[Preimplantation genetic testing]. / Gynécologie-obstétrique: Tests préimplantatoires de l'embryonen médecine de reproduction.

Preimplantation genetic testing avoids the transmission of monogenic diseases or structural chromosome abnormality to the offspring in fertile couples. Furthermore, it allows screening for aneuploidies (PGT-A, Preimplantation genetic testing for aneuploidy), with the aim of selecting one euploid embryo before transfer in infertile couples undergoing in vitro fertilization (IVF). Indeed, aneuploidies are frequent and explain most IVF failures and early miscarriages. The indications for PGT-A remain controversial, due to the lack of clear evidence of improved outcomes after IVF. Cost-effectiveness studies and follow-up of neonatal outcomes are needed. Finally, each situation requires counseling taking into account ethical considerations.

Les tests préimplantatoires permettent à un couple fertile d'éviter la transmission d'une maladie monogénique ou d'une anomalie chromosomique structurelle à sa descendance. Mais ils peuvent également dépister des aneuploïdies (PGT-A, Preimplantation genetic testing for aneuploidy), avec pour but la sélection d'un embryon euploïde avant transfert in utero pour les couples infertiles réalisant une fécondation in vitro (FIV). En effet, les aneuploïdies, très fréquentes, sont à l'origine de la majorité des échecs d'implantation après FIV et des avortements spontanés précoces. Les indications du PGT-A restent néanmoins controversées en l'absence de preuve évidente d'une amélioration des résultats en FIV. Des études coût/efficacité et un suivi des issues néonatales sont nécessaires. Enfin, chaque situation nécessite un counseling en intégrant les aspects éthiques.

Mutations in the polyglutamylase gene TTLL5, expressed in photoreceptor cells and spermatozoa, are associated with cone-rod degeneration and reduced male fertility.

Hereditary retinal degenerations encompass a group of genetic diseases characterized by extreme clinical variability. Following next-generation sequencing and autozygome-based screening of patients presenting with a peculiar, recessive form of cone-dominated retinopathy, we identified five homozygous variants [p.(Asp594fs), p.(Gln117*), p.(Met712fs), p.(Ile756Phe), and p.(Glu543Lys)] in the polyglutamylase-encoding gene TTLL5, in eight patients from six families. The two male patients carrying truncating TTLL5 variants also displayed a substantial reduction in sperm motility and infertility, whereas those carrying missense changes were fertile. Defects in this polyglutamylase in humans have recently been associated with cone photoreceptor dystrophy, while mouse models carrying truncating mutations in the same gene also display reduced fertility in male animals. We examined the expression levels of TTLL5 in various human tissues and determined that this gene has multiple viable isoforms, being highly expressed in testis and retina. In addition, antibodies against TTLL5 stained the basal body of photoreceptor cells in rat and the centrosome of the spermatozoon flagellum in humans, suggesting a common mechanism of action in these two cell types. Taken together, our data indicate that mutations in TTLL5 delineate a novel, allele-specific syndrome causing defects in two as yet pathogenically unrelated functions, reproduction and vision.

Focus on intracytoplasmic morphologically selected sperm injection (IMSI): a mini-review.

Intracytoplasmic sperm injection (ICSI) is the recommended treatment in many cases of male-factor infertility. Several studies have demonstrated a positive correlation between optimal sperm morphology and positive ICSI outcomes. In fact, spermatozoa with severe abnormalities of the head are well documented to be associated with low fertilisation, implantation and pregnancy rates. However, a spermatozoon which is classified as 'normal' by microscopic observation at low magnification could contain ultrastructural defects that impair both the fertilisation process and embryonic development. The intracytoplasmic morphologically selected sperm injection (IMSI) procedure changed the perception of how a spermatozoon suitable for injection should appear. Sperm selection is carried out at ×6000 magnification, allowing improved assessment of the sperm nucleus. Currently, standardized clinical indications for IMSI are lacking and the candidates are selected on the grounds of their medical history or of a careful analysis of the sperm suspension. Further prospective randomized studies are needed to confirm the advantages of IMSI in specific groups of patients. In addition to providing a brief overview of the IMSI procedure, this study aims to review the literature, which explains the theoretical basis and the clinical outcomes of this technique. Several reports show that IMSI is associated with improved implantation and clinical pregnancy rates as well as lower abortion rates when compared to ICSI. Although a possible correlation between the sperm's abnormal nucleus shape, increased DNA fragmentation and negative laboratory and clinical outcomes has been long investigated, the results are conflicting.

