Changes in cytokine and sequestosome-1 levels during twin pregnancy progression: Association with outcome.

BACKGROUND: Twin pregnancies are associated with complications and adverse outcomes. The number of twin pregnancies has increased in the last decades, due to the use of assisted reproductive techniques and delayed childbearing. Analysis of changes that occur during twin pregnancy progression and their association with outcome will lead to improved clinical interventions. OBJECTIVE: We evaluated if the plasma concentration of select cytokines and the level of sequestosome-1 (p62) in peripheral blood mononuclear cells (PBMCs) during each trimester of twin gestations was predictive of pregnancy outcome. STUDY DESIGN: This prospective, observational study was conducted at Careggi University Hospital, Florence, Italy. Plasma from 82 women with twin pregnancies was collected in each trimester for measurement of interleukin (IL)-1ß, IL-6, IL-10, IL-12 and tumor necrosis factor (TNF)-α. The intracellular PBMC concentration of p62, a protein involved in autophagy, kinase activity and cell differentiation, was also determined. RESULTS: IL-1ß (p < 0.001), IL-6 (p < 0.001), TNF-α (p < 0.001) and p62 (p < 0.05) increased from the 1st to the 2nd to the 3rd trimester. The TNF-α level was correlated with the IL-1ß concentration in the 1st and 3rd trimesters p < 0.01) and with the IL-6 concentration in each of the three trimesters (p < 0.01). The intracellular p62 level in PBMCs was negatively correlated with the concentration of IL-1ß in the 2nd trimester (p < 0.05) and negatively correlated with the IL-6 level in the 3rd trimester (p < 0.05). The TNF-α level was significantly higher in the 2nd (p < 0.05) and 3rd (p < 0.001) trimester in women with a spontaneous preterm delivery. The TNF-α concentrations in the 2nd (p < 0.05) and 3rd (p < 0.01) trimester, respectively, and 3rd trimester IL-6 (p < 0.01), were negatively associated with gestational age at delivery. The concentration of IL-6 was highest in the 2nd (p < 0.05) and 3rd (p < 0.05) trimesters in women who utilized assisted reproductive technologies. An elevated IL-1ß level in the 3rd trimester was associated with gestational diabetes mellitus (p < 0.05). CONCLUSION: Variations in cytokine levels between individual women during the three trimesters of twin gestations are predictive of spontaneous preterm delivery and the onset of gestational diabetes.

Unveiling the Role of Tryptophan 2,3-Dioxygenase in the Angiogenic Process.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO1) and tryptophan-2,3-dioxygenase (TDO) are the two principals enzymes involved in the catabolization of tryptophan (Trp) into kynurenine (Kyn). Despite their well-established role in the immune escape, their involvement in angiogenesis remains uncertain. We aimed to characterize TDO and IDO1 in human umbilical venular endothelial cells (HUVECs) and human endothelial colony-forming cells (ECFCs). METHODS: qRT-PCR and immunofluorescence were used for TDO and IDO1 expression while their activity was measured using ELISA assays. Cell proliferation was examined via MTT tests and in in vitro angiogenesis by capillary morphogenesis. RESULTS: HUVECs and ECFCs expressed TDO and IDO1. Treatment with the selective TDO inhibitor 680C91 significantly impaired HUVEC proliferation and 3D-tube formation in response to VEGF-A, while IDO1 inhibition showed no effect. VEGF-induced mTor phosphorylation and Kyn production were hindered by 680C91. ECFC morphogenesis was also inhibited by 680C91. Co-culturing HUVECs with A375 induced TDO up-regulation in both cell types, whose inhibition reduced MMP9 activity and prevented c-Myc and E2f1 upregulation. CONCLUSIONS: HUVECs and ECFCs express the key enzymes of the kynurenine pathway. Significantly, TDO emerges as a pivotal player in in vitro proliferation and capillary morphogenesis, suggesting a potential pathophysiological role in angiogenesis beyond its well-known immunomodulatory effects.

Exploring Plasma-Level Gut Microbiota Mediators and Pro-Inflammatory Markers in Pregnant Women with Short Cervix and Gestational Diabetes Mellitus.

