When the Embryo Meets the Endometrium: Identifying the Features Required for Successful Embryo Implantation.

Evaluation of the optimal number of embryos, their quality, and the precise timing for transfer are critical determinants in reproductive success, although still remaining one of the main challenges in assisted reproduction technologies (ART). Indeed, the success of in vitro fertilization (IVF) treatments relies on a multitude of events and factors involving both the endometrium and the embryo. Despite concerted efforts on both fronts, the overall success rates of IVF techniques continue to range between 25% and 30%. The role of the endometrium in implantation has been recently recognized, leading to the hypothesis that both the "soil" and the "seed" play a central role in a successful pregnancy. In this respect, identification of the molecular signature of endometrial receptivity together with the selection of the best embryo for transfer become crucial in ART. Currently, efforts have been made to develop accurate, predictive, and personalized tests to identify the window of implantation and the best quality embryo. However, the value of these tests is still debated, as conflicting results are reported in the literature. The purpose of this review is to summarize and critically report the available criteria to optimize the success of embryo transfer and to better understand current limitations and potential areas for improvement.

An improved in vitro model simulating the feto-maternal interface to study developmental effects of potentially toxic compounds: The example of titanium dioxide nanoparticles.

The study of developmental effect of xenobiotics in humans is limited and often relies on epidemiological data. Whether and to which extent potentially toxic compounds may cross the placental barrier, and whether adverse effects on embryo development are the consequence of direct or indirect placental-mediated action is debated. The availability of in vitro models simulating the feto-maternal interface could contribute to elucidate this issue. Here, we report the development of a novel in vitro model using murine blastocyst derived trophoblast stem cells (TSC) to mimic the placental barrier and mouse embryoid bodies (EBs) to represent the embryonic tissues. We demonstrate that this model can be used for translocation studies, as well as embryotoxicity assessment of titanium dioxide nanoparticles (TiO2NPs). By evaluating trans-epithelial electrical resistance, translocation of fluorescein isothiocyanate-dextran beads and expression of junctional complex proteins, we show that TSCs cultured on transwell inserts under differentiating condition form syncytia. We also show that TiO2NPs administered in the upper transwell compartment are able to reach the lower compartment and interfere with EB differentiation when no TSC are cultured on the insert. On the contrary, when TSC are present, NPs translocate to a lesser extent and do not affect EB development. These results indicate that the proposed in vitro model is suitable to study the correlation between translocation and toxicity of TiO2NPs and suggest a direct effect of the particles on EB development. We propose that this model could be exploited to study developmental effect of other xenobiotics.

Placental Dysfunction in Assisted Reproductive Pregnancies: Perinatal, Neonatal and Adult Life Outcomes.

Obstetric and newborn outcomes of assisted reproductive technology (ART) pregnancies are associated with significative prevalence of maternal and neonatal adverse health conditions, such as cardiovascular and metabolic diseases. These data are interpreted as anomalies in placentation involving a dysregulation of several molecular factors and pathways. It is not clear which extent of the observed placental alterations are the result of ART and which originate from infertility itself. These two aspects probably act synergically for the final obstetric risk. Data show that mechanisms of inappropriate trophoblast invasion and consequent altered vascular remodeling sustain several clinical conditions, leading to obstetric and perinatal risks often found in ART pregnancies, such as preeclampsia, fetal growth restriction and placenta previa or accreta. The roles of factors such as VEGF, GATA3, PIGF, sFLT-1, sEndoglin, EGFL7, melatonin and of ART conditions, such as short or long embryo cultures, trophectoderm biopsy, embryo cryopreservation, and supraphysiologic endometrium preparation, are discussed. Inflammatory local conditions and epigenetic influence on embryos of ART procedures are important research topics since they may have important consequences on obstetric risk. Prevention and treatment of these conditions represent new frontiers for clinicians and biologists involved in ART, and synergic actions with researchers at molecular levels are advocated.

Circulating EGFL7 distinguishes between IUGR and PE: an observational case-control study.

