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BACKGROUND Testicular tissue freezing is proposed for fertility preservation to (pre)pubertal boys with cancer before highly gonadotoxic treatment. Studies accurately comparing human (pre)pubertal testicular tissue quality before freezing and after thawing are exceptional. No study has reported this approach in a systematic manner and routine care. OBJECTIVES To assess the impact of a control slow freezing protocol on testicular tissue architecture and integrity of (pre)pubertal boys after thawing. MATERIALS AND METHODS (Pre)pubertal boys (n = 87) with cancer from 8 Reproductive Biology Laboratories of the French CECOS network benefited from testicular tissue freezing before hematopoietic stem cell transplantation. Seminiferous tubule cryodamage was determined histologically by scoring morphological alterations and by quantifying intratubular spermatogonia and the expression of DNA replication and repair marker in frozen-thawed testicular fragments. RESULTS A significant increase in nuclear and epithelial score alterations was observed after thawing (p

BACKGROUND Many studies reported that reproductive desire could be high among transgender individuals. In France, fertility preservation and sperm donation were very little proposed to transgender individuals until recently, mainly because the Bioethics Law allows the use of assisted reproductive technologies only in infertile couples and prohibits surrogacy. OBJECTIVES To evaluate the distribution of care on the French territory concerning fertility preservation and sperm donation in transgender individuals. MATERIALS AND METHODS A multicentric national survey was carried out between January 2019 and October 2020 in 28 assisted reproductive technology centres of the French CECOS (Centres d'Etudes et de Conservation des Oeufs et du Sperme) network. Each centre was questioned to find out how many transgender individuals came, were informed and cared for fertility preservation and sperm donation. RESULTS Concerning fertility preservation, 71.4% of centres received transgender individuals and performed gamete cryopreservation; 581 transgender individuals consulted for fertility preservation. Transgender women were more likely to desire (p

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?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.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/µl. The limit of quantification was 1 copy of the SARS-CoV-2 genome/µl. The method was effective regardless of semen quality (normal and altered sperm parameters), number of spermatozoa or the cryoprotectant media used to freeze spermatozoa.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.

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.

Resumen La frecuencia de la endometriosis esta en fuerte progresion, particularmente en las mujeres infertiles. La etiologia es desconocida. La fisiopatologia se comprende muy mal. Las formas anatomoclinicas son diversas. Estos elementos de incertidumbre hacen delicado el tratamiento. Este tema ha sido objeto de mas de 10.000 publicaciones registradas en PubMed. En este articulo, los autores han tratado de hacer una sintesis de los conocimientos y proponen un algoritmo de tratamiento teniendo en cuenta los datos conocidos en materia de cirugia, tratamientos medicos y tecnicas de reproduccion asistida.

International audience; Abstract Background Although widely used, slow freezing considerably modifies the functions of human spermatozoa. Cryopreservation induces nuclear sperm alterations and cryo-capacitation, reducing the chances of pregnancy. Hypotaurine is naturally present in the male and female genital tracts and has capacitating, osmolytic and anti-oxidant properties. The analysis were performed on surplus semen of men with normal ( n = 19) or abnormal ( n = 14) sperm parameters. Spermatozoa were selected by density gradient centrifugation before slow freezing. For each sample, these steps were performed in parallel with (“H+” arm) or without (“H-” arm) hypotaurine supplementation. After thawing, we measured total and progressive mobility, vitality, acrosome integrity, markers of capacitation signaling pathway and nuclear quality. For the latter, we focused on sperm chromatin packaging, DNA fragmentation and the presence of vacuoles in the sperm nucleus. Results Post-thaw spermatozoa selected and frozen in the presence of hypotaurine had a higher vitality (+ 16.7%, p

