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.

Maternal exposure to zinc oxide nanoparticles causes cochlear dysfunction in the offspring.

Introduction: Zinc oxide nanoparticles (ZnO NPs) have been engineered and are largely used in material science and industry. This large and increasing use justifies a careful study about the toxicity of this material for human subjects. The concerns regard also the reproductive toxicity and the fetotoxicity. Materials and methods: The effect of the exposure to ZnO NPs on the cochlear function was studied in a group of pregnant CD1 mice and in their offspring. This study is part of a larger toxicological study about the toxicity of ZnO NPs during pregnancy. Four groups were analyzed and compared, exposed and non-exposed dams and their offspring. The cochlear function was quantitatively assessed by means of Distortion Product Otoacoustic Emissions (DPOAEs). Results and discussion: A large statistically significant difference was found between the non-exposed dams offspring and the exposed dams offspring (p = 1.6 · 10-3), whose DPOAE levels were significantly lower than those of non-exposed dams offspring and comparable to those of the adults. The DPOAE levels of the exposed and non-exposed dams were very low and not significantly different. This occurrence is related to the fact that these mice encounter a rapid aging process. Conclusion: Our findings show that maternal exposure to ZnO NPs does not reflect in overt toxicity on fetal development nor impair offspring birth, however it may damage the nervous tissue of the inner ear in the offspring. Other studies should confirm this result and identify the mechanisms through which ZnO NPs may affect ear development.

Predicting the cytotoxicity of nanomaterials through explainable, extreme gradient boosting.

Nanoparticles (NPs) are a wide class of materials currently used in several industrial and biomedical applications. Due to their small size (1-100 nm), NPs can easily enter the human body, inducing tissue damage. NP toxicity depends on physical and chemical NP properties (e.g., size, charge and surface area) in ways and magnitudes that are still unknown. We assess the average as well as the individual importance of NP atomic descriptors, along with chemical properties and experimental conditions, in determining cytotoxicity endpoints for several nanomaterials. We employ a multicenter cytotoxicity nanomaterial database (12 different materials with first and second dimensions ranging between 2.70 and 81.2 nm and between 4.10 and 4048 nm, respectively). We develop a regressor model based on extreme gradient boosting with hyperparameter optimization. We employ Shapley additive explanations to obtain good cytotoxicity prediction performance. Model performances are quantified as statistically significant Spearman correlations between the true and predicted values, ranging from 0.5 to 0.7. Our results show that i) size in situ and surface areas larger than 200 nm and 50 m2/g, respectively, ii) primary particles smaller than 20 nm; iii) irregular (i.e., not spherical) shapes and iv) positive Z-potentials contribute the most to the prediction of NP cytotoxicity, especially if lactate dehydrogenase (LDH) assays are employed for short experimental times. These results were moderately stable across toxicity endpoints, although some degree of variability emerged across dose quantification methods, confirming the complexity of nano-bio interactions and the need for large, systematic experimental characterization to reach a safer-by-design approach.

A practical approach to the management of thyroid dysfunction during pregnancy.

Pregnancy has an important impact on the thyroid gland and its function. Thyroid activity changes as a consequence of the novel physiological state of pregnancy and requires a complex hormonal and metabolic adaptation, which is possible only in the presence of a perfectly functioning thyroid gland. In fact, thyroid function is crucial for the success of the implantation and the progression of pregnancy. Abnormal thyroid function is very common among childbearing age women, explaining the high incidence of thyroid diseases that occur during pregnancy. Aim of this work is to analyze the adaptive events that characterize the thyroid function during pregnancy, exploring their hormonal, metabolic and molecular mechanisms. Moreover, the interpretation of the laboratory data necessary to monitor the thyroid functioning during normal pregnancy or in the presence of thyroid abnormalities will be discussed.

Pre-validation of a reporter gene assay for oxidative stress for the rapid screening of nanobiomaterials.

Engineered nanomaterials have been found to induce oxidative stress. Cellular oxidative stress, in turn, can result in the induction of antioxidant and detoxification enzymes which are controlled by the nuclear erythroid 2-related factor 2 (NRF2) transcription factor. Here, we present the results of a pre-validation study which was conducted within the frame of BIORIMA ("biomaterial risk management") an EU-funded research and innovation project. For this we used an NRF2 specific chemically activated luciferase expression reporter gene assay derived from the human U2OS osteosarcoma cell line to screen for the induction of the NRF2 mediated gene expression following exposure to biomedically relevant nanobiomaterials. Specifically, we investigated Fe3O4-PEG-PLGA nanomaterials while Ag and TiO2 "benchmark" nanomaterials from the Joint Research Center were used as reference materials. The viability of the cells was determined by using the Alamar blue assay. We performed an interlaboratory study involving seven different laboratories to assess the applicability of the NRF2 reporter gene assay for the screening of nanobiomaterials. The latter work was preceded by online tutorials to ensure that the procedures were harmonized across the different participating laboratories. Fe3O4-PEG-PLGA nanomaterials were found to induce very limited NRF2 mediated gene expression, whereas exposure to Ag nanomaterials induced NRF2 mediated gene expression. TiO2 nanomaterials did not induce NRF2 mediated gene expression. The variability in the results obtained by the participating laboratories was small with mean intra-laboratory standard deviation of 0.16 and mean inter laboratory standard deviation of 0.28 across all NRF2 reporter gene assay results. We conclude that the NRF2 reporter gene assay is a suitable assay for the screening of nanobiomaterial-induced oxidative stress responses.

