Efficient pathway for men fertility preservation in testicular cancer or lymphoma: a cross-sectional study of national 2018 data.

BACKGROUND: In 15-49 years-old men, the main cancers are testicular cancer (TC) and lymphomas (L): freezing of ejaculated sperm is primarily used for male fertility preservation (FP) before cancer treatment. Our objective was to analyze the French FP rate in 15-49 years-old men diagnosed with TC or L in 2018. We designed a national descriptive cross-sectional study of sperm banking rate in men with a diagnosis of TC, Hodgkin L (HL) or non-Hodgkin L (NHL). From the French National Cancer Institute (INCa) 2018 data, we extracted the estimated incidence of TC and L in metropolitan France. From the 2018 activity report of CECOS network (Centers for Study and Banking of Eggs and Sperm), we extracted the number of men with TC or L who banked ejaculated sperm. We estimated the proportion of 15-49 years-old men diagnosed with TC or L who banked sperm. RESULTS: Among 15-49 years-old men, INCa estimated 38,048 new cancer diagnoses in metropolitan France in 2018: 2,630 TC and 3,913 L (943 HL and 2,970 NHL). The CECOS network provided data from 26/27 metropolitan centers (96% response rate): 1,079 sperm banking for men with TC, 375 for HL and 211 for NHL. We estimated that the 2018 sperm banking rate in France was 41% for TC, 40% for HL, and 7% for NHL. CONCLUSIONS: To our knowledge, our paper is the first cross-sectional study with multicenter and national data analyzing FP rate in cancer men: it suggests an efficient pathway for men to FP before cancer treatment, compared to previously published studies. Although sperm banking rate in 15-49 years-old men could definitely be improved, further studies should evaluate the information given to patients before gonadotoxic treatments, the factors associated with the absence of sperm banking and whether this lack of referral induces a loss of chance for these men.

RéSUMé: CONTEXTE: Chez les hommes de 15 à 49 ans, les principaux cancers sont le cancer du testicule (CT) et les lymhomes (L): la congélation de spermatozoïdes éjaculés est utilisée en première intention pour leur préservation de fertilité (PF) avant traitement du cancer. Notre objectif était d'analyser le taux de PF chez les hommes de 15 à 49 ans diagnostiqués avec un CT ou un L en 2018 en France. Nous avons réalisé une étude nationale transversale descriptive du taux de congelation de spermatozoïdes chez les hommes âgés de 15 à 49 ans diagnostiqués avec un CT, un L de Hodgkin (LH) ou un L non-Hodgkinien (LNH). A partir des données de l'Institut National du Cancer (INCa) de 2018, nous avons extrait l'incidence estimée de CT et de L en France métropolitaine. A partir des données du bilan d'activité 2018 de la Federation Française des CECOS (Centre d'Etude et de Conservation des Oeufs et du Sperme), nous avons extrait le nombre d'hommes avec un CT ou un L qui ont congelé leurs spermatozoïdes. Nous avons enfin estimé la proportion d'hommes de 15 à 49 ans diagnostiqués avec un CT ou un L qui ont congelé leurs spermatozoïdes. RéSULTATS: Chez les hommes de 15 à 49 ans, l'INCa a estimé en 2018 38 048 nouveaux cas de cancers diagnostiqués en France métropolitaine en 2018: 2 630 CT et 3 913 L (943 LH et 2 970 LNH). Le réseau des CECOS a produit les résultats issus de 26/27 centres métropolitains (taux de réponse de 96%): 1 079 congélations de sperme pour des hommes atteints de CT, 375 pour LH et 211 pour LNH. Nous avons estimé que le taux de congelation de spermatozoïdes de 2018 en France était de 41% pour le CT, 40% pour le LH et 7% pour le LNH. CONCLUSIONS: A notre connaissance, notre travail est la première étude transversale multicentrique de données nationales analysant le taux de PF chez les hommes atteints de cancer: il suggère un parcours patient efficace pour la PF des hommes avant traitement d'un cancer, par rapport aux études précédemment publiées. Bien que le taux de PF chez les hommes puisse certainemen être amélioré, des études futures devraient évaluer l'information donnée aux patients avant traitement gonadotoxique, les facteurs associés à l'absence de PF et si le défaut d'adressage au CECOS induit un perte de chance pour ces hommes. MOTS-CLéS: Chimiothérapie, Radiothérapie, Oncofertiité, Azoospermia, Paternité.

