A systematic review and evidence assessment of monogenic gene-disease relationships in human female infertility and differences in sex development.

BACKGROUND: As in other domains of medicine, high-throughput sequencing methods have led to the identification of an ever-increasing number of gene variants in the fields of both male and female infertility. The increasing number of recently identified genes allows an accurate diagnosis for previously idiopathic cases of female infertility and more appropriate patient care. However, robust evidence of the gene-disease relationships (GDR) allowing the proper translation to clinical application is still missing in many cases. OBJECTIVE AND RATIONALE: An evidence-based curation of currently identified genes involved in female infertility and differences in sex development (DSD) would significantly improve both diagnostic performance and genetic research. We therefore performed a systematic review to summarize current knowledge and assess the available GDR. SEARCH METHODS: PRISMA guidelines were applied to curate all available information from PubMed and Web of Science on genetics of human female infertility and DSD leading to infertility, from 1 January 1988 to 1 November 2021. The reviewed pathologies include non-syndromic as well as syndromic female infertility, and endocrine and reproductive system disorders. The evidence that an identified phenotype is caused by pathogenic variants in a specific gene was assessed according to a standardized scoring system. A final score (no evidence, limited, moderate, strong, or definitive) was assigned to every GDR. OUTCOMES: A total of 45 271 publications were identified and screened for inclusion of which 1078 were selected for gene and variant extraction. We have identified 395 genes and validated 466 GDRs covering all reported monogenic causes of female infertility and DSD. Furthermore, we present a genetic diagnostic flowchart including 105 genes with at least moderate evidence for female infertility and suggest recommendations for future research. The study did not take into account associated genetic risk factor(s) or oligogenic/polygenic causes of female infertility. WIDER IMPLICATIONS: We have comprehensively reviewed the existing research on the genetics of female infertility and DSD, which will enable the development of diagnostic panels using validated genes. Whole genome analysis is shifting from predominantly research to clinical application, increasing its diagnostic potential. These new diagnostic possibilities will not only decrease the number of idiopathic cases but will also render genetic counselling more effective for infertile patients and their families.

Improving preimplantation genetic diagnosis for Fragile X syndrome: two new powerful single-round multiplex indirect and direct tests.

Fragile X syndrome (FraX) is caused by the expansion of an unstable CGG repeat located in the Fragile X mental retardation 1 gene (FMR1) gene. Preimplantation genetic diagnosis (PGD) can be proposed to couples at risk of transmitting the disease, that is, when the female carries a premutation or a full mutation. We describe two new single-cell, single-round multiplex PCR for indirect and direct diagnosis of FraX on biopsied embryos. These tests include five unpublished, highly heterozygous simple sequence repeats, and the co-amplification of non-expanded CGG repeats for the direct test. Heterozygosity of the new markers ranged from 69 to 81%. The mean rate of non-informative marker included in the tests was low (26% and 23% for the new indirect and direct tests, respectively). This strategy allows offering a PGD for FraX to 96% of couples requesting it in our centre. A conclusive genotype was obtained in all cells with a rate of cells presenting an allele dropout ranging from 17% for the indirect test to 26% for the direct test. The new indirect test was applied for eight PGD cycles: 32 embryos were analysed, 9 were transferred and 3 healthy babies were born. By multiplexing these highly informative markers, robustness of the diagnosis is improved and the loss of potentially healthy embryos (because they are non-diagnosed or misdiagnosed) is limited. This may increase the chances of success of couples requesting a PGD for FraX, in particular, when premature ovarian insufficiency in premutated women leads to a reduced number of embryos available for analysis.

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.

Tex 19 paralogs exhibit a gonad and placenta-specific expression in the mouse.

We have previously suggested that TEX19, a mammalian-specific protein of which two paralogs exist in rodents, could be implicated in stem cell self-renewal and pluripotency. We have established here the expression profiles of Tex19.1 and Tex19.2 during mouse development and adulthood. We show that both genes are coexpressed in the ectoderm and then in primordial germ cells (PGCs). They are also coexpressed in the testis from embryonic day 13.5 to adulthood, whereas only Tex19.1 transcripts are detected in the developing and adult ovary as well as in the placenta and its precursor tissue, the ectoplacental cone. The presence of both Tex19.1 and Tex19.2 in PGCs, gonocytes and spermatocytes opens the possibility that these two genes could play redundant functions in male germ cells. Furthermore, the placental expression of Tex19.1 can explain why Tex19.1 knockout mice show embryonic lethality, in addition to testis defects.

