by a substantial activation of pericellular proteolysis, and further confirm that both processes are suppressed upon collagen oxidation. An important observation was that this abrogated remodeling cannot be prevented by the EGCG pretreatment. Conversely, the detailed morphometric analysis showed that oxidized FITC-collagen tends to accumulate beneath cells and around cell nuclei, suggesting the activation of alternative routes for its removal, such as internalization and/or transcytosis. Morphometric analysis also revealed that both processes are supported by EGCG pretreatment.

A study of the transformation of umbilical cord mesenchymal stem cells by interferon-gamma.

OBJECTIVES: Mesenchymal stem cells (MSCs) exist in almost all tissues. Their unique nature is completed by their immunomodulatory functions, holding promise for the treatment of many diseases. An inflammatory environment precedes the immunosuppressive abilities of MSCs and this study was intended to better understand how umbilical cord MSCs (UCMSCs) react to the process of inflammation, regarding their basic characteristics and behavior when primed with the key pro-inflammatory cytokine, Interferon-γ (IFNγ). MATERIALS AND METHODS: Human MSCs from the umbilical cord were isolated, expanded, and treated with IFNγ. Primed cells were analyzed to define their ability to form colonies, their morphology, differentiation potential, proliferation, and apoptosis rate. RESULTS: UCMSCs treated with IFNγ changed their fibroblast-like morphology and retained the expression of typical MSCs markers. IFNγ treated UCMSCs had significantly higher MFI levels regarding the expression of HLA-I (980.43 ± 556.64) and PD-L1 (598.04 ± 416.90) compared with the control cells (144.97 ± 78.5 and 122.05 ± 103.83, respectively; P<0.01). Under the influence of IFNγ, the cells had a lower population doubling time compared with the control cultures (50.345 ± 9.155 versus 61.135 ± 21.110, respectively; P<0.01) and higher numbers of colony-forming unit-fibroblasts (26.0 ± 12.2 versus 10.2 ± 8.0, respectively; P<0.05). The primed MSCs could not undergo osteogenic and adipogenic differentiation. IFNγ increased the percentage of cells in the apoptotic state on day eight (29.470 ± 6.59 versus 15.708 ± 6.190, respectively; P<0.01). CONCLUSION: The properties of UCMSCs can be influenced by the pro-inflammatory cytokine IFNγ.

New target cells of the immunomodulatory effects of progesterone.

It is well known that the reproductive steroid hormones, particularly progesterone, in addition to its widely recognized effects on endometrial epithelial and stromal cells and spiral arteries, affect the activities of T cells and natural killer cells in the deciduas, thus inducing active immune tolerance against the fetal antigens. The immunomodulatory effects of progesterone on T cells, B cells and natural killer cells have been discussed extensively in the literature. The aim of the present review is to sum up and discuss the results from this and other laboratories of investigations on the effects of progesterone on dendritic cells and adult stem cells, which are some of the other cell populations present at the fetal-maternal interface and possibly are related to the immunoregulation during pregnancy. These cells have been shown to have a number of specific functions but their involvement in the entire process of regulation of the immune response in pregnancy is still under discussion. The present review focuses on facts showing that the progesterone is a kind of 'regulator of regulators' in the decidua, thus creating the most favourable conditions for the development of the semi-allogeneic fetus in successful pregnancy.

Assessment of presence and characteristics of multipotent stromal cells in human endometrium and decidua.

This review discusses the presence and characteristics of multipotent stromal cells in human endometrium and decidua. A number of research groups have reported the isolation and characterization of multipotent stromal cells from the basal layer of the endometrium, and in a single case just from the menstrual blood, i.e. the superficial functional layer. Similarly, multipotent pre-decidual stromal cells are isolated from early decidua and characterized accordingly. Multipotent endometrial stromal cells and multipotent decidual stromal cells are shown to express the basic features of adult stem cells, which are clonogenicity, self-renewal, a potential to differentiate into adipogenic, osteogenic, chrondrogenic, endothelial-like cells and a specific set of surface molecules (CD73, CD90 and CD105). So far, it is not clear whether the same population of multipotent stromal cells is isolated from the basal endometrium or early decidua because it has been shown that in some cases the differentiation potential of endometrial stromal cells is more restricted in comparison to the decidual stromal cells. It is reasonable to assume that it is one cell population under different control by hormonal, paracrine and autocrine factors. Thus far, the functions of these cells have not been convincingly revealed.