A 16-year bicentric retrospective analysis of ovarian tissue cryopreservation in pediatric units: indications, results, and outcome.

Background: Cancer treatments of the last decades improve the survival rate of children and adolescents. However, chemo- and radiotherapy result in gonadal damage, leading to acute ovarian failure and sterility. The preservation of fertility is now an integral part of care of children requiring gonadotoxic treatments. Ovarian tissue cryopreservation (OTC) is an effective fertility preservation option that allows long-term storage of primordial follicles, subsequent transplantation, and restoration of endocrine function and fertility. The efficacy of this technique is well-demonstrated in adults but the data are scarce for pediatric patients. Currently, OTC represents the only possibility of preserving the potential fertility in prepubertal girls. Procedure: This is a retrospective study of OTC practice of two French centers from January 2004 to May 2020. A total of 72 patients from pediatric units underwent cryopreservation of ovarian tissue before gonadotoxic therapy for malignant or non-malignant diseases. The ovarian cortex was cut into fragments and the number of follicles per square millimeter was evaluated histologically. The long-term follow-up includes survival rate and hormonal and fertility status. Results: The mean age of patients at OTC was 9.3 years [0.2-17] and 29.2% were postpubertal; 51 had malignant diseases and 21 had non-malignant diseases. The most frequent diagnoses included acute leukemia, hemoglobinopathies, and neuroblastoma. Indication for OTC was stem cell transplantation for 81.9% (n = 59) of the patients. A third of each ovary was collected for 62.5% (n = 45) of the patients, a whole ovary for 33.3% (n = 24) of the patients, and a third of one ovary for 4.2% (n = 3) of the patients. An average of 17 fragments [5-35] per patient was cryoconserved. A correlation was found between the age of the patients and the number of fragments (p < 0.001). More fragments were obtained from partial bilateral harvesting than from whole ovary harvesting (p < 0.05). Histological analysis of ovarian tissue showed a median of 6.0 primordial follicles/mm2 [0.0-106.5] and no malignant cells were identified. A negative correlation was found between age and follicular density (p < 0.001). Median post-harvest follow-up was 92 months [1-188]. A total of 15 girls had died, 11 were still under treatment for their pathology, and 46 were in complete remission. Of all patients, 29 (40.2%) were subjected to a hormonal status evaluation and 26 were diagnosed with premature ovarian insufficiency (POI) (p < 0.001). One patient had undergone thawed ovarian tissue transplantation. Conclusion: OTC should be proposed to all girls with high risk of developing POI following gonadotoxic therapies in order to give them the possibility of fertility and endocrine restoration.

Testicular germ cell tumour risk by occupation and industry: a French case-control study - TESTIS.

OBJECTIVE: Testicular germ cell tumours (TGCT) are the most common cancer in men of working age and its incidence has increased notably over the past 40 years. Several occupations have been identified as potentially associated with TGCT risk. The aim of this study was to further explore the relationship between occupations, industries and TGCT risk in men aged 18-45 years. METHODS: The TESTIS study is a multicenter case-control study conducted between January 2015 and April 2018 in 20 of 23 university hospital centers in metropolitan France. A total of 454 TGCT cases and 670 controls were included. Full job histories were collected. Occupations were coded according to the International Standard Classification of Occupation 1968 version (ISCO-1968) and industry according to the 1999 version of Nomenclature d'Activités Française (NAF-1999). For each job held, ORs and 95% CIs were estimated using conditional logistic regression. RESULTS: A positive association was observed between TGCT and occupation as agricultural, animal husbandry worker (ISCO: 6-2; OR 1.71; 95% CI (1.02 to 2.82)), as well as salesman (ISCO: 4-51; OR 1.84; 95% CI (1.20 to 2.82)). An increased risk was further observed among electrical fitters and related, electrical and electronics workers employed for 2 years or more (ISCO: 8-5; OR≥2 years 1.83; 95% CI (1.01 to 3.32)). Analyses by industry supported these findings. CONCLUSIONS: Our findings suggest that agricultural, electrical and electronics workers, and salesmen workers experience an increased risk of TGCT. Further research is needed to identify the agents or chemicals in these high-risk occupations which are relevant in the TGCT development. TRIAL REGISTRATION NUMBER: NCT02109926.

Timeline of Developmental Defects Generated upon Genetic Inhibition of the Retinoic Acid Receptor Signaling Pathway.