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

Background Idiopathic central precocious puberty (ICPP) is supposed to be non-existent in a context of testicular destruction that is typically present in Klinefelter syndrome (KS). Herein, we describe a rare case of ICPP in a Klinefelter patient (47,XXY) with 2 maternal X chromosomes. Moreover, we highlight the differences in gonadotropin levels in comparison to males with ICPP and a normal karyotype. Case presentation An 8 years old boy with a history of cryptorchidism was evaluated for precocious puberty (Tanner staging: P2/G3). Both testes measured 25x35mm. His hormonal profile confirmed a central origin of precocious puberty with high serum testosterone (4.3 ng/ml), luteinizing hormone [LH (3.5 UI/l)] and follicle stimulating hormone [FSH (7.7 UI/l)] levels. Luteinizing hormone-releasing hormone (LHRH) test amplified LH and FSH secretion to 24 and 14 UI/l respectively. Brain magnetic resonance imaging (MRI) was normal. No MKRN3 mutation was detected. He was treated for ICPP for two years. During puberty, he suffered from hypergonadotropic hypogonadism leading to the diagnosis of KS (47,XXY karyotype). Chromosomal analysis by fluorescent multiplex polymerase chain reaction (PCR) using X chromosome microsatellite markers identified 2 maternal X chromosomes. Analysing 8 cases of KS developing ICPP (our reported case and 7 other published cases) revealed that these KS patients with ICPP have higher LH and FSH levels during ICPP episode than in ICPP patients with a normal karyotype (ICPP with KS vs ICPP with a normal karyotype: LH levels 9.4 ± 12 vs 1.1 ± 0.6 UI/l; FSH levels 23.1 ± 38.5 vs 2.7 ± 1.5 UI/l). Furthermore, their response to gonadotropin-releasing hormone (GnRH) stimulation is characterized by excessive LH and FSH secretion (LH levels post-GnRH: 58 ± 48 vs 15.5 ± 0.8 UI/l; FSH levels post-GnRH: 49.1 ± 62.1 vs 5.7 ± 3.9 UI/l). Conclusions ICPP in boys is extremely rare. The pathophysiology of ICPP in KS is unknown. However, maternal X supplementary chromosome and early testicular destruction may play a significant role in the initiation of ICPP, in part explaining the relative “overrepresentation of ICPP in KS. Thus, karyotype analysis could be considered for boys suffering from ICPP, especially if testicular size is smaller or gonadotropins are significantly elevated.

Objectives Patients with RA have a higher prevalence of infertility than the general population. This study sought to examine the impact of RA disease activity and treatments on ovarian reserve measured by serum anti-Müllerian hormone (AMH) levels in the ESPOIR cohort. We sought to better define the indications for fertility preservation. Methods Patients and serum analysis data were derived from the French national cohort ESPOIR. Enrolled patients (n = 102; 18–37-year-olds) fulfilled ACR/EULAR 2010 criteria for RA. Serum AMH levels were measured at T0, T6, T12, T24 and T36 months post-diagnosis. The impacts of RA activity (DAS28 and CRP level) and treatments (MTX only or with other medications) were evaluated at each study visit. Results A gradual decrease in patients’ serum AMH levels was observed over time, in line with the descending curve described for healthy women. Serum AMH levels of RA patients in comparison with the values considered normal for age did not reveal any significant differences (P > 0.05). We did not observe any impact of RA treatments. We demonstrated an inverse correlation between AMH variation and disease activity (DAS28: r = –0.27, P = 0.003; CRP: r = –0.16, P = 0.06). Conclusion This is the first study to determine serum AMH levels of a large cohort of RA patients over 36 months. Rapid disease activity control appears to be required to limit changes in the ovarian reserve. Fertility preservation is not likely to be necessary if inflammation is promptly controlled. ClinicalTrials.gov Identifier NCT03666091.

Resume Introduction Les cancers des adolescents et jeunes adultes presentent des specificites epidemiologiques particulieres. L’amelioration de leur survie doit s’accompagner d’une prise en consideration accrue des effets indesirables des traitements, parmi lesquels la possible diminution de la fertilite. L’objectif de l’etude etait de decrire l’acces a la preservation de la fertilite de ces patients au CHU de Clermont-Ferrand sur une periode de trois ans. Methodes Au cours de cette etude descriptive retrospective, differentes donnees sociodemographiques et cliniques ont ete collectees. Resultats Cent cinquante nouveaux cas de cancers ont ete diagnostiques chez les patients de 15–24 ans. Quarante-quatre pour cent ont beneficie d’au moins une consultation au centre d’etude et de conservation des œufs et du sperme humains, 29 % chez les filles et 58 % chez les garcons (p Discussion Nous avons pu observer des disparites dans l’acces a la preservation de la fertilite selon le sexe. Malgre l’existence de recommandations, des progres restent a faire. La mise en place de plateformes clinicobiologiques devrait permettre une meilleure sensibilisation des patients et professionnels de sante, et ainsi de promouvoir l’acces aux techniques de preservation de la fertilite des jeunes patients atteints de cancers.

