In Situ Labeling of the Aqueous Compartment of Extracellular Vesicles with Luminescent Gold Nanoclusters.

Extracellular vesicles (EVs) are well-known membrane-limited particles secreted by both healthy and cancerous cells. They are considered as biomarkers for early cancer diagnosis and are involved in many pathologies and physiological pathways. They could serve as diagnostic tools in liquid biopsies, as therapeutics in regenerative medicine, or as drug delivery vehicles. Our aim is here to encapsulate luminescent nanoprobes in the aqueous compartment of human EVs extracted from reproductive fluids. The analysis and labeling of the EVs content with easily detectable luminescent nanoparticles could enable a powerful tool for early diagnosis of specific diseases and also for the design of new therapeutics. In this view, gold nanoclusters (AuNCs) appear as an attractive alternative as nontoxic fluorophore probes because of their luminescence properties, large window of fluorescence lifetimes (1 ns-1 µs), ultrasmall size (<2 nm), good biocompatibility, and specific ability as X-ray photosensitizers. Here, we investigated an attractive method that uses fusogenic liposomes to deliver gold nanoclusters into EVs. This approach guarantees the preservation of the EVs membrane without any breakage, thus maintaining compartmental integrity. Different lipid compositions of liposomes preloaded with AuNCs were selected to interact electrostatically with human EVs and compared in terms of fusion efficiency. The mixture of liposomes and EVs results in membrane mixing as demonstrated by FRET experiments and fusion revealed by flux cytometry and cryo-TEM. The resulting fused EVs exhibit typical fluorescence of the AuNCs together with an increased size in agreement with fusion. Moreover, the fusion events in mixtures of EVs and AuNCs preloaded liposomes were analyzed by using cryo-electron microscopy. Finally, the ratio of released AuNCs during the fusion between the fusogenic liposomes and the EVs was estimated to be less than 20 mol % by Au titration using ICP spectroscopy.

[First-line management of infertile couple. Guidelines for clinical practice of the French College of Obstetricians and Gynecologists 2022]. / Prise en charge de première intention du couple infertile : mise à jour des RPC 2010 du CNGOF.

OBJECTIVE: To update the 2010 CNGOF clinical practice guidelines for the first-line management of infertile couples. MATERIALS AND METHODS: Five major themes (first-line assessment of the infertile woman, first-line assessment of the infertile man, prevention of exposure to environmental factors, initial management using ovulation induction regimens, first-line reproductive surgery) were identified, enabling 28 questions to be formulated using the Patients, Intervention, Comparison, Outcome (PICO) format. Each question was addressed by a working group that had carried out a systematic review of the literature since 2010, and followed the Grading of Recommendations Assessment, Development and Evaluation (GRADE®) methodology to assess the quality of the scientific data on which the recommendations were based. These recommendations were then validated during a national review by 40 national experts. RESULTS: The fertility work-up is recommended to be prescribed according to the woman's age: after one year of infertility before the age of 35 and after 6months after the age of 35. A couple's initial infertility work-up includes a single 3D ultrasound scan with antral follicle count, assessment of tubal permeability by hysterography or HyFOSy, anti-Mullerian hormone assay prior to assisted reproduction, and vaginal swabbing for vaginosis. If the 3D ultrasound is normal, hysterosonography and diagnostic hysteroscopy are not recommended as first-line procedures. Chlamydia trachomatis serology does not have the necessary performance to predict tubal patency. Post-coital testing is no longer recommended. In men, spermogram, spermocytogram and spermoculture are recommended as first-line tests. If the spermogram is normal, it is not recommended to check the spermogram. If the spermogram is abnormal, an examination by an andrologist, an ultrasound scan of the testicles and hormonal test are recommended. Based on the data in the literature, we are unable to recommend a BMI threshold for women that would contraindicate medical management of infertility. A well-balanced Mediterranean-style diet, physical activity and the cessation of smoking and cannabis are recommended for infertile couples. For fertility concern, it is recommended to limit alcohol consumption to less than 5 glasses a week. If the infertility work-up reveals no abnormalities, ovulation induction is not recommended for normo-ovulatory women. If intrauterine insemination is indicated based on an abnormal infertility work-up, gonadotropin stimulation and ovulation monitoring are recommended to avoid multiple pregnancies. If the infertility work-up reveals no abnormality, laparoscopy is probably recommended before the age of 30 to increase natural pregnancy rates. In the case of hydrosalpinx, surgical management is recommended prior to ART, with either salpingotomy or salpingectomy depending on the tubal score. It is recommended to operate on polyps>10mm, myomas 0, 1, 2 and synechiae prior to ART. The data in the literature do not allow us to systematically recommend asymptomatic uterine septa and isthmoceles as first-line surgery. CONCLUSION: Based on strong agreement between experts, we have been able to formulate updated recommendations in 28 areas concerning the initial management of infertile couples.

