Paternal Age Amplifies Cryopreservation-Induced Stress in Human Spermatozoa.

The global fall in male fertility is a complicated process driven by a variety of factors, including environmental exposure, lifestyle, obesity, stress, and aging. The availability of assisted reproductive technology (ART) has allowed older couples to conceive, increasing the average paternal age at first childbirth. Advanced paternal age (APA), most often considered male age ≥40, has been described to impact several aspects of male reproductive physiology. In this prospective cohort study including 200 normozoospermic patients, 105 of whom were ≤35 years (non-APA), and 95 of whom were ≥42 years (APA), we assessed the impact of paternal age on different endpoints representative of sperm quality and cryopreservation tolerance. Non-APA patients had superior fresh semen quality; DNA fragmentation was notably increased in APA as compared to non-APA individuals (21.7% vs. 15.4%). Cryopreservation further increased the DNA fragmentation index in APA (26.7%) but not in non-APA patients. Additionally, APA was associated with increased mtDNAcn in both fresh and frozen/thawed sperm, which is indicative of poorer mitochondrial quality. Cryopreservation negatively impacted acrosome integrity in both age groups, as indicated by reduced incidences of unreacted acrosome in relation to fresh counterparts in non-APA (from 71.5% to 57.7%) and APA patients (from 75% to 63%). Finally, cryopreservation significantly reduced the phosphorylation status of proteins containing tyrosine residues in sperm from young males. Therefore, the present findings shed light on the effects of paternal age and cryopreservation on sperm quality and serve as valuable new parameters to improve our understanding of the mechanisms underlying sperm developmental competence that are under threat in current ART practice.

Efficacy of Lactiplantibacillus plantarum PBS067, Bifidobacterium animalis subsp. lactis BL050, and Lacticaseibacillus rhamnosus LRH020 in the Amelioration of Vaginal Microbiota in Post-Menopausal Women: A Prospective Observational Clinical Trial.

The menopausal transition marks a significant physiological shift in women. Menopause-related symptoms can significantly affect a woman's quality of life and probiotics have emerged as a promising avenue. This study aims to investigate the benefits of probiotics in improving vaginal well-being and microbiota composition in post-menopausal women. A prospective observational clinical trial was carried out enrolling 50 post-menopausal healthy women, aged between 45 and 65 years old, taking a supplement containing Lactiplantibacillus plantarum PBS067, Bifidobacterium animalis subsp. lactis BL050, and Lacticaseibacillus rhamnosus LRH020 (3B CFU/day) for 28 days. Vaginal swabs were collected to evaluate microbiota fluctuation and the inflammatory pattern was recorded. A Vaginal Health Index was provided to evaluate vaginal well-being throughout the trial. Clinical outcomes revealed a decrease in menopausal symptoms. Significant improvements were observed across various parameters: a 50% enhancement in the VHI score (p < 0.0001), alongside substantial reductions in inflammatory cytokine levels. An 87.8% decrease in IL-6, 57.6% in IL-1ß, and 40.8% in TNF-α was observed (p < 0.05). Moreover, the probiotic intervention facilitated the restoration of vaginal microbiota, evidenced by an increase in lactobacilli abundance. In conclusion, the combination of these specific probiotic strains, previously clinically tested in childbearing-age women, showed to be effective also for post-menopausal women.

Exposure to Cumulus Cell Secretome Improves Sperm Function: New Perspectives for Sperm Selection In Vitro.

In the literature, there is a well-known correlation between poor semen quality and DNA sperm integrity, which can turn into negative outcomes in terms of embryo development and clinical pregnancy. Sperm selection plays a pivotal role in clinical practice, and the most widely used methods are mainly based on sperm motility and morphology. The cumulus oophorus complex (COC) during natural fertilization represents a barrier that spermatozoa must overcome to reach the zona pellucida and fertilize the oocyte. Spermatozoa that can pass through the COC have better structural and metabolic characteristics as well as enhanced acrosome reaction (AR). The present study aimed to evaluate the exposure of sperm to cumulus cell secretome during swim-up treatment (SUC) compared with the routinely used swim-up method (SU). To determine the effectiveness of this method, biological factors critical for the ability of sperm to fertilize an oocyte, including capacitation, AR, tyrosine phosphorylation signature, DNA integrity, and mitochondrial functionality, were assessed. The SUC selection assures recovery of high-quality spermatozoa, with enhanced mitochondrial functionality and motility compared with both SU-selected and unselected (U) sperm. Furthermore, using this modified swim-up procedure, significantly reduced sperm DNA damage (p < 0.05) was detected. In conclusion, the SUC approach is a more physiological and integrated method for sperm selection that deserves further investigation for its translation into clinical practice.

