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International audience; Purpose: Infertility in adolescents and young adult (AYA) survivors of malignant disease remains a major long-term adverse effect, but semen collection for fertility preservation in fertility centers is not always feasible and makes AYAs uncomfortable. We evaluated the feasibility of collecting sperm samples on the ward versus in fertility centers. Methods: Consecutive hospitalized AYA-aged male patients in the Hematology AYA unit (Saint-Louis Hospital, France) between August 2010 and June 2016 with hematological disease and indication of semen collection (n = 95) were included in this retrospective study. Semen quality was analyzed according to World Health Organization guidelines and was compared according to semen collection place: on the ward (n = 46) or in fertility center (n = 49). Results: The median age was median age 19.1 years (range: 13.7-33.3; interquartile range: 17.1-22.8) and 85 patients successfully collected semen. Sperm collection failure was ∼11% and was comparable between the two modalities as were main sperm quality characteristics (semen volume, sperm concentration, total sperm count, progressive motility and vitality, sperm morphology, and multiple anomalies index). Oligospermia was significantly higher in the samples obtained in fertility center (47.7%) than on the ward (26.8%), p = 0.047. Average frozen straws were comparable, 12.2 ± 6.4 on the ward versus 11.9 ± 6.3 in fertility center. Conclusion: Semen collection on the ward is feasible and would be particularly interesting for AYA male patients without altering semen quality characteristics.

The aim of this prospective study was to evaluate outcome benefits expected in repeated implantation failure (RIF) patients (n = 217) after customized embryo transfer based upon identification of the receptivity window by transcriptomic approach using the Win-Test. In this test, the expression of 11 endometrial genes known to be predictive of endometrial receptivity is assessed by RT-PCR in biopsies collected during the implantation window (6–9 days after the spontaneous luteinizing hormone surge during natural cycles, 5–9 days after progesterone administration during hormone replacement therapy cycles). Then, patients underwent either customized embryo transfer (cET, n = 157 patients) according to the Win-Test results or embryo transfer according to the classical procedure (control group, n = 60). Pregnancy and live birth rates were compared in the two groups. The Win-Test showed that in 78.5% of women, the receptivity window lasted less than 48 h, although it could be shorter ( 48 h, 12%). This highlighted that only in 20% of patients with RIF the endometrium would have been receptive if the classical embryo transfer protocol was followed. In the other 80% of patients, the receptivity window was delayed by 1–3 days relative to the classical timing. This suggests that implantation failure could be linked to inadequate timing of embryo transfer. In agreement, both implantation (22.7% vs. 7.2%) and live birth rates per patient (31.8% vs. 8.3%) were significantly higher in the cET group than in the control group. cET on the basis of the Win-Test results could be proposed to improve pregnancy and live birth rates. ClinicalTrials.gov ID: NCT04192396; December 5, 2019, retrospectively registered. Electronic supplementary material The online version of this article (10.1007/s43032-020-00252-0) contains supplementary material, which is available to authorized users.

International audience; Human ovarian tissue cryopreservation (OTC) is increasingly used worldwide to preserve female fertility in prepubertal girls and women at risk of premature ovarian insufficiency (POI) in the context of urgent gonadotoxic treatments or ovarian surgery. Fertility preservation is challenging because there is no consensus regarding patient management, preservation fertility strategies, or even technical laboratory protocols, which implies that each procedure must be adapted to the characteristics of the patient profile and its own risk-benefit ratio. During OTC, mature/immature oocytes can be aspirated directly from large/small antral follicles within ovarian tissue samples and/or be released into culture media from growing follicles during ovarian tissue dissection in prepubertal girls and women. In this manuscript, we present a protocol that combines ovarian tissue freezing with the cryopreservation of mature/immature oocytes retrieved from ovarian tissue samples, improving the reproductive potential of fertility preservation. Appropriate collection, handling, and storage of ovarian tissue and oocytes before, during, and after the cryopreservation will be described. The subsequent use and safety of cryopreserved/thawed ovarian tissue samples and oocytes will also be discussed, as well as the optimal timing for in vitro maturation of immature oocytes. We recommend the systematic use of this protocol in fertility preservation of prepubertal girls and women as it increases the whole reproductive potential of fertility preservation (i.e., oocyte vitrification in addition of OTC) and also improves the safety and use of fertility preservation (i.e., thawing of oocytes versus ovarian graft), maximizing the chance of successful childbirth for the patients at risk of POI.