The composition of the gut microbiota (GM) undergoes significant changes during pregnancy, influenced by metabolic status, energy homeostasis, fat storage, and hormonal and immunological modifications. Moreover, dysbiosis during pregnancy has been associated with preterm birth, which is influenced by factors such as cervical shortening, infection, inflammation, and oxidative stress. However, dysbiosis also affects the levels of lipopolysaccharide-binding protein (LBP), short-chain fatty acids (SCFAs), and free fatty acids (FFA) in other tissues and the bloodstream. In this study, we investigated the plasmatic levels of some pro-inflammatory cytokines, such as matrix metalloproteinases-8 (MMP-8), interleukin-8 (IL-8), heat shock protein 70 (Hsp70), and microbial markers in pregnant women with a short cervix (≤25 mm) compared to those with normal cervical length (>25 mm). We examined the differences in the concentration of these markers between the two groups, also assessing the impact of gestational diabetes mellitus. Understanding the relationship between GM dysbiosis, inflammatory mediators, and cervical changes during pregnancy may contribute to the identification of potential biomarkers and therapeutic targets for the prevention and management of adverse pregnancy outcomes, including preterm birth.

Tryptophan degradation enzymes expression in the placenta and the Kynurenine/Tryptophan ratio in maternal plasma after elective cesarean section.

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) metabolize tryptophan in the kynurenine pathway. We evaluated these enzymes' mRNA expression in maternal and fetal sides of the placenta of uncomplicated, unlabored full-term pregnancies after elective cesarean section and compared it with that of placentas obtained from vaginal delivery. Tryptophan and kynurenine plasmatic levels after cesarean section were measured, to investigate their possible correlation with IDO1 and TDO mRNA (TDO2) expression. The results suggested that IDO1 and TDO2 expression was higher in the maternal side of the placenta and that labor significantly affects TDO2 expression and the plasma Kynurenine/Tryptophan ratio.

Tryptophan degradation enzymes and Angiotensin (1-7) expression in human placenta.

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are key enzymes for tryptophan degradation, regulating immune tolerance during pregnancy. The intrauterine renin-angiotensin system is also involved in the progression of a healthy pregnancy. Angiotensin(1-7) maintains the integrity of fetal membranes via counteracting the pro-inflammatory actions of Angiotensin II. No data are available on placental Angiotensin(1-7) co-expression with TDO. We aimed to characterize TDO mRNA expression and its localization in different areas of the placenta of physiological pregnancies delivered at term; its co-expression with Angiotensin(1-7) and its correlation with the plasma kynurenine/tryptophan (Kyn/Trp) ratio was investigated. This prospective observational study included a nonconsecutive series of 20 singleton uncomplicated pregnancies delivered vaginally. TDO mRNA was expressed in both maternal and fetal sides of the placentas and TDO protein also in the villi and it was co-expressed with IDO1 in almost half of the placental cells at these sites. The percentage of TDO+ and IDO1+ cells appeared to be influenced by maternal pre-gestational smoking and newborn weight. A strong correlation was found between the percentage of TDO+ and IDO1+ cells in the villi. TDO+ cells also expressed Angiotensin(1-7), with a higher percentage on the fetal side and in the villi compared to the maternal one. Kyn/Trp plasma ratio was not correlated with IDO and TDO expression nor with the patient's characteristics. Collectively, our data indicate that TDO is detectable in placental tissue and is co-expressed with IDO and with Angiotensin(1-7)+ on the fetal side and in the villi.

Dexamethasone Promotes a Stem-Like Phenotype in Human Melanoma Cells via Tryptophan 2,3 Dioxygenase.

In addition to its well-established immunosuppressive actions, tryptophan 2,3-dioxygenase (TDO) appears to elicit direct effects on tumor cell function. Although TDO has been associated with cancer stemness, its involvement in melanoma stem cell biology remains largely unknown. Since we showed that by upregulating TDO, dexamethasone (dex) promotes proliferation and migration of SK-Mel-28 human melanoma cells, we sought to investigate dex effects on melanoma spherogenesis and stemness, and whether these events are mediated by TDO. We demonstrate here that dex significantly upregulates TDO in A375, a more aggressive melanoma cell line, confirming that dex effects are not limited to SK-Mel-28 cells. Moreover, dex stimulates spherogenesis of both cell lines, which is mediated by TDO, evident by its suppression with 680C91, a TDO inhibitor. The formed melanospheres appear to be enriched with embryonic stem cell marker mRNAs, the expression of which is potentiated by dex. Expression of cancer stem cell markers (CD133, CD44, ganglioside GD2) was significantly increased in A375 spheres, as detected by flow cytometry. Taken together, our results suggest that TDO could represent a promising target in the management of melanoma and that dex, routinely used as a co-medication also in advanced melanoma, may stimulate melanoma cell function/tumor-supporting properties, a rather debilitating and undesired side effect.