Vaginal colonization of women after oral administration of Lactobacillus crispatus strain NTCVAG04 from the human microbiota.

BACKGROUND: The genomic approach has deeply changed the microbiology perspective, mainly concerning the microbioma identification. In this regard, some microbes colonize the healthy vagina. Vaginitis is a common gynecological ailment and includes bacterial vaginosis (BV), usually caused by local dysbiosis, such as a microbiota imbalance. Lactobacilli are the most prevalent bacteria colonizing the healthy vagina, so guaranteeing local eubiosis. In particular, vaginal colonization by L. crispatus is associated with low susceptibility to BV. Therefore, probiotics, such as life bacteria providing health advantages, are a current strategy in the prevention or treatment of vaginitis, including BV. However, there is a low level of evidence that probiotics after ingestion could really colonize the vagina. In particular, no study evidenced that L. crispatus after ingestion can colonize vagina. Therefore, the current study explored the capacity of Biovaginil® (NTC, Milan, Italy) dietary supplement containing Lactobacillus crispatus NTCVAG04 and vitamin A to colonize the gut and vagina in women with a history of vaginitis/vaginosis. METHODS: Twenty fertile females (mean age 34.0 years) were enrolled in the study. Rectal and vaginal swabs were collected at baseline and after the first and second cycle of Biovaginil®. Each cycle lasted 14 days within two consecutive menstrual periods. RESULTS: Seven women were excluded from the analysis because the samples were technically not evaluable. One woman dropped out because of mild adverse event. At the end of the study, nine women (75%) had positive rectal swab for L. crispatus NTCVAG04, and 8 of them also had positive vaginal swab. CONCLUSIONS: The current study provided the first evidence that L. crispatus NTCVAG04, administered by two Biovaginil® courses, colonized both the gut and vagina. Moreover, the L. crispatus NTCVAG04 strain could be considered the archetype of a new class of oral probiotics that actively colonize the vagina, and that could be called "colpobiotics."

Bitter Taste Receptors and Endocrine Disruptors: Cellular and Molecular Insights from an In Vitro Model of Human Granulosa Cells.

Endocrine disrupting chemicals (EDCs) are compounds that interfere with the synthesis, transport and binding action of hormones responsible for reproduction and homeostasis. Some EDCs compounds are activators of Taste bitter Receptors, a subclass of taste receptors expressed in many extraoral locations, including sperm and follicular somatic cells. This makes TAS2Rs attractive molecules to study and investigate to shed light on the effect of EDCs on female reproduction and fertility. This study aims to assess the effect of selected EDCs [namely Biochanin A (BCA), caffeine, Daidzein, Genistein and Isoflavone] on hGL5, an immortalized cell line exhibiting characteristics coherent with primary follicular granulosa cells. After demonstrating that this model expresses all the TAS2Rs (TAS2R3, TAS2R4, TAS2R14, TAS2R19, TAS2R43) specifically expressed by the primary human granulosa cells, we demonstrated that BCA and caffeine significantly affect mitochondrial footprint and intracellular lipid content, indicating their contribution in steroidogenesis. Our results showed that bitter taste receptors may be involved in steroidogenesis, thus suggesting an appealing mechanism by which these compounds affect the female reproductive system.

Cellular and Molecular Mechanisms of In Vivo and In Vitro SARS-CoV-2 Infection: A Lesson from Human Sperm.