Cryoconservation of gametes : how to perform ? The patients can preserve their gametes when they are exposed to potential gonadotoxic pathology or treatment. In this context, the French bioethical law clearly states the obligation to inform the patients about the risks for their fertility and the possibilities to cryopreserve their gametes. Regional platforms of fertility preservation allow notably for the coordination of the oncology teams and the CECOS. For the men, sperm freezing is achieved by a slow and controlled temperature protocol. For the women, the oocytes are usually vitrified after hormonal stimulation and ovarian punction. For both, the gametes are cryopreserved in straws and stored in liquid nitrogen until use in assisted reproductive treatment (ART). Each year, the CECOS keeping the gametes interrogates patients on their wish to continue, or not, the cryoconservation. The gametes can only be used in ART by the patients only during their lifetime and with their consent, without alterations related to the duration of storage.Conservation des gamètes : quelles modalités ? Les patient(e)s peuvent conserver leurs gamètes lorsqu’ils( ou elles) sont exposé(e)s à une pathologie ou un traitement potentiellement gonadotoxique. Dans ce contexte, la loi de bioéthique énonce très clairement l’obligation d’informer les patients des risques pour leur fertilité et des possibilités de conservation de leurs gamètes. Les plateformes régionales de préservation de la fertilité assurent la coordination entre les équipes d’oncologie et les centres d’étude et de conservation des oeufs et du sperme humains (CECOS) pour la mise en oeuvre de cette préservation. Pour les hommes, la congélation des spermatozoïdes est réalisée par une descente en température lente et contrôlée. Pour les femmes, la cryoconservation des ovocytes se fait par vitrification après stimulation hormonale et ponction ovarienne. Dans les deux cas, les gamètes sont conservés dans des paillettes qui sont stockées dans une cuve d’azote liquide jusqu’à utilisation en assistance médicale à la procréation (AMP). Le CECOS conservant les gamètes interroge chaque année par courrier les patients concernés sur leur souhait de poursuivre, ou non, la cryoconservation. Les gamètes pourront être utilisés en AMP par les patients de leur vivant et après leur consentement, sans altération liée à la durée de stockage.

Long-term preservation of muscle mitochondria for consequent functional analysis is an important and still unresolved challenge in the clinical study of metabolic diseases and in the basic research of mitochondrial physiology. We here present a method for cryopreservation of mitochondria in various muscle types including human biopsies. Mitochondrial function was analyzed after freeze–thawing permeabilized muscle fibers using glycerol and dimethyl sulfoxide as cryoprotectant. Using optimal freeze–thawing conditions, high rates of adenosine 5 ′ -diphosphate-stimulated respiration and high respiratory control were observed, showing intactness of mitochondrial respiratory function after cryopreservation. Measurement of adenosine 5 ′ -triphosphate (ATP) formation showed normal rates of ATP synthesis and ATP/O ratios. Intactness of the outer mitochondrial membrane and functional coupling between mitochondrial creatine kinase and oxidative phosphorylation were verified by respiratory cytochrome c and creatine tests. Simultaneous confocal imaging of mitochondrial flavoproteins and nicotinamide adenine dinucleotide revealed normal intracellular arrangement and metabolic responses of mitochondria after freeze–thawing. The method therefore permits, after freezing and long-term storage of muscle samples, mitochondrial function to be estimated and energy metabolism to be monitored in situ. This will significantly expand the scope for screening and exchange of human biopsy samples between research centers, thus providing a new basis for functional analysis of mitochondrial defects in various diseases.

International audience; Objective: To evaluate the effect of induced blastocoele shrinkage before vitrification in a closed carrier device.Patients and methods: Prior to vitrification, blastocyst cavity was artificially shrinked by laser pulse or not treated according to a 2:1 randomized procedure. A total of 185 warming cycles from April 2011 to March 2013 have been analyzed. Clinical pregnancy rate and survival rate were compared between the two groups. The mean (±SD) women age was 33.5±5.7 years for both groups.Results: The pregnancy rate in the group with artificial reduction of the cavity was higher ([32/67] 47.7%) than in the control group but not significantly ([43/113] 38%). The survival rate in the artificial shrinkage group was significantly higher compared with the control group : 99% (102/103) and 91.8% (168/183) respectively (P=0.01).Discussion and conclusion: This study reveals that artificial shrinkage of blastocoelic cavity by laser pulse before vitrification in a closed carrier device improves survival rate after warming.