[An overview of pluripotent stem cell lines]. / Tour d'horizon des lignées de cellules souches pluripotentes.

More than 20 years ago, the finding of a population of cells with the ability of self-renewal and differentiation inside teratocarcinomas (embryonic carcinoma cells) would allow their direct derivation from preimplantation embryos (embryonic stem cells, ESC). The phenomenal pluripotency properties of those cells and the therapeutical potential of their human counterparts triggered a massive interest from the scientific community. The research on the field of pluripotent stem cells improved a lot and many ES-like pluripotent stem cells of several embryonic and adult sources were described. Next step has been the reprogramming of terminally differentiated cells into embryonic cells, with the aim to produce patient-specific stem cells. The recent breakthrough has been the in vitro reprogramming of adult cells into ES cell-like cells named induced pluripotent stem cells (iPSC), using four transcription factors (Oct4, Sox2, Klf4, c-Myc). Even though some challenges remain, we are now one step closer to the eventuality to use these cells for clinical purposes. In this review we propose to analyse the several pluripotent stem cells existing today.

"OpenLAB": A 2-hour PCR-based practical for high school students.

The Strasbourg University PhD school in Life and Health Sciences launched an initiative called "OpenLAB." This project was developed in an effort to help high school teenagers understand theoretical and abstract concepts in genetics. A second objective of this program is to help students in defining their future orientation and to attract them to biology. The general idea is a 2-hour PCR-based practical that is developed around a fictitious criminal investigation. The practical is taught by PhD graduate students who bring all the required reagents and modern equipment into the classroom. Running the PCR provides free time dedicated to discussions with students about their future plans after the high school diploma. A specific website and a powerpoint presentation were developed to provide appropriate scientific information. Starting on a modest scale in Strasbourg in December 2008, "OpenLAB" was rapidly and well received all around, visiting 53 classes spread over a 200 km area in Alsace until May 2009. It permitted interactions with almost one thousand students in their last year of high school, with the prospect to visit 20% more classes this school year. Our experience, along with feedback from students and their teachers, suggests that it is possible to reach out to many students and have a strong impact with a rather limited budget.

Highly dynamic and sex-specific expression of microRNAs during early ES cell differentiation.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the mammalian blastocyst. Cellular differentiation entails loss of pluripotency and gain of lineage-specific characteristics. However, the molecular controls that govern the differentiation process remain poorly understood. We have characterized small RNA expression profiles in differentiating ES cells as a model for early mammalian development. High-throughput 454 pyro-sequencing was performed on 19-30 nt RNAs isolated from undifferentiated male and female ES cells, as well as day 2 and 5 differentiating derivatives. A discrete subset of microRNAs (miRNAs) largely dominated the small RNA repertoire, and the dynamics of their accumulation could be readily used to discriminate pluripotency from early differentiation events. Unsupervised partitioning around meloids (PAM) analysis revealed that differentiating ES cell miRNAs can be divided into three expression clusters with highly contrasted accumulation patterns. PAM analysis afforded an unprecedented level of definition in the temporal fluctuations of individual members of several miRNA genomic clusters. Notably, this unravelled highly complex post-transcriptional regulations of the key pluripotency miR-290 locus, and helped identify miR-293 as a clear outlier within this cluster. Accordingly, the miR-293 seed sequence and its predicted cellular targets differed drastically from those of the other abundant cluster members, suggesting that previous conclusions drawn from whole miR-290 over-expression need to be reconsidered. Our analysis in ES cells also uncovered a striking male-specific enrichment of the miR-302 family, which share the same seed sequence with most miR-290 family members. Accordingly, a miR-302 representative was strongly enriched in embryonic germ cells derived from primordial germ cells of male but not female mouse embryos. Identifying the chromatin remodelling and E2F-dependent transcription repressors Ari4a and Arid4b as additional targets of miR-302 and miR-290 supports and possibly expands a model integrating possible overlapping functions of the two miRNA families in mouse cell totipotency during early development. This study demonstrates that small RNA sampling throughout early ES cell differentiation enables the definition of statistically significant expression patterns for most cellular miRNAs. We have further shown that the transience of some of these miRNA patterns provides highly discriminative markers of particular ES cell states during their differentiation, an approach that might be broadly applicable to the study of early mammalian development.

The pluripotency-associated gene Dppa4 is dispensable for embryonic stem cell identity and germ cell development but essential for embryogenesis.

