Development of a biodegradable microstent for minimally invasive treatment of Fallopian tube occlusions

Obstructions of the Fallopian tube represent one of the most common reasons for an unfulfilled desire to have children. Microstent technology opens up new therapeutic possibilities to restore the natural lumen of the Fallopian tube within a single treatment. Within the current work we developed a self-expandable biodegradable microstent for gynecological applications. Based on a novel microstent design, prototypes were manufactured from poly-L-lactide tubing by means of fs-laser cutting. Microstent prototypes were characterized morphologically by means of scanning electron microscopy and biaxial laser scanning. As manufactured, a microstents outside diameter of about 2.3 mm and a strut thickness/width of about 114 µm/103 µm was measured. Mechanical characterization of microstents included bending as well as crimping and release behavior. After crimping to a minimum diameter of 0.8 mm and consecutive release, a microstent recovery to a diameter of 1.8 mm was found. Therefore, proof-of-concept for the self-expandable microstent could be successfully provided. © 2020 by Walter de Gruyter Berlin/Boston 2020

The effect of Fusobacterium nucleatum on leukocyte-trophoblast interactions in vitro

The success of pregnancy depends on precisely adjusted, local immune mechanisms. In early pregnancy, fetal trophoblast cells implant into the endometrium to build and anchor the placenta. Simultaneously, they mediate fetal tolerance and defense against infections. To cover these versatile requirements, local immune factors must be in balance. A too tolerogenic milieu can lead to an inadequate placentation; while a too inflammatory milieu can cause rejection of the semi-allogenic fetus. Bacterial infections can provoke these inflammatory pregnancy complications as well. Therefore, the pregnant uterus was long thought to be sterile. Descriptions of a placental microbiome opened a scientific discourse, which is unsolved due to contrary studies. The colonization of the non-pregnant endometrium is, however, confirmed. It is supposed to affect both, uterine pathologies and fertility. Precise data are lacking. Aim of this work was to assess if and under which circumstances a bacterial colonization would be tolerable. One of the described species in placental and endometrial samples is Fusobacterium nucleatum. It is an opportunistic bacterium, which is known from the human oral cavity and associated with the development of colon carcinomas. F. nucleatum supports tumorigenesis by the induction of epithelial proliferation, survival, migration and invasion as well as angiogenesis and tumor tolerance. Since similar processes are required for implantation and placentation, F. nucleatum might support these as well. In this work, the effects of F. nucleatum on leukocyte-trophoblast-interactions, especially of macrophages and innate lymphoid cells type 3 (ILC3), were assessed. The monocytic cells (THP-1) were differentiated into inflammatory M1 (IFN-γ) or tissue-repairing and tolerogenic M2a (IL-4) and M2c (TGF-β) macrophages. Inactivated F. nucleatum, LPS or E. coli was added. Only small concentrations of inactivated bacteria were used (bacteria:leukocyte ratio of 0.1 or 1), since it was not the aim to analyze infections. Conditioned medium of treated leukocytes was added to trophoblastic cells (HTR-8/SVneo). Migratory, invasive and tube formation behavior of trophoblastic cells was quantified. Treated M1 macrophages impaired trophoblast function, whereas M2a macrophages induced trophoblast invasion. M2c macrophages supported trophoblast migration and tube formation if treated with the smaller, but not with the higher concentration of F. nucleatum. This treatment induced the accumulation of HIF-1α and the secretion of VEGF-A in M2c macrophages as well. Moreover, the higher concentration of F. nucleatum caused rather inflammatory responses (NF-κB activation and cytokine expression). The activation of the HIF-1α-VEGF-A axis under the influence of TGF-β might serve as a mild immune stimulation by low abundant commensal bacteria supporting placentation. In contrast to macrophages, the function of ILC3s during pregnancy is still unknown. In general, ILC3s are located in mucosal tissue, such as the gut. They participate in tolerance mechanisms and form the local micromilieu by the secretion of cytokines and the presentation of antigens. In order to characterize local, uterine ILC3s, murine ILC3s were compared to peripheral, splenic ILC3s. Uterine ILC3s were more activated and produced higher levels of IL-17 compared to splenic ILC3s. However, uterine ILC3s barely expressed MHCII on their surface. A reduced antigen presentation potential was confirmed in human ILC3s differentiated from cord blood stem cells by the addition of TGF-β or hCG. The treatment with bacteria increased MHCII expression, but not to the initial level. The higher bacterial concentration induced IL-8 secretion and led to an increased trophoblast invasion. ILC3s were less sensitive to bacterial stimulation than macrophages. Recent studies on the uterine or placental presence of bacteria during pregnancy are discrepant. The results of this project indicate that bacteria or bacterial residues might serve as a mild stimulus under certain circumstances to support implantation without negative effects. The current discussion must therefore not only be expanded by additional studies, but especially include differentiated local conditions. In this context, the sheer