International audience; Our knowledge of the organization of the cell is linked, to a great extent, to light and electron microscopy. Choosing either photons or electrons for imaging has many consequences on the image obtained, as well as on the experiment required in order to generate the image. One apparent effect on the experimental side is in the sample preparation, which can be quite elaborate for electron microscopy. In recent years, rapid freezing, cryo-preparation and cryo-electron microscopy have been more widely used because they introduce fewer artefacts during preparation when compared with chemical fixation and room temperature processing. In addition, cryo-electron microscopy allows the visualization of the hydrated specimens. In the present review, we give an introduction to the rapid freezing of biological samples and describe the preparation steps. We focus on bulk samples that are too big to be directly viewed under the electron microscope. Furthermore, we discuss the advantages and limitations of freeze substitution and cryo-electron microscopy of vitreous sections and compare their application to the study of bacteria and mammalian cells and to tomography.

Background The objective of the present study is to assess viability tests and to evaluate follicle ovarian tissue quality after freezing-thawing procedures. Methods Ewe's ovaries were harvested at the slaughterhouse, after dissection each ovarian specimen was divided into two groups: fresh tissue (control group) and frozen tissue. In the first part of the study, the follicles viability was assessed by trypan blue staining, calcein AM/ethidium homodimer-1 staining (LIVE/DEAD viability/cytotoxicity kit, Molecular Probes) and morphology in the two groups. In the second part of the study the quality of the whole ovarian tissue was evaluated by the quantification of the release of lactate dehydrogenase measurement (Cytotoxicity Detection kit ROCHE), DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) in primordial and primary follicles (ApopDETEK Kit system Enzo) and morphology in the two groups. 100 Follicles (primordial and primary) were counted on both fresh and frozen hemiovary to assess this various tests. Results Ovarian follicle viability assessment was similar using trypan blue or calcein/ethidium staining. Follicles showed a decreased viability after freezing-thawing. After cryopreservation, a significant correlation between the percentage of normal follicles and viability rate was found using trypan blue (r = 0.82, p < 0.05) or calcein AM/ethidium homodimer-1 staining (r = 0.76, p < 0.05). Increased cytotoxicity showed by enhancement of LDH release was found after cryopreservation (21.60 +/- 1.1% vs 52.2 +/- 7.7%). A significant negative correlation between the percentage of morphologically normal follicles and cytotoxicity was observed. No significant difference in DNA fragmentation rate between frozen and control groups was found (26 ± 8.2% vs 38 ± 4.5%). Conclusion We suggest the use of trypan blue staining for the histological assessment of viability, the use of LDH assay for the cytotoxicity assessement and finally the use of DNA fragmentation assessment to valid different freezing-thawing protocols.

Geraniums (Pelargonium spp.) are among the most popular bedding and pot plants (25% of the French domestic market). On one hand, as vegetatively propagated plants, Pelargonium are submitted to pathogen pressure. On the other hand, innovation via interspecific hybridisation faces some difficulties. In this chapter, the two first protocols (from seeds and meristems) explain how in vitro plants free of virus could be obtained. The development of this technique is the long-term preservation of genetic resources via meristem cryopreservation. The third protocol describes propagation of Pelargonium with limited risks of variation. This technique also allows the constitution and the maintenance of a plant-stock from which explants can be taken for other studies. The two last protocols describe plant regenerations from leaf discs and mesophyll protoplasts, used for gene transfer and somatic hybridisation. These protocols were established mainly with Pelargonium x hortorum cultivars, but we propose possible solutions for the other species: P. x peltatum, P. x domesticum, P. capitatum and P. graveolens.

