Extracellular vesicles secreted by human aneuploid embryos present a distinct transcriptomic profile and upregulate MUC1 transcription in decidualised endometrial stromal cells.

STUDY QUESTION: Do extracellular vesicles (EVs) secreted by aneuploid human embryos possess a unique transcriptomic profile that elicits a relevant transcriptomic response in decidualized primary endometrial stromal cells (dESCs)? SUMMARY ANSWER: Aneuploid embryo-derived EVs contain transcripts of PPM1J, LINC00561, ANKRD34C, and TMED10 with differential abundance from euploid embryo-derived EVs and induce upregulation of MUC1 transcript in dESCs. WHAT IS KNOWN ALREADY: We have previously reported that IVF embryos secrete EVs that can be internalized by ESCs, conceptualizing that successful implantation to the endometrium is facilitated by EVs. Whether these EVs may additionally serve as biomarkers of ploidy status is unknown. STUDY DESIGN SIZE DURATION: Embryos destined for biopsy for preimplantation genetic testing for aneuploidy (PGT-A) were grown under standard conditions. Spent media (30 µl) were collected from euploid (n = 175) and aneuploid (n = 140) embryos at cleavage (Days 1-3) stage and from euploid (n = 187) and aneuploid (n = 142) embryos at blastocyst (Days 3-5) stage. Media samples from n = 35 cleavage-stage embryos were pooled in order to obtain five euploid and four aneuploid pools. Similarly, media samples from blastocysts were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy. PARTICIPANTS/MATERIALS SETTING METHODS: EVs were isolated from pools of media by differential centrifugation and EV-RNA sequencing was performed following a single-cell approach that circumvents RNA extraction. ESCs were decidualized (estradiol: 10 nM, progesterone: 1 µM, cAMP: 0.5 mM twice every 48 h) and incubated for 24 h with EVs (50 ng/ml). RNA sequencing was performed on ESCs. MAIN RESULTS AND THE ROLE OF CHANCE: Aneuploid cleavage stage embryos secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc = -5.13, P = 0.011), LINC00561 (log2fc = -7.87, P = 0.010), and ANKRD34C (log2fc = -7.30, P = 0.017) and more abundant in TMED10 (log2fc = 1.63, P = 0.025) compared to EVs of euploid embryos. Decidualization per se induced downregulation of MUC1 (log2fc = -0.54, P = 0.0028) in ESCs as a prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualized ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2fc = 0.85, P = 0.0201). LARGE SCALE DATA: Raw data have been uploaded to GEO (accession number GSE234338). LIMITATIONS REASONS FOR CAUTION: The findings of the study will require validation utilizing a second cohort of EV samples. WIDER IMPLICATIONS OF THE FINDINGS: The discovery that the transcriptomic profile of EVs secreted from aneuploid cleavage stage embryos differs from that of euploid embryos supports the possibility to develop a non-invasive methodology for PGT-A. The upregulation of MUC1 in dESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure. STUDY FUNDING/COMPETING INTERESTS: The study was supported by a Marie Sklodowska-Curie Actions fellowship awarded to SM by the European Commission (CERVINO grant agreement ID: 79620) and by a BIRTH research grant from Theramex HQ UK Ltd. The authors have no conflicts of interest to declare.

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.

The relationship between CYP19A1 gene expression in luteinized granulosa cells and follicular estradiol output in women with endometriosis.

Endometriosis was claimed to negatively affect the intrafollicular environment, hindering oocyte competence. Previous studies evaluated expression levels of cytochrome P450 aromatase (CYP19A) in granulosa and cumulus oophorus cells collected from endometriosis women, but results are controversial. To further investigate the intrafollicular environment whose alteration may potentially disturb ovarian steroidogenesis in endometriosis, gene expression of CYP19A and of its upstream enzymes, StAR and 3ßHSD was assessed in luteinized granulosa cells isolated from follicular fluids (FF) collected during Assisted Reproduction Technology (ART) procedures in women with stage III-IV disease and from subjects without the condition. In a subgroup of patients, cumulus oophorus cells (COCs) were also assessed for CYP19A, StAR and 3ßHSD gene expression. No difference in mRNA expression of CYP19A1, StAR and 3ßHSD in both granulosa cells and COCs was observed between the two groups of patients. No significant difference was also found between estradiol FF levels detected in endometriosis patients (median=873, IQR=522-1221 ng/ml)) and control patients (median=878, IQR=609-1137 ng/ml). To gain more insight into the intrafollicular regulation of CYP19A in patients with endometriosis, associations between expression of the analyzed genes, systemic and follicular 17ß-estradiol levels and ART outcomes were assessed. While in the control group, levels of CYP19A1, StAR and 3ßHSD transcripts significantly correlated with follicular estradiol levels (adjusted R² of 0.60), no significant association was detected in affected women (adjusted R² of 0.23). After stratification of the populations based on the presence of the disease, CYP19A1 expression was shown to correlate with the number of oocytes retrieved [ß:- 1.214;95%CI: - 2.085 - (-0.343); p = 0.007] in the control group while this association was not present in patients with endometriosis [ß:- 0.003; 95%CI:- 0.468-0.461; p = 0.988)]. These results do not support data from the literature indicating a reduced aromatase expression in granulosa cells of affected women, but they highlight a potential subtle mechanism affecting the ovulation process in these women.