Oxidative Stress-Induced Endothelial Dysfunction in Cardiovascular Diseases.

Cardiovascular disease (CVD) is a major cause of mortality worldwide. A better understanding of the mechanisms underlying CVD is key for better management or prevention. Oxidative stress has been strongly implicated in the pathogenesis of CVD. Indeed, several studies demonstrated that reactive oxygen species (ROS), via different mechanisms, can lead to endothelial cell (EC) dysfunction, a major player in the etiology of several CVDs. ROS appears to modulate a plethora of EC biological processes that are critical for the integrity of the endothelial function. This review seeks to dissect the role of oxidative stress-induced endothelial dysfunction in CVD development, with emphasis on the underlying mechanisms and pathways. Special attention is given to ROS-induced reduction of NO bioavailability, ROS-induced inflammation, and ROS-induced mitochondrial dysfunction. A better understanding and appraisal of these pathways may be essential to attenuate oxidative stress or reverse EC dysfunction, and hence, reduce CVD burden.

Tryptophan metabolism and immune regulation in the human placenta.

The placenta represents the maternal-fetal vascular interface. It is capable of supplying the bioenergetic needs of the developing conceptus. It is composed of different cell types that engage in highly varied functions, ranging from attachment, invasion and vascular remodeling to cell fusion, hormone production, and nutrient transport. A deep knowledge of the immunological mechanisms responsible for maintaining an active tolerance towards an allogeneic fetus and the anti-inflammatory properties of the placenta can be useful to clarify the pathogenesis of adverse events in pregnancy. While the systemic mechanisms of this immunological regulation in pregnancy have been well studied, the metabolic processes involved in the placental immune response are still poorly understood. The aim of this review is to summarize the most important information concerning the immune regulation in pregnancy, focusing on the role of tryptophan (Trp) catabolism performed by indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) in the placenta.

Dexamethasone Induces the Expression and Function of Tryptophan-2-3-Dioxygenase in SK-MEL-28 Melanoma Cells.

Tryptophan-2,3-dioxygenase (TDO) is one of the key tryptophan-catabolizing enzymes with immunoregulatory properties in cancer. Contrary to expectation, clinical trials showed that inhibitors of the ubiquitously expressed enzyme, indoleamine-2,3-dioxygenase-1 (IDO1), do not provide benefits in melanoma patients. This prompted the hypothesis that TDO may be a more attractive target. Because the promoter of TDO harbors glucocorticoid response elements (GREs), we aimed to assess whether dexamethasone (dex), a commonly used glucocorticoid, modulates TDO expression by means of RT-PCR and immunofluorescence and function by assessing cell proliferation and migration as well as metalloproteinase activity. Our results show that, in SK-Mel-28 melanoma cells, dex up-regulated TDO and its downstream effector aryl hydrocarbon receptor (AHR) but not IDO1. Furthermore, dex stimulated cellular proliferation and migration and potentiated MMP2 activity. These effects were inhibited by the selective TDO inhibitor 680C91 and enhanced by IDO1 inhibitors. Taken together, our results demonstrate that the metastatic melanoma cell line SK-Mel-28 possesses a functional TDO which can also modulate cancer cell phenotype directly rather than through immune suppression. Thus, TDO appears to be a promising, tractable target in the management or the treatment of melanoma progression.

Establishment and characterization of a new spontaneously immortalized ER-/PR-/HER2+ human breast cancer cell line, DHSF-BR16.

Invasive ductal carcinoma (IDC) constitutes the most frequent malignant cancer endangering women's health. In this study, a new spontaneously immortalized breast cancer cell line, DHSF-BR16 cells, was isolated from the primary IDC of a 74-years old female patient, treated with neoadjuvant chemotherapy and disease-free 5-years after adjuvant chemotherapy. Primary breast cancer tissue surgically removed was classified as ER-/PR-/HER2+, and the same phenotype was maintained by DHSF-BR16 cells. We examined DHSF-BR16 cell morphology and relevant biological and molecular markers, as well as their response to anticancer drugs commonly used for breast cancer treatment. MCF-7 cells were used for comparison purposes. The DHSF-BR16 cells showed the ability to form spheroids and migrate. Furthermore, DHSF-BR16 cells showed a mixed stemness phenotype (i.e. CD44+/CD24-/low), high levels of cytokeratin 7, moderate levels of cytokeratin 8 and 18, EpCAM and E-Cadh. Transcriptome analysis showed 2071 differentially expressed genes between DHSF-BR16 and MCF-7 cells (logFC > 2, p-adj < 0.01). Several genes were highly upregulated or downregulated in the new cell line (log2 scale fold change magnitude within - 9.6 to + 12.13). A spontaneous immortalization signature, mainly represented by extracellular exosomes-, plasma membrane- and endoplasmic reticulum membrane pathways (GO database) as well as by metabolic pathways (KEGG database) was observed in DHSF-BR16 cells. Also, these cells were more resistant to anthracyclines compared with MCF-7 cells. Overall, DHSF-BR16 cell line represents a relevant model useful to investigate cancer biology, to identify both novel prognostic and drug response predictive biomarkers as well as to assess new therapeutic strategies.

