Cryopreservation of in vitro matured oocytes is still considered as an experimental alternative to mature oocyte vitrification after ovarian stimulation. Here, we investigated whether rescue-IVM should be performed before or after vitrification. For this, 101 immature oocytes (germinal vesicle stage) from women undergoing ICSI were used. Oocytes were divided into three groups: freshly in vitro matured oocytes (IVM), freshly in vitro matured oocytes subsequently vitrified (IVM + VIT) and vitrified/warmed GV oocytes then in vitro matured (VIT + IVM). Oocyte maturation rates and kinetics were assessed using time-lapse technology. Spindle dimensions and polarity, chromosome alignment and cytoplasmic F-actin filament length and density were determined using confocal microscopy and quantitative image analyses. No differences in IVM rates (fresh IVM: 63.16

The COVID-19 pandemic, driven by SARS-CoV-2, led authorities to recommend halting assisted reproductive technology programs, focusing instead on fertility preservation, for cancer patients. The presence of SARS-CoV-2 in semen remains controversial. This multicentric prospective cohort study, conducted across 12 university medical centers, aimed to determine if SARS-CoV-2 is present in spermatozoa/seminal plasma in cancer patients by RT-PCR and to assess its impact on standard semen parameters. The levels of cytokines and TNF-alpha were measured in seminal fluid by ELISA. We enrolled 129 men who underwent sperm cryopreservation between July 7, 2020, and June 30, 2021. The 63 were included and tested for COVID-19 in nasal swab samples by RT-PCR and/or by serology. All patients were asymptomatic on the day of semen collection: 50 were uninfected, 8 had a positive nasal swab (PCR+) and 5 were seropositive. SARS-CoV-2 RNA was not detected in the seminal fluid or spermatozoa. Ejaculate volume was significantly lower in the PCR+ group compared to the uninfected group (median [IQR]: 2.6 mL [1.6-3.4] vs. 4.6 mL [2.6-5.2] p < 0.05). Total and progressive motility were lower in the PCR+ group compared to the seropositive group (32.5% [25.0-45.0] vs. 50% [49.0-55.0] p < 0.05, and 22.5% [10.0; 32.5] vs. 44.5% [40-49] p < 0.05). Higher TNF-alpha level was observed in the PCR+ group (1.9 pg/mL [0-3.9]) compared to the uninfected group (0 pg/mL [0-0.4]) p < 0.05. Although SARS-CoV-2 was not detected in the sperm samples of cancer patients who were PCR+, the infection appears to impact sperm parameters, likely due to inflammation.

Correlations were reported between sperm telomere length (STL) and male fertility, sperm DNA fragmentation, and oxidation. Sperm freezing is widely used for assisted reproductive techniques, fertility preservation, and sperm donation. However, its impact on STL remains unknown. For this study, semen surplus from patients who underwent routine semen analysis were used. The impact of slow freezing on STL was analyzed by performing qPCR before and after freezing. Sperm populations with different STL were evaluated using Q-FISH. The relationship between sperm DNA oxidation, DNA fragmentation, and STL was assessed in fresh and frozen sperm samples. No significant impact of slow freezing on STL was observed, neither measured by qPCR nor Q-FISH. However, Q-FISH allowed for the distinguishing of sperm populations with different STLs within individual sperm samples. Slow freezing induced different STL distributions for some of the analyzed sperm samples, but no correlation was found between STL and sperm DNA fragmentation or oxidation. Slow freezing does not alter STL despite increasing sperm DNA oxidation and fragmentation. As STL alterations could be transmitted to offspring, the lack of impact of the slow freezing method on STL ensures the safety of this procedure.