Despite the major target of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, being the respiratory system, clinical evidence suggests that the male reproductive system may represent another viral target organ. Revealing the effect of SARS-CoV-2 infection on testis and sperm is a priority for reproductive biology, as well as for reproductive medicine. Here, we confirmed that the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is highly expressed on human testis and ejaculated sperm; moreover, we provide evidence for the expression of the co-receptors transmembrane protease/serine (TMPRSS2), Basigin (BSG), and Catepsin L (CTSL). Human sperm were readily infected, both in vivo and in vitro, by SARS-CoV-2, as demonstrated by confocal and electron microscopy. The demonstration that the seminiferous epithelium and sperm support SARS-CoV-2 viral replication suggests the possibility that the spermatogenetic process may be detrimentally affected by the virus, and at the same time, supports the need to implement safety measures and guidelines to ensure specific care in reproductive medicine.

PCOS Physiopathology and Vitamin D Deficiency: Biological Insights and Perspectives for Treatment.

Recent literature has stressed the importance of vitamin D (VD) in polycystic ovary syndrome (PCOS). Women with PCOS are deficient in VD, particularly those with a higher weight. Hypovitaminosis is a risk factor for glucose intolerance, and reduced levels of VD is associated with insulin resistance and increased diabetes risk. Since women with PCOS and hirsutism seem to have lower levels of VD than women with PCOS without hirsutism, a correlation between VD deficiency and hyperandrogenism may be suggested. Interestingly, VD is crucial for many human physiological functions, including to counteract inflammation and oxidative stress. Some studies evaluated effects of VD supplementation on glucose homeostasis variables, hormonal status, lipid concentrations, and biomarkers of inflammation and oxidative stress among VD-deficient women. Moreover, VD has been shown to play a role in egg quality and fertility. This review aims to show the relationship between VD and the endocrine and metabolic profile of PCOS patients, as well as its implications for their fertility. The supplement of VD to the common therapy can lead to an improvement of the insulin resistance and lipid metabolism, a reduction of circulating androgens, as well as a better response to the induction of ovulation in PCOS women.

Multi-Omics Integrative Approach of Extracellular Vesicles: A Future Challenging Milestone.

In the era of multi-omic sciences, dogma on singular cause-effect in physio-pathological processes is overcome and system biology approaches have been providing new perspectives to see through. In this context, extracellular vesicles (EVs) are offering a new level of complexity, given their role in cellular communication and their activity as mediators of specific signals to target cells or tissues. Indeed, their heterogeneity in terms of content, function, origin and potentiality contribute to the cross-interaction of almost every molecular process occurring in a complex system. Such features make EVs proper biological systems being, therefore, optimal targets of omic sciences. Currently, most studies focus on dissecting EVs content in order to either characterize it or to explore its role in various pathogenic processes at transcriptomic, proteomic, metabolomic, lipidomic and genomic levels. Despite valuable results being provided by individual omic studies, the categorization of EVs biological data might represent a limit to be overcome. For this reason, a multi-omic integrative approach might contribute to explore EVs function, their tissue-specific origin and their potentiality. This review summarizes the state-of-the-art of EVs omic studies, addressing recent research on the integration of EVs multi-level biological data and challenging developments in EVs origin.

Positive Effect of a New Combination of Antioxidants and Natural Hormone Stimulants for the Treatment of Oligoasthenoteratozoospermia.

Oligoasthenoteratozoospermia (OAT) accounts for about 90% of male infertility; in many cases this disorder may be associated with oxidative stress, a condition that decreases the success of fertilization. Therefore, the empirical treatment of male infertility is often based on the use of antioxidants. The aim of the present study was to assess the effectiveness of three months' administration of a new nutraceutical preparation on hormone profile, sperm parameters and fertilization capability in men undergoing in vitro fertilization (IVF). A total of 36 OAT patients were daily treated for 3 months with a dose of a formulation containing: Inositol, L-Carnitine, Vitamins C, D, E, Coenzyme Q10 and Selenium. Selected parameters were analysed before (T0) and after (T1) treatment, and IVF outcomes were evaluated. We observed an improvement of sperm concentration, motility, morphology and vitality; blood level of testosterone also showed an increase. A significant increase of fertilization rate was detected in 14 couples, whose male partner were treated with the nutraceutical preparation. The present results indicate that a formulation containing antioxidant and energy supply substances was effective in the treatment of sperm alterations and led to significant recovery of fertilizing capacity.