Summary Background Plasma citrulline is currently used in clinical practice as a marker of small bowel functional mass. Behaviour of plasma citrulline after bariatric surgery and its link with post-operative outcome are still poorly understood. Objective Primary objective was to compare plasma citrulline 12 months after two types of bariatric surgery with pre-operative concentrations. Secondary objectives were to search for correlation between plasma citrulline variation and body weight and fat mass loss. Design This is an ancillary study of the BARIASPERM study. Forty-six adult men (mean age 38.9 ± 7.9 years) who underwent gastric bypass (GB, n = 20) or sleeve gastrectomy (SG, n = 26) were included in this prospective study. Plasma citrulline was measured at baseline, 6 months and 12 months after surgery, as well as total body weight and fat mass measured by dual x-ray absorptiometry (DEXA). Results Plasma citrulline increased significantly 12 months after surgery, both after gastric bypass and sleeve gastrectomy (respectively 30.2% [18.3–42.2] and 17.8% [5.8–29.7]). The increase was significantly higher after GB than after SG (p = 0.02) while total body weight and fat mass loss were not significantly different between GB and SG. The increase in plasma citrulline levels tended to be positively correlated with both weight and fat mass loss however the association did not reach statistical significance (p = 0.07 and p = 0.06 respectively). Conclusion These results confirm the increase in plasma citrulline after GB published in two previous small studies. Citrulline also significantly increased after SG, and in spite of similar weight loss obtained with both surgery types, citrulline increase was higher after GB than SG. This suggests different modifications of intestinal functional mass after these two different techniques.

International audience; 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.

BACKGROUND We showed that in men with a constitutional chromosomal abnormality, DNA fragmentation was significantly higher in chromosomally unbalanced spermatozoa than in spermatozoa with a normal or balanced chromosomal content. These results could be explained by a phenomenon already described in infertile men: abortive apoptosis. OBJECTIVES To determine whether magnetic-activated cell separation could select spermatozoa with lower levels of DNA fragmentation and unbalanced chromosome content in men carrying a structural chromosomal abnormality. MATERIALS AND METHODS The spermatozoa of ten males with a chromosomal rearrangement were separated into two populations using magnetic-activated cell separation (annexin V (-) and annexin V (+) fractions), in order to study meiotic segregation by fluorescence in situ hybridization, the percentage of spermatozoa with an externalization of phosphatidylserine by annexin V staining and DNA fragmentation by TdT-mediated dUTP nick-end labeling on the whole ejaculate and on selected spermatozoa in the same patient. RESULTS For all patients, the percentage of spermatozoa with externalization of phosphatidylserine decreased in the annexin V (-) fraction and increased in the annexin V (+) fraction as compared to the frozen-thawed semen sample. The rates of DNA fragmentation were statistically much lower in the annexin V (-) fraction when compared to the rate before magnetic-activated cell separation for all but one patient. Conversely, we observed a statistically significantly higher rate of DNA fragmentation in the annexin V (+) fraction for six patients. After magnetic-activated cell separation, there was a significant increase of normal/balanced spermatozoa in the fraction of annexin V (-) for all patients. Conversely, we observed a significant decrease in the fraction of annexin V (+) for seven patients. DISCUSSION AND CONCLUSIONS Magnetic-activated cell separation is a promising tool for increasing the selection of healthy spermatozoa, with a decrease in the number of spermatozoa with externalization of phosphatidylserine, DNA fragmentation, and chromosome unbalance, for use in assisted reproductive technologies such as intracytoplasmic sperm injection for males with a chromosomal structural abnormality.

