Do endometrial natural killer and regulatory T cells differ in infertile and clinical pregnancy patients? An analysis in patients undergoing frozen embryo transfer cycles.

PROBLEM: Clinical significance of endometrial and peripheral blood natural killer (NK) and regulatory T cells (Tregs) during frozen embryo transfer (FET) cycles has not been well characterized. DESIGN: Retrospective cohort study. METHOD OF STUDY: Endometrial tissue was collected from infertility patients prior to a frozen embryo transfer cycle as part of an endometrial receptivity analysis (ERA® ) biopsy or endometrial scratch test. Uterine NK (uNK) and Treg cell density was compared based on pregnancy status in the subsequent frozen embryo transfer cycle. Peripheral blood was also collected from a separate cohort of patients undergoing frozen embryo transfer. Treg cell density was compared by the presence or the absence of a clinical pregnancy in each phase of the cycle. RESULTS: In the 33 luteal phase biopsies there were more endometrial Tregs, similar uNK and a trend toward lower CD16+ uNK cells in women with a future ongoing clinical pregnancy compared to non-pregnant women. There were no differences in uNK and Treg density in natural scratch cycles vs programmed cycles or in non-receptive vs receptive endometrium (ERA® cycles). In the peripheral blood analysis, the pregnant group had higher peripheral blood Tregs on the day of serum ß-hCG time point when compared to the non-pregnant group. CONCLUSION: Higher levels of endometrial Tregs and lower levels of CD16+ uNK cells are positive prognostic factors for infertile women prior to frozen embryo transfer. Our work on phenotypic and proportional analyses of endometrial immune cells may complement the ERA® in predicting improved pregnancy rates in patients with implantation failure.

Human chorionic gonadotropin-mediated modulation of pregnancy-compatible peripheral blood natural killer cells in frozen embryo transfer cycles.

PROBLEM: To evaluate pregnancy-compatible phenotypic and functional changes in peripheral blood natural killer (pNK) cells during frozen embryo transfer (FET) cycles. METHOD OF STUDY: Peripheral blood was collected from patients undergoing frozen embryo transfer cycles at three separate time points in the cycle. pNK cell phenotype was analyzed by flow cytometry. Impact of pregnancy status on pNK cell cytotoxicity was characterized by two methods: (1) a three-dimensional endovascular tube formation approach and (2) a NK cell-specific K562 cell kill assay. RESULTS: A total of 35 patients were enrolled, 15 with clinical pregnancies and 20 with negative serum ß-hCG levels. Overall percentage of CD45+ CD3- CD56+ pNK cell did not change during the FET cycle. Pregnancy resulted in an increase in CD45+ CD3- CD56+ pNK cell population on the day of serum ß-hCG. pNK cells from non-pregnant patients caused significant tube disruption when compared to pregnant patients. Addition of serum from pregnant women reduced the tube disruption by pNK cells from non-pregnant patients. pNK cells from pregnant patients showed significantly lower cytotoxicity toward K562 cells in serum-free conditions. The addition of pregnancy serum decreased non-pregnant pNK cell cytotoxicity. Pregnancy status had no impact on VEGF-A and VEGF-C serum levels. Recombinant hCG added to non-pregnant serum resulted in a significant reduction in non-pregnant pNK cell-mediated K562 cell kill. CONCLUSION: There was no difference in pNK cell populations based on timing of the FET cycle. However, pregnancy increased the percentage of CD45+ CD3- CD56+ pNK cells. Additionally, pNK cells from pregnant women have reduced cytotoxicity and this is possibly mediated by hCG.

Novel predictive and therapeutic options for better pregnancy outcome in frozen embryo transfer cycles.

Since 1978, in the first decades of in vitro fertilization (IVF), the use of ovarian hyperstimulation allowed for the development and transfer of multiple embryos. As IVF technology improved, the number of multiple pregnancies increased, which led to gradual reduction in the number of embryos that were transferred. Embryo freezing (vitrification) was recommended to allow subsequent transfer if the fresh cycle was unsuccessful. However, experimentation has continued to improve pregnancy outcomes. We discuss here the significance of frozen embryo transfer cycle and the impact of uterine and peripheral immunity dominated by NK cells and regulatory T cells and human chorionic gonadotropin on pregnancy outcome in this innovative mode of IVF therapy.

Immunobiology of Gestational Diabetes Mellitus in Post-Medawar Era.

Although the concepts related to fetal immune tolerance proposed by Sir Peter Medawar in the 1950s have not withstood the test of time, they revolutionized our current understanding of the immunity at the maternal-fetal interface. An important extension of the original Medawar paradigm is the investigation into the underlying mechanisms for adverse pregnancy outcomes, including recurrent spontaneous abortion, preterm birth, preeclampsia and gestational diabetes mellitus (GDM). Although a common pregnancy complication with systemic symptoms, GDM still lacks understanding of immunological perturbations associated with the pathological processes, particularly at the maternal-fetal interface. GDM has been characterized by low grade systemic inflammation that exacerbates maternal immune responses. In this regard, GDM may also entail mild autoimmune pathology by dysregulating circulating and uterine regulatory T cells (Tregs). The aim of this review article is to focus on maternal-fetal immunological tolerance phenomenon and discuss how local or systemic inflammation has been programmed in GDM. Specifically, this review addresses the following questions: Does the inflammatory or exhausted Treg population affecting the Th17:Treg ratio lead to the propensity of a pro-inflammatory environment? Do glycans and glycan-binding proteins (mainly galectins) contribute to the biology of immune responses in GDM? Our understanding of these important questions is still elementary as there are no well-defined animal models that mimic all the features of GDM or can be used to better understand the mechanistic underpinnings associated with this common pregnancy complication. In this review, we will leverage our preliminary studies and the literature to provide a conceptualized discussion on the immunobiology of GDM.