It has been established for almost 30 years that the retinoic acid receptor (RAR) signalling pathway plays essential roles in the morphogenesis of a large variety of organs and systems. Here, we used a temporally controlled genetic ablation procedure to precisely determine the time windows requiring RAR functions. Our results indicate that from E8.5 to E9.5, RAR functions are critical for the axial rotation of the embryo, the appearance of the sinus venosus, the modelling of blood vessels, and the formation of forelimb buds, lung buds, dorsal pancreatic bud, lens, and otocyst. They also reveal that E9.5 to E10.5 spans a critical developmental period during which the RARs are required for trachea formation, lung branching morphogenesis, patterning of great arteries derived from aortic arches, closure of the optic fissure, and growth of inner ear structures and of facial processes. Comparing the phenotypes of mutants lacking the 3 RARs with that of mutants deprived of all-trans retinoic acid (ATRA) synthesising enzymes establishes that cardiac looping is the earliest known morphogenetic event requiring a functional ATRA-activated RAR signalling pathway.

Pathogenesis of Anorectal Malformations in Retinoic Acid Receptor Knockout Mice Studied by HREM.

Anorectal malformations (ARMs) are relatively common congenital abnormalities, but their pathogenesis is poorly understood. Previous gene knockout studies indicated that the signalling pathway mediated by the retinoic acid receptors (RAR) is instrumental to the formation of the anorectal canal and of various urogenital structures. Here, we show that simultaneous ablation of the three RARs in the mouse embryo results in a spectrum of malformations of the pelvic organs in which anorectal and urinary bladder ageneses are consistently associated. We found that these ageneses could be accounted for by defects in the processes of growth and migration of the cloaca, the embryonic structure from which the anorectal canal and urinary bladder originate. We further show that these defects are preceded by a failure of the lateral shift of the umbilical arteries and propose vascular abnormalities as a possible cause of ARM. Through the comparisons of these phenotypes with those of other mutant mice and of human patients, we would like to suggest that morphological data may provide a solid base to test molecular as well as clinical hypotheses.

Meiosis occurs normally in the fetal ovary of mice lacking all retinoic acid receptors.

Gametes are generated through a specialized cell differentiation process, meiosis, which, in ovaries of most mammals, is initiated during fetal life. All-trans retinoic acid (ATRA) is considered as the molecular signal triggering meiosis initiation. In the present study, we analyzed female fetuses ubiquitously lacking all ATRA nuclear receptors (RAR), obtained through a tamoxifen-inducible cre recombinase-mediated gene targeting approach. Unexpectedly, mutant oocytes robustly expressed meiotic genes, including the meiotic gatekeeper STRA8. In addition, ovaries from mutant fetuses grafted into adult recipient females yielded offspring bearing null alleles for all Rar genes. Thus, our results show that RAR are fully dispensable for meiotic initiation, as well as for the production of functional oocytes. Assuming that the effects of ATRA all rely on RAR, our study goes against the current model according to which meiosis is triggered by endogenous ATRA in the developing ovary. It therefore revives the search for the meiosis-inducing substance.

Ovarian Cortex Transplantation.

STUDY OBJECTIVE: To describe a laparoscopic technique for the transplantation of a cryopreserved ovarian cortex. DESIGN: Educational video. SETTING: University Hospital of Strasbourg, France. INTERVENTIONS: A 28-year-old nulliparous woman presented with anaplastic T lymphoma and was then treated with chemotherapy. Before the treatment, the ovarian cortex was collected by laparoscopy to preserve fertility. Remission was achieved, but the patient suffered from premature ovarian failure. At the age of 32 years, she wished to become pregnant. The patient was thus included in the research protocol Development of Ovarian Tissue Autograft in Order to Restore Ovarian Function, and the transplantation site was chosen accordingly. The cortex was stored in liquid nitrogen at -196°C after slow congelation. To restore ovarian function and because of pregnancy desire, we transplanted the cryopreserved ovarian cortex in the right ovary and inside a pocket of the peritoneum of the left ovarian fossa. The first step included adhesiolysis to treat small adhesions developed after the first surgery. On the right, the ovarian cortex was opened by an antimesial incision with cold scissors. The cryopreserved ovarian cortex was placed through the cortex of the right ovary and fixed with stitches. On the left side, the peritoneum of the ovarian fossa was opened, and a subperitoneal pocket was dissected. The cortex was inserted. It was then closed with absorbable sutures or with a hemostatic pad. Six months after her surgery, the patient had natural cycles. We monitored an ovulation of both the sides. She underwent 3 in vitro fertilizations but with failures of embryo transfer. She conceived spontaneously a year after the surgery. She gave birth to a healthy child weighing 3300 g. CONCLUSION: For patients who have suffered from premature ovarian failure owing to chemotherapy, ovarian cortex transplantation can restore the ovulatory function, allow in vitro fertilization, and permit, as in our case, a spontaneous pregnancy.