Isolated intrauterine growth restriction (IUGR) and preeclampsia (PE) share common placental pathogenesis. Differently from IUGR, PE is a systemic disorder which may also affect liver and brain. Early diagnosis of these conditions may optimize maternal and fetal management. Aim of this study was to assess whether Epidermal Growth Factor-Like domain 7 (EGFL7) dosage in maternal blood discriminates between isolated IUGR and PE. A total of 116 women were enrolled in this case-control study: 12 non-pregnant women, 34 healthy pregnant women, 34 women presenting with isolated IUGR and 36 presenting with PE. Levels of circulating EGFL7 and other known pro- and anti-angiogenic factors were measured by ELISA at different gestational ages (GA). Between 22-25 weeks of gestation, EGFL7 levels in early-onset PE (e-PE) plasma samples were significantly higher than those measured in controls or isolated IUGR samples (69.86 ± 6.17 vs. 19.8 ± 2.5 or 18.8 ± 2.8 µg/ml, respectively). Between 26-34 weeks, EGFL7 levels remained significantly higher in e-PE compared to IUGR. At term, circulating and placental EGFL7 levels were comparable between IUGR and late-onset PE (l-PE). In contrast, circulating levels of PlGF were decreased in both IUGR- and PE- complicated pregnancies, while levels of both sFLT-1 and sENDOGLIN were increased in both conditions. In conclusion, EGFL7 significantly discriminates between isolated IUGR and PE.

Silica encapsulation of ZnO nanoparticles reduces their toxicity for cumulus cell-oocyte-complex expansion.

BACKGROUND: Metal oxide nanoparticles (NPs) are increasingly used in many industrial and biomedical applications, hence their impact on occupational and public health has become a concern. In recent years, interest on the effect that exposure to NPs may exert on human reproduction has grown, however data are still scant. In the present work, we investigated whether different metal oxide NPs interfere with mouse cumulus cell-oocyte complex (COC) expansion. METHODS: Mouse COCs from pre-ovulatory follicles were cultured in vitro in the presence of various concentrations of two types of TiO2 NPs (JRC NM-103 and NM-104) and four types of ZnO NPs (JRC NM-110, NM-111, and in-house prepared uncoated and SiO2-coated NPs) and the organization of a muco-elastic extracellular matrix by cumulus cells during the process named cumulus expansion was investigated. RESULTS: We show that COC expansion was not affected by the presence of both types of TiO2 NPs at all tested doses, while ZnO NM-110 and NM-111 induced strong toxicity and inhibited COCs expansion at relatively low concentration. Medium conditioned by these NPs showed lower toxicity, suggesting that, beside ion release, inhibition of COC expansion also depends on NPs per se. To further elucidate this, we compared COC expansion in the presence of uncoated or SiO2-coated NPs. Differently from the uncoated NPs, SiO2-coated NPs underwent slower dissolution, were not internalized by the cells, and showed an overall lower toxicity. Gene expression analysis demonstrated that ZnO NPs, but not SiO2-coated ZnO NPs, affected the expression of genes fundamental for COC expansion. Dosimetry analysis revealed that the delivered-to-cell mass fractions for both NPs was very low. CONCLUSIONS: Altogether, these results suggest that chemical composition, dissolution, and cell internalization are all responsible for the adverse effects of the tested NPs and support the importance of a tailored, safer-by-design production of NPs to reduce toxicity.

Positive Impact of Levothyroxine Treatment on Pregnancy Outcome in Euthyroid Women with Thyroid Autoimmunity Affected by Recurrent Miscarriage.

Impaired thyroid hormone availability during early pregnancy is associated with recurrent miscarriage (RM) and adverse pregnancy outcomes. The main cause of thyroid dysfunction is thyroid-related autoimmunity (TAI), characterized by a significantly higher serum level of thyroid-stimulating hormone (TSH) compared to that of women without thyroid autoimmunity. TAI is associated with a significantly increased risk of miscarriage, and the incidence of TAI in women experiencing RM is higher compared to normal fertile women. In the present study, we have performed a retrospective analysis comparing the ability to conceive, the number of miscarriages and full-term pregnancies between 227 euthyroid women with autoimmune thyroid disease affected by RM and treated with levothyroxine (LT4) as adjuvant therapy, and a control group of 230 untreated women. We have observed a significant improvement of full-term pregnancies in treated women (59%) compared to untreated women (13%, p < 0.0001). Compared to the control group, treated women had a lower percentage of miscarriages (12% vs. 30%) and improved capacity to conceive (57% vs. 29%). Using age as a variable, the outcome in women younger than 35 years was not influenced by the LT4 therapy. Whereas, in women over 35 years, supplementation with LT4 significantly reduced the miscarriage rate (p < 0.05). We can conclude that a transient impairment of TH availability, not easily detectable before pregnancy, could be an important cause of RM in a subset of euthyroid women with autoimmune thyroid disease. This transient impairment may be reverted using adjuvant treatment with low doses of LT4.