presence of bacteria or bacterial components must not be equated with an infection representing a known hazard.Der Erfolg einer Schwangerschaft hängt von präzise abgestimmten, lokalen Immunvorgängen ab. Zu Beginn implantieren sich fetale Trophoblastzellen in das Endometrium, die die Plazenta bilden und verankern. Ihre Invasion wird von maternalen Immunzellen begleitet. Gleichzeitig vermitteln sie fetale Toleranz und bieten Schutz gegen Infektionen. Um diesen vielseitigen Ansprüchen gerecht zu werden, müssen lokale Immunfaktoren gut ausbalanciert werden. Ein zu tolerogenes Milieu kann zu einer unzureichenden Plazentation und damit zur Unterversorgung des Fetus führen; ein zu stark inflammatorisches Milieu kann die Abstoßung des semi-allogenen Embryos auslösen. Auch bakterielle Infektionen können die Ursache für inflammatorische Schwangerschaftskomplikationen sein. Daher wurde der schwangere Uterus lange für steril gehalten. Beschreibungen eines plazentalen Mikrobioms eröffneten einen wissenschaftlichen Diskurs, der durch konträre Studienergebnisse bis heute ungeklärt bleibt. Die Kolonisation des nicht-schwangeren Endometriums gilt dagegen als bestätigt. Dem endometrialen Mikrobiom wird zudem eine bisher unterschätzte Bedeutung in uterinen Krankheitsbildern aber auch in der Fertilität zugeschrieben, wobei die Datenlage bisher lückenhaft ist. In dieser Arbeit sollte untersucht werden, ob und unter welchen Umständen eine Besiedlung zu Beginn der Schwangerschaft tolerierbar wäre. Unter den in Plazenta- und Endometriumproben beschriebenen Spezies findet sich Fusobacterium nucleatum. Es ist ein opportunistisches Bakterium, welches aus der Mundhöhle bekannt und mit der Entwicklung von Kolonkarzinomen assoziiert ist. F. nucleatum unterstützt die Tumorgenese durch die Induktion von Epithelproliferation, -überleben, -migration und -invasion, sowie Angiogenese und Tumortoleranz. Da ähnliche Prozesse für die Implantation erforderlich sind, könnte F. nucleatum diese ebenfalls unterstützen. In dieser Arbeit wurden die Effekte von F. nucleatum auf die Leukozyt-Trophoblast-Interaktion, insbesondere von Makrophagen und Innate Lymphoid Cells Type 3 (ILC3), betrachtet. Dafür wurde die Monozytenzelllinie THP-1 zu pro-inflammatorischen M1- bzw. zu Gewebe-reparierenden und Toleranz-fördernden M2a- und M2c-Makrophagen differenziert. Anschließend wurden sie mit F. nucleatum, LPS bzw. E. coli behandelt. Da keine Infektion sondern eine geringe bakterielle Kolonisation untersucht werden sollte, wurden die Makrophagen mit verschiedenen kleinen Konzentrationen behandelt (Bakterien:Leukozyten-Verhältnis von 0,1 und 1). Das konditionierte Medium der behandelten Leukozyten wurde verwendet, um extravillöse Trophoblastzellen (HTR-8/SVneo) zu stimulieren. Schließlich wurde ihr Migrations-, Invasions- und Tube Formation-Verhalten untersucht. Behandelte M1-Makrophagen schränkten die Trophoblastfunktion ein, während behandelte M2a-Makrophagen die Trophoblastinvasion unterstützten. Behandelte M2c-Makrophagen förderten Trophoblastmigration und Tube Formation, wenn sie mit der kleineren Konzentration F. nucleatum, nicht aber mit der höheren Konzentration, behandelt wurden. Die niedrigere F. nucleatum-Konzentration erhöhte sowohl die Akkumulation von HIF-1α als auch die Sekretion von VEGF-A in den M2c-Makrophagen. Dagegen verursachte die höhere Bakterienkonzentration eine verstärkt inflammatorische Reaktion (NF-κB-Aktivierung und Zytokinproduktion). Durch die Aktivierung der HIF-1α-VEGF-A-Achse unter dem Einfluss von TGF-β könnte die kontrollierte Immunstimulation durch Kommensalbakterien nicht nur die Trophoblastfunktion, sondern auch die Angiogenese während der Plazentation unterstützen. Im Gegensatz zu Makrophagen ist die Funktion von lokalen ILC3s während der Schwangerschaft noch weitestgehend unbekannt. ILC3s sind insbesondere in Schleimhäuten, wie dem Darm, zu finden. Dort zeichnen sie sich durch eine starke Sekretion von Zytokinen, aber auch durch Antigenpräsentation. So beeinflussen sie das lokale Mikromilieu und unterstützen Toleranzmechanismen. Um uterine ILC3s zu charakterisieren, wurden murine ILC3s aus Uterus und Milz stimuliert und analysiert. Verglichen mit murinen Milz-ILC3s waren uterine ILC3s stärker aktiviert und produzierten höhere IL-17-Level, exprimierten allerdings kaum MHCII auf ihrer Oberfläche. Ein reduziertes Antigenpräsentationspotential konnte in humanen ILCs, die aus Nabelschnurstammzellen differenziert wurden, durch die Zugabe des Schwangerschaftshormons hCG bzw. TGF-β bestätigt werden. Die Behandlung mit Bakterien erhöhte die MHCII-Expression, allerdings nicht auf das Ausgangslevel. Außerdem induzierte nur die höhere bakterielle Konzentration die IL-8-Sekretion und führte zu einer verstärkten Trophoblastinvasion. ILC3s scheinen daher weniger sensitiv auf bakterielle Stimulation zu reagieren als Makrophagen. Aktuelle Studien zur uterinen bzw. plazentalen Präsenz von Bakterien während der Schwangerschaft stehen sich derzeit diskrepant gegenüber. Die Ergebnisse dieser Arbeit legen nahe, dass unter bestimmten Umständen Bakterien oder bakterielle Bestandteile einen sanften Stimulus geben könnten, um die Vorgänge der Implantation zu unterstützen, ohne einen negativen Effekt zu zeigen. Die aktuelle Diskussion muss also nicht nur durch zusätzliche Studien erweitert werden, sondern insbesondere differenziert lokale Gegebenheiten einbeziehen. Die schiere Präsenz von Bakterien oder bakteriellen Bestandteilen darf in diesem Zusammenhang nicht mit einer Infektion, die eine bekannte Gefahr darstellt, gleichgesetzt werden