Abstract Study question Did the use of universal vitrification/warming media have an impact on the clinical pregnancy rates in oocyte donation cycles compared with oocyte-specific vitrification/warming media? Summary answer Using universal media for oocyte vitrification and warming yields similar clinical pregnancy rates but higher oocyte survival rates than oocyte-specific media. What is known already Vitrification is now the gold standard for oocyte and embryo cryopreservation. Several commercial kits are available, some are designed for specific cell types (e.g., oocytes, zygotes, cleavage-stage embryos or blastocysts) and others are suitable for several stages, therefore termed “universal”. While the composition and the exposition protocol of stage specific media are optimized for specific cell types, “universal” media display a single composition, therefore exposition protocols need to be adapted to each specific developmental stage to ensure optimal cell survival rates. The potential use of the universal media allows to optimize the management of media in ART laboratories. Study design, size, duration This is a retrospective monocentric study. We compared the results obtained in our center for oocyte vitrification in donor cycles with the successive use of oocyte-specific vitrification/warming media (RapidVit/Warm™ Oocyte, Vitrolife) and afterwards universal vitrification/warming media (RapidVit/Warm™ Omni, Vitrolife). We analyzed 111 oocyte recipient ICSI cycles performed between March 2016 and July 2020 (86 recipient couples) and their 81 corresponding oocyte donation cycles (79 donors) with these media. Participants/materials, setting, methods Oocyte recipient ICSI cycles were divided in three groups depending on the combination of vitrification and warming media used for the oocyte vitrification and warming procedures: “specific/specific” (S/S), “specific/universal” (S/U) and “universal/universal” (U/U). The primary outcome was the clinical pregnancy rate per embryo transfer. Secondary outcomes were the oocyte survival rates, fertilization rates, cleavage and blastocyst rates, live birth and miscarriage rates. Main results and the role of chance The age of the donors (31.7+/-3.6 yo) and the recipients (35.1+/-4.5) was similar for the three groups (p > 0.05). Total FSH dose was similar for the 3 groups (1814.2+/-505UI) (p > 0.05) Clinical pregnancy rates were similar when universal vitrification/warming media were used (25.6% for S/U; 25.8% for U/U) compared with oocyte-specific vitrification/warming media (12.2% for S/S, p = 0.25). We observed higher oocyte survival rates when universal media were used (93.7% for U/U, 85.5% for S/U) compared with the use of the oocyte-specific media (75.6% for S/S, p < 0.0001). Fertilization (74.9% for U/U; 68% for S/U; 68.1% for S/S), cleavage (97.9% for U/U; 94.6% for S/U; 89.3% for S/S), and blastulation rates (46.6% for U/U; 50.3% for S/U; 41.7% for S/S) were not different between the three groups (p > 0.05). There was no difference in miscarriage (6.5% for U/U; 10.2% for S/U; 4.9% for S/S) and live birth rates (19.4% for U/U; 15.4% for S/U; 7.3% for S/S) between the 3 groups (p > 0.05). Limitations, reasons for caution The preliminary results of this retrospective study need to be confirmed by a larger prospective study. Wider implications of the findings The use of vitrification/warming universal media, which improves the management of media used for vitrification of oocytes and embryos in ART laboratory, allows to obtain similar clinical pregnancy rates for oocyte recipient donors. Trial registration number not applicable

Research question: Is it possible to validate an accurate and reliable method for direct detection of SARS-CoV-2 by reverse transcription polymerase chain reaction (RT-PCR) in human semen fractions?Design: This qualitative improvement study aimed to provide a prospective validation of SARS-CoV-2 detection in male semen. The SARS-CoV-2 genome was detected by multiplex real-time RT-PCR on patient samples that underwent routine semen analyses for infertility at the Center for Reproductive Medicine at the University Hospital of Clermont-Ferrand. Samples comprised surplus semen collected for treatment with assisted reproductive technology. Seminal fluid and spermatozoa fractions were isolated with density gradient centrifugation and cryopreserved. Positive samples were prepared with a standard of inactivated SARS-CoV-2 particles.Results: The analytical method was validated in both seminal fluid and spermatozoa fractions. In both semen fractions, the assay was repeatable, reproducible and showed high sensitivity with a limit of detection of 0.33 SARS-CoV-2 genome copies/pl. The limit of quantification was 1 copy of the SARS-CoV-2 genome/pl. The method was effective regardless of semen quality (normal and altered sperm parameters), number of spermatozoa or the cryoprotectant media used to freeze spermatozoa.Conclusion: This validated RT-PCR assay provided accurate and reliable screening of SARS-CoV-2 in seminal fluid and spermatozoa fractions. This method is essential to ensure protection against viral contamination in the cryobanking process.

Abstract Study question What are the optimal vitrification method (semi-automated vs. manual) and oocyte stage for in vitro -matured oocyte cryopreservation using meiosis kinetics and chromosome segregation as readouts? Summary answer The semi-automated vitrification method does not impact oocyte nuclear maturation quality compared with the manual method. Immature oocyte cryopreservation should be performed after IVM. What is known already Fertility preservation using oocyte vitrification should be performed before oncological treatments. The reference protocol consists in collecting mature oocytes after ovarian stimulation. Nevertheless, ovarian stimulation sometimes yields immature oocytes or cannot be performed (e.g., emergency oncological treatment). An IVM step is therefore required but it is not clearly demonstrated whether IVM should be performed before or after vitrification. Oocyte vitrification is usually performed with manual methods. A semi-automated vitrification device (Gavi®, Genea Biomedx) showing high performances for embryo was recently released. To our knowledge, no study has analysed its efficiency on oocyte vitrification. Study design, size, duration 200 immature oocytes collected from ICSI cycles from January 2020 will be used. Oocytes will be divided in five groups (40 oocytes/group): freshly matured oocytes (group 1 control), oocytes vitrified after IVM by a manual technique (group 2a) or by Gavi® (group 2b) and oocytes vitrified prior IVM (groups 3a and 3b). We assess oocyte nuclear maturation quality by evaluating IVM kinetics by time-lapse (Geri®) and the accuracy of homologous chromosomes segregation by CGH array. Participants/materials, setting, methods Since January 2020, 124 out of 200 immature oocytes have been included for this study. These oocytes provide from women under 37 years old without ovulatory disorder after signing an informed consent. The kinetics of meiotic resumption (germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) timings), is determined by time-lapse technology (Geri®, Genea Biomedx). The accuracy of the homologous chromosome segregation during the first meiotic division will be assessed by CGH-Array. Main results and the role of chance The clinico-biological characteristics (age, BMI, smoking and total FSH dose) are comparable between the five groups (p > 0.05). No significant difference in post-thawing oocyte survival rate is observed between the two vitrification methods (semi-automated 58% vs. manual 64%). A significant difference in the overall oocyte survival rate is observed according to the stage of vitrification with a significantly higher survival rate if oocytes are vitrified at the mature stage (93% (2a+2b) vs. 61% (3a+3b), p = 0.02). The IVM rate is significantly higher if oocytes are matured freshly (86% in group 1 (fresh IVM, n = 7) and 93% in group 2a+2b (IVM before vitrification, n = 27)) compared to post vitrification (71% in group 3a+3b, n = 28), p = 0.03. The vitrification technique does not seem to impact IVM rate since it reached 64% (group 3a, n = 11) and 76% (group 3b, n = 16) (p = 0.4). GVBD and PBE timings are not significantly different between the 5 groups, suggesting that neither the oocyte stage of vitrification nor the vitrification technique affects maturation kinetics. Similarly, our preliminary results of polar bodies and oocytes chromosomal profiles assessed by CGH-Array demonstrate a similar rate of aneuploidy (monosomy or trisomy) between the groups. Limitations, reasons for caution Our preliminary results of CGH array should be confirmed with the analysis of a larger number of IVM oocytes. Wider implications of the findings Vitrification of immature oocytes should be performed by semi-automated or manual methods after IVM. Tour knowledge, this is the first study comparing the efficiency of both semi-automated and manual vitrification methods on immature and in vitro-matured oocytes. Trial registration number NCT03680937