International audience; Methyldrostanolone (2alpha,17alpha-dimethyl-17beta-hydroxy-5alpha-androstan-3-one) was synthesized from drostanolone (17beta-hydroxy-2alpha-methyl-5alpha-androstan-3-one) and identified in commercial products. Cultures of cryopreserved human hepatocytes were used to study the biotransformation of drostanolone and its 17-methylated derivative. For both steroids, the common 3alpha- (major) and 3beta-reduced metabolites were identified by GC-MS analysis of the extracted culture medium and the stereochemistry confirmed by incubation with 3alpha-hydroxysteroid dehydrogenase. Structures corresponding to hydroxylated metabolites in C-12 (minor) and C-16 were proposed for other metabolites based upon the evaluation of the mass spectra of the pertrimethylsilyl (TMS-d(0) and TMS-d(9)) derivatives. Finally, on the basis of the GC-MS and (1)H NMR data and through chemical synthesis of the 17-methylated model compounds, structures could be proposed for metabolites hydroxylated in C-2. All the metabolites extracted from hepatocyte culture medium were present although in different relative amounts in urines collected following the administration to a human volunteer, therefore confirming the suitability of the cryopreserved hepatocytes to generate characteristic metabolites and study biotransformation of new steroids.

International audience; The droplet-vitrification method was adapted to Pelargonium apices by optimizing the duration of the loading solution (LS) as well as the plant vitrification solution 2 (PVS2). The excised apices were dehydrated in two steps (20 min in LS and 15 min in PVS2) and then immersed directly in liquid nitrogen (LN). After thawing and unloading in the recovery solution at room temperature for 15 min, apices were plated onto semi-solid Murashige and Skoog medium. This simple protocol without any pretreatment was successfully applied to eight cultivars with a survival level ranging between 55.6 - 96.2 % and a regrowth level between 9.1% - 70.6%. These results prove the feasibility of the long-term storage of Pelargonium germplasm through cryopreservation

International audience; The aim of this study was to demonstrate that embryo transfer can be used to produce CAEV-free kids from CAEV-infected biological mothers when appropriate procedure is implemented. Twenty-eight goats that had tested positive for CAEV using PCR on vaginal secretions were used as embryo donors. Embryos with intact-ZP were selected and washed 10 times; they were then frozen and used for transfer into CAEV-free recipient goats. Nineteen of the 49 recipient goats gave birth, producing a total of 23 kids. Three blood samples were taken from each recipient goat, 10 days before, during, and 10 days after parturition; these were tested for CAEV antibodies using ELISA and for CAEV proviral DNA using PCR. The mothers were then euthanized. Tissue samples were taken from the lungs, udder, and retromammary and prescapular lymph nodes. The kids were separated from their mothers at birth. Seven of them died. At 4 months of age, 16 kids were subjected to drug-induced immunosuppression. Blood samples were taken every month from birth to 4 months of age; samples were then taken on days 15, 21, and 28 after the start of the immunosuppressive treatment. The kids were then euthanized and tissue samples taken from the carpal synovial membrane, lung tissue, prescapular lymph nodes, inguinal and retromammary lymph nodes, and uterus. All samples from the 19 recipient goats and 23 kids were found to be negative for CAEV antibodies and/or CAEV proviral DNA. Under acute conditions for infection this study clearly demonstrates that embryo transfer can be safely used to produce CAEV-free neonates from infected CAEV donors.

N.O and S.L.G. contributed equally as first authors; International audience; The distinction between S?ry syndrome (SS) and benign erythrodermic inflammatory diseases (EID) is difficult to make both clinically and on skin biopsies, since histomorphology can provide nonspecific results. New markers of circulating malignant S?ry cells have been recently described, especially CD158k/KIR3DL2 and T-plastin, but it has not been yet determined whether they could help in the diagnosis of erythroderma in skin samples. In this study, 13 frozen skin specimens from 10 SS patients and 26 from EID were analyzed for CD158k/KIR3DL2 expression using immunohistochemistry with AZ158 mAb, which also recognizes the monomeric CD158e/KIR3DL1 receptor. Although positive in all SS samples, immunohistochemistry appeared to not reliably discriminate between SS and EID. Therefore in all samples disclosing a significant staining with AZ158 mAb, CD158k/KIR3DL2, CD158e/KIR3DL1 and T-plastin mRNA expression were analyzed on the same skin specimen using conventional and/or quantitative real-time reverse transcription (RT)-PCR. Interestingly, only CD158k/KIR3DL2 transcripts were found to be significantly overexpressed in skin biopsies from patients with SS (P