Bitter Taste Receptors Expression in Human Granulosa and Cumulus Cells: New Perspectives in Female Fertility.

Bitter taste receptors (TAS2RS) expression is not restricted to the oral cavity and the presence of these receptors in the male reproductive system and sperm provides insights into their possible role in human reproduction. To elucidate the potential role of TAS2Rs in the female reproductive system, we investigated the expression and localization of bitter taste receptors and the components of signal transduction cascade involved in the pathway of taste receptors in somatic follicular cells obtained from women undergoing assisted reproductive techniques. We found that TAS2R genes are expressed in both cumulus (CCs) and granulosa (GCs) cells, with TAS2R14 being the most highly expressed bitter receptor subtype. Interestingly, a slight increase in the expression of TAS2R14 and TAS2R43 was shown in both GCs and CCs in young women (p < 0.05), while a negative correlation may be established between the number of oocytes collected at the pickup and the expression of TAS2R43. Regarding α-gustducin and α-transducin, two Gα subunits expressed in the taste buds on the tongue, we provide evidence for their expression in CCs and GCs, with α-gustducin showing two additional isoforms in GCs. Finally, we shed light on the possible downstream transduction pathway initiated by taste receptor activation in the female reproductive system. Our study, showing for the first time the expression of taste receptors in the somatic ovarian follicle cells, significantly extends the current knowledge of the biological role of TAS2Rs for human female fertility.

Main actors behind the endometrial receptivity and successful implantation.

Embryo implantation occurs during a short period of time, the implantation window, in the mid-secretory phase of the menstrual cycle. The cross-talk between the endometrium and the embryo, at the stage of blastocyst, is a necessary condition for successful implantation. Till now, no single molecule or receptor has been identified to play an essential role on embryo implantation but a huge number of mediators, including cytokines, lipids, adhesion molecules, growth factors, and others, are reported to support the establishment of pregnancy. Therefore, the aim of this review is not only to describe the different actors involved in the implantation process, but also to try to characterize the relationships between these factors as well as their time-regulated activation. Moreover, the availability of in vitro culture systems to study the interactions between embryo and endometrium as well as the paracrine communication regulated by exosomal vesicles will be investigated, as an innovative approach for a more precise characterization of the interactions between the different molecules involved in this process. The in-depth knowledge of all these complex mechanisms will allow to address the reasons of implantation failure and infertility, thus providing new avenues for promoting the successful establishment of a pregnancy.

Extracellular matrix remodeling and inflammatory pathway in human endometrium: insights from uterine leiomyomas.

OBJECTIVE: To compare the expression profiles of matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of matrix metalloproteinases, TIMPs) in the endometrium of women with and without type 3 leiomyomas and to understand their relationship with inflammatory status. DESIGN: Molecular and in silico studies. SETTING: University hospital. PATIENT(S): Patients with type 3 leiomyomas ranging from 3 to 10 cm in diameter (n = 18) and control age-matched women undergoing surgery for ovarian cysts (n = 18) who underwent endometrial biopsies. INTERVENTION(S): Endometrial biopsies. MAIN OUTCOME MEASURE(S): To evaluate the expression levels of MMPs and TIMPs in the endometrium, quantitative reverse transcriptase polymerase chain reaction and Western blot were performed. With the use of immunofluorescence analysis, the investigated proteins were localized in the tissues. The expression levels of inflammatory mediators such as IL-1ß, IL-6, IL-10, TGF, COX1, COX2, STAT3, and VEGF were evaluated by quantitative reverse transcriptase polymerase chain reaction, and their relationships were detected by the STRING approach. RESULT(S): The endometrium of women with type 3 leiomyomas exhibited differential expression of MMPs and TIMPs, particularly MMP2, MMP11, and MMP14, as well as different topographic distribution, suggesting that leiomyomas may influence the endometrial molecular profile. Significant decreases in IL-1ß, IL-6, and IL-10 expression, along with increases in COX1 and COX2, as well as VEGF, were highlighted. The STRING approach suggests that this altered gene expression profile may affect the Th17 cell differentiation pathway. CONCLUSION(S): The differential expression and localization of MMPs and TIMPs observed in women with type 3 leiomyomas, along with the reported derangement in the expression of key molecules involved in the inflammatory pathway, may contribute to changes in endometrial receptivity in these patients.