The expression and role of glycans at the feto-maternal interface in humans.

During pregnancy, both the maternal endometrium and the blastocyst have highly glycosylated proteins with glycosylations controlled in a specific manner. Carbohydrates play a fundamental role in cell-cell and cell-matrix recognition and are involved in defining the structure and integrity of tissues. The uterus' secretions, which are rich in glycoproteins and glycogen and the presence of a functional glycocalyx on the uterine epithelium, establish a favourable milieu, which is essential for the correct implantation and subsequent development of the blastocyst. Likewise, carbohydrate residues such as fucose and sialic acid present at the placental level are determinant in creating an immuno-environment, which supports the mother's tolerance towards the fetal antigens. In this review, we explore the literature concerning the role of important glycan-epitopes at the feto-maternal interface in the human species. Moreover, we also show some unpublished interesting results on changes of glycan residues in human placenta tissues from the first trimester of 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.

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.

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.

Expression of Taste Receptor 2 Subtypes in Human Testis and Sperm.

Taste receptors (TASRs) are expressed not only in the oral cavity but also throughout the body, thus suggesting that they may play different roles in organ systems beyond the tongue. Recent studies showed the expression of several TASRs in mammalian testis and sperm, indicating an involvement of these receptors in male gametogenesis and fertility. This notion is supported by an impaired reproductive phenotype of mouse carrying targeted deletion of taste receptor genes, as well as by a significant correlation between human semen parameters and specific polymorphisms of taste receptor genes. To better understand the biological and thus clinical significance of these receptors for human reproduction, we analyzed the expression of several members of the TAS2Rs family of bitter receptors in human testis and in ejaculated sperm before and after in vitro selection and capacitation. Our results provide evidence for the expression of TAS2R genes, with TAS2R14 being the most expressed bitter receptor subtype in both testis tissue and sperm cells, respectively. In addition, it was observed that in vitro capacitation significantly affects both the expression and the subcellular localization of these receptors in isolated spermatozoa. Interestingly, α-gustducin and α-transducin, two Gα subunits expressed in taste buds on the tongue, are also expressed in human spermatozoa; moreover, a subcellular redistribution of both G protein α-subunits to different sub-compartments of sperm was registered upon in vitro capacitation. Finally, we shed light on the possible downstream transduction pathway initiated upon taste receptor activation in the male reproductive system. Performing ultrasensitive droplets digital PCR assays to quantify RNA copy numbers of a distinct gene, we found a significant correlation between the expression of TAS2Rs and TRPM5 (r = 0.87), the cation channel involved in bitter but also sweet and umami taste transduction in taste buds on the tongue. Even if further studies are needed to clarify the precise functional role of taste receptors for successful reproduction, the presented findings significantly extend our knowledge of the biological role of TAS2Rs for human male fertility.

Proteostasis network alteration in lysosomal storage disorders: Insights from the mouse model of Krabbe disease.

In Krabbe disease, a mutation in GALC gene causes widespread demyelination determining cell death by apoptosis, mainly in oligodendrocytes and Schwann cells. Less is known on the molecular mechanisms induced by this deficiency. Here, we report an impairment in protein synthesis and degradation and in proteasomal clearance with a potential accumulation of the misfolded proteins and induction of the endoplasmic reticulum stress in the brain of 6-day-old twitcher mice (TM) (model of Krabbe disease). In particular, an imbalance of the immunoproteasome function was highlighted, useful for shaping adaptive immune response by neurological cells. Moreover, our data show an involvement of cytoskeleton remodeling in Krabbe pathogenesis, with a lamin meshwork disaggregation in twitcher oligodendrocytes in 6-day-old TM. This study provides interesting protein targets and mechanistic insight on the early onset of Krabbe disease that may be promising options to be tested in combination with currently available therapies to rescue Krabbe phenotype.

Increased expression of neurogenic factors in uterine fibroids.