Two functionally redundant sources of retinoic acid secure spermatogonia differentiation in the seminiferous epithelium.

In mammals, all-trans retinoic acid (ATRA) is instrumental to spermatogenesis. It is synthesized by two retinaldehyde dehydrogenases (RALDH) present in both Sertoli cells (SCs) and germ cells (GCs). In order to determine the relative contributions of each source of ATRA, we have generated mice lacking all RALDH activities in the seminiferous epithelium (SE). We show that both the SC- and GC-derived sources of ATRA cooperate to initiate and propagate spermatogenetic waves at puberty. In adults, they exert redundant functions and, against all expectations, the GC-derived source does not perform any specific roles despite contributing to two-thirds of the total amount of ATRA present in the testis. The production from SCs is sufficient to maintain the periodic expression of genes in SCs, as well and the cycle and wave of the SE, which account for the steady production of spermatozoa. The production from SCs is also specifically required for spermiation. Importantly, our study shows that spermatogonia differentiation depends upon the ATRA synthesized by RALDH inside the SE, whereas initiation of meiosis and expression of STRA8 by spermatocytes can occur without ATRA.

Luteal phase stimulation, the future of fertility preservation? Retrospective cohort study of luteal phase versus follicular phase stimulation.

RESEARCH QUESTION: Is luteal phase stimulation capable of improving fertility preservation? MATERIALS AND METHODS: We performed a retrospective cohort study of consecutive ovarian stimulations, during July 2012 and September 2018 at Strasbourg University Teaching Hospital in France. Enrollment criteria were patients aged below 40 who had been referred to our center following a diagnosis of cancer or requiring gonadotoxic treatment. All patients enrolled had regular menstrual cycles and normal ovulation. Non-enrollment criteria were an expected low ovarian response (defined by an anti-Müllerian hormone (AMH) level <0.75µg/L and/or an antral follicle count inferior (AFC) inferior than 5), polycystic ovarian syndrome, amenorrhea, prior history of infertility or gonadotoxic treatment. The primary endpoint is the number of mature oocytes (metaphase II) obtained. Secondary outcomes were oocyte yields obtained, stimulation duration, initial gonadotropin dose and total gonadotropin dose. RESULTS: A total of 100 patients were included: 20 in luteal phase and 80 in follicular phase. A larger number of mature oocytes was obtained in luteal phase versus follicular phase (13.1+/8.0 versus 9.2+/-5.8 with p=0.01). A greater amount of total (mature and immature) oocytes was obtained in luteal phase versus follicular phase with a significant difference (16.8+/-9.3 versus 11.8+/-7.3 with p=0.01). No difference was found for the initial and total doses of gonadotropin. CONCLUSIONS: Luteal phase stimulation has the advantage of a better flexibility with positives results as to the number of oocytes obtained in fertility preservation.

Roles of Retinoic Acid in Germ Cell Differentiation.

The modalities of gametogenesis differ markedly between sexes. Female are born with a definitive reserve of oocytes whose size is crucial to ensure fertility. Male fertility, in contrast, relies on a tightly regulated balance between germ cell self-renewal and differentiation, which operates throughout life, according to recurring spatial and temporal patterns. Genetic and pharmacological studies conducted in the mouse and discussed in this review have revealed that all-trans retinoic acid and its nuclear receptors are major players of gametogenesis and are instrumental to fertility in both sexes.

A no-stop mutation in MAGEB4 is a possible cause of rare X-linked azoospermia and oligozoospermia in a consanguineous Turkish family.