Dppa4 (developmental pluripotency-associated 4) has been identified in several high-profile screens as a gene that is expressed exclusively in pluripotent cells. It encodes a nuclear protein with an SAP-like domain and appears to be associated preferentially with transcriptionally active chromatin. Its exquisite expression pattern and results of RNA interference experiments have led to speculation that Dppa4, as well as its nearby homolog Dppa2, might play essential roles in embryonic stem (ES) cell function and/or germ cell development. To rigorously assess suggested roles, we have generated Dppa4-deficient and Dppa4/Dppa2 doubly deficient ES cells, as well as mice lacking Dppa4. Contrary to predictions, we find that Dppa4 is completely dispensable for ES cell identity and germ cell development. Instead, loss of Dppa4 in mice results in late embryonic/perinatal death and striking skeletal defects with partial penetrance. Thus, surprisingly, Dppa4-deficiency affects tissues that apparently never transcribed the gene, and at least some loss-of-function defects manifest phenotypically at an embryonic stage long after physiologic Dppa4 expression has ceased. Concomitant with targeted gene inactivation, we have introduced into the Dppa4 locus a red fluorescent marker (tandem-dimer red fluorescent protein) that is compatible with green fluorescent proteins and allows noninvasive visualization of pluripotent cells and reprogramming events.

Nuclear proteome analysis of undifferentiated mouse embryonic stem and germ cells.

Embryonic stem cells (ESCs) and embryonic germ cells (EGCs) provide exciting models for understanding the underlying mechanisms that make a cell pluripotent. Indeed, such understanding would enable dedifferentiation and reprogrammation of any cell type from a patient needing a cell therapy treatment. Proteome analysis has emerged as an important technology for deciphering these biological processes and thereby ESC and EGC proteomes are increasingly studied. Nevertheless, their nuclear proteomes have only been poorly investigated up to now. In order to investigate signaling pathways potentially involved in pluripotency, proteomic analyses have been performed on mouse ESC and EGC nuclear proteins. Nuclei from ESCs and EGCs at undifferentiated stage were purified by subcellular fractionation. After 2-D separation, a subtractive strategy (subtracting culture environment contaminating spots) was applied and a comparison of ESC, (8.5 day post coïtum (dpc))-EGC and (11.5 dpc)-EGC specific nuclear proteomes was performed. A total of 33 ESC, 53 (8.5 dpc)-EGC, and 36 (11.5 dpc)-EGC spots were identified by MALDI-TOF-MS and/or nano-LC-MS/MS. This approach led to the identification of two isoforms (with and without N-terminal acetylation) of a known pluripotency marker, namely developmental pluripotency associated 5 (DPPA5), which has never been identified before in 2-D gel-MS studies of ESCs and EGCs. Furthermore, we demonstrated the efficiency of our subtracting strategy, in association with a nuclear subfractionation by the identification of a new protein (protein arginine N-methyltransferase 7; PRMT7) behaving as proteins involved in pluripotency.

Tex19, a mammalian-specific protein with a restricted expression in pluripotent stem cells and germ line.

Although the properties of embryonic stem (ES) cells make these cells very attractive in the field of replacement therapy, the molecular mechanisms involved in the maintenance of their pluripotency are not fully characterized. Starting from the observation that most pluripotent markers are also expressed by spermatogonia stem cells, we identified Tex19 as a new potential pluripotency marker. We show that Tex19 is a mammalian-specific protein duplicated in mouse and rat, renamed Tex19.1 and Tex19.2, whereas only one form is found in human. In mouse, both forms are localized on chromosome 11 and transcribed in opposite directions. Tex19 proteins are well conserved, showing two highly conserved domains that do not present any similarity with any other known domains. We show that Tex19.2 is specifically detected in the male somatic gonad lineage, whereas Tex19.1 expression is very similar to that of Oct4. Transcripts are maternally inherited, and expression starts as soon as the early embryo and later is limited to the germ line. Tex19.1 transcripts were also detected in mouse pluripotent stem cells, and expression of Tex19.1, like that of Oct4, decreases after murine embryonic stem and germ cell differentiation. Human TEX19 was more closely related to murine Tex19.1 and was also detected in adult testis and in undifferentiated ES cells. By immunofluorescence, we found that Tex19.1 protein localizes to the nucleus of mouse ES and inner cell mass cells. All these results suggest that Tex19.1, as well as human TEX19, could be a new factor involved in the maintenance of self-renewal or pluripotency of stem cells.