Understanding the heart-brain axis response in COVID-19 patients: A suggestive perspective for therapeutic development.

In-depth characterization of heart-brain communication in critically ill patients with severe acute respiratory failure is attracting significant interest in the COronaVIrus Disease 19 (COVID-19) pandemic era during intensive care unit (ICU) stay and after ICU or hospital discharge. Emerging research has provided new insights into pathogenic role of the deregulation of the heart-brain axis (HBA), a bidirectional flow of information, in leading to severe multiorgan disease syndrome (MODS) in patients with confirmed infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Noteworthy, HBA dysfunction may worsen the outcome of the COVID-19 patients. In this review, we discuss the critical role HBA plays in both promoting and limiting MODS in COVID-19. We also highlight the role of HBA as new target for novel therapeutic strategies in COVID-19 in order to open new translational frontiers of care. This is a translational perspective from the Italian Society of Cardiovascular Researches.

Insulin resistance and obesity affect monocyte-derived dendritic cell phenotype and function.

AIM: Cardiovascular disease (CVD) is prevalent in women after menopause, which may be associated with obesity, insulin resistance and metaflammation. Despite the recognized role of immunological mechanisms in vascular remodeling, the role of dendritic cells (DCs) is still unclear. The aim was to characterize monocyte-derived DCs (Mo-DC) in post-menopausal patients with type 2 diabetes (T2DM) and obese woman, without clinical manifestations of atherosclerosis. METHODS: Obese post-menopausal women with or without T2DM were enrolled and were compared to age-matched healthy women. DCs obtained from patients were phenotypically and functionally characterized by flow cytometry and mixed lymphocyte reaction. MRNA integrins expression was assessed by real time RT-PCR; circulating fetuin-A and adiponectin levels were measured by ELISA. RESULTS: Phenotypic dysregulation of Mo-DC reported was related to a defective allogenic lymphocyte stimulation and to an increased mRNA of CD11c, CD18 and DC-SIGN/CD209 which regulate their adhesion to vascular wall cells. Fetuin-A and adiponectin levels were significantly altered and negatively correlated. Hyperglycaemia significantly impaired CD14+ transdifferentiation into Mo-DC. CONCLUSIONS: These data show a dysfunction of Mo-DCs obtained from precursors isolated from T2DM obese post-menopausal woman without any documented clinical CV event. Association of obesity to diabetes seems to worsen DC's phenotype and function and increase vascular inflammation.

Identification of Vaginal Microbial Communities Associated with Extreme Cervical Shortening in Pregnant Women.

The vaginal microbiota plays a critical role in pregnancy. Bacteria from Lactobacillus spp. are thought to maintain immune homeostasis and modulate the inflammatory responses against pathogens implicated in cervical shortening, one of the risk factors for spontaneous preterm birth. We studied vaginal microbiota in 46 pregnant women of predominantly Caucasian ethnicity diagnosed with short cervix (<25 mm), and identified microbial communities associated with extreme cervical shortening (≤10 mm). Vaginal microbiota was defined by 16S rRNA gene sequencing and clustered into community state types (CSTs), based on dominance or depletion of Lactobacillus spp. No correlation between CSTs distribution and maternal age or gestational age was revealed. CST-IV, dominated by aerobic and anaerobic bacteria different than Lactobacilli, was associated with extreme cervical shortening (odds ratio (OR) = 15.0, 95% confidence interval (CI) = 1.56-14.21; p = 0.019). CST-III (L. iners-dominated) was also associated with extreme cervical shortening (OR = 6.4, 95% CI = 1.32-31.03; p = 0.02). Gestational diabetes mellitus (GDM) was diagnosed in 10/46 women. Bacterial richness was significantly higher in women experiencing this metabolic disorder, but no association with cervical shortening was revealed by statistical analysis. Our study confirms that Lactobacillus-depleted microbiota is significantly associated with an extremely short cervix in women of predominantly Caucasian ethnicity, and also suggests an association between L. iners-dominated microbiota (CST III) and cervical shortening.