Oxygen regulates ILC3 antigen presentation potential and pregnancy-related hormone actions

Early pregnancy is marked by placentation and embryogenesis, which take place under physiological low oxygen concentrations. This oxygen condition is crucial for many aspects of placentation, trophoblast function, vascularization and immune function. Recently, a new family of innate lymphoid cells has been found to be expressed at the fetomaternal interface. Among these, type 3 innate lymphoid cells (ILC3) are important antigen presenting cells in the context of MHC-II. The expression of MHC-II on ILC3s during pregnancy is reduced. We tested the hypothesis that low oxygen concentrations reduce the potential of ILC3s to present antigens promoting fetal tolerance. Using an in vitro approach, NCR+ ILC3s generated from cord blood stem cell precursors were incubated under different O2 concentrations in the presence or absence of the pregnancy-related hormones hCG and TGF-β1. The expression of MHC-II, accessory molecules and an activation marker were assessed by flow cytometry. We observed that 1% O2 reduced the expression of the MHC-II molecule HLA-DR as compared to 21% O2 and modulated the relative effects of hCG and TGF-β1. Our data indicate that low oxygen concentrations reduce the antigen presentation potential of NCR+ ILC3s and suggest that it may promote fetal tolerance during the first trimester of pregnancy

Comparison of angiogenic potential in vitrified vs. slow frozen human ovarian tissue

Abstract Vitrification of ovarian tissue is a promising alternative approach to the traditional slow freezing method. Few empirical investigations have been conducted to determine the angiogenic profiles of these two freezing methods. In this study we aimed to answer the question whether one of the cryopreservation methods should be preferred based on the secretion of angiogenic factors. Tissue culture with reduced oxygen (5%) was conducted for 48 h with samples of fresh, slow frozen/thawed and vitrified/rapid warmed ovarian cortex tissue from 20 patients. From each patient, tissue was used in all three treatment groups. Tissue culture supernatants were determined regarding cytokine expression profiles of angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin binding epidermal growth factor, hepatocyte growth factor, Leptin, Platelet-derived growth factor B, placental growth factor and vascular endothelial growth factor A via fluoroimmunoassay. Apoptotic changes were assessed by TUNEL staining of cryosections and supplemented by hematoxylin and eosin and proliferating cell nuclear antigen staining. Comparing the angiogenic expression profiles of vitrified/rapid warmed tissue with slow frozen/thawed tissue samples, no significant differences were observed. Detection of apoptotic DNA fragmentation via TUNEL indicated minor apoptotic profiles that were not significantly different comparing both cryopreservation methods. Vitrification of ovarian cortical tissue does not appear to impact negatively on the expression profile of angiogenic factors and may be regarded as an effective alternative approach to the traditional slow freezing method

Development of a biodegradable microstent for minimally invasive treatment of Fallopian tube occlusions

Obstructions of the Fallopian tube represent one of the most common reasons for an unfulfilled desire to have children. Microstent technology opens up new therapeutic possibilities to restore the natural lumen of the Fallopian tube within a single treatment. Within the current work we developed a self-expandable biodegradable microstent for gynecological applications. Based on a novel microstent design, prototypes were manufactured from poly-L-lactide tubing by means of fs-laser cutting. Microstent prototypes were characterized morphologically by means of scanning electron microscopy and biaxial laser scanning. As manufactured, a microstents outside diameter of about 2.3 mm and a strut thickness / width of about 114 μm / 103 μm was measured. Mechanical characterization of microstents included bending as well as crimping and release behavior. After crimping to a minimum diameter of 0.8 mm and consecutive release, a microstent recovery to a diameter of 1.8 mm was found. Therefore, proof-of-concept for the self-expandable microstent could be successfully provided