Numerous tests have been proposed to evaluate sperm DNA integrity. To assess the sperm chromatin dispersion (SCD) test in an andrology laboratory, twenty-five men attending Clermont-Ferrand (France) University Hospital's Center for Reproductive Medicine were recruited. Sperm DNA damage was measured in the same semen samples using the SCD test and the Terminal Uridine Nick-end Labeling by flow cytometry technique (TUNEL/FCM) after density gradient centrifugation.SCD test reliability between readings, readers or slides was clearly established with very high agreement between measurements (Intraclass correlation coefficient (ICC) at 0.97, 0.95 and 0.98 respectively). Despite very good agreement between the SCD test and TUNEL/FCM (ICC at 0.94), the SCD test tended to slightly but significantly underestimate DNA damage compared with TUNEL (Andrology laboratories using the SCD test to measure sperm DNA damage need to know that it appears to give slightly underestimated measurements compared to TUNEL/FCM. However, this systematic underestimation is very small in amplitude. Both techniques give almost perfectly congruent results. Our study underlines the importance for each laboratory to validate its method to assess sperm DNA damage before implementing it in routine andrology lab practice.Plusieurs tests sont disponibles pour évaluer l’intégrité de l’ADN spermatique. Afin d’évaluer l’applicabilité de la technique de dispersion de la chromatine spermatique (SCD) dans un laboratoire d’andrologie, nous avons recruté 25 patients pris en charge au Centre de Médecine de la Reproduction du centre hospitalo-universitaire de Clermont-Ferrand (France). L’altération de l’ADN spermatique a été mesurée en ayant recours au test SCD et au test Terminal Uridine Nick-end Labeling en cytométrie en flux (TUNEL/CMF) dans les mêmes échantillons pour les deux techniques, après avoir réalisé un gradient de densité.Pour le test SCD, la concordance entre les lectures, les lecteurs et les lames a été clairement établie avec un accord quasiment parfait entre les mesures (Coefficient de corrélation intra-classe (CCI) respectivement à 0,97, 0,95 et 0,98). Malgré une bonne concordance entre le test SCD et le test TUNEL/CMF (CCI à 0,94), le test SCD tend à sous-estimer légèrement mais de façon significative l’altération de l’ADN spermatique en comparaison avec le test TUNEL (les laboratoires d’andrologie utilisant le test SCD pour mesurer l’altération de l’ADN spermatique doivent savoir qu’il donne apparemment des valeurs légèrement sous-estimées en comparaison du test TUNEL/CMF. Cependant, cette sous-estimation systématique est. de faible amplitude et les deux techniques donnent des résultats presque parfaitement concordants dans notre étude. Cette dernière montre bien que chaque laboratoire doit valider sa méthode sur site pour évaluer l’altération de l’ADN spermatique avant de le mettre en place en pratique quotidienne en andrologie.

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Background Implantation disorders are regarded differently in different groups undergoing in vitro fertilization (IVF) because of heterogeneous patient populations. To avoid this problem, this study was based solely on the first attempts of a single embryo transfer in patients under the age of 35. Methods This was a retrospective comparative study with 2 groups of patients: a group with endometriosis (n = 288) and a group without endometriosis (n = 1,368), registered from January 2010 to December 2013 in the IVF center of the University Hospital of Clermont-Ferrand. This study compared the 2 groups and subgroups according to ovarian response and embryo quality, and analyzed oocyte number, embryo number, transfer rate, implantation rate and delivery rate per ovum pick-up and per transfer. Results In the cases of endometriosis, the number of collected oocytes (8.37 ± 7.01 vs. 10.13 ± 6.53 pConclusions Endometriosis has no impact on the embryo transfer in IVF, particularly when the embryo quality is satisfactory.

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International audience; A close relationship exists between cholesterol and female reproductive physiology. Indeed, cholesterol is crucial for steroid synthesis by ovary and placenta, and primordial for cell structure during folliculogenesis. Furthermore, oxysterols, cholesterol-derived ligands, play a potential role in oocyte maturation. Anomalies of cholesterol metabolism are frequently linked to infertility. However, little is known about the molecular mechanisms. In parallel, increasing evidence describing the biological roles of liver X receptors (LXRs) in the regulation of steroid synthesis and inflammation, two processes necessary for follicle maturation and ovulation. Both of the isoforms of LXRs and their bona fide ligands are present in the ovary. LXR-deficient mice develop late sterility due to abnormal oocyte maturation and increased oocyte atresia. These mice also have an ovarian hyper stimulation syndrome in response to gonadotropin stimulation. Hence, further studies are necessary to explore their specific roles in oocyte, granulosa, and theca cells. LXRs also modulate estrogen signaling and this could explain the putative protective role of the LXRs in breast cancer growth. Altogether, clinical studies would be important for determining the physiological relevance of LXRs in reproductive disorders in women

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.