Treatment of pregnancies complicated by intrauterine growth restriction with nitric oxide donors increases placental expression of Epidermal Growth Factor-Like Domain 7 and improves fetal growth: A pilot study.

Intrauterine growth restriction (IUGR) is a pathological condition of pregnancy with high perinatal mortality and morbidity, characterized by inadequate fetal growth associated to altered maternal hemodynamics with impaired uteroplacental blood flow and placental insufficiency. To date, iatrogenic premature delivery remains the elective therapeutic strategy. However, in recent years the possibility of a therapeutic approach with vasodilators and myorelaxants, such as nitric oxide (NO) donors, has gained interest. NO controls many endothelial cell functions, including angiogenesis and vascular permeability, by regulating the expression of angiogenic factors, such as Vascular Endothelial Growth Factor. In the present study, we investigated if treatment of pregnancies complicated by IUGR with NO donors affects the expression of Epidermal Growth Factor-Like Domain 7 (EGFL7), a secreted endothelial factor, previously demonstrated to be expressed by both endothelial and trophoblast cells and involved in proper placental development. NO donor treatment induced placental levels of EGFL7 and, in association with oral fluids, significantly improved fetal growth. Ex vivo experiments confirmed that NO donors increased expression and secretion of EGFL7 by villous explants. To specifically investigate the potential response of trophoblast cells to NO, we treated HTR8-sVneo cells with NO donors and observed induction of EGFL7 expression. Altogether, our findings indicate that NO induces endothelial and trophoblast expression of EGFL7 in the placenta and improves fetal growth, suggesting a correlation between placental levels of EGFL7 and pregnancy outcome.

Molecular Signaling Regulating Endometrium-Blastocyst Crosstalk.

Implantation of the embryo into the uterine endometrium is one of the most finely-regulated processes that leads to the establishment of a successful pregnancy. A plethora of factors are released in a time-specific fashion to synchronize the differentiation program of both the embryo and the endometrium. Indeed, blastocyst implantation in the uterus occurs in a limited time frame called the "window of implantation" (WOI), during which the maternal endometrium undergoes dramatic changes, collectively called "decidualization". Decidualization is guided not just by maternal factors (e.g., estrogen, progesterone, thyroid hormone), but also by molecules secreted by the embryo, such as chorionic gonadotropin (CG) and interleukin-1ß (IL-1 ß), just to cite few. Once reached the uterine cavity, the embryo orients correctly toward the uterine epithelium, interacts with specialized structures, called pinopodes, and begins the process of adhesion and invasion. All these events are guided by factors secreted by both the endometrium and the embryo, such as leukemia inhibitory factor (LIF), integrins and their ligands, adhesion molecules, Notch family members, and metalloproteinases and their inhibitors. The aim of this review is to give an overview of the factors and mechanisms regulating implantation, with a focus on those involved in the complex crosstalk between the blastocyst and the endometrium.

Increased circulating levels of Epidermal Growth Factor-like Domain 7 in pregnant women affected by preeclampsia.

Proper placental development is crucial to establish a successful pregnancy. Defective placentation is the major cause of several pregnancy complications, including preeclampsia (PE). We have previously demonstrated that the secreted factor Epidermal Growth Factor-like Domain 7 (EGFL7) is expressed in trophoblast cells of the human placenta and that it regulates trophoblast migration and invasion, suggesting a role in placental development. In the present study, we demonstrate that circulating levels of EGFL7 are undetectable in nonpregnant women, increase during pregnancy and decline toward term. Close to term, circulating levels of EGFL7 are significantly higher in patients affected by PE when compared to normal pregnancies. Consistent with these results, villus explant cultures obtained from placentas affected by PE display increased release of EGFL7 in the culture medium when compared to those from normal placentas. Our results suggest that increased release of placenta-derived EGFL7 and increased circulating levels of EGFL7 are associated with the clinical manifestation of PE.