PURPOSE: The purpose of this study was to identify mutations that cause non-syndromic male infertility using whole exome sequencing of family cases. METHODS: We recruited a consanguineous Turkish family comprising nine siblings with male triplets; two of the triplets were infertile as well as one younger infertile brother. Whole exome sequencing (WES) performed on two azoospermic brothers identified a mutation in the melanoma antigen family B4 (MAGEB4) gene which was confirmed via Sanger sequencing and then screened for on control groups and unrelated infertile subjects. The effect of the mutation on messenger RNA (mRNA) and protein levels was tested after in vitro cell transfection. Structural features of MAGEB4 were predicted throughout the conserved MAGE domain. RESULTS: The novel single-base substitution (c.1041A>T) in the X-linked MAGEB4 gene was identified as a no-stop mutation. The mutation is predicted to add 24 amino acids to the C-terminus of MAGEB4. Our functional studies were unable to detect any effect either on mRNA stability, intracellular localization of the protein, or the ability to homodimerize/heterodimerize with other MAGE proteins. We thus hypothesize that these additional amino acids may affect the proper protein interactions with MAGEB4 partners. CONCLUSION: The whole exome analysis of a consanguineous Turkish family revealed MAGEB4 as a possible new X-linked cause of inherited male infertility. This study provides the first clue to the physiological function of a MAGE protein.

Tex19 paralogs are new members of the piRNA pathway controlling retrotransposon suppression.

Tex19 genes are mammalian specific and duplicated to give Tex19.1 and Tex19.2 in some species, such as the mouse and rat. It has been demonstrated that mutant Tex19.1 males display a variable degree of infertility whereas they all upregulate MMERVK10C transposons in their germ line. In order to study the function of both paralogs in the mouse, we generated and studied Tex19 double knockout (Tex19DKO) mutant mice. Adult Tex19DKO males exhibited a fully penetrant phenotype, similar to the most severe phenotype observed in the single Tex19.1KO mice, with small testes and impaired spermatogenesis, defects in meiotic chromosome synapsis, persistence of DNA double-strand breaks during meiosis, lack of post-meiotic germ cells and upregulation of MMERVK10C expression. The phenotypic similarities to mice with knockouts in the Piwi family genes prompted us to check and then demonstrate, by immunoprecipitation and GST pulldown followed by mass spectrometry analyses, that TEX19 paralogs interact with PIWI proteins and the TEX19 VPTEL domain directly binds Piwi-interacting RNAs (piRNAs) in adult testes. We therefore identified two new members of the postnatal piRNA pathway.

Development of a methodology for in vivo follow-up of hepatocellular carcinoma in hepatocyte specific Trim24-null mice treated with myo-inositol trispyrophosphate.

BACKGROUND: Genetically induced hepatocellular carcinoma (HCC) models are generally used to investigate carcinogenesis pathways, but very few attempts were made to valorize them for pharmacological testing. This study describes a micro-computed tomography (micro-CT) - based methodology for the diagnostic and lifelong follow-up of HCC in the hepatocyte-specific Trim24-null mouse line. Myo-inositol trispyrophosphate (ITPP) was tested as anti-cancer drug. METHODS: Partial hepatectomy was performed in 2 months-old Trim24-null mice, in order to accelerate the carcinogenesis process. HCC diagnosis was obtained by micro-CT scan with double contrast agent: 10 µl/g Fenestra™ LC was injected intraperitoneally 6 h prior to imaging and 10 µl/g Fenestra™ VC was injected intravenously 15 min prior to imaging. Twenty three hepatocyte-specific Trim24-null mice were considered for ITPP testing (3 mg/g/week intraperitoneally during 10 months in 12 mice, versus 11 controls). Lifelong follow-up was performed using micro-CT. Comparative analysis was performed using unpaired t test with Welch correction and survival curves were compared by log-rank test. Gene expression analysis was performed using the RT q-PCR technique. RESULTS: Double contrast micro-CT scan allowed HCC diagnosis as hypodense, isodense or hyperdense nodules. Positive predictive value was 81.3 %. Negative predictive value was 83.3 %. Tumor growth could be objectified by micro-CT scan before the ITPP treatment was started, and at 3 and 9 months follow-up. Significant progression of tumor volume was demonstrated in the both groups, with no difference between groups (p > 0.05). In the ITPP group, a mild decrease in tumor doubling time was first observed (31.9 +/- 12 days, p > 0.05) followed by a significant increase (59.8 +/- 18.3 days, p = 0.008). However, tumor doubling time was not different between groups (p > 0.05). Median survival after treatment initiation was 223 days (controls) versus 296 days (ITPP group, p = 0.0027). HIF1α, VEGF, glutamine synthase, osteopontin expression levels were not significantly modified at the end of follow-up. In the ITPP group, the p53 expression profile was inversed as compared to the control group, higher in non-tumor livers than in tumors. CONCLUSION: ITPP treatment allowed for a two-month survival improvement, with better tolerance of tumor burden and apoptosis increase in non-tumor, pathological livers.