Telomere Length and Male Fertility.

Over the past decade, telomeres have attracted increasing attention due to the role they play in human fertility. However, conflicting results have been reported on the possible association between sperm telomere length (STL) and leukocyte telomere length (LTL) and the quality of the sperm parameters. The aim of this study was to run a comprehensive study to investigate the role of STL and LTL in male spermatogenesis and infertility. Moreover, the association between the sperm parameters and 11 candidate single nucleotide polymorphisms (SNPs), identified in the literature for their association with telomere length (TL), was investigated. We observed no associations between sperm parameters and STL nor LTL. For the individual SNPs, we observed five statistically significant associations with sperm parameters: considering a p < 0.05. Namely, ACYP2-rs11125529 and decreased sperm motility (p = 0.03); PXK-rs6772228 with a lower sperm count (p = 0.02); NAF1-rs7675998 with increased probability of having abnormal acrosomes (p = 0.03) and abnormal flagellum (p = 0.04); ZNF208-rs8105767 and reduction of sperms with normal heads (p = 0.009). This study suggests a moderate involvement of telomere length in male fertility; however, in our analyses four SNPs were weakly associated with sperm variables, suggesting the SNPs to be pleiotropic and involved in other regulatory mechanisms independent of telomere homeostasis, but involved in the spermatogenic process.

Emerging role of embryo secretome in the paracrine communication at the implantation site: a proof of concept.

OBJECTIVE: To assess the role of embryo secretome in modifying the molecular profile of glycodelin A (GdA) in endometrial organoids (ORG) mimicking the implantation window. To verify whether the use of embryo-conditioned culture medium at the time of the embryo transfer may increase in vitro fertilization outcome. DESIGN: Molecular study with human endometrial ORG and embryo-conditioned culture medium. Retrospective study using prospectively recorded data. SETTING: University hospital. PATIENT(S): For isolation and culture of endometrial glandular ORG, endometrial biopsy specimens from five white women of proven fertility undergoing laparoscopy for tubal sterilization. A total of 75 women undergoing intracytoplasmic sperm injection for tubal and/or male infertility factor. INTERVENTIONS(S): In vitro fertilization. MAIN OUTCOME MEASURE(S): Pinopodes presence in human endometrial ORG. Glycodelin A expression profile by means of two-dimensional electrophoresis. In vitro fertilization outcome. RESULT(S): This in vitro study demonstrated that the treatment of endometrial ORG with the secretome of medium conditioned by the growing embryo increased the GdA relative abundance and induced a different glycoform pattern. Biochemical and clinical pregnancy rate significantly increased when the spent medium was loaded during the transfer (17.5% vs. 36.6% and 16.5% vs. 35.1%, respectively). CONCLUSION(S): This study demonstrated that the secretome of implanting embryos is able to induce the expression as well as to determine the relative abundance and the glycosilation profile of endometrial GdA, a protein having a key role in the embryo-endometrial cross talk. Moreover, a significant increase in pregnancy rate was observed when the embryo transfer was performed by using the culture medium conditioned by the growing embryo.

Oxidative Stress Measurement in Frozen/Thawed Human Sperm: The Protective Role of an In Vitro Treatment with Myo-Inositol.