Relevance to investigate different stages of pregnancy to highlight toxic effects of nanoparticles: The example of silica.

Amorphous silica nanoparticles (SiO2NPs) have been recognized as safe nanomaterial, hence their use in biomedical applications has been explored. Data, however, suggest potential toxicity of SiO2 NPs in pregnant individuals. However, no studies relating nanoparticle biokinetic/toxicity to the different gestational stages are currently available. In this respect, we have investigated the possible embryotoxic effects of three-size and two-surface functionalization SiO2NPs in mice. After intravenous administration of different concentrations at different stages of pregnancy, clinical and histopathological evaluations, performed close to parturition, did not show signs of maternal toxicity, nor effects on placental/fetal development, except for amino-functionalized 25 nm NPs. Biodistribution was studied by ICP-AES 24 h after administration, and demonstrates that all particles distributed to placenta and conceptuses/fetuses, although size, surface charge and gestational stage influenced biodistribution. Our data suggest the need of comprehensive toxicological studies, covering the entire gestation to reliably assess the safety of nanoparticle exposure during pregnancy.

Thyroid hormone regulates protease expression and activation of Notch signaling in implantation and embryo development.

A clinical association between thyroid dysfunction and pregnancy complications has been extensively reported; however, the molecular mechanisms through which TH might regulate key events of pregnancy have not been elucidated yet. In this respect, we performed in vivo studies in MMI-induced hypothyroid pregnant mice, evaluating the effect of hypothyroidism on the number of implantation sites, developing embryos/resorptions and pups per litter, at 4.5, 10.5, 18.5 days post-coitum (dpc) and at birth. We also studied the expression of major molecules involved in implantation and placentation, such as the proteases ISPs, MMPs, TIMPs and Notch pathway-related genes. Our results demonstrate that hypothyroidism may have a dual effect on pregnancy, by initially influencing implantation and by regulating placental development at later stages of gestation. To further elucidate the role of TH in implantation, we performed in vitro studies by culturing 3.5 dpc blastocysts in the presence of TH, with or without endometrial cells used as the feeder layer, and studied their ability to undergo hatching and outgrowth. We observed that, in the presence of endometrial feeder cells, TH is able to anticipate blastocyst hatching by upregulating the expression of blastocyst-produced ISPs, and to enhance blastocyst outgrowth by upregulating endometrial ISPs and MMPs. These results clearly indicate that TH is involved in the bidirectional crosstalk between the competent blastocyst and the receptive endometrium at the time of implantation.

Silver nanoparticles inhaled during pregnancy reach and affect the placenta and the foetus.

Recently, interest for the potential impact of consumer-relevant engineered nanoparticles on pregnancy has dramatically increased. This study investigates whether inhaled silver nanoparticles (AgNPs) reach and cross mouse placental barrier and induce adverse effects. Apart from their relevance for the growing use in consumer products and biomedical applications, AgNPs are selected since they can be unequivocally identified in tissues. Pregnant mouse females are exposed during the first 15 days of gestation by nose-only inhalation to a freshly produced aerosol of 18-20 nm AgNPs for either 1 or 4 h, at a particle number concentration of 3.80 × 107 part./cm-3 and at a mass concentration of 640 µg/m³. AgNPs are identified and quantitated in maternal tissues, placentas and foetuses by transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy and single-particle inductively coupled plasma mass spectrometry. Inhalation of AgNPs results in increased number of resorbed foetuses associated with reduced oestrogen plasma levels, in the 4 h/day exposed mothers. Increased expression of pregnancy-relevant inflammatory cytokines is also detected in the placentas of both groups. These results prove that NPs are able to reach and cross the mouse placenta and suggest that precaution should be taken with respect to acute exposure to nanoparticles during pregnancy.

Different expression of VEGF and EGFL7 in human hepatocellular carcinoma.