Production and characterization of a novel monoclonal antibody against progesterone-induced blocking factor (PIBF).

Progesterone-induced blocking factor (PIBF) has been described as an active factor intimately involved in regulation of the immune response in pregnancy. It has been shown that PIBF biased the cytokine balance to Th2-type in pregnancy and inhibited the activity of NK cells. The biological roles of PIBF would be better defined if methods for its detection and measurement in biological fluids are available. However, so far, reliable antibodies have not been developed to be used as specific probes. A monoclonal antibody designated as MAB 3A6 was produced and characterized. MAB 3A6 reacts specifically with PIBF. It can detect this protein in biological fluids when tested by immunoblot and recognizes PIBF expressed on the surface of lymphocytes of pregnant women stimulated in vitro with progesterone. The characteristics of MAB 3A6 makes it the possible basis for development of a clinically applicable assay to assess the presence and concentration of PIBF in biological samples.

Osteogenic differentiation of mesenchymal stem cells using hybrid nanofibers with different configurations and dimensionality.

Novel, hybrid fibrinogen/polylactic acid (FBG/PLA) nanofibers with different configuration (random vs aligned) and dimensionality (2-D vs 3-D environment) were used to control the overall behavior and the osteogenic differentiation of human adipose-derived mesenchymal stem cells (ADMSCs). Aligned nanofibers in both the 2-D and 3-D configurations are proved to be favored for osteodifferentiation. Morphologically, we found that on randomly configured nanofibers, the cells developed a stellate-like morphology with multiple projections; however, time-lapse analysis showed significantly diminished cell movements. Conversely, an elongated cell shape with advanced cell spreading and extended actin cytoskeleton accompanied with significantly increased cell mobility were observed when cells attached on aligned nanofibers. Moreover, a clear tendency for higher alkaline phosphatase activity was also found on aligned fibers when ADMSCs were switched to osteogenic induction medium. The strongest accumulation of Alizarin red (AR) and von Kossa stain at 21 days of culture in osteogenic medium were found on 3-D aligned constructs while the rest showed lower and rather undistinguishable activity. Quantitative reverse transcription-polymerase chain reaction analysis for Osteopontin (OSP) and RUNX 2 generally confirmed this trend showing favorable expression of osteogenic genes activity in 3-D environment particularly in aligned configuration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2065-2074, 2017.

Biochemical and immunochemical characterization of boar sperm flagellar protein with role in hyperactivation/capacitation process.

Investigations on specific and functionally active sperm antigens could bring about the elucidation of the mechanisms of gamete interaction and help the search for new approaches in prognosis and regulation of fertility. Previously, we reported that the monoclonal antibody (Mab) 3G4 against capacitated boar spermatozoa was capable of inhibiting boar sperm-porcine zona pellucida binding due to its inhibitory effect on sperm hyperactivation and capacitation. The cell and tissue specificity of Mab 3G4 was demonstrated in indirect immunofluorescence (IIF) and ELISA experiments against spermatozoa from different vertebrate species, as well as against extracts of boar reproductive and somatic organs. In the present IIF experiments, it was shown that Mab 3G4 recognized an antigen determinant on the flagellar midpiece region of ejaculated and capacitated boar spermatozoa. It was speculated that the Mab 3G4-corresponding antigen participates in pyruvate/lactate metabolism because of its specific localization in the sperm structure, which is responsible for producing forward motility and its involvement in processes that require the metabolism of pyruvate and lactate. As a possible approach toward investigating the participation of Ag 3G4 in pyruvate/lactate metabolism, Mab 3G4's effect on lactate dehydrogenase (LDH) was examined. Using an electrophoretic approach we provided evidence that Mab 3G4 stimulates LDH activity in the Triton X-100 and NP40 protein fractions of capacitated boar spermatozoa. In addition, we found that LDH isoenzymes stimulated by Mab 3G4 are of gametic C type. In Western blot, under nonreducing conditions, Mab 3G4 identified a single protein band with a molecular weight of 140 kDa. The biochemical and immunochemical experiments provided evidence supporting the involvement of 3G4 antigen in the sperm pyruvate/lactate metabolism.