The fertility preservation when it is threatened is a right enshrined in the french law on bioethics. It is most often performed before gonadotoxic treatments for cancers with a long survival. When the patient has a limited life expectancy, is the preservation of fertility lawful? The authors present the arguments for and against the preservation of fertility in this particular situation and give driving they adopt in their team.

Research question Is sperm fluorescence in-situ hybridization (FISH) useful to evaluate the risk of chromosomally unbalanced gametes in interchromosomal reciprocal insertion (IRI) carriers? How do these imbalances lead to recurrent miscarriages? Design This study reports a clinical and molecular study of a rare familial balanced IRI resulting in recurrent spontaneous miscarriage. Sperm FISH was performed to estimate the number of unbalanced gametes. Results A 31-year-old healthy male (proband) and his 28-year-old female partner were referred to the Genetics Department for three spontaneous miscarriages occurring during the first trimester of pregnancy. FISH analysis of the proband with the LSI TRA/D (14q11.2) and DiGeorge N25 (22q11.2) break-apart probes showed the presence of a balanced IRI between 14q11.2 and 22q11.2 chromosomal regions. This IRI was also identified in the proband's father. Sperm FISH with the same probes showed that more than 40% of gametes of the proband were unbalanced for either 14q11.2 or 22q11.2, despite normal sperm parameters. FISH analysis of a product of conception indicated that unbalanced gametes result in a non-viable fetus. Conclusions This study shows the value of sperm FISH analysis in improving genetic reproductive advice for IRI carriers. Disruption of critical genes through this rearrangement and their consequent functional impairment could result in recurrent miscarriages. In this case, several genes located in the 14q11.2 region, particularly RNase 3, would be good candidates to explain the lethality of the imbalances.

A prospective study on randomized patients was conducted to determine how morphokinetic parameters are altered in embryos grown in sequential versus global culture media. Eleven morphokinetic parameters of 160 single embryos transferred were analyzed by time lapse imaging involving two University-affiliated in vitro fertilization (IVF) centers. We found that the fading of the two pronuclei occurred earlier in global (22.56±2.15 hpi) versus sequential media (23.63±2.71 hpi; p=0.0297). Likewise, the first cleavage started earlier at 24.52±2.33 hpi vs 25.76±2.95 hpi (p=0.0158). Also, the first cytokinesis was shorter in global medium, lasting 18±10.2 minutes in global versus 36±37.8 minutes in sequential culture medium (p

International audience; Neuroblastoma (NB) is the most common type of extracranial solid tumor in children with a high prevalence in toddlers. For childhood cancer survivors, preservation of reproductive potential is an important factor for quality of life. The optimization of NB minimal residual disease (MRD) detection in testicular tissue is crucial to evaluate the risk of malignant cell reintroduction. The first step in the present study was to assess the accuracy of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect tyrosine hydroxylase (TH), paired-like homeobox 2b (PHOX2B) and doublecortin (DCX) mRNA expression in frozen/thawed testicular tissues of patients with non-obstructive azoospermia (NOA) contaminated (in vitro model) with an increasing number of IMR-32 and SK-N-SH NB cells. Testicular tissues were frozen by slow or snap freezing. The second step was to determine the expression levels of these markers in testicular samples from 4 pre-pubertal males (2 with stage IV NB and 2 with non-NB malignancy). The yield of extracted RNA was similar in testicular samples frozen by slow or snap freezing. In the in vitro model, TH and DCX transcripts were detected in uncontaminated testicular tissues, whereas PHOX2B mRNA was not detected. There was a strong positive association between the number of NB cells used for contamination and PHOX2B transcript levels. For IMR-32 and SK-N-SH NB cell lines, specificity and sensitivity rates of detection were 100% for PHOX2B following in vitro contamination with 10 tumor cells. In testicular samples from pre-pubertal males with and without NB, PHOX2B mRNA expression was not observed, but high expression levels of TH and DCX mRNA were detected, which were similar to expression detected in the in vitro model. Among the markers used in blood and bone marrow for NB MRD studies, the detection of PHOX2B transcripts by RT-qPCR may provide an accurate assessment of NB cells in testicular tissues from males who require fertility preservation.