BACKGROUND: Vascular endothelial growth factor (VEGF) is one of several angiogenic factors expressed in cirrhosis and during progression to malignancy, that seem to play a major role in hepatocellular carcinoma development. Lately, another angiogenic factor, epidermal growth factor-like domain multiple 7 (EGFL7), has attracted interest due to its possible relationship with hepatocellular carcinoma metastasis. AIMS: To evaluate expression of VEGF and EGFL7 in human hepatocellular carcinoma, compared to corresponding cirrhotic surrounding tissue. METHODS: Tumoural and cirrhotic tissue was harvested from 12 consecutive patients undergoing surgical resection. VEGF and EGFL7 were assessed by immunofluorescence and quantitative reverse transcriptase-polymerase chain reaction, compared with normal controls. RESULTS: Both angiogenic factors were over-expressed in cirrhotic livers compared to normal controls. VEGF and EGFL7 expressions did not differ according to disease aetiology, nodule size or other clinical variables. While VEGF expression was constant, regardless of tumour differentiation stage and unchanged compared to surrounding cirrhotic tissue, EGFL7 expression increased in less differentiated hepatocellular carcinoma. CONCLUSIONS: The preferential expression of EGFL7 in less differentiated hepatocellular carcinoma compared to VEGF, suggests a possible important role of this angiogenic factor in a later oncogenic and infiltrative/metastatic phase.

Novel expression of EGFL7 in placental trophoblast and endothelial cells and its implication in preeclampsia.

The mammalian placenta is the site of nutrient and gas exchange between the mother and fetus, and is comprised of two principal cell types, trophoblasts and endothelial cells. Proper placental development requires invasion and differentiation of trophoblast cells, together with coordinated fetal vasculogenesis and maternal vascular remodeling. Disruption in these processes can result in placental pathologies such as preeclampsia (PE), a disease characterized by late gestational hypertension and proteinuria. Epidermal Growth Factor Like Domain 7 (EGFL7) is a largely endothelial-restricted secreted factor that is critical for embryonic vascular development, and functions by modulating the Notch signaling pathway. However, the role of EGFL7 in placental development remains unknown. In this study, we use mouse models and human placentas to begin to understand the role of EGFL7 during normal and pathological placentation. We show that Egfl7 is expressed by the endothelium of both the maternal and fetal vasculature throughout placental development. Importantly, we uncovered a previously unknown site of EGFL7 expression in the trophoblast cell lineage, including the trophectoderm, trophoblast stem cells, and placental trophoblasts. Our results demonstrate significantly reduced Egfl7 expression in human PE placentas, concurrent with a downregulation of Notch target genes. Moreover, using the BPH/5 mouse model of PE, we show that the downregulation of Egfl7 in compromised placentas occurs prior to the onset of characteristic maternal signs of PE. Together, our results implicate Egfl7 as a possible factor in normal placental development and in the etiology of PE.

Biodistribution and toxicity of pegylated single wall carbon nanotubes in pregnant mice.

BACKGROUND: Single wall carbon nanotubes (SWCNTs) are considered promising nanoparticles for industrial and biomedical applications; however their potential toxicity in several biological systems, including the feto-placental unit, has been demonstrated. Functionalization of SWCNTs with polyethylene glycol chains (PEG-SWCNTs) dramatically reduces their toxicity, and for this reason PEG-SWCNTs are candidates for biomedical applications. However, no data are available on their safety for the developing embryo, in spite of the clinical and social relevance of this topic. The purpose of this study is therefore to investigate the safety of PEG-SWCNTs for their use as biomedical carriers in pregnancy. METHODS: For toxicological studies, amino-functionalized PEG-SWCNT were intravenously injected in CD1 pregnant mice at different doses (range 0.1-30 µg/mouse), in single or multiple administrations. For biodistribution studies, fluorescently labeled PEG-SWCNTs were obtained by acylation of terminal PEG amino groups with near infrared emitting fluorochromes (PEG-SWCNT-750) and injected at the dosage of 10 µg/mouse, at either day 5.5 (when the placenta is still developing) or day 14.5 of gestation (when the maturation of the placenta is complete). RESULTS: We found no adverse effects both on embryos and dams up to the dose of 10 µg/mouse. At the dose of 30 µg/mouse, occasional teratogenic effects, associated with placental damage, were detected both when administered as a single bolus (1 out of 10 dams; 1 malformed embryo) or as multiple doses (2 out of 10 dams; 5 malformed embryos). The difference in the prevalence of dams with malformed embryos between the 30 µg exposed group and controls approached the statistical significance (p = 0.06). Hepatic damage in dams was seen only in the multiple exposure group (4 out of 10; p = 0.04 when compared with the single exposure group or controls). PEG-SWCNT-750 reached the conceptus when administered early in pregnancy. At later stages, PEG-SWCNT-750 were detected in the placenta and the yolk sac, but not in the embryo. CONCLUSIONS: PEG-SWCNTs may cause occasional teratogenic effects in mice beyond a threshold dose. Such effect might depend on their ability to reach the feto-placenta unit. Although not automatically transferable to humans, these data should be considered if exposing women during pregnancy.