Humoral immune response in prostate cancer patients after immunization with gene-based vaccines that encode for a protein that is proteasomally degraded.

Prostate-specific membrane antigen (PSMA), whose expression is upregulated in poorly differentiated, metastatic, and hormone refractory prostate cancer, could be targeted by gene-based vaccines. The aim of this study was to characterize the humoral immune response against PSMA in prostate carcinoma patients who have been vaccinated against PSMA with gene-based vaccines. Sera from prostate cancer patients who had been immunized repeatedly with plasmid DNA and a recombinant adenoviral vector, both carrying an expression cassette for human PSMA, and sera from healthy donors were tested for anti-PSMA antibodies by Western blot analysis and immunofluorescence. PSMA-producing LNCaP cells, recombinant PSMA protein, and a specific antibody against PSMA were used as positive controls. Specific anti-PSMA antibodies were detected by both Western blot and immunofluorescence in the sera of patients who had been vaccinated against PSMA with plasmid and recombinant adenoviral vectors. The specificity of the anti-PSMA antibodies was confirmed by preincubation and blocking experiments. Positive reactions were detected in 86% of the vaccinated prostate cancer patients. Anti-PSMA antibodies were not detected either in the patients' sera prior to vaccination or in the sera from healthy men and women. These data demonstrate that PSMA, a specific marker for prostate cancer, is a target for humoral immune response induced by gene-based PSMA vaccination. Detection of anti-PSMA antibodies by immunoblot analysis and by indirect immunofluorescence could be used to monitor the vaccination effect.

FGF-1 and S100A13 possibly contribute to angiogenesis in endometriosis.

Endometriosis is referred often as an angiogenic disease. The pivotal role of angiogenesis in the pathophysiology of this disease has been confirmed by many studies. This process has several steps, and VEGF is probably the most important in its initiation. There are others involved in its continuation and maintenance of the tight balance between a quiescent and activated blood vessel state. In the process of formation of new blood capillaries and arterioles, many different factors are involved in sometimes distinct pathways. Such factors are TGF-beta and endoglin--the latter being one of the main modulators of the TGF-beta signaling pathway. Endoglin is now not only established as a marker of active neo-angiogenesis and activated endothelium, but also turns to be an active player in the very process of endometriotic angiogenesis. Its signaling pathway of hypoxic activation is tightly interconnected with that of VEGF, and also some of the FGFs. FGF-1 and S100A13 are members of two distinct families of proteins -- the FGFs, growth and angiogenic factors, and that of the S100 proteins, -- Ca(2+)-binding proteins involved in cell function regulation, motility and signaling. These two particular members are quite unique in having no signal peptide sequence and being involved in common export pathway. Our hypothesis is that these two factors are involved in vascular remodeling in endometriotic angiogenesis, playing a role in vascular wall formation and migration of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). We believe also that endoglin is tightly involved in the new arteriolar formation in endometriosis, being expressed in VSMCs but not on the ECs of the middle-sized vessels.

Endoglin (cd105) and S100A13 as markers of active angiogenesis in endometriosis.