Despite its widespread use, sperm cryopreservation induces serious detrimental alterations in sperm function; indeed, it is commonly associated with decreased sperm viability and motility, and DNA fragmentation. Mechanisms of human sperm cryodamage are thought to be multifactorial, but oxidative stress seems to have a prominent role. A huge amount of data supported the cryoprotective effect of different antioxidants able to minimize the detrimental effects of reactive oxygen species (ROS) and improve the quality of spermatozoa. Among others, myo-inositol is one of the most powerful and has been reported to be effective in improving sperm quality and motility when used both in vivo and in vitro. This study aimed to determine the in vitro impact of myo-inositol in ameliorating sperm oxidative status during sperm cryopreservation. In particular, we demonstrated a significant improvement of sperm parameters (vitality and motility) when myo-inositol was added after sperm thawing (p < 0.05). Moreover, we showed that myo-inositol induces a significant increase in oxygen consumption, the main index of oxidative phosphorylation efficiency and ATP production. Finally, by means of 2D-electrophoresis, we demonstrated a significant decrease in the level of carbonyl groups, the main structural changes occurring in conditions of oxidative stress (p < 0.05). In conclusion, the sperm cryopreservation procedure we developed, assuring the reduction of ROS-induced sperm modifications, may improve the in vitro procedure currently used in ART laboratory for sperm cryostorage.

Characterization of the Age-Dependent Changes in Antioxidant Defenses and Protein's Sulfhydryl/Carbonyl Stress in Human Follicular Fluid.

The oxidative stress, characterized by the imbalance between pro-oxidants and antioxidants molecules, seems to be involved in the pathogenesis of female subfertility. In particular, the presence of different markers of oxidative stress has been reported in human follicular fluid (FF) surrounding oocytes. Based on its distinctive composition and on the close proximity to the oocyte, FF creates a unique microenvironment having a direct impact on oocyte quality, implantation, and early embryo development. An imbalance in reactive oxygen species (ROS) production in ovarian follicular fluid may have a negative effect on these processes and, as a consequence, on female fertility. Therefore, the aim of this study was to evaluate the redox state of the FF through various methodological approaches. By means of 2D-electrophoresis we demonstrated that the main structural changes occurring in the proteins of the follicular fluid of normovulatory women were correlated to the age of the patients and to the antioxidant defenses present in the FF. Measurement of these parameters could have clinical relevance, since the assessment of the oxidative stress rate may be helpful in evaluating in vitro fertilization potential.

Relationship between the metabolic and lipid profile in follicular fluid of women undergoing in vitro fertilization.

Among the follicular fluid (FF) components promoting the development of the oocyte are included glycoproteins, several fatty acids, and steroid hormones synthesized by the dominant follicle. For this, the analysis of the metabolites present in FF can determine the quality of the oocyte. FF composition is in part determined by local follicular metabolic processes and in part a plasma transudate. Since the causes of impaired fertility may be due to a metabolic imbalance, metabolomics is useful to identify low molecular weight metabolites. Oxidative stress is involved in human infertility and the use of metabolomics can be crucial to identify which other metabolites besides reactive oxygen species are involved in oxidative stress correlated to infertility. To obtain new information on the study of signaling molecules in FF, the knowledge of the lipid content will be important to improve information on the understanding of follicular development. The objective of this study is to identify (a) a metabolic profile and a lipid profile of FF in women undergoing in vitro fertilization and (b) to correlate the previous information obtained regarding adiponectin and oxidative stress with the metabolic and lipid profile obtained in the present study. As result, we found an increase in oxidative stress due to both an increase of androgens and an accumulation of lipids in the follicular environment and we suggest that this might be one of the causes of reduced fertility.

Respiratory Mitochondrial Efficiency and DNA Oxidation in Human Sperm after In Vitro Myo-Inositol Treatment.