Different effects of tryptophan 2,3-dioxygenase inhibition on SK-Mel-28 and HCT-8 cancer cell lines.

PURPOSE: Indoleamine 2,3-dioxygenase-1 (IDO1) and more recently, tryptophan 2,3-dioxygenase (TDO), are tryptophan-catabolizing enzymes with immunoregulatory properties in cancer. IDO1 is more expressed than TDO in many tumours including melanomas; however, IDO inhibitors did not give expected results in clinical trials, highlighting the need to consider TDO. We aimed to characterize both TDO expression and function in a melanoma cell line, named SK-Mel-28, with the purpose to compare it with a colon cancer cell line, HCT-8, and with a human endothelial cell line (HUVEC). METHODS: TDO expression was assessed as real time-PCR and western blot, for mRNA and protein expression, respectively. While cell proliferation was assessed as cell duplication, cell apoptosis and cell cycle were analysed by means of flow cytometry. RESULTS: SK-Mel-28 cells showed higher TDO levels compared to HCT-8 and to HUVEC cells. A selective TDO inhibitor, 680C91, significantly impaired cell proliferation in a concentration-dependent manner, by inducing cell arrest during the G2 phase for SK-Mel-28 and HUVEC cells, while an early apoptosis was increasing in HCT-8 cells. No toxic effects were observed. These data demonstrated that TDO is highly expressed in SK-Mel-28 cells and may be involved in the regulation of their proliferation. CONCLUSION: TDO may directly modulate cancer cell function rather than immune suppression and can be considered as a target for melanoma progression together with IDO1.

Obesity Therapy: How and Why?

BACKGROUND: Obesity represents the second preventable mortality cause worldwide, and is very often associated with type 2 Diabetes Mellitus (T2DM). The first line treatment is lifestyle modification to weight-loss, but for those who fail to achieve the goal or have difficulty in maintaining achieved results, pharmacological treatment is needed. Few drugs are available today, because of their side effects. OBJECTIVE: We aim to review actual pharmacological management of obese patients, highlighting differences between Food and Drug Administration - and European Medicine Agency-approved molecules, and pointing out self-medications readily obtainable and widely distributed. METHODS: Papers on obesity, weight loss, pharmacotherapy, self- medication and diet-aid products were selected using Medline. Research articles, systematic reviews, clinical trials and meta-analyses were screened. RESULTS: Anti-obesity drugs with central mechanisms, such as phentermine and lorcaserin, are available in USA, but not in Europe. Phentermine/topiramate and naltrexone/bupropion combinations are now available, even though the former is still under investigation from EMA. Orlistat, with peripheral mechanisms, represents the only drug approved for weight reduction in adolescents. Liraglutide has been approved at higher dose for obesity. Anti-obesity drugs, readily obtainable from the internet, include crude-drug products and supplements for which there is often a lack of compliance to national regulatory standards. CONCLUSIONS: Mechanisms of weight loss drugs include the reduction of energy intake or the increase in energy expenditure and sense of satiety as well as the decrease of hunger or the reduction in calories absorption. Few drugs are approved, and differences exist between USA and Europe. Moreover, herbal medicines and supplements often sold on the internet and widely used by obese patients, present a risk of adverse effects.

T-cell immunoglobulin mucin-3 and galectin-9 levels in peripheral blood mononuclear cells predict fetal acidemia in twin pregnancies.

