NK and T Cell Subtypes in the Endometrium of Patients with Recurrent Pregnancy Loss and Recurrent Implantation Failure: Implications for Pregnancy Success.

BACKGROUND: RPL and RIF are challenges in reproductive medicine. The immune system plays a pivotal role in endometrial receptivity, successful implantation, and pregnancy complications. Immunological changes have been associated with RPL and RIF. Understanding immune dysregulation especially in NK and T cell subtypes may lead to better diagnostic concepts and treatments. From July 2019 to August 2020 patients with RPL and RIF underwent a standardized diagnostic procedure including endometrial biopsies. Immune cell analysis was performed using flow cytometry. Patients were contacted in March 2023 and interviewed concerning their pregnancy outcomes following diagnostics. RESULTS: Out of 68 patients undergoing endometrial biopsies, 49 patients were finally included. Live birth rates were high with 72% in RPL and 86% in RIF. Immune cell analysis revealed that patients with RPL had more cytotoxic CD56dimCD16high cells, while RIF patients had more CD56+ uNK cells. RPL patients with pregnancy complications showed increased NKT cell percentages. CONCLUSION: Our findings suggest specific immune changes in RPL and RIF patients, offering potential therapeutic targets. Tailored immunotherapy based on endometrial immunophenotyping might be an option, but further research is needed.

Uterine microbiota plasticity during the menstrual cycle: Differences between healthy controls and patients with recurrent miscarriage or implantation failure.

In contrast to the former notion of a sterile womb, sequencing techniques have proven a bacterial colonization of the uterus. However, timing of microbiota analysis regarding possible intra-cycle variations as well as specific alterations in patients with recurrent miscarriage (RM) or recurrent implantation failure (RIF) remain unknown. In total, n = 20 RM-, n = 20 RIF-patients and n = 10 healthy controls were included in this prospective study. In every subject, uterine flushing was performed during follicular, ovulatory and luteal phase. Bacterial DNA was isolated and 16S amplicon sequencing analysis of the V3-V4 region was carried out. Diversity measures were compared between samples from the disease groups and the control group separately for each timepoint of the menstrual cycle and over time. In the control group a significant decrease of species richness and evenness was shown around ovulation which remained at this lower level during the luteal phase (Shannon index), indicating a more uniform distribution of microbiota (p < 0.05). This loss of diversity during the menstrual cycle was not apparent in RIF and RM patients. A higher similarity was seen in taxonomic distribution between RM and RIF patients compared to the control group. Longitudinal dynamics included increases in Firmicutes (controls and RM only) and a concomitant loss of Proteobacteria in controls that was not present in RIF and RM. We demonstrate longitudinal intra-cycle-dependent changes in the endometrial microbiota of healthy controls. An increased diversity in both patient groups could be the cause or consequence of a micro-environment that is more prone to pregnancy failures.

Dubious effects of methadone as an "anticancer" drug on ovarian cancer cell-lines and patient-derived tumor-spheroids.

BACKGROUND: The opioid agonist D,L-methadone exerts analgesic effects via the mu opioid receptor, encoded by OPRM1 and therefore plays a role in chronic pain management. In preclinical tumor-models D,L-methadone shows apoptotic and chemo-sensitizing effects and was therefore hyped as an off-label "anticancer" drug without substantiation from clinical trials. Its effects in ovarian cancer (OC) are completely unexplored. METHODS: We analyzed OPRM1-mRNA expression in six cisplatin-sensitive, two cisplatin-resistant OC cell-lines, 170 OC tissue samples and 12 non-neoplastic control tissues. Pro-angiogenetic, cytotoxic and apoptotic effects of D,L-methadone were evaluated in OC cell-lines and four patient-derived tumor-spheroid models. RESULTS: OPRM1 was transcriptionally expressed in 69% of OC-tissues and in three of eight OC cell-lines. D,L-methadone exposure significantly reduced cell-viability in five OC cell-lines irrespective of OPRM1 expression. D,L-methadone, applied alone or combined with cisplatin, showed no significant effects on apoptosis or VEGF secretion in cell-lines. Notably, in two of the four spheroid models, treatment with D,L-methadone significantly enhanced cell growth (by up to 121%), especially after long-term exposure. This is consistent with the observed attenuation of the inhibitory effects of cisplatin in three spheroid models when adding D,L-methadone. The effect of methadone treatment on VEGF secretion in tumor-spheroids was inconclusive. CONCLUSIONS: Our study demonstrates that certain OC samples express OPRM1, which, however, is not a prerequisite for D,L-methadone function. As such, D,L-methadone may exert also detrimental effects by stimulating the growth of certain OC-cells and abrogating cisplatin's therapeutic effect.

Different Background: Natural Killer Cell Profiles in Secondary versus Primary Recurrent Pregnancy Loss.