Background Ovarian tissue cryopreservation (OTC) is the only option available to preserve fertility in prepubertal females with neuroblastoma (NB), a childhood solid tumor that can spread to the ovaries, with a risk of reintroducing malignant cells after an ovarian graft. Procedure We set out to determine whether the analysis of TH (tyrosine hydroxylase), PHOX2B (paired-like homeobox 2b), and DCX (doublecortin) transcripts using quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) could be used to detect NB contamination in ovarian tissue. Analyses were performed on benign ovarian tissue from 20 healthy women between November 2014 and September 2015 at the University Hospital of Clermont-Ferrand. Pericystic benign ovarian tissues were collected and contaminated with increasing numbers of human NB cells (cell lines IMR-32 and SK-N-SH) before detection using RT-qPCR. Results TH and DCX transcripts were detected in uncontaminated ovarian tissue from all the donors, hampering the detection of small numbers of tumor cells. By contrast, PHOX2B was not detected in any uncontaminated ovarian fragment. PHOX2B levels were significantly increased from 10 NB cells. Our study is the first to evaluate minimal residual disease detection using NB mRNAs in human ovarian tissue. Only PHOX2B was a reliable marker of NB cells contaminating ovarian tissue. Conclusions These results are encouraging and offer hope in the near future for grafting ovarian tissue in women who survive cancer, whose fertility has been jeopardized by treatment, and who could benefit from OTC without oncological risk.

International audience; 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.

Resume Objectifs La cryoconservation du tissu ovarien (CTO) constitue un moyen de preserver la fertilite des femmes et jeunes filles devant suivre un traitement potentiellement gonadotoxique (chimiotherapie, radiotherapie…). Cette etude a pour objectif d’analyser les indications et le devenir des patientes qui ont beneficie d’une CTO dans notre centre de biologie et medecine de la reproduction apres 17 annees de pratique. Patientes et methodes Etude retrospective realisee chez 46 patientes ayant beneficie d’une CTO avant un traitement sterilisant dans le service d’AMP- CECOS du CHU de Clermont-Ferrand, de janvier 1997 a decembre 2009. Resultats L’âge moyen des patientes lors du prelevement etait de 19,5 ans. Les patientes etaient mineures dans 52 % des cas au moment du prelevement. Par ordre de frequence decroissante, les pathologies presentees par les patientes au moment de la CTO etaient hematologiques, des tumeurs ovariennes, des sarcomes ou tumeurs neuroectodermiques (PNET) et des neoplasies mammaires. Dans 93,5 % des cas, le prelevement du cortex ovarien a ete pratique par cœlioscopie. Suite au prelevement de tissu ovarien, 82,6 % des patientes ont ete traitees par chimiotherapie. Une greffe de moelle osseuse a ete realisee chez 48 % des patientes. Au moment du recueil des donnees, 57 % des patientes pour qui un suivi de la fonction ovarienne avait ete realise, presentaient une insuffisance ovarienne prematuree. Huit patientes ont presente une grossesse apres l’arret du traitement. Il s’agissait d’une grossesse naturelle pour cinq d’entre elles. Les trois autres patientes ont obtenu une grossesse apres recours a l’assistance medicale a la procreation (fecondation in vitro et don d’ovocytes). Discussion et conclusion Nous rapportons un suivi a long terme des patientes prises en charge dans notre centre pour un prelevement et une CTO. Notre etude a pour originalite d’evaluer l’ensemble des aspects de prise en charge, de la decision de proposer une CTO aux patientes a leur devenir plusieurs annees apres le prelevement. Il s’agit donc d’une approche multidisciplinaire a la fois oncologique, gynecologique et pediatrique de la CTO qui est souvent etudiee de facon restrictive dans la litterature. Enfin, il semble essentiel d’instaurer une surveillance medicale specifique pour ces patientes. Ce suivi doit permettre une evaluation satisfaisante du developpement pubertaire et de la fonction ovarienne, la prise en charge de la carence œstrogenique et de l’infertilite secondaire, l’accompagnement des patientes dans leur desir de maternite.

