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International audience

International audience

International audience

International audience; Study question:How to ensure a reliable and accurate detection of SARS-CoV-2 in seminal plasma and spermatozoa fractions of human sperm samples? Summary answer:This RT-PCR assay showed high sensibility, repeatability and reproducibility for SARS-CoV-2 detection in seminal plasma and spermatozoa fractions, with a detection limit of 17 genomes/reaction. What is known already:SARS-CoV-2 pandemic brings numerous concerns, such as the safety of gametes for patients undergoing assisted reproductive technologies, fertility preservation or sperm donation. Transient viremia and expression of SARS-CoV-2 receptors in testis and accessory glands bring the question of the presence of the virus in sperm samples. Moreover, the contamination during sperm collection may be possible. The few available studies about this issue mostly showed the absence of SARS-CoV-2 detection in semen of COVID-19 patients, except one reported study. All these studies performed SARS-CoV-2 detection with RT-PCR assays approved for naso-pharyngeal swabs, without a process specifically validated for semen fractions. Study design, size, duration:Method validation was conducted between July 2020 and January 2021. SARS-CoV-2 direct detection was performed according to the French Society of Microbiology guidelines (SFM). Repeatability (n=6), reproducibility (n=3), limit of quantification (n=2) and of detection (n=6) were evaluated in seminal plasma (SP) and spermatozoa samples isolated after density gradient centrifugation and cryopreserved. In addition, variability of the whole analytical method efficiency was evaluated in samples of men with normal (n=6) or altered sperm parameters (n=6).Participants/materials, setting, methods:Samples were surplus semen obtained from men undergoing routine semen analysis after granting informed consent. Assays were performed on SP and frozen spermatozoa fractions. After automated RNA extraction (MGISP-960, MGI-Tech®), real-time RT-PCR was performed using the one-step multiplex TaqPath COVID-19 kit (ThermoFisher®) targeting three viral regions (ORF1, nucleocapsid-N and spike-S proteins). An exogenous internal control was added before RNA extraction. Positive samples and dilution ranges were prepared with a standard (SARS-CoV-2 inactivated virus, QnosticTM Randox®). Main results and the role of chance:RT-PCR assay applied for human sperm samples has been previously validated and is routinely used for SARS-CoV-2 detection in naso-pharyngeal swabs. We evaluated the efficiency of RNA extraction and RT-PCR for SARS-CoV-2 detection in semen fractions. The qualitative and quantitative performance of the whole analytical method was validated with an accuracy profile for SP and spermatozoa fractions. Overall, for repeatability, the standard deviation (SD) of the cycle threshold (Ct) was lower than 0.40 for the strong positive sample and 0.50 for the low positive one. An exception was observed for the S target of the low positive SP samples (SD=3) which was consistent with S being the less sensitive target of the assay. For reproducibility, SD of the Ct was lower than 0.30 for the strong positive sample and 0.80 for the low positive, except for the S target of the low positive (SD=1.5). The linearity range was determined for N target, the most sensitive target of the RT-PCR assay. It layed between 5200 and 52 SARS-CoV-2 genomes/reaction. The limit of detection of the RT-PCR assay was 17 viral genomes/reaction. Equal efficiency of the assay was observed for SP and spermatozoa independently of semen parameters (normal and altered sperm parameters). Limitations, reasons for caution:Our detection method was validated for the whole process: RNA extraction (reagents and system), RT-PCR (reagents and thermocycler QuantStudio 5TM) and for both SP and frozen spermatozoa fractions. Variability might be observed with a different extraction system or a different type of biological sample.Wider implications of the findings:This validated RT-PCR assay enables accurate and reliable screening of SARS-CoV-2 in SP and spermatozoa fractions, mandatory to investigate the presence of the virus in semen samples of patients undergoing assisted reproductive techniques, fertility preservation or sperm donation, and to ensure viral safety in the cryobanking process during covid-19 pandemic.

International audience

Cancers of adolescents and young adults have particular epidemiological specificities. The improvement in their survival should be accompanied by an increased consideration of the treatments' side effects, among which the potential decrease in fertility. The objective of the study was to describe the access to fertility preservation of these patients at the University Hospital of Clermont-Ferrand over a period of 3 years.During this retrospective descriptive study, various socio-demographic and clinical data were collected.One hundred and fifty new cases of cancers were diagnosed in patients aged 15 to 24 years. Forty-four percent received at least one fertility consultation, 29 % for girls and 58 % for boys (P0.001). The number of cases that did not result in fertility preservation was significantly higher for girls than boys (P=0.005). Fertility preservation was mainly achieved by cryopreservation of ovarian tissue in female adolescents, ovocytes in young women and sperm in boys.We observed sex disparities in access to fertility preservation. Despite the existence of recommendations, progress remains to be made. The establishment of clinico-biological platforms should allow a better awareness of patients and professionals, and thus promote access to fertility preservation techniques for young patients with cancer.