Purposes: The pH in the umbilical artery at delivery provides information on the fetal environment and is related to postnatal outcomes. The ability to predict fetal acidemia at delivery would improve clinical management and neonatal well-being. We hypothesized that an alteration in maternal immunity would accompany placental changes that precede a decrease in pH in the fetal circulation in twin gestations.Methods: Peripheral blood mononuclear cells (PBMCs), obtained from 86 women with twin pregnancies, were lysed and assayed for concentrations of T-cell immunoglobulin mucin domain 3 (Tim-3) and galectin-9 (gal-9) by ELISA. Tim-3-gal-9 interaction is a primary mechanism promoting immune suppression. At delivery, the pH of arterial cord blood was determined.Results: In eight women (9.3%), the pH in the placental arteries from both twins was <7.15, indicating fetal acidosis. In the remaining 78 women the arterial pH was ≥7.15 in both twins. The median Tim-3 level was 361 pg/ml when arterial pH was <7.15 and 199 pg/ml when pH was ≥7.15 (p = .003). Similarly, gal-9 was 31.2 versus 12.4 ng/ml when pH was <7.15 or ≥7.15, respectively (p = .001). A Tim-3 concentration >260 pg/ml predicted arterial pH <7.15 with a sensitivity of 87.5%, specificity of 79.5% and negative predictive value of 98.4%. A gal-9 level >18.4 predicted arterial pH <7.15 with a sensitivity of 100%, specificity of 73.8% and a negative predictive value of 100%.Conclusion: We conclude that elevations in Tim-3 and gal-9 in PBMCs during gestation predict the subsequent occurrence of a pH <7.15 in the fetal arteries at delivery in twin gestations.

Insulin-like growth factor binding protein-1 predicts preterm premature rupture of membranes in twin pregnancies.

PURPOSE: Mechanisms leading to preterm premature rupture of membranes (PPROM) remain incompletely defined. Based on the elevated occurrence of PPROM in twin gestations and recent studies of the involvement of insulin-like growth factor binding protein-1 (IGFBP-1) in the inhibition of collagen production we hypothesized that serum IGFBP-1 levels might be predictive of susceptibility to PPROM in women with twins. METHODS: In this prospective study peripheral blood was obtained from 58 women with twin gestations prior to 20 weeks gestation and sera analyzed by ELISA for concentrations of IGFBP-1. Demographic and clinical outcome data were subsequently obtained and associations between IGFBP-1 and PPROM were analyzed by the Mann-Whitney test and receiver operator curve (ROC) analysis. RESULTS: Eight of our subjects developed PPROM. They did not differ from the other women in demographics, medical history or current pregnancy outcome parameters. However, median IGFBP-1 levels were higher in women who subsequently developed PPROM (59.3 ng/ml) than in the other women (46.6 ng/ml) (p = 0.042). Using a cutoff value of 53.9 ng/ml the circulating IGFBP-1 level predicted development of PPROM with a sensitivity of 74%, specificity of 75%, a negative predictive value of 97% and a positive predictive value of 20%. CONCLUSIONS: Pending validation in larger studies the findings suggest that determination of serum IGFBP-1 levels in women with twin pregnancies may predict the later development of PPROM.

Unique Associations between Insulin-Like Growth Factor Binding Protein-1, Insulin-Like Growth Factor-1 and T Cell Immunoglobulin Mucin 3 in Successful Twin Pregnancies Conceived with Donor Oocytes.

Background and Objectives: To investigate if pregnancies conceived using an oocyte donor necessitate an alteration in immune regulation, we compared concentrations of insulin-like growth factor binding protein (IGFBP)-1, insulin-like growth factor (IGF)-1 and T cell immunoglobulin mucin-3 (Tim-3) in women with ongoing successful twin pregnancies conceived spontaneously, using assisted reproductive technologies that utilized homologous oocytes or with donor oocytes. Differences in levels of these immune modulatory proteins may be magnified and easier to detect in twin as compared to singleton pregnancies. Methods: In this prospective study IGFBP-1 and IGF-1 were measured in sera and Tim-3 in lysates of peripheral blood mononuclear cells (PBMCs) by ELISA. Results: Median IGFBP-1 levels were lower in women with donor oocytes (41.4 ng/ml) as compared to those with a spontaneous conception (51.2 ng/mL) or who conceived with various assisted reproduction protocols using homologous oocytes (52.4 ng/mL) (p < 0.001). IGF-1 and Tim-3 levels were comparable in each group. The IGFBP-1 level was inversely correlated to the IGF-1 concentration only in women with donor oocytes (p = 0.032). IGFBP-1 and Tim-3 levels were similarly negatively correlated in the donor oocyte group (p = 0. 012). Women in the assisted reproduction group who conceived following intracytoplasmic sperm injection were the only other group in which IGFBP-1 and Tim-3 were negatively correlated (p = 0.018). Conclusions: Down-regulation of IGFBP-1 production in pregnancies conceived with donor oocytes may reduce the extent of pro-inflammatory immunity and contribute to successful outcome in totally allogeneic pregnancies.