(1) Background: Prior studies suggested a significant impact of previous live births on peripheral natural killer cells (pNK) in patients with recurrent pregnancy loss (RPL). Patients with primary RPL (pRPL, no live birth) showed higher numbers of pNK than secondary RPL patients (sRPL, ≥ 1 live birth). (2) Methods: To further determine immunological differences between RPL patients and controls, we analysed pNK subpopulations and activation markers in pRPL (n = 47), sRPL (n = 24) and controls with previous live birth (sCtrl, n = 25) and nullipara (pCtrl, n = 60) within a prospective study. Percentages and numbers of CD56dimCD16bright cells, subpopulations and activation markers (CD57+, CD62L+, NKG2D+, NKp46+) were measured in non-pregnant RPL patients and n = 85 controls (n = 60 pCtrl, n = 25 sCtrl) in the mid-luteal phase by flow cytometry. (3) Results: Compared to sRPL patients, sCtrls showed higher CD56+ and CD56dimCD16bright numbers. Further, sRPL patients showed lower numbers of CD56dimCD16brightNKG2D+ and CD56dimCD16brightNKp46+ than sCtrls. (4) Conclusion: We suggest a chronic immune stimulation leading to a lower NK-cell count in sRPL patients with a lower NK cytotoxicity. This underlines the necessity to investigate pNK subpopulations as well as pRPL and sRPL separately to delineate the immune alterations in RPL.

Differential integrin expression in pre-implantation embryos developing under in vivo and in vitro conditions.

Implantation failure is a major problem in human assisted reproduction, which persists regardless the optimization of endometrial receptivity and selection of genetically and morphologically healthy embryos. Since embryo-endometrium interaction depends on cell junctional, cell adhesion and cell-substratum adhesion molecules, the present study inquired whether in vitro growing murine embryos display similar to the in vivo growing embryos patterns of adhesion molecules. To this extend aVb3 expression and distribution in zygotes and 2-cell stage embryos were studied. The results demonstrated that only the in vivo growing embryos displayed specifically polarized aVb3 distribution, indicating their potential successful interaction with endometrium. Based on previous studies showing that L-carnitine (L-Cn) could affect embryonic development, it was demonstrated that the addition of L-Cn to the culture medium, could lead the in vitro growing embryos to acquire aVb3 expression and distribution similar to the in vivo growing embryos. Visualization of the effect of L-Cn using third harmonic generation imaging showed decreased lipid droplet levels in 2-cell-stage embryos, observation that correlates with an active energetic state of the growing embryos. Thus, the application of L-Cn to the culture medium could assist pre-implantation-state embryos to acquire aVb3 expression and distribution similar to the in vivo developing conditions.

Protection Against Lipopolysaccharide-Induced Immunosuppression by IgG and IgM.

Lipopolysaccharide (LPS) is commonly used in murine sepsis models, which are largely associated with immunosuppression and collapse of the immune system. After adapting the LPS treatment to the needs of locally bred BALB/c mice, the present study explored the potential role of IgG and IgM in reversing LPS endotoxemia. The established protocol consisted of five daily intraperitoneal injections of 0.2 µg/g LPS, which was tolerable by half of the manipulated animals. Such a protocol allowed longer survival, necessary in the prospect of therapeutic treatment application. This treatment significantly decreased CD4+, CD8+, CD3z+, and CD19+ cells, while increasing myeloid-derived suppressor cells (MDSCs; CD11b+Gr1+), CD25+ and Foxp3+ cells. These results were accompanied by increased arginase-1 activity in spleen cell lysates and production of IL-6, TNF-α, IL-18, and C-reactive protein (CRP) in the serum. The applied LPS protocol did not alter serum procalcitonin levels. MDSCs isolated from the spleen of LPS-treated animals (LPS-MDSCs) decreased proliferation of naive T cells in coculture experiments. The application of IgG and IgM to the naive T cell/LPS-MDSCs cocultures significantly decreased CD25+, Foxp3+, and CD3z+ cells, indicating an anti-suppressive effect of immunoglobulins. The in vivo application of IgG and IgM significantly decreased the percent of CD11b+Gr1+, CD25+, Foxp3+ cells, and arginase-1 activity in the spleen of LPS-treated animals, while decreasing IL-6, TNF-α, and CRP levels in the serum, allowing survival to all animals tested. In conclusion, these results reveal a novel mode of action of IgG/IgM in LPS endotoxemia, strengthening thus the use of immunoglobulin treatment is septic patients.

l-Carnitine affects preimplantation embryo development toward infertility in mice.