International audience; Since the improvement of cancer diagnosis and treatment, survival rates of these patients increase. Gonadal damages are frequent consequences of cancer treatments with different evidence of impaired fertility. In this context, fertility preservation should be proposed to patients exposed to potentially gonadotoxic treatments. Different preservation approaches may be proposed depending on patient age, sex, cancer type and type of treatment. The indications of fertility preservation depend on sexual maturity. In young girls, ovarian cortex cryopreservation is the only technique feasible in order to preserve their reproductive potential. Vitrification of oocytes which needs ovarian stimulation or oocytes in vitro maturation is becoming more commonly performed for pubertal women to preserve their fertility. Ovarian cortex freezing could be offered to emergency fertility preservation of adult female cancer patients. In prepubertal boys, testicular tissue cryopreservation is the only line treatment for fertility preservation. For future use, various approaches are being evaluated such as spermatogonial stem cell injection or in vitro maturation. Cryopreservation of spermatozoa is, today, an established and successful technique for male adults. When there are no spermatozoa in ejaculate, sperm can be retrieved after treatment of testicular biopsy. The French bioethics law clearly indicates that fertility preservation should be proposed to patients exposed to potentially gonadotoxic treatment. Today, many approaches are possible. Fertility preservation indications are based on multidisciplinary consultations within platforms for the fertility preservation in order to optimize the patient care.; Avec les progrès diagnostiques et thérapeutiques, la survie des patients atteints de cancer s'est améliorée ces dernières années. La toxicité gonadique reste une conséquence relativement fréquente des traitements utilisés avec différents niveaux d'altération de la fertilité. Dans cecontexte, la préservation de la fertilité doit être proposée aux patient(e) exposé(e)s à un traitement potentiellement gonadotoxique. Différentes options sont proposées selon l'âge du patient, son sexe, l'urgence thérapeutique et l'évaluation des retentissements potentiels sur lafertilité. Chez les femmes prépubères, la congélation de tissu ovarien est la seule option possible. Après la puberté, la vitrification ovocytaire est de plus en plus utilisée mais nécessite une stimulation hormonale ou une maturation in vitro des ovocytes. Lorsque l'urgence thérapeutiquene permet pas le délai d'une stimulation, la congélation de cortex ovarien est l'option privilégiée. Chez les garçons prépubères, la congélation de tissu testiculaire est proposée. Pour l'utilisation ultérieure du tissu cryoconservé, différentes techniques sont en cours d'évaluation telles quel'injection de cellules souches ou la maturation in vitro. Concernant l'homme adulte, la congélation de spermatozoïdes est pratiquée depuis des années et son efficacité en Assistance médicale à la procréation est aujourd'hui clairement démontrée. La loi de bioéthique précise clairement que la préservation de la fertilité doit être proposée aux patients exposés à un traitement potentiellement gonadotoxique. De nombreuses méthodes sont aujourd'hui possibles. Les indications de préservation dans le cadre du cancer relèvent d'une concertation multidisciplinaire au sein des plateformes clinicobiologiques de préservation de la fertilité afin d'optimiser la prise en charge despatients.

International audience

Ewing sarcoma (EWS) is a common pediatric solid tumor with high metastatic potential. Due to toxic effects of treatments on reproductive functions, the cryopreservation of ovarian tissue (OT) or testicular tissue (TT) is recommended to preserve fertility. However, the risk of reintroducing residual metastatic tumor cells should be evaluated before fertility restoration. Our goal was to validate a sensitive and specific approach for EWS minimal residual disease (MRD) detection in frozen germinal tissues. Thawed OT (n = 12) and TT (n = 14) were contaminated with tumor RD-ES cells (10, 100, and 1000 cells) and EWS-FLI1 tumor-specific transcript was quantified with RT-qPCR. All contaminated samples were found to be positive, with a strong correlation between RD-ES cell numbers and EWS-FLI1 levels in OT (r = 0.93) and TT (r = 0.96) (p &lt, 0.001). No transcript was detected in uncontaminated control samples. The invasive potential of Ewing cells was evaluated using co-culture techniques. After co-culturing, tumor cells were detected in OT/TT with histology, FISH, and RT-qPCR. In addition, four OT and four TT samples from children with metastatic EWS were tested, and no MRD was found using RT-qPCR and histology. We demonstrated the high sensitivity and specificity of RT-qPCR to detect EWS MRD in OT/TT samples. Clinical trial: NCT 02400970.