Semen samples are known to contain abnormal amounts of reactive oxygen species (ROS) and oxygen free radicals; therefore, the identification of antioxidant molecules able to counteract the oxidative damage caused by ROS is foresight. Indeed, improving semen quality in terms of motility and reduction in DNA damage, can significantly improve the fertilization potential of sperm in vitro. To this regard, myo-inositol, based on its antioxidant properties, has been reported to be effective in improving sperm quality and motility in oligoasthenozoospermic patients undergoing assisted reproduction techniques when used as a dietary supplementation. Moreover, in vitro treatment demonstrated a direct relationship between myo-inositol, mitochondrial membrane potential and sperm motility. This experimental study aimed to evaluate the effects of myo-inositol (Andrositol-lab) in vitro treatment on sperm motility, capacitation, mitochondrial oxidative phosphorylation and DNA damage. Our results demonstrate that myo-inositol induces a significant increase in sperm motility and in oxygen consumption, the main index of oxidative phosphorylation efficiency and ATP production, both in basal and in in vitro capacitated samples. Moreover, we provide evidence for a significant protective role of myo-inositol against oxidative damage to DNA, thus supporting the in vitro use of myo-inositol in assisted reproductive techniques. Even if further studies are needed to clarify the mechanisms underlying the antioxidant properties of myo-inositol, the present findings significantly extend our knowledge on human male fertility and pave the way to the definition of evidence-based guidelines, aiming to improve the in vitro procedure currently used in ART laboratory for sperm selection.

Organoids of Human Endometrium: A Powerful In Vitro Model for the Endometrium-Embryo Cross-Talk at the Implantation Site.

Embryo implantation has been defined as the "black box" of human reproduction. Most of the knowledge on mechanisms underlining this process derives from animal models, but they cannot always be translated to humans. Therefore, the development of an in vitro/ex vivo model recapitulating as closely and precisely as possible the fundamental functional features of the human endometrial tissue is very much desirable. Here, we have validated endometrial organoids as a suitable 3D-model to studying epithelial endometrial interface for embryo implantation. Transmission and scanning electron microscopy analyses showed that organoids preserve the glandular organization and cell ultrastructural characteristics. They also retain the responsiveness to hormonal treatment specific to the corresponding phase of the menstrual cycle, mimicking the in vivo glandular-like aspect and functions. Noteworthy, organoids mirroring the early secretive phase show the development of pinopodes, large cytoplasmic apical protrusions of the epithelial cells, traditionally considered as reliable key features of the implantation window. Moreover, organoids express glycodelin A (GdA), a cycle-dependent marker of the endometrial receptivity, with its quantitative and qualitative features accounting well for the profile detected in the endometrium in vivo. Accordingly, organoids deriving from the eutopic endometrium of women with endometriosis show a GdA glycosylation pattern significantly different from healthy organoids, confirming our prior data on endometrial tissues. The present results strongly support the idea that organoids may closely recapitulate the molecular and functional characteristics of their cells/tissue of origin.

Expression of Matrix Metalloproteinases and Their Inhibitors in Endometrium: High Levels in Endometriotic Lesions.

Endometriosis is a condition defined as presence of endometrium outside of the uterine cavity. These endometrial cells are able to attach and invade the peritoneum or ovary, thus forming respectively the deep infiltrating endometriosis (DIE) and the ovarian endometrioma (OMA), the ectopic lesions feature of this pathology. Endometriotic cells display high invasiveness and share some features of malignancy with cancer cells. Indeed, the tissue remodeling underlining lesion formation is achieved by matrix metalloproteinases (MMPs) and their inhibitors. Therefore, these molecules are believed to play a key role in development and pathogenesis of endometriosis. This study investigated the molecular profile of metalloproteinases and their inhibitors in healthy (n = 15) and eutopic endometrium (n = 19) in OMA (n = 10) and DIE (n = 9); moreover, we firstly validated the most reliable housekeeping genes allowing accurate gene expression analysis in these tissues. Gene expression, Western blot, and immunofluorescence analysis of MMP2, MMP3, and MMP10 and their tissue inhibitors TIMP1 and TIMP2 demonstrated that these enzymes are finely tuned in these tissues. In OMA lesions, all the investigated MMPs and their inhibitors were significantly increased, while DIE expressed high levels of MMP3. Finally, in vitro TNFα treatment induced a significant upregulation of MMP3, MMP10, and TIMP2 in both healthy and eutopic endometrial stromal cells. This study, shedding light on MMP and TIMP expression in endometriosis, confirms that these molecules are altered both in eutopic endometrium and endometriotic lesions. Although further studies are needed, these data may help in understanding the molecular mechanisms involved in the extracellular matrix remodeling, a crucial process for the endometrial physiology.