Abstract 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. Trial registration number EudraCT 2020-A01409–30

Introduction > Cancers of adolescents and young adults hove particular epidemiological specificities. The improvement in their survival should be accompanied by on 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. Methods > During this retrospective descriptive study, various socio-demographic and clinical data were collected. Results > 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 (P < 0.001). 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 cryopre-servation of ovarian tissue in female adolescents, ovocytes in young women and sperm in boys. Discussion > 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.

This prospective study aimed to determine the effects of dry nitrogen cryostorage on human sperm characteristics in comparison with liquid nitrogen cryostorage. For this purpose, 42 men undergoing routine semen analysis (21 normozoospermia and 21 with altered semen parameters) were analyzed. After slow freezing, half of the straws of each sample were randomly stored in liquid and dry tanks, at the top and bottom levels of the latter. After 6 months storage, thawed samples were treated by density gradient centrifugation and sperm characteristics were compared. There was no difference in sperm progressive motility (15.1% ± 14.2% vs. 15.1% ± 12.7%; p = 0.76), sperm vitality (25.5% ± 17.7% vs. 26.2% ± 19%; p = 0.71), percentages of acrosome-reacted spermatozoa (38% ± 8.5% vs. 38.5% ± 7.4%; p = 0.53) and DNA fragmentation spermatozoa (27.3% ± 12.4% vs. 28.5% ± 12.9%, p = 0.47) after cryostorage in the dry or the liquid nitrogen tank. Moreover, we did not observe differences between either cryostorage system for normal and altered sperm samples. This lack of difference was also observed whatever the floor level of cryostorage in the dry tank. The temperature measurement of the dry tank showed a stable temperature at -194 °C throughout storage whatever the storage floor level, guaranteeing the stability of the low temperatures suitable for human sperm storage. Because of its greater safety, dry storage without contact with the liquid phase should be preferred and can be a useful alternative for the cryostorage of human sperm samples.

Les cancers des adolescents et jeunes adultes présentent des spécificités épidémiologiques particulières. L’amélioration de leur survie doit s’accompagner d’une prise en considération accrue des effets indésirables des traitements, parmi lesquels la possible diminution de la fertilité. L’objectif de l’étude était de décrire l’accès à la préservation de la fertilité de ces patients au CHU de Clermont-Ferrand sur une période de trois ans. Au cours de cette étude descriptive rétrospective, différentes données sociodémographiques et cliniques ont été collectées. Cent cinquante nouveaux cas de cancers ont été diagnostiqués chez les patients de 15–24 ans. Quarante-quatre pour cent ont bénéficié d’au moins une consultation au centre d’étude et de conservation des œufs et du sperme humains, 29 % chez les filles et 58 % chez les garçons (p < 0,001). Le nombre de cas n’ayant pas abouti à une préservation de la fertilité était significativement plus élevé chez les filles que chez les garçons (p = 0,005). La préservation de la fertilité a été réalisée majoritairement par cryoconservation de tissu ovarien chez les adolescentes, d’ovocytes chez les jeunes femmes et de sperme chez les garçons. Nous avons pu observer des disparités dans l’accès à la préservation de la fertilité selon le sexe. Malgré l’existence de recommandations, des progrès restent à faire. La mise en place de plateformes clinicobiologiques devrait permettre une meilleure sensibilisation des patients et professionnels de santé, et ainsi de promouvoir l’accès aux techniques de préservation de la fertilité des jeunes patients atteints de cancers. 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 (P < 0.001). 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.

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 < 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: NCT02400970.

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.