Resume Objectifs Si l’association entre endometriose et infertilite est bien connue, son retentissement sur l’issue obstetricale est encore peu etudie. Notre objectif etait de determiner l’impact de l’endometriose sur les issues obstetricales des singletons. L’objectif secondaire etait de determiner si la severite de la maladie avait une influence sur ces issues. Patientes et methodes Nous avons realise une etude retrospective monocentrique portant sur une population de 1204 femmes infertiles dont 258 endometriosiques ayant obtenu, par assistance medicale a la procreation, une grossesse unique evolutive au-dela du stade embryonnaire. Deux analyses ont ete realisees. La premiere comparait les femmes endometriosiques aux femmes presentant une autre cause d’infertilite. La seconde observait les complications obstetricales en fonction des stades AFS-R de l’endometriose. Resultats Le taux global d’enfants vivants a la naissance etait de 95,8 %. En presence d’endometriose, il existait une augmentation significative de l’incidence des accouchements prematures, surtout avant 32 SA (6,2 % vs 3,1 % dans le groupe « sans endometriose », p = 0,03), des metrorragies (5,3 % vs 2,2 %, p = 0,01) et du placenta praevia (4,9 % vs 0,9 %, p Discussion et conclusion L’endometriose est un facteur de risque obstetrical independamment de l’infertilite qu’elle entraine. Le score AFS-R ne semble pas etre representatif du pronostic obstetrical au-dela du premier trimestre de grossesse chez les femmes porteuses d’endometriose.

El desarrollo de las tecnicas de reproduccion asistida ha permitido la aparicion de las donaciones de gametos y de embriones, asi como la creacion de bancos de semen, de embriones, e incluso de ovocitos desde hace poco. En este articulo se detallan las modalidades de reclutamiento y de seleccion de donantes, asi como las indicaciones de cada tipo de donacion. Tambien se exponen las distintas tecnicas disponibles (inseminacion, fecundacion in vitro simple o con microinyeccion, transferencia sincronica, congelacion de ovocitos, transferencia de embriones congelados), asi como las modalidades de su eleccion y sus resultados. Estos metodos suscitan muchas cuestiones eticas y juridicas: filiacion, secreto, anonimato, remuneracion, proteccion de los donantes, actitud ante las parejas homosexuales y las mujeres solas, riesgo de consanguinidad, etcetera. Existe una gran variabilidad en cuanto a las respuestas en funcion de criterios religiosos, culturales, filosoficos o simplemente personales. En la practica, estas cuestiones han dado origen a legislaciones muy distintas entre los paises. Esta disparidad ha dado lugar en la actualidad a la edad de oro del turismo medico. Por ultimo, se describira la situacion de la legislacion francesa en 2012.

Resume Objectif Les buts de cette etude etait d’evaluer l’efficacite du drilling ovarien, de comparer la technique par fertiloscopie a la technique par cœlioscopie, et de rechercher des facteurs pronostiques de succes. Patientes et methodes L’etude retrospective a porte sur 154 drillings ovariens realises entre juin 1998 et decembre 2010 ou le drilling a ete propose apres echec du clomifene et avant stimulation par FSH chez des patientes presentant un PCOS. Resultats Le taux d’ovulation post-drilling est de 62 % ; le taux de grossesse evolutive spontanee est de 31 % et le taux total de grossesse evolutive incluant les stimulations secondaires est de 58 %. Il n’est pas retrouve de difference significative entre la cœlioscopie et la fertiloscopie. Il a ete note plus de complications peroperatoires en fertiloscopie et 20 % des fertiloscopies ont du etre convertie en cœlioscopie dont la moitie pour complications ou difficultes techniques. Les seuls facteurs predictifs preoperatoires retrouves sont une euthyroidie qui augmente les chances de grossesses en general (incluant les stimulations secondaires) et un taux de FSH plus bas. Par contre, il apparait que les chances de grossesse en stimulation sont fortement diminuees si le drilling n’a pas induit d’ovulation versus les cas ou il y a eu ovulation mais pas de grossesse (28,8 % versus 58,1 %, p Discussion et conclusion Les resultats obtenus en fertiloscopie sont comparables a ceux de la cœlioscopie, ce qui suggere un avantage a cette technique en termes de cout, de confort et de duree d’hospitalisation. Aucun critere de selection des patientes utilisable en pratique courante n’a pu etre mis en evidence. L’etude suggere que l’absence d’ovulation apres drilling pourrait etre une indication directe de FIV.