International audience; BACKGROUND AND OBJECTIVE: Amniotic membranes are used with success in ophthalmology to treat corneal wounds and ulcers. In this pilot study, we attempt to assess the tolerance of amniotic membranes in the management of resistant venous and/or arterial vascular ulcers. MATERIAL AND METHODS: We prospectively included 8 patients, 7 males and 1 female, mean age 69.5+/- 9.6 years, with venous and/or arterial ulcers resistant after 6 months with usual medical care and/or after revascularisation failure. Amniotic membranes were applied on a weekly basis with the fetal side on the ulcer, covered by a secondary bandage. The primary end-point was evaluation of tolerance of amniotic membranes on vascular ulcers. The secondary end-points were a >50% reduction of ulcer's area, a significant (P< or =0.05) improvement of pain visual scale score and the quality of life assessed by the SF-36 questionnaire. RESULTS: Tolerance was excellent in all cases. We observed no adverse effect. We observed complete healing at weeks 19 and 26 for 2 patients and a >50% reduction of ulcer area at weeks 26, 31 and 32 for 3 patients. A sixth patient had an ulcer area reduction

International audience; Numerous parameters related to the patients and the conditions of the sample collection can affect the properties of a biological sample. The pre-analytical parameters are especially important to deal with in the setting of multicenter clinical trials. Here, we report the preliminary results of a trial conducted by the cooperative group GOELAMS (Groupe Ouest-Est des Leucémies aiguës et Autres Maladies du Sang) to define the pre-analytical parameters that affect the proteomic analysis of serum samples in patients with B-cell non Hodgkin lymphoma included in clinical trials.

International audience; Decellularized allograft tissues have been identified as a potential extracellular matrix scaffold for tissue-engineered vascular substitutes. In order to improve the thromboresistance, it is necessary to pre-coat the intra-luminal vessel surface. Recently a new surface modification technique appeared, based on the alternate adsorption of positive and negative charged polyelectrolytes. Our objective was to develop an alternative vascular scaffold made of decellularized human umbilical arteries treated with a PAH/PSS polyelectrolyte multilayered film. The vessels luminal surfaces covered with the multilayer film were observed by electronic scanning microscopy. Our observations showed that the luminal surface is completely devoid of ECs following treatment with trypsin. A top view of the coated artery indicated that the multilayer uniformly covered internal surface of the vessels. The successful of the multilayer correct deposition and retention on the arterial wall were controlled by confocal microscopy using a fluorescent polyelectrolyte (rhodamine-PAH). The data suggest that decellularized cryopreserved arteries represent a potential scaffold for further vascular tissue engineering efforts. Moreover, the multilayer films can be used to coat biological surfaces and following the terminated layer (PAH or PSS), favour the cell adhesion or cell resistance.

International audience; The study of mechanical properties of the arterial wall is an important step in the comprehension of the vascular physiopathological functioning. However, cryopreserving biological tissues using very low temperatures can induce biological and structural modifications which may involve complications (dilatation, bursting, stenosis) after reimplantation. Many procedures of mechanical tests (traction, dilatation) developed in research allow us to comprehend and analyse rheological behaviour of the arterial wall. The study presented in this article offers a new perspective to detect changes of mechanical properties of cryopreserved arterial samples. In fact, the original idea is to couple a mechanical test bed (uniaxial traction of arterial rings) with spectroscopic measurements (autofluorescence) for the purpose of correlating mechanical modifications and spectral variations. Ultimately, this new approach could lead to develop a device allowing atraumatic and contactless optical examinations of arterial graft to determine its mechanical state before reimplantation.