l-Carnitine (l-Cn), despite the beneficial role as energy-generating substance delivering long-chain fatty acids to the ß-oxidation pathway in mitochondria, has been accused to cause an endometriosis-like state to BALB/c mice manifested by increased inflammatory cytokines in serum and peritoneal fluid, accumulation of immune cells in the peritoneal cavity and uterine walls and most importantly, correlating to infertility. Exploring this type of infertility, the effect of l-Cn on preimplantation embryo development, ovarian integrity and systemic maternal immunity was studied. Using nonlinear microscopy analysis, which was shown to be a powerful tool for determining embryo quality by quantitatively estimating the lipid body (LB) content of the cells, it was shown that in vitro and in vivo administration of l-Cn significantly decreased LB mean area in zygotes. Daily intraperitoneal administration of 2.5mg l-Cn for 3, 4 and 7days to mice significantly decreased the percent of normal zygotes. However, only the 7-day treatment persisted by affecting 2- and 8-cell stage embryos, while almost abolishing blastocyst development. Such effects were accompanied by abnormal ovarian histology, showing increased numbers of corpora luteus and elevated progesterone concentration in the serum. In addition, it was shown that the 7-day l-Cn treatment pushed maternal systemic immunity toward inflammation and immunosuppression by increasing CD11b-, CD25- and CD11bGr1-positive cells in spleen, which opposed the necessity for immunostimulation at these early stages of pregnancy. In conclusion, the results presented here demonstrated that elevated doses of l-Cn affect early stages of embryo development, leading to infertility.

Characterization of antigen-binding and MHC class II-bearing T cells with suppressive activity in response to tolerogenic stimulus.

Antigen specific non-responsiveness is generally developed through clonal deletion, anergy, and suppression. The term "suppression" is being considered as a functional immune deficit that can be adoptively transferred by regulatory/suppressor T cells. Following tolerance induction protocols the aim of the present study was to characterize the T cells involved in antigen-specific suppression. After defining the immunogenic and tolerogenic protocols in vitro and in vivo, it was shown that CD90(+) cells from tolerogenic mice were able to reduce specific antibody production when adaptively transferred to immunized mice. These cells were shown to highly express CD25 and Foxp3, co-localizing with CD4 and MHC class II antigens (MHCII), while a small percentage of these cells (8-14%) was binding free antigen. Further isolation of CD90(+)MHCII(+) and CD90(+)HSA(+) from mice having received the tolerogenic treatment and adaptive transfer to immunized mice showed that CD90(+)MHCII(+) cells were able to suppress antigen-specific response only when transferred along with the second antigenic challenge, while CD90(+)HSA(+) cells were suppressive only when given along with the first antigenic challenge. The suppressive effect of these two sub-populations could also be obtained in in vitro spleen cell proliferation assays in response to the HSA antigenic stimulus. Both in vitro and in vivo tolerogenic treatments were shown to correlate with soluble MHCII production in culture supernatants or serum respectively. Increase of MHCII in the serum could only be detected upon adaptive transfer of CD90(+)HSA(+) cells, whereas transfer of CD90(+)MHCII(+) cells resulted in increased levels of IL-10 and IFN-γ in the serum. These results defined at least two different levels of suppression, one during the onset which was antigen-specific and MHC restricted, and another non-specific at the end of an immune response.

Following the course of pre-implantation embryo patterning by non-linear microscopy.

Embryo patterning is subject to intense investigation. So far only large, microscopically obvious structures like polar body, cleavage furrow, pro-nucleus shape can be evaluated in the intact embryo. Using non-linear microscopic techniques, the present work describes new methodologies to evaluate pre-implantation mouse embryo patterning. Third Harmonic Generation (THG) imaging, by detecting mitochondrial/lipid body structures, could provide valuable and complementary information as to the energetic status of pre-implantation embryos, time evolution of different developmental stages, embryo polarization prior to mitotic division and blastomere equivalence. Quantification of THG imaging detected highest signalling in the 2-cell stage embryos, while evaluating a 12-18% difference between blastomeres at the 8-cell stage embryos. Such a methodology provides novel, non-intrusive imaging assays to follow up intracellular structural patterning associated with the energetic status of a developing embryo, which could be successfully used for embryo selection during the in vitro fertilization process.

The crystal structure of cobalt-substituted pseudoazurin from Alcaligenes faecalis.

The Cu(II) center at the active site of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by Co(II) via denaturing of the protein, chelation and removal of copper by EDTA and refolding of the apo-protein, followed by addition of an aqueous solution of CoCl(2). Sitting drop vapour diffusion experiments produced green hexagonal crystals, which belong to space group P6(5), with unit cell dimensions a = b = 50.03, c = 98.80 Å. Diffraction data, collected at 291 K on a copper rotating anode X-ray source, were phased by the anomalous signal of the cobalt atom. The structure was built automatically, fitted manually and subsequently refined to 1.86 Å resolution. The Co-substituted protein exhibits similar overall geometry to the native structure with copper. Cobalt binds more strongly to the axial Met86-Sδ and retains the tetrahedral arrangement with the four ligand atoms, His40-Nδ(1), Cys78-Sγ, His81-Nδ(1), and 86Met-Sδ, although the structure is less distorted than the native copper protein. The structure reported herein, is the first crystallographic structure of a Co(II)-substituted pseudoazurin.