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.

The process of ovarian aging: it is not just about oocytes and granulosa cells.

Ovarian age is classically considered the main cause of female reproductive infertility. In women, the process proceeds as an ongoing decline in the primordial follicle stockpile and it is associated with reduced fertility in the mid-thirties, irregular menstruation from the mid-forties, cessation of fertility, and, eventually, menopause in the early fifties. Reproductive aging is historically associated with changes in oocyte quantity and quality. However, besides the oocyte, other cellular as well as environmental factors have been the focus of more recent investigations suggesting that ovarian decay is a complex and multifaceted process. Among these factors, we will consider mitochondria and oxidative stress as related to nutrition, changes in extracellular matrix molecules, and the associated ovarian stromal compartment where immune cells of both the native and adaptive systems seem to play an important role. Understanding such processes is crucial to design treatment strategies to  slow down ovarian aging and consequently prolong reproductive lifespan and, more to this, alleviaingt side effects of menopause on the musculoskeletal, cardiovascular, and nervous systems.

Human adipose-derived stromal cells transplantation prolongs reproductive lifespan on mouse models of mild and severe premature ovarian insufficiency.

BACKGROUND: Although recent studies have investigated the ability of Mesenchymal Stromal Cells (MSCs) to alleviate short-term ovarian damage in animal models of chemotherapy-induced Premature Ovarian Insufficiency (POI), no data are available on reproductive lifespan recovery, especially in a severe POI condition. For this reason, we investigated the potential of MSCs isolated from human adipose tissue (hASCs), since they are easy to harvest and abundant, in ameliorating the length and performance of reproductive life in both mild and severe chemotherapy-induced murine POI models. METHODS: Mild and severe POI models were established by intraperitoneally administering a light (12 mg/kg busulfan + 120 mg/kg cyclophosphamide) or heavy (30 mg/kg busulfan + 120 mg/kg cyclophosphamide) dose of chemotherapy, respectively, in CD1 mice. In both cases, a week later, 1 × 106 hASCs were transplanted systemically through the tail vein. After four additional weeks, some females were sacrificed to collect ovaries for morphological evaluation. H&E staining was performed to assess stroma alteration and to count follicle numbers; immunofluorescence staining for αSMA was used to analyse vascularization. Of the remaining females, some were mated after superovulation to collect 2-cell embryos in order to evaluate their pre-implantation developmental capacity in vitro, while others were naturally mated to monitor litters and reproductive lifespan length. F1 litters' weight, ovaries and reproductive lifespan were also analysed. RESULTS: hASC transplantation alleviated ovarian weight loss and size decrease and reduced alterations on ovarian stroma and vasculature, concurrently preventing the progressive follicle stockpile depletion caused by chemotherapy. These effects were associated with the preservation of the oocyte competence to develop into blastocyst in vitro and, more interestingly, with a significant decrease of chemotherapy-induced POI features, like shortness of reproductive lifespan, reduced number of litters and longer time to plug (the latter only presented in the severe POI model). CONCLUSION: Human ASC transplantation was able to significantly reduce all the alterations induced by the chemotherapeutic treatment, while improving oocyte quality and prolonging reproductive functions, thus counteracting infertility. These results, strengthened by the use of an outbred model, support the potential applications of hASCs in women with POI, nowadays mainly induced by anticancer therapies.

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.

Clinical consequences of defective decidualization.

Decidualization is characterized by a series of genetic, metabolic, morphological, biochemical, vascular and immune changes occurring in the endometrial stroma in response to the implanting embryo or even before conception and involves the stromal cells of the endometrium. It is a fundamental reproductive event occurring in mammalian species with hemochorial placentation. A growing body of experimental and clinical evidence strongly suggests that defective or disrupted decidualization contributes to the establishment of an inappropriate maternal-fetal interface. This has relevant clinical consequences, ranging from recurrent implantation failure and recurrent pregnancy loss in early pregnancy to several significant complications of advanced gestation. Moreover, recent evidence indicates that selected diseases of the endometrium, such as chronic endometritis and endometriosis, can have a detrimental impact on the decidualization response in the endometrium and may help explain some aspects of the reduced reproductive outcome associated with these conditions. Further research efforts are needed to fully understand the biomolecular mechanisms ans events underlying an abnormal decidualization response. This will permit the development of new diagnostic and therapeutic strategies aimed to improve the likelihood of achieveing a successful pregnancy.

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.

No small matter: a perspective on nanotechnology-enabled solutions to fight COVID-19.