Sperm DNA methylation dynamics after chemotherapy: a longitudinal study of a patient with testicular germ cell tumor treatment.

BACKGROUND: An important issue for young men affected by testicular germ cell tumor (TGCT) is how TGCT and its treatment will affect, transiently or permanently, their future reproductive health. Previous studies have reported that xenobiotics can induce changes on human sperm epigenome and have the potential to promote epigenetic alterations in the offspring. OBJECTIVES: Here, we report the first longitudinal DNA methylation profiling of frozen sperm from a TGCT patient before and up to 2 years after a bleomycin, etoposide, and cisplatin (BEP) chemotherapy. MATERIALS AND METHODS: A TGCT was diagnosed in a 30-year-old patient. A cryopreservation of spermatozoa was proposed before adjuvant BEP treatment. Semen samples were collected before and after chemotherapy at 6, 9, 12, and 24 months. The DNA methylation status was determined by RRBS to detect DNA differentially methylated regions (DMRs). RESULTS: The analysis revealed that among the 74 DMRs showing modified methylation status 6 months after therapy, 17 remained altered 24 months after treatment. We next associated DMRs with differentially methylated genes (DMGs), which were subsequently intersected with loci known to be important or expressed during early development. DISCUSSION AND CONCLUSION: The consequences of the cancer treatment on the sperm epigenome during the recovery periods are topical issues of increasing significance as epigenetic modifications to the paternal genome may have deleterious effects on the offspring. The altered methylated status of these DMGs important for early development might modify their expression pattern and thus affect their function during key stages of embryogenesis, potentially leading to developmental disorders or miscarriages.

Myelinosome Organelles in the Retina of R6/1 Huntington Disease (HD) Mice: Ubiquitous Distribution and Possible Role in Disease Spreading.

Visual deficit is one of the complications of Huntington disease (HD), a fatal neurological disorder caused by CAG trinucleotide expansions in the Huntingtin gene, leading to the production of mutant Huntingtin (mHTT) protein. Transgenic HD R6/1 mice expressing human HTT exon1 with 115 CAG repeats recapitulate major features of the human pathology and exhibit a degeneration of the retina. Our aim was to gain insight into the ultrastructure of the pathological HD R6/1 retina by electron microscopy (EM). We show that the HD R6/1 retina is enriched with unusual organelles myelinosomes, produced by retinal neurons and glia. Myelinosomes are present in all nuclear and plexiform layers, in the synaptic terminals of photoreceptors, in the processes of retinal neurons and glial cells, and in the subretinal space. In vitro study shows that myelinosomes secreted by human retinal glial Müller MIO-M1 cells transfected with EGFP-mHTT-exon1 carry EGFP-mHTT-exon1 protein, as revealed by immuno-EM and Western-blotting. Myelinosomes loaded with mHTT-exon1 are incorporated by naive neuronal/neuroblastoma SH-SY5Y cells. This results in the emergence of mHTT-exon1 in recipient cells. This process is blocked by membrane fusion inhibitor MDL 28170. Conclusion: Incorporation of myelinosomes carrying mHTT-exon1 in recipient cells may contribute to HD spreading in the retina. Exploring ocular fluids for myelinosome presence could bring an additional biomarker for HD diagnostics.

Extra-cellular vesicles of the male genital tract: new actors in male fertility?

Extracellular Vesicles (EVs) are membrane-limited particles containing proteins, lipids, metabolites and nucleic acids that are secreted by healthy and cancerous cells. These vesicles are very heterogeneous in size and content and mediate a variety of biological functions. Three subtypes of EV have been described in the male genital tract: microvesicles, myelinosomes and exosomes. Each type of EVs depends on the location of secretion such as the testis, prostate or epididymis. It has been shown that EVs can fuse together and deliver information to recipient cells, for example spermatozoa in the male genital tract. Cryo-electron microscopy remains the reference technique for determining EV morphology, but quantifying the absolute concentration of these EVs in biological fluids remains a challenge from a clinical point of view. The field of bio detection has considerably increased with the introduction of nanomaterials in biosensors and will provide a better understanding of the impact of these EVs. However, functional modifications of male gametes result from interactions with the components of the intraluminal fluid all along the genital tract and depend on the secretion and absorption of proteins and lipids from the local microenvironment. We cannot therefore exclude the possibility of epigenetic modulation of the information that will be transmitted to the embryo and therefore to the next generation via EVs.