International audience; Freezing of prokaryotic and eukaryotic microorganisms is the main interest in the study of cold stress responses of living organisms. In parallel, applications which arise from this approach are of two types: (i) optimization of the frozen starters used in food processing; and (ii) improvement of the ex situ preservation of microorganisms in collections. Currently, cryopreservation of microorganisms in collections is carried out in cryotubes, and bibliographical references related to freezing microorganisms packaged in straws are scarce. In this context, a preliminary study was completed to evaluate the technological potential of ionomeric resin straws compared to polycarbonate cryo-tubes. Survival under freezing stress was tested on three microorganisms selected for their biotechnological interest: two lactic acid bacteria, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus and a deuteromycete fungus, Geotrichum candidum. The stress was carried out by repeated freezing-thawing cycles to artificially accelerate the lethal effect of freezing on the microorganisms. Two main results were obtained: (i) the survival rate values (per freezing-thawing cycle) seems to depend on the thermal type of the studied microorganism, and (ii) there was no, under our experimental conditions, significant difference between straws and tubes. However, conservation in the resin straws lead to a slight increase in the survival of L. cremoris and G. candidum compared to microtubes. In those conditions, straws seems an alternative system to securely store frozen microorganisms with three main characteristics: (i) a high resistance to thermal stress, (ii) a safe closing by hermetic weld, and (iii) a system for inviolable identification.

Mitochondria play a critical role in myocardial cold ischemia-reperfusion (CIR) and induction of apoptosis. The nature and extent of mitochondrial defects and cytochrome c (Cyt c) release were determined by high-resolution respirometry in permeabilized myocardial fibers. CIR in a rat heart transplant model resulted in variable contractile performance, correlating with the decline of ADP-stimulated respiration. Respiration with succinate or N,N,N′, N′-tetramethyl- p-phenylenediamine dihydrochloride (substrates for complexes II and IV) was partially restored by added Cyt c, indicating Cyt c release. In contrast, NADH-linked respiration (glutamate+malate) was not stimulated by Cyt c, owing to a specific defect of complex I. CIR but not cold ischemia alone resulted in the loss of NADH-linked respiratory capacity, uncoupling of oxidative phosphorylation and Cyt c release. Mitochondria depleted of Cyt c by controlled hypoosmotic shock provided a kinetic model of homogenous Cyt c depletion. Comparison to Cyt c control of respiration in CIR-injured myocardial fibers indicated heterogeneity of Cyt c release. The complex I defect and uncoupling correlated with heterogeneous Cyt c release, the extent of which increased with loss of cardiac performance. These results demonstrate a complex pattern of multiple mitochondrial damage as determinants of CIR injury of the heart.

Objectives : To measure the strength at tearing of pelvic ligaments used in the cure of prolapse and urinary incontinence. Material and methods : We performed our measurements on pelvis ligaments from cadaveric specimens. We dissected 29 human female pelvis cadavers of which storage conditions differed. Ten were frozen, 10 fresh and 9 were stored in formalin. In each cadaver we dissected pre-vertebral ligaments at promontory and right and left symmetrical ligaments. These were the iliopectineal, sacrospinous and arcus tendineus of pelvic fascia. A subjective clinical evaluation of the ligament properties was performed by visual observation as well as finger palpation. Ligaments were classified into three groups. Group A contained high quality ligaments, in terms of thickness and apparent strength following finger palpation. Ligaments of doubtful quality were classified in group B and low apparent quality ligaments in group C. Then the ligaments were stitched by a suture taking the entire ligament and a force was applied on the vagina axis until tearing. The device used for strength measurement during traction was a SAMSON type force gauge, model EASY, serial number SMS-R-ES 300N manufactured by Andilog that was developed for the purpose of our study. Measurements were given in Newton (N). Results : There was a great variability in the values obtained at tearing with minimal values at around 20 N and maximal values at 200 N. Individually measured, ligament strength varied between individuals, and for the same patient between the type of ligaments and the side. The pre-vertebral ligament was on average the strongest. There was no significant difference according to the storage condition except for the pre-vertebral ligament in formalin cadavers. For bilateral ligaments, there was no difference between the left and right side. The iliopectineal ligament was statistically significantly stronger than the sacrospinous and arcus tendineus of pelvic fascia. There was a correlation between subjective evaluation and objective strength measurements. Discussion : No papers have been published on the strength of pelvic ligaments at tearing. These are however routinely used in the cure of prolapse and urinary incontinence. Our results show that there is a great variability in strength between individuals, and for a same patient between the types of ligaments and side. These observations could explain some of the surgical intervention failures and demonstrate the importance of per-operative strength evaluation. Per-operative subjective evaluation of strength is related to objective measurements and could be used to determine the type of ligaments to be used for surgical suspension. Freezing does not damage pre-vertebral ligament strength and further studies are required to evaluate elasticity of pelvic ligaments.

International audience