Screening of nanoparticle embryotoxicity using embryonic stem cells.

Due to the increasing use of engineered nanoparticles in many consumer products, rapid and economic tests for evaluating possible adverse effects on human health are urgently needed. In the present chapter the use of mouse embryonic stem cells as a valuable tool to in vitro screen nanoparticle toxicity on embryonic tissues is described. This in vitro method is a modification of the embryonic stem cell test, which has been widely used to screen soluble chemical compounds for their embryotoxic potential. The test offers an alternative to animal experimentation, reducing experimental costs and ethical issues.

Physico-chemical properties mediating reproductive and developmental toxicity of engineered nanomaterials.

With the increasing production of engineered nanomaterials (ENMs) exploited in many consumer products, a wider number of people is expected to be exposed to such materials in the near future, both in occupational and environmental settings. This has raised concerns about the possible implications on public health. In particular, very recently the scientific community has focused on the effect that ENMs might exert on the reproductive apparatus and on embryonic development. Indications that ENMs might have adverse effects on cells of the germ line and on the developing embryos have been reported. In the present minireview we will perform a critical analysis of the published work on reproductive and developmental toxicity of the most commonly used nanoparticles with a major focus on mammalian models. We will place emphasis on the main physico-chemical characteristics that can affect NP behaviour in biological systems, i.e. presence of contaminants and nanoparticle destabilization, size, dosage, presence of functional groups, influence of the solvent used for their suspension in biological media, aggregation/agglomeration, intrinsic chemical composition and protein corona/opsonisation. The importance of this specific field of nanotoxicology is documented by the rapidly increasing number of published papers registered in the last three years, which might be a consequence of the growing concerns on the possible interference of ENMs with reproductive ability and pregnancy outcome, in a time in which reproductive age has increased and the possibility to bear children appears reduced.

Low doses of pristine and oxidized single-wall carbon nanotubes affect mammalian embryonic development.

Several in vitro and in vivo studies suggest local and systemic effects following exposure to carbon nanotubes. No data are available, however, on their possible embryotoxicity in mammals. In this study, we tested the effect of pristine and oxidized single-wall carbon nanotubes (SWCNTs) on the development of the mouse embryo. To this end, SWCNTs (from 10 ng to 30 µg/mouse) were administered to female mice soon after implantation (postcoital day 5.5); 10 days later, animals were sacrificed, and uteri, placentas, and fetuses examined. A high percentage of early miscarriages and fetal malformations was observed in females exposed to oxidized SWCNTs, while lower percentages were found in animals exposed to the pristine material. The lowest effective dose was 100 ng/mouse. Extensive vascular lesions and increased production of reactive oxygen species (ROS) were detected in placentas of malformed but not of normally developed fetuses. Increased ROS levels were likewise detected in malformed fetuses. No increased ROS production or evident morphological alterations were observed in maternal tissues. No fetal and placental abnormalities were ever observed in control animals. In parallel, SWCNT embryotoxicity was evaluated using the embryonic stem cell test (EST), a validated in vitro assay developed for predicting embryotoxicity of soluble chemical compounds, but never applied in full to nanoparticles. The EST predicted the in vivo data, identifying oxidized SWCNTs as the more toxic compound.