Retinoic Acid Receptors Control Spermatogonia Cell-Fate and Induce Expression of the SALL4A Transcription Factor.

All-trans retinoic acid (ATRA) is instrumental to male germ cell differentiation, but its mechanism of action remains elusive. To address this question, we have analyzed the phenotypes of mice lacking, in spermatogonia, all rexinoid receptors (RXRA, RXRB and RXRG) or all ATRA receptors (RARA, RARB and RARG). We demonstrate that the combined ablation of RXRA and RXRB in spermatogonia recapitulates the set of defects observed both upon ablation of RAR in spermatogonia. We also show that ATRA activates RAR and RXR bound to a conserved regulatory region to increase expression of the SALL4A transcription factor in spermatogonia. Our results reveal that this major pluripotency gene is a target of ATRA signaling and that RAR/RXR heterodimers are the functional units driving its expression in spermatogonia. They add to the mechanisms through which ATRA promote expression of the KIT tyrosine kinase receptor to trigger a critical step in spermatogonia differentiation. Importantly, they indicate also that meiosis eventually occurs in the absence of a RAR/RXR pathway within germ cells and suggest that instructing this process is either ATRA-independent or requires an ATRA signal originating from Sertoli cells.

Exome sequencing reveals a nonsense mutation in TEX15 causing spermatogenic failure in a Turkish family.

Infertility is a global healthcare problem, and despite long years of assisted reproductive activities, a significant number of cases remain idiopathic. Our currently restricted understanding of basic mechanisms driving human gametogenesis severely limits the improvement of clinical care for infertile patients. Using exome sequencing, we identified a nonsense mutation leading to a premature stop in the TEX15 locus (c.2130T>G, p.Y710*) in a consanguineous Turkish family comprising eight siblings in which three brothers were identified as infertile. TEX15 displays testis-specific expression, maps to chromosome 8, contains four exons and encodes a 2789-amino acid protein with uncertain function. The mutation, which should lead to early translational termination at the first exon of TEX15, co-segregated with the infertility phenotype, and our data strongly suggest that it is the cause of spermatogenic defects in the family. All three affected brothers presented a phenotype reminiscent of the one observed in KO mice. Indeed, previously reported results demonstrated that disruption of the orthologous gene in mice caused a drastic reduction in testis size and meiotic arrest in the first wave of spermatogenesis in males while female KO mice were fertile. The data from our study of one Turkish family suggested that the identified mutation correlates with a decrease in sperm count over time. A diagnostic test identifying the mutation in man could provide an indication of spermatogenic failure and prompt patients to undertake sperm cryopreservation at an early age.

Role of retinoic acid receptor (RAR) signaling in post-natal male germ cell differentiation.

All-trans retinoic acid (atRA), the active metabolite of vitamin A, plays critical functions in spermatogenesis, a complex, highly organized and regulated process comprising three phases. During the proliferative phase, undifferentiated spermatogonia divide to maintain a stem cell population and expand a progenitor cell population, of which a fraction enters the differentiation pathway yielding primary spermatocytes. During the meiotic phase, primary spermatocytes undergo recombination, segregation and reduction by half of chromosomes to produce haploid round spermatids. During the morphogenetic, post-meiotic phase, spermatids differentiate and elongate to ultimately form spermatozoa. Studies performed during the past 20 years have significantly improved our knowledge on the location of the proteins transducing the atRA signal, on the target genes of atRA and on its mechanism of action. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

ONSL and OSKM cocktails act synergistically in reprogramming human somatic cells into induced pluripotent stem cells.