The aim of the present study was to evaluate the expression of the neo-angiogenic marker endoglin and its localization in tissues of normal and endometriotic patients as well as to compare it with one new angiogenic marker candidate - S100A13. Human recombinant S100A13 and endoglin 35mer synthetic peptide of the intracellular domain were used for the production of rabbit polyclonal antisera. The antisera were characterized for specificity, using immunoenzyme assay (ELISA), Western blot and immunohistochemistry. Formalin-fixed, paraffin-embedded tissue sections from normal endometrium, adenomyosis, ovarian endometriosis, eutopic endometrium from different endometriotic specimens were tested by immunohistochemistry. No endoglin specific staining was observed on the microvessels of the normal endometrium. In adenomyosis and ovarian endometriosis, the expression pattern was different - endoglin was expressed in all microvessels, with an even stronger expression in the myometrial compartment. Weak endoglin-positive staining was detected in the microvessels of eutopic endometrium specimens from different endometriosis cases. In comparison to endoglin, S100A13 exhibited a moderate expression in endometrial glands of normal endometrium, but strong expression in endometriotic specimens. No S100A13 extensive staining of the microvessels was observed in normal endometrium, while in endometriosis, it exhibited very intense staining in microvascular endothelia and less intense in the perivascular area of middle to large-sized vessels. This study for the first time shows over-expression of S100A13 in endometriosis. These data show that the expression of endoglin and S100A13 corresponds to the activation of the endothelial cells in the process of endometriotic angiogenesis, suggesting a beneficial role for these two molecules as markers for actively progressing endometriotic process.

Potential cellular conformations of the CCN3(NOV) protein.

AIM: To study the cellular distribution of CCN3(NOV) and to determine if the carboxyterminus of CCN3 is hidden or masked due to high affinity interactions with other partners. CCN3 was detected using affinity purified antibodies (anti-K19M-AF) as well as a Protein A purified anti-K19M antibodies (anti-K19M IgG) against a C-terminal 19-aminoacid peptide (K19M) of human CCN3 protein. The antibodies were applied in indirect immunofluorescence tests and immunoenzyme assays on glial tumor cell line, G59, and its CCN3-transfected variant G59/540 and the adrenocortical cell line, NCI-H295R. RESULTS: Anti-K19M-AF antibodies reacted against K19M peptide in ELISA and recognized two bands of 51 kDa and 30 kDa in H295R (adrenocortical carcinoma) cell culture supernatants by immunoblotting. H295R culture supernatants which contained CCN3 as shown by immunoblotting did not react with anti-CCN3 antibodies in liquid phase. Anti-CCN3 antibodies stained the surface membranes of non-permeabilized H295R and cytoplasm in permeabilized H295R cells. Similarly, anti-CCN3 stained surface membranes of G59/540, but did not react with G59 cells. Prominent cytoplasmic staining was observed in G59/540, as well as the cell footprints of G59/540 and H295R were strongly labeled. CONCLUSIONS: The K19M-AF antibody directed against the C-terminal 19-aminoacid peptide of CCN3 recognized the secreted protein under denaturing conditions. However, the C-terminal motif of secreted CCN3 was not accessible to K19M-AF in liquid phase. These anti-CCN3 antibodies stained CCN3 protein which was localized to cytoplasmic stores, cell membranes and extracellular matrix. This would suggest that cytoplasmic and cell membrane bound CCN3 has an exposed C-terminus while secreted CCN3 has a sequestered C-terminus which could be due to interaction with other proteins or itself (dimerization). Thus the K19M-AF antibodies revealed at least two conformational states of the native CCN3 protein.

Effects of low dose hormone replacement therapy on markers of inflammation in postmenopausal women.