RéSUMé: Les Vésicules Extracellulaires (VE) sont des constituants d'origine membranaire contenant des protéines solubles ou membranaires, des lipides, des métabolites ou des acides nucléiques. Ces vésicules sont très hétérogènes en taille et en contenu. Trois catégories de VE ont été décrites dans le tractus génital mâle: les microvésicules, les myélinosomes et les exosomes. Les types de VE sont différents selon le lieu de production testiculaire, prostatique ou encore épididymaire. Il a été montré que les VE peuvent fusionner et délivrer une information à la cellule réceptrice, en l'occurrence le spermatozoïde dans le tractus génital mâle. La cryo-microscopie électronique reste la technique de référence pour déterminer la morphologie des VE mais la quantification de la concentration absolue de ces VE dans les liquides biologiques reste un challenge dans le cadre d'une approche clinique. Le domaine de la biodétection s'est. considérablement développé avec l'introduction des nanomatériaux dans les biocapteurs et va permettre de mieux comprendre l'impact de ces VE. Or les modifications fonctionnelles des gamètes mâles résultent d'interactions avec les composants du liquide intraluminal tout le long du tractus génital et dépendent de la sécrétion et de l'absorption de protéines et de lipides du microenvironnement local. On ne peut donc pas exclure la possibilité d'une modulation épigénétique des informations qui seront transmises à l'embryon donc à la génération suivante via les VE.

MERTK-Mediated LC3-Associated Phagocytosis (LAP) of Apoptotic Substrates in Blood-Separated Tissues: Retina, Testis, Ovarian Follicles.

Timely and efficient elimination of apoptotic substrates, continuously produced during one's lifespan, is a vital need for all tissues of the body. This task is achieved by cells endowed with phagocytic activity. In blood-separated tissues such as the retina, the testis and the ovaries, the resident cells of epithelial origin as retinal pigmented epithelial cells (RPE), testis Sertoli cells and ovarian granulosa cells (GC) provide phagocytic cleaning of apoptotic cells and cell membranes. Disruption of this process leads to functional ablation as blindness in the retina and compromised fertility in males and females. To ensure the efficient elimination of apoptotic substrates, RPE, Sertoli cells and GC combine various mechanisms allowing maintenance of tissue homeostasis and avoiding acute inflammation, tissue disorganization and functional ablation. In tight cooperation with other phagocytosis receptors, MERTK-a member of the TAM family of receptor tyrosine kinases (RTK)-plays a pivotal role in apoptotic substrate cleaning from the retina, the testis and the ovaries through unconventional autophagy-assisted phagocytosis process LAP (LC3-associated phagocytosis). In this review, we focus on the interplay between TAM RTKs, autophagy-related proteins, LAP, and Toll-like receptors (TLR), as well as the regulatory mechanisms allowing these components to sustain tissue homeostasis and prevent functional ablation of the retina, the testis and the ovaries.

[Myelinosomes: A new pathway of protein quality control]. / Les myélinosomes : une nouvelle voie du contrôle de qualité des protéines.

Maintenance of cell proteostasis relies on two degradation pathways: proteasome and autophagy. Here we describe a new proteostasis pathway avoiding degradation of abnormal proteins yet carrying them outside the cell using nanovesicles called myelinosomes. These myelinosomes are produced in pathological or stress situations in relation with genetic or environmental factors. Myelinosome vesicles are nano-sized multi-stacked membrane structures, resembling myelin sheath. It has recently been shown in two models of genetic diseases (Huntington's disease and cystic fibrosis) that myelinosomes are important for eliminating mutant proteins in an unusual secretory process, thus preventing their accumulation and aggregation in cells.