Correlation between heart valve interstitial cell stiffness and transvalvular pressure: implications for collagen biosynthesis.

It has been speculated that heart valve interstitial cells (VICs) maintain valvular tissue homeostasis through regulated extracellular matrix (primarily collagen) biosynthesis. VICs appear to be phenotypically plastic, inasmuch as they transdifferentiate into myofibroblasts during valve development, disease, and remodeling. Under normal physiological conditions, transvalvular pressures (TVPs) on the right and left side of the heart are vastly different. Hence, we hypothesize that higher left-side TVPs impose larger local tissue stress on VICs, which increases their stiffness through cytoskeletal composition, and that this relation affects collagen biosynthesis. To evaluate this hypothesis, isolated ovine VICs from the four heart valves were subjected to micropipette aspiration to assess cellular stiffness, and cytoskeletal composition and collagen biosynthesis were quantified by using the surrogates smooth muscle alpha-actin (SMA) and heat shock protein 47 (HSP47), respectively. VICs from the aortic and mitral valves were significantly stiffer (P < 0.001) than those from the pulmonary and tricuspid valves. Left-side isolated VICs contained significantly more (P < 0.001) SMA and HSP47 than right-side VICs. Mean VIC stiffness correlated well (r = 0.973) with TVP; SMA and HSP47 also correlated well (r = 0.996) with one another. Assays were repeated for VICs in situ, and, as with in vitro results, left-side VIC protein levels were significantly greater (P < 0.05). These findings suggest that VICs respond to local tissue stress by altering cellular stiffness (through SMA content) and collagen biosynthesis. This functional VIC stress-dependent biosynthetic relation may be crucial in maintaining valvular tissue homeostasis and also prove useful in understanding valvular pathologies.

Comparison of three myofibroblast cell sources for the tissue engineering of cardiac valves.

The objective of this study was to evaluate the capacity of three clinically useful tissue sources: tricuspid valve leaflet (TVL), carotid artery (CA), and jugular vein (JV), to generate myofibroblasts for potential use in a tissue-engineered cardiac valve replacement. Tissue biopsies of clinically appropriate sizes obtained from juvenile sheep were used for this work. Cells obtained from all three tissue sources exhibited a myofibroblast phenotype in vitro, as demonstrated by their immunoreactivity with antibodies directed against vimentin, alpha-smooth muscle actin, fibronectin, and chondroitin sulfate. Protein synthesis characteristics were defined for the key extracellular matrix components: collagen, glycosaminoglycans, and elastin. Among the three sources, JV generated the highest numbers of cells, and JV cells produced the largest amount of collagen per cell. These data suggest that venous tissue, with its relative ease of accessibility, may generate myofibroblasts potentially useful for the interstitial cellular component of a tissue-engineered cardiac valve.

Tricuspid valve biopsy: a potential source of cardiac myofibroblast cells for tissue-engineered cardiac valves.

BACKGROUND AND AIMS OF THE STUDY: As progress is made in the development of a tissue-engineered cardiac valve, the need for a reliable cell source is particularly important. A technique has been developed for the reliable biopsy of tricuspid valve leaflets. Expanding the harvested cells in culture is feasible and provides a source of leaflet cells that are structurally and functionally similar to the pulmonary and aortic valve leaflet cells that they may replace. METHODS: Thirteen sheep underwent tricuspid valve biopsy. Transthoracic echocardiography (TTE) was performed to evaluate function and guide the subsequent biopsy. Myofibroblasts were isolated from the biopsy samples, expanded in culture through 10 passages, and evaluated with immunocytochemistry for valve cell markers. Two animals were sacrificed acutely, two animals died during the immediate postoperative period, and nine animals survived for four weeks or more. RESULTS: All preoperative and pre-explantation echocardiograms were normal. Both animals sacrificed acutely showed that the tricuspid valve leaflet was indeed biopsied with this technique. Two perioperative deaths occurred; one animal died secondary to injury of the chorda tendinea with subsequent destruction of the posterior leaflet; another died from disruption of the superior vena cava that led to irreversible cardiac tamponade. At sacrifice (2 to 17 weeks), all other animals showed intact tricuspid valves with normal leaflet anatomy. All cultured biopsies generated myofibroblasts that were immunocytochemically positive for alpha smooth muscle actin, chondroitin sulfate, vimentin and fibronectin. CONCLUSION: Biopsy of the tricuspid valve to obtain recipient cardiac valve leaflet cells is possible, and the technique is simple and reliable. Biopsy of the leaflet does not compromise function. Interstitial cells can be harvested and expanded in culture. Cellular structure and function is preserved and is similar to that of other cardiac leaflet cells. Tricuspid valve leaflet biopsies are a potential source for harvesting cells to be used in the development of a tissue-engineered cardiac valve.