Resume Objectif Evolution des taux d’accouchement et des taux de grossesse multiple en Assistance Medicale a la Procreation (AMP) consecutive a la mise en application du SET (Single Embryo Transfer) dans un centre d’AMP. Patientes et methodes Etude retrospective realisee a partir de donnees recueilles de maniere prospective entre le 01/01/2001 et le 31/12/2010 dans le centre d’AMP du CHU de Clermont Ferrand. Les criteres etudies sont le nombre moyen d’embryon transfere, les taux d’accouchement et de grossesse multiple apres transfert d’embryon frais et d’embryon congele. Les resultats ont ete analyses de facon globale puis stratifies en fonction de l’âge des patientes. Resultats Le taux d’accouchement cumule (apres transfert d’embryon frais et d’embryon congele) a atteint 27,3 % en 2010 tandis que le taux de grossesse multiple a diminue de 30 % en 2001 a 7,9 % en 2010. Le nombre moyen d’embryon transfere est passe de 2,29 en 2001 a 1,55 en 2011. En 2010, un SET a ete realise dans 85 % des 1res tentatives et 34,4 % des 2es tentatives chez les patientes de moins de 35 ans. Conclusions La mise en pratique du SET ne modifie pas les taux d’accouchement mais diminue de maniere significative le nombre de grossesses multiples. Le SET devrait etre realise de maniere systematique lors des premieres et deuxiemes tentatives chez les patientes de moins de 35 ans lorsqu’au moins deux embryons de bonne qualite ont ete obtenus.

To evaluate the efficiency of an original slow freezing protocol on the quality and function of human ovarian cortex.Human ovarian tissues were cryopreserved using a freezing medium supplemented with propanediol and raffinose as cryoprotectants and antioxidants (L-glutamine, taurine). Samples were then frozen using a faster cooling rate than the usual one. Viability and morphology of follicles, DNA fragmentation in follicles and stroma as well as histology of the vascular endothelium were analyzed before and after freezing/thawing. Moreover, a functional analysis was performed based on the evaluation of follicular growth and development in thawed ovarian tissues that were cultured in vitro.Our freezing/thawing protocol allows preservation of a high proportion of viable follicles and the preservation of the different follicle developmental stages (p0.05 versus fresh control). 70.5 ± 5.2 % of follicles retained an intact morphology after cryopreservation (p=0.04). Stroma cells but not follicles exhibited a slight increase of DNA fragmentation after thawing (p0.05). Microvessel endothelium within thawed tissues appeared to be preserved. Granulosa cells showed signs of proliferation in follicles cultured for 12 days. Secretion of 17β-oestradiol significantly increased during in vitro culture.This protocol leads to good preservation of ovarian integrity and functionality post-thawing and thus appears as a suitable technique of ovarian tissue cryopreservation in clinical settings. Further research could be extended to optimize conditions of in vitro culture.

International audience; Significantly improved survival rates in children and adolescents with cancer have put fertility preservation high on the pediatric oncology agenda. Here we report a retrospective single-center study of 13 years experience of ovarian tissue cryopreservation (OTC) before sterilizing treatment in order to define the safety/benefits of OTC and study clinical/hormonal outcomes in girls. From 2000 to 2013, OTC was performed in 36 girls: eight had non-malignant disease and 28 had malignant disease. Laparoscopy was used to collect a third of each ovary that was frozen by a slow cooling protocol. Indications for OTC were 13 auto-, 19 allo-stem-cell-transplantation and 4 sterilizing chemotherapy. Ovarian tissue harvested by intraumbilical laparoscopy led to no major postoperative complications and did not delay chemotherapy. Histological analysis of ovarian tissue showed an average of 9 primordial follicles/mm(2) [0-83] and no malignant cells were identified. Median post-harvest follow-up was 36 months [1-112]: 26 girls were alive in complete remission and 10 had died. Hormonal results were evaluable for 27 patients (median age 17 yrs [5-26]): 16 patients were in premature ovarian insufficiency. OTC sampling one third of each ovary appears to be an appropriate approach to preserve fertility in children without consequences on subsequent therapeutic program.