The advent of human induced pluripotent stem cells (hiPSC) is revolutionizing many research fields including cell-replacement therapy, drug screening, physiopathology of specific diseases and more basic research such as embryonic development or diseases modeling. Despite the large number of reports on reprogramming methods, techniques in use remain globally inefficient. We present here a new optimized approach to improve this efficiency. After having tested different monocistronic vectors with poor results, we adopted a polycistronic cassette encoding Thomson's cocktail OCT4, NANOG, SOX2 and LIN28 (ONSL) separated by 2A peptides. This cassette was tested in various vector backbones, based on lentivirus or retrovirus under a LTR or EF1 alpha promoter. This allowed us to show that ONSL-carrier retrovectors reprogrammed adult fibroblast cells with a much higher efficiency (up to 0.6%) than any other tested. We then compared the reprogramming efficiencies of two different polycistronic genes, ONSL and OCT4, SOX2, KLF4 and cMYC (OSKM) placed in the same retrovector backbone. Interestingly, in this context ONSL gene reprograms more efficiently than OSKM but OSKM reprograms faster suggesting that the two cocktails may reprogram through distinct pathways. By equally mixing RV-LTR-ONSL and RV-LTR-OSKM, we indeed observed a remarkable synergy, yielding a reprogramming efficiency of >2%. We present here a drastic improvement of the reprogramming efficiency, which opens doors to the development of automated and high throughput strategies of hiPSC production. Furthermore, non-integrative reprogramming protocols (i.e. mRNA) may take advantage of this synergy to boost their efficiency.

The mammalian-specific Tex19.1 gene plays an essential role in spermatogenesis and placenta-supported development.

STUDY QUESTION: What is the consequence of Tex19.1 gene deletion in mice? SUMMARY ANSWER: The Tex19.1 gene is important in spermatogenesis and placenta-supported development. WHAT IS KNOWN ALREADY: Tex19.1 is expressed in embryonic stem (ES) cells, primordial germ cells (PGCs), placenta and adult gonads. Its invalidation in mice leads to a variable impairment in spermatogenesis and reduction of perinatal survival. STUDY DESIGN, SIZE, DURATION: We generated knock-out mice and ES cells and compared them with wild-type counterparts. The phenotype of the Tex19.1 knock-out mouse line was investigated during embryogenesis, fetal development and placentation as well as during adulthood. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used a mouse model system to generate a mutant mouse line in which the Tex19.1 gene was deleted in the germline. We performed an extensive analysis of Tex19.1-deficient ES cells and assessed their in vivo differentiation potential by generating chimeric mice after injection of the ES cells into wild-type blastocysts. For mutant animals, a morphological characterization was performed for testes and ovaries and placenta. Finally, we characterized semen parameters of mutant animals and performed real-time RT-PCR for expression levels of retrotransposons in mutant testes and ES cells. MAIN RESULTS AND THE ROLE OF CHANCE: While Tex19.1 is not essential in ES cells, our study points out that it is important for spermatogenesis and for placenta-supported development. Furthermore, we observed an overexpression of the class II LTR-retrotransposon MMERVK10C in Tex19.1-deficient ES cells and testes. LIMITATIONS, REASONS FOR CAUTION: The Tex19.1 knock-out phenotype is variable with testis morphology ranging from severely altered (in sterile males) to almost indistinguishable compared with the control counterparts (in fertile males). This variability in the testis phenotype subsequently hampered the molecular analysis of mutant testes. Furthermore, these results were obtained in the mouse, which has a second isoform (i.e. Tex19.2), while other mammals possess only one Tex19 (e.g. in humans). WIDER IMPLICATIONS OF THE FINDINGS: The fact that one gene has a role in both placentation and spermatogenesis might open new ways of studying human pathologies that might link male fertility impairment and placenta-related pregnancy disorders. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM) (Grant Avenir), the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, the Université de Strasbourg, the Association Française contre les Myopathies (AFM) and the Fondation pour la Recherche Médicale (FRM) and Hôpitaux Universitaires de Strasbourg.The authors have nothing to disclose.

Neurons and cardiomyocytes derived from induced pluripotent stem cells as a model for mitochondrial defects in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy. FRDA is due to expanded GAA repeats within the first intron of the gene encoding frataxin, a conserved mitochondrial protein involved in iron-sulphur cluster biosynthesis. This mutation leads to partial gene silencing and substantial reduction of the frataxin level. To overcome limitations of current cellular models of FRDA, we derived induced pluripotent stem cells (iPSCs) from two FRDA patients and successfully differentiated them into neurons and cardiomyocytes, two affected cell types in FRDA. All FRDA iPSC lines displayed expanded GAA alleles prone to high instability and decreased levels of frataxin, but no biochemical phenotype was observed. Interestingly, both FRDA iPSC-derived neurons and cardiomyocytes exhibited signs of impaired mitochondrial function, with decreased mitochondrial membrane potential and progressive mitochondrial degeneration, respectively. Our data show for the first time that FRDA iPSCs and their neuronal and cardiac derivatives represent promising models for the study of mitochondrial damage and GAA expansion instability in FRDA.