Title: Les myélinosomes : une nouvelle voie du contrôle de qualité des protéines. Abstract: Deux voies de dégradation des protéines mal repliées sont classiquement décrites : la voie du protéasome et la voie de l'autophagie. Nous décrivons ici une nouvelle voie de protéostase cellulaire ne dégradant pas la protéine anormale mais l'expulsant hors de la cellule grâce à des nanovésicules appelées myélinosomes. Ces myélinosomes sont produits par la cellule dans des situations pathologiques ou de stress en lien avec des facteurs génétiques ou environnementaux. Sur le plan morphologique, les myélinosomes sont caractérisés par des membranes osmiophiles denses aux électrons dont l'arrangement empilé est semblable à celui de la myéline et présente jusqu'à 30 feuillets selon le type de cellule. Dans deux modèles, au moins, de maladies génétiques (la maladie de Huntington et la mucoviscidose), les myélinosomes sont importants pour éliminer les protéines mutées par un processus sécrétoire inhabituel, évitant ainsi leur agrégation dans les cellules.

A novel action of follicle-stimulating hormone in the ovary promotes estradiol production without inducing excessive follicular growth before puberty.

In cyclic females, FSH stimulates ovarian estradiol (E2) production and follicular growth up to the terminal stage. A transient elevation in circulating FSH and E2 levels occurs shortly after birth. But what could be the action of FSH on the ovary during this period, and in particular how it stimulates ovarian steroidogenesis without supporting terminal follicular maturation is intriguing. By experimentally manipulating FSH levels, we demonstrate in mice that the mid-infantile elevation in FSH is mandatory for E2 production by the immature ovary, but that it does not stimulate follicle growth. Importantly, FSH increases aromatase expression to stimulate E2 synthesis, however it becomes unable to induce cyclin D2, a major driver of granulosa cell proliferation. Besides, although FSH prematurely induces luteinizing hormone (LH) receptor expression in granulosa cells, LH pathway is not functional in these cells to induce their terminal differentiation. In line with these results, supplying infantile mice with a superovulation regimen exacerbates E2 production, but it does not stimulate the growth of follicles and it does not induce ovulation. Overall, our findings unveil a regulation whereby high postnatal FSH concentrations ensure the supply of E2 required for programming adult reproductive function without inducing follicular maturation before puberty.

Impact of Hodgkin or non-Hodgkin lymphoma and their treatments on sperm aneuploidy: a prospective study by the French CECOS network.

OBJECTIVE: To assess sperm production and aneuploidy in Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) before and after treatments. DESIGN: Multicenter, prospective, longitudinal study of lymphoma patients analyzed before treatment and after 3, 6, 12, and 24 months. SETTING: University hospitals. PATIENT(S): Forty-five HL and 13 NHL patients were investigated before and after treatment. Treatment regimens were classified in two groups: ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) with or without (±) radiotherapy, and CHOP (doxorubicin, cyclophosphamide, vincristine, prednisone)/MOPP-ABV (mechlorethamine, oncovin, procarbazine, prednisone-doxorubicin, bleomycin, vinblastine). A control group of 29 healthy men was also studied. INTERVENTION(S): Semen analyses and aneuploidy study by FISH were performed at each time point. MAIN OUTCOME MEASURE(S): Comparison of mean sperm characteristics and percentage of sperm aneuploidy rates before and after treatment. RESULT(S): Before treatment, HL and NHL men had altered semen characteristics and higher sperm aneuploidy rates (median 0.76 [interquartile range 0.56-0.64]) than the control group (0.54 [0.46-0.74]). After treatment, sperm production was significantly lowered 3 and 6 months after ABVD ± radiotherapy or CHOP/MOPP-ABV. After ABVD ± radiotherapy, the aneuploidy rate increased significantly only at 3 months, and values obtained 1 or 2 years later were lower than pretreatment values. In contrast, in the CHOP/MOPP-ABV treatment group, semen characteristics and aneuploidy rate did not return to normal levels until 2 years after treatment. CONCLUSION(S): Lymphoma itself has consequences on sperm aneuploidy frequency before treatment. Moreover, lymphoma treatments have deleterious effects on sperm chromosomes related to treatment type and time since treatment. Patient counseling is essential concerning the transient but significant sperm aneuploidy induced by lymphoma and its treatments.

Predictive factors of healthy term birth after single blastocyst transfer.