There is an urgent need for safe and effective approaches to combat COVID-19. Here, we asked whether lessons learned from nanotoxicology and nanomedicine could shed light on the current pandemic. SARS-CoV-2, the causative agent, may trigger a mild, self-limiting disease with respiratory symptoms, but patients may also succumb to a life-threatening systemic disease. The host response to the virus is equally complex and studies are now beginning to unravel the immunological correlates of COVID-19. Nanotechnology can be applied for the delivery of antiviral drugs or other repurposed drugs. Moreover, recent work has shown that synthetic nanoparticles wrapped with host-derived cellular membranes may prevent virus infection. We posit that nanoparticles decorated with ACE2, the receptor for SARS-CoV-2, could be exploited as decoys to intercept the virus before it infects cells in the respiratory tract. However, close attention should be paid to biocompatibility before such nano-decoys are deployed in the clinic.

Defective proteasome biogenesis into skin fibroblasts isolated from Rett syndrome subjects with MeCP2 non-sense mutations.

Rett Syndrome (RTT) is a rare X-linked neurodevelopmental disorder which affects about 1: 10000 live births. In >95% of subjects RTT is caused by a mutation in Methyl-CpG binding protein-2 (MECP2) gene, which encodes for a transcription regulator with pleiotropic genetic/epigenetic activities. The molecular mechanisms underscoring the phenotypic alteration of RTT are largely unknown and this has impaired the development of therapeutic approaches to alleviate signs and symptoms during disease progression. A defective proteasome biogenesis into two skin primary fibroblasts isolated from RTT subjects harbouring non-sense (early-truncating) MeCP2 mutations (i.e., R190fs and R255X) is herewith reported. Proteasome is the proteolytic machinery of Ubiquitin Proteasome System (UPS), a pathway of overwhelming relevance for post-mitotic cells metabolism. Molecular, transcription and proteomic analyses indicate that MeCP2 mutations down-regulate the expression of one proteasome subunit, α7, and of two chaperones, PAC1 and PAC2, which bind each other in the earliest step of proteasome biogenesis. Furthermore, this molecular alteration recapitulates in neuron-like SH-SY5Y cells upon silencing of MeCP2 expression, envisaging a general significance of this transcription regulator in proteasome biogenesis.

Length-dependent toxicity of TiO2 nanofibers: mitigation via shortening.

Length and aspect ratio represent important toxicity determinants of fibrous nanomaterials. We have previously shown that anatase TiO2 nanofibers (TiO2 NF) cause a dose-dependent decrease of cell viability as well as the loss of epithelial barrier integrity in polarized airway cell monolayers. Herein we have investigated the impact of fiber shortening, obtained by ball-milling, on the biological effects of TiO2 NF of industrial origin. Long TiO2 NF (L-TiO2 NF) were more cytotoxic than their shortened counterparts (S-TiO2 NF) toward alveolar A549 cells and bronchial 16HBE cells. Moreover, L-TiO2 NF increased the permeability of 16HBE monolayers and perturbed the distribution of tight-junction proteins, an effect also mitigated by fiber shortening. Raw264.7 macrophages efficiently internalized shortened but not long NF, which caused cell stretching and deformation. Compared with L-TiO2 NF, S-TiO2 NF triggered a more evident macrophage activation, an effect suppressed by the phagocytosis inhibitor cytochalasin B. Conversely, a significant increase of inflammatory markers was detected in either the lungs or the peritoneal cavity of mice exposed to L-TiO2 NF but not to S-TiO2 NF, suggesting that short-term macrophage activation in vitro may not be always a reliable indicator of persistent inflammation in vivo. It is concluded that fiber shortening mitigates NF detrimental effects on cell viability and epithelial barrier competence in vitro as well as inflammation development in vivo. These data suggest that fiber shortening may represent an effective safe-by-design strategy for mitigating TiO2 NF toxic effects.

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.

Molecular organization and mechanical properties of the hyaluronan matrix surrounding the mammalian oocyte.

Successful ovulation and oocyte fertilization are essential prerequisites for the beginning of life in sexually reproducing animals. In mammalian fertilization, the relevance of the protein coat surrounding the oocyte plasma membrane, known as zona pellucida, has been widely recognized, while, until not too long ago, the general belief was that the cumulus oophorus, consisting of follicle cells embedded in a hyaluronan rich extracellular matrix, was not essential. This opinion was based on in vitro fertilization procedures, in which a large number of sperms are normally utilized and the oocyte can be fertilized even if depleted of cumulus cells. Conversely, in vivo, only very few sperm cells reach the fertilization site, arguing against the possibility of a coincidental encounter with the oocyte. In the last two decades, proteins required for HA organization in the cumulus extracellular matrix have been identified and the study of fertility in mice deprived of the corresponding genes have provided compelling evidence that this jelly-like coat is critical for fertilization. This review focuses on the advances in understanding the molecular interactions making the cumulus environment suitable for oocyte and sperm encounter. Most of the studies on the molecular characterization of the cumulus extracellular matrix have been performed in the mouse and we will refer essentially to findings obtained in this animal model.