OBJECTIVE: Concentrations of soluble fractions of cell adhaesion molecules [sCAM], C-reactive protein (CRP) and serum amyloid A (SAA) are predictive for future cardiovascular events in postmenopausal women. The effect of hormone replacement therapy (HRT) on these inflammatory markers is not uniform. In the presented study the effect of a low-dose HRT preparation, on sCAM, CRP and SAA in healthy postmenopausal women was evaluated. METHODS: Serum levels of intracellular adhesion molecules (sICAM-1), vascular cell adhesion molecules (sVCAM-1), P-selectin, CRP and SAA were measured at baseline and after 12 weeks of treatment with continuous combined HRT containing 1 mg micronized 17beta-estradiol and 0.5 mg norethisterone acetate. The concentrations of total cholesterol, triglycerides LDL-cholesterol and HDL-c were measured as well. RESULTS: The studied low dose continuous combined therapy significantly reduced the concentration of sICAM-1, sVCAM-1 and P-selectin by 25.3, 20 and 34%, respectively. Despite statistically not significant the concentration of CRP and SAA increased by 35.6 and 9.4%, respectively. A statistically significant decrease in the triglycerides and TC/HDL-cholesterol ratio has been found. CONCLUSIONS: The outcomes of the presented study suggest that HRT with low dose continuous combined HRT containing 1 mg micronized 17beta-estradiol and 0.5 mg norethisterone acetate have divergent effects on studied parameters of vascular inflammation. These effects are similar to effects of other studied HRT combinations. To our best knowledge this is the first study evaluating the effect of HRT on the concentration of SAA.

Recurrent implantation failure: the role of the endometrium.

The success rate of reproductive treatment methods depends on many different factors. The most important and discussed ones in the literature are maternal age, the causes of infertility, the ovarian response to stimulation, the influence of the male factor and sperm quality, embryo quality and the various uterine pathologies. Some couples fail repeatedly after transferring good quality embryos without any obvious reason and this becomes a major continuing problem after IVF/ICSI procedures. It can be speculated that in these couples, insufficiency of the endometrium might be a possible reason for implantation failure. This review article summarized current literature describing the consecutive endomertial procedures involved in successful embryo implantation. It is believed that efforts to align criteria for definition of recurrent implantation failure (RIF) and attempts to classify different RIF types would develop guidelines for treatment procedures which would result in an increase in patients' opportunities to conceive.

Secretion of immunoregulatory cytokines by mesenchymal stem cells.

According to the minimal criteria of the International Society of Cellular Therapy, mesenchymal stem cells (MSCs) are a population of undifferentiated cells defined by their ability to adhere to plastic surfaces when cultured under standard conditions, express a certain panel of phenotypic markers and can differentiate into osteogenic, chondrogenic and adipogenic lineages when cultured in specific inducing media. In parallel with their major role as undifferentiated cell reserves, MSCs have immunomodulatory functions which are exerted by direct cell-to-cell contacts, secretion of cytokines and/or by a combination of both mechanisms. There are no convincing data about a principal difference in the profile of cytokines secreted by MSCs isolated from different tissue sources, although some papers report some quantitative but not qualitative differences in cytokine secretion. The present review focuses on the basic cytokines secreted by MSCs as described in the literature by which the MSCs exert immunodulatory effects. It should be pointed out that MSCs themselves are objects of cytokine regulation. Hypothetical mechanisms by which the MSCs exert their immunoregulatory effects are also discussed in this review. These mechanisms may either influence the target immune cells directly or indirectly by affecting the activities of predominantly dendritic cells. Chemokines are also discussed as participants in this process by recruiting cells of the immune systems and thus making them targets of immunosuppression. This review aims to present and discuss the published data and the personal experience of the authors regarding cytokines secreted by MSCs and their effects on the cells of the immune system.

Stem cells in the reproductive system.

This review article summarizes current knowledge on regulation, functions, and capacities of stem cells in the female and male reproductive tract. Major locations in which pluripotent cells reside and from where they can be isolated are the ovaries, the endometrium, the decidua, and the testis. They include oocytes, embryonic stem cells, trophoblast stem cells, and spermatogonial stem cells, but also several side populations, which can be obtained after certain isolation and culture procedures. The potential of pluripotent cells in the reproductive tract to differentiate is manifold, but heterogenous, depending upon their respective origin. As stem cells have a potential for future application in transplantation and regenerative medicine, this article also reviews the literature on major histocompatibility complex expression on stem cells of the reproductive tract, because of its immunogenic effects, but also because of its potential expression of HLA-G, a potent immunomodulator mainly associated with trophoblast cells.