BACKGROUND: Blastocyst culture and elective single embryo transfer programmes are increasingly used to reduce multiple pregnancies after IVF. To optimize the results, there is a need to better select embryos, to implement efficient cryopreservation programmes and to refine selection criteria. In the present study, we set out to identify relevant clinical predictors of healthy term birth (HTB) after single blastocyst transfer (SBT). DESIGN: analysis of prospectively collected database. SETTING: University IVF centre in Nantes, France. In 872 infertile women undergoing their first IVF cycle with SBT between January 2007 and December 2008, multivariable analysis and logistic regression were used to identify predictive factors of HTB, i.e. delivery of a live born term singleton of ≥ 2500 g, surviving at least 28 days with no reported congenital anomaly. RESULTS: Of 304 deliveries, there were 16 twin pairs (5.5%) and no high order deliveries. The rate of HTB was 266/872 (30.5%). Univariate analysis showed that the probability of HTB was significantly higher in women under 35 years [odds ratio (OR):1.75, 0.95 confidence interval (CI): 1.2-2.5, P = 0.001], in women with a BMI < 30 kg/m² (OR: 3.0, 0.95 CI: 1.5-5.9, P = 0.001), in non-smoking women (OR: 2.2, 0.95 CI: 1.5-3.2, P < 0.0001), and after Day 5 compared with after Day 6 transfer (OR: 2.65, 0.95 CI: 1.8-3.8, P < 0.0001). Multivariable analysis showed that BMI, smoking and day of embryo transfer were independent predictors of HTB, regardless of female age. CONCLUSIONS: After SBT, female obesity and smoking reduce the chance of HTB, independent of female age. Day 6 transfer should be avoided.

Progressive alcohol-induced sperm alterations leading to spermatogenic arrest, which was reversed after alcohol withdrawal.

This is a report of a 6-year follow-up of a male patient's semen parameters during heavy chronic alcohol intoxication and after withdrawal. A slowly progressive negative impact of alcohol could be observed: isolated moderate teratozoospermia was firstly noted followed by oligoasthenoteratospermia. Then a severe worsening resulted in cryptozoospermia and ultimately in azoospermia. At this moment, the histological analysis of a testicular biopsy revealed a maturation arrest of the germinal cells at the pachytene stage with no mature sperm cells. Alcohol withdrawal was then obtained, allowing a very fast and drastic improvement of semen characteristics; strictly normal semen parameters were observed after no more than 3 months. Taking into consideration these data, patients should be questioned about their alcohol intake before assisted reproductive technology and should be informed about this adverse effect. Moreover, this case report emphasizes how quickly benefits can be obtained after withdrawal, even in the case of heavy chronic alcohol intake.

Lack of association between genetic polymorphisms in enzymes associated with folate metabolism and unexplained reduced sperm counts.

BACKGROUND: The metabolic pathway of folate is thought to influence DNA stability either by inducing single/double stranded breaks or by producing low levels of S-adenosyl-methionine leading to abnormal gene expression and chromosome segregation. Polymorphisms in the genes encoding enzymes in the folate metabolism pathway show distinct geographic and/or ethnic variations and in some cases have been linked to disease. Notably, the gene Methylenetetrahydrofolate reductase (MTHFR) in which the homozygous (TT) state of the polymorphism c.665C>T (p.A222V) is associated with reduced specific activity and increased thermolability of the enzyme causing mild hyperhomocysteinemia. Recently several studies have suggested that men carrying this polymorphism may be at increased risk to develop infertility. METHODOLOGY/PRINCIPAL FINDINGS: We have tested this hypothesis in a case/control study of ethnic French individuals. We examined the incidence of polymorphisms in the genes MTHFR (R68Q, A222V and E429A), Methionine synthase reductase MTRR; (I22M and S175L) and Cystathionine beta-synthase (CBS; G307S). The case population consisted of DNA samples from men with unexplained azoospermia (n = 70) or oligozoospermia (n = 182) and the control population consisted of normospermic and fertile men (n = 114). We found no evidence of an association between the incidence of any of these variants and reduced sperm counts. In addition haplotype analysis did not reveal differences between the case and control populations. CONCLUSIONS/SIGNIFICANCE: We could find no evidence for an association between reduced sperm counts and polymorphisms in enzymes involved in folate metabolism in the French population.

Mutational analysis of the WNT gene family in women with Mayer-Rokitansky-Kuster-Hauser syndrome.

The aim of this study is to determine if Müllerian agenesis has a genetic basis linked to the WNT genes. Genomic DNA analyses for mutations in the coding sequences of four members of this family in a series of 11 women with Mayer-Rokitansky-Kuster-Hauser syndrome found four variants in the coding sequence of these genes, but causal mutations were not observed. This supports the hypothesis that mutations in the coding sequence of WNT4, WNT5A, WNT7A, and WNT9B are not responsible for the Mayer-Rokitansky-Kuster-Hauser syndrome.