STUDY QUESTION: Are selective markers for the neuronal differentiation such as microtubule-associated protein 2 (MAP-2) and synaptophysin (SYP) as well as the nerve growth factor (NGF) expressed by fibroids, myometrium and eutopic endometrium? SUMMARY ANSWER: Neuronal markers NGF, MAP-2 and SYP are highly expressed in fibroids compared with matched myometrium, and this neurogenic pathway is upregulated by tumor necrosis factor (TNF) alpha in cultured smooth muscle cells (SMCs). WHAT IS KNOWN ALREADY: Uterine fibroids or leiomyomas are the most common benign tumors, accounting for approximately one-third of hysterectomies. The present trend is to improve the medical treatment avoiding surgery, also for fertility sparing; hence, the pathogenic mechanisms are investigated, aiming to develop new therapeutic strategy. STUDY DESIGN, SIZE, DURATION: This laboratory-based case-control study is focused on fibroids and myometrial specimens obtained between 2015 and 2017 from 15 women of reproductive age at the proliferative phase of the menstrual cycle. Leiomyomas, matched myometrium and endometrium from each woman were analyzed. Control endometrium was obtained from women undergoing surgery for ovarian cyst (n = 15). PARTICIPANTS/MATERIALS, SETTING, METHODS: qRT-PCR, western blotting and immunostaining were applied to evaluate the expression of neurogenic markers; the effects of TNF on NGF, MAP-2 and SYP expression in cultured SMCs from leiomyomas and matched myometrium were analyzed. MAIN RESULTS AND THE ROLE OF CHANCE: qRT-PCR analyses using tissues from clinical patients showed that the levels of NGF, MAP-2 and SYP mRNA were significantly higher in uterine leiomyomas compared with their matched myometrium (P < 0.05), whereas only NGF was significantly increased in eutopic endometrium compared with healthy endometrium. In primary SMCs, isolated from fibroids or from the adjacent myometrium, NGF, MAP-2 and SYP mRNA expression were significantly increased by TNF treatment (P < 0.05). Finally, human endometrial stromal cells prepared from the endometrium of patients affected by uterine fibroids display higher TNF expression (P < 0.001). LIMITATIONS, REASONS FOR CAUTION: qRT-PCR analysis and immunofluorescence validation are robust methods demonstrating a clear upregulation of neurogenic factors in leiomyomas, even though additional studies are needed to establish a correlation between increased neuronal gene expression and degree of pain, as well as the involvement of inflammation mediators in the development of the neurogenic unhinge. Therefore, more in vivo studies are needed to confirm the results achieved from primary cultured SMCs. WIDER IMPLICATIONS OF THE FINDINGS: The increased expression of neurogenic factors in uterine fibroids and endometrium may contribute to explain the painful stimuli. Accordingly, these neurogenic pathways may represent potential therapeutic avenues to treat the fibroid-related disorders. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by research grants from the University of Siena. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Matrix metalloproteinases and their inhibitors in human cumulus and granulosa cells as biomarkers for oocyte quality estimation.

OBJECTIVE: To study the molecular profile of metalloproteinases and their tissue inhibitors in granulosa and cumulus cells in a subset of fertile and infertile women. DESIGN: Molecular study with granulosa and cumulus cells. SETTING: University hospital. PATIENT(S): Forty-four women undergoing assisted reproductive techniques for female infertility factor, with partners having a normal spermiogram and 15 normally fertile women with male partner affected by severe oligoasthenoteratozoospermia or nonobstructive azoospermia. INTERVENTION(S): In vitro fertilization. MAIN OUTCOME MEASUREMENT(S): We investigated gene expression level of metalloproteinases (MMP2, MMP9, MMP11) and their tissue inhibitors (TIMP1, TIMP2) by means of quantitative reverse-transcription polymerase chain reaction, protein quantification by means of Western blot, and localization by means of immunofluorescence. RESULT(S): We firstly validated HPRT1 as the most reliable housekeeping gene enabling correct gene expression analysis in both granulosa and cumulus cells. Gene expression, Western blot, and immunofluorescence analysis of MMP2, MMP9, and MMP11 and their tissue inhibitors TIMP1 and TIMP2 demonstrated that these enzymes are finely tuned in these cells. MMP9 is specifically expressed only in granulosa, whereas MMP2 is more expressed in cumulus and granulosa cells in cases of reduced ovarian response and decreased fertilization rate. CONCLUSION(S): This study sheds light on MMP and TIMP expression in granulosa and cumulus cells, and it may help in understanding the fine regulation of oocyte maturation inside the follicle. Although further studies are needed to fully understand the molecular mechanisms involved in these processes, our findings may be useful in the identification of biomarkers of oocyte maturation, competence acquiring, and fertilization.

Dysregulation of GdA Expression in Endometrium of Women With Endometriosis: Implication for Endometrial Receptivity.

Glycodelin-A (GdA) has been proposed to represent a potential biomarker of endometrial function, but little is known about its expression during the different phases of the menstrual cycle and under pathological conditions. In the light of its potential importance also in embryo implantation, we aimed to evaluate the expression profile of GdA as well as the presence of different glycosylated glycoforms and the immunolocalization in endometrial tissue from women with endometriosis and in women with proven fertility, at different times during the menstrual cycle. Our results showed that GdA is synthesized by endometrial epithelial and stromal cells, both in healthy endometrium and eutopic endometrium from women with endometriosis, with a profile including several glycosylated glycoforms, differentially expressed in each phase of the menstrual cycle. During the secretory phase, a significant increase in GdA protein expression, with a different glycoforms profile, was observed in endometriotic eutopic endometrium. Protein localization in eutopic endometrial tissue resulted significantly different in comparison with endometrium from women with proven fertility. This study indicate that GdA is a complex glycoprotein including up to 6 different glycoforms specifically expressed during the different phase of the menstrual cycle; in pathologic conditions such as endometriosis, the expression profile is altered possibly related to the impaired endometrial receptivity.