Clinical outcome after IMSI procedure in an unselected infertile population: a pilot study.

BACKGROUND: To date the IMSI procedure represents the only real-time and unstained method available to discard spermatozoa with ultrastructural defects. Several studies demonstrated that IMSI provides positive results in couples with severe male factor infertility or repeated ICSI failures. Aim of this pilot study is to evaluate the differences between IMSI and ICSI in terms of IVF outcomes in an unselected infertile patient population. METHODS: Three hundred and thirty-two couples were analyzed: 281 couples underwent conventional ICSI procedure and 51 underwent IMSI technique. RESULTS: No statistically significant differences were found between implantation rate (ICSI: 16,83%; IMSI: 16,67%), fertilization rate (ICSI: 77,27%; IMSI: 80,00%) and pregnancy rate (ICSI: 25,30%; IMSI: 23,50%). Both groups were comparable when considering live birth rate (ICSI: 11,39%; IMSI:13,72%), ongoing pregnancy rate (ICSI: 7,47%; IMSI: 5,88%) and miscarriage rate (ICSI: 17,78; IMSI: 5,26%). The subgroup analyses did not show a statistical difference between ICSI and IMSI neither in male factor infertility subgroup nor in patients with more than one previous ICSI attempt. A trend towards better laboratory and clinical outcomes was detected in the male factor infertility subgroup when IMSI was applied. CONCLUSIONS: Our preliminary results show that the IMSI technique does not significantly improve IVF outcomes in an unselected infertile population.

The tyrosinase enhancer is activated by Sox10 and Mitf in mouse melanocytes.

The terminal differentiation of melanocytes is associated with the transcriptional activation of genes responsible for pigment production such as tyrosinase. Pigment cell-specific transcription factors, such as Mitf, as well as specific proximal and distal regulatory elements (DRE) are implicated in the tight control of tyrosinase expression during development and adulthood. Proper tyrosinase expression in melanocytes depends upon the presence of a DRE that is located at -15 kb and provides enhancer activity via a central element termed core-enhancer. In this report, we show that the transcription factors Sox10, Mitf and USF-1 are able to activate the core-enhancer in luciferase reporter assays. Comparative sequence analysis identified evolutionarily motifs resembling Sox10 binding sites that were required for full enhancer activity in melanoma cells and in tyrosinase::lacZ transgenic mice. Sox10 was able to bind the DRE in vitro and mutation of the conserved motifs abolished the enhancer transactivation mediated by Sox10. In addition, two highly conserved CAGCTG E-box motifs were identified that were also required for enhancer activity and for transactivation by Mitf. The results suggest that Sox10 directly, and Mitf, most likely indirectly, activate the tyrosinase enhancer, underlining the contribution of Sox10 to tyrosinase gene regulation in melanocytes.

Distinct distal regulatory elements control tyrosinase expression in melanocytes and the retinal pigment epithelium.

Pigment cells of mammals are characterized by two different developmental origins: cells of the retinal pigment epithelium (RPE) originate from the optic cup of the developing forebrain, whereas melanocytes arise from the neural crest. The pigmentation gene tyrosinase is expressed in all pigment cells but differentially regulated in melanocytes and RPE. The tyrosinase promoter does not confer strong expression in pigment cells in vivo, while inclusion of a distal regulatory element at position -15 kb is necessary and sufficient to provide strong expression in melanocytes. Nevertheless, the regulatory elements responsible for correct spatial and temporal tyrosinase expression in the RPE remained unidentified so far. In this report, we show that a 186 kb BAC containing the tyrosinase gene provides transgene expression in both RPE and melanocytes indicating the presence of regulatory sequences required for expression in the RPE. A deletion analysis of the BAC was performed demonstrating that a RPE-regulatory element resides between -17 and -75 kb. Using multi-species comparative genomic analysis we identified three conserved sequences within this region. When tested in transgenic mice one of these sequences located at -47 kb targeted expression to the RPE. In addition, deletion of this regulatory element within a tyrosinase::lacZ BAC provided evidence that this sequence is not only sufficient but also required for correct spatial and temporal expression in the RPE. The identification of this novel element demonstrates that tyrosinase gene expression is controlled by separate distal regulatory sequences in melanocytes and RPE.

A conserved transcriptional enhancer that specifies Tyrp1 expression to melanocytes.

Pigment cells of mammals originate from two different lineages: melanocytes arise from the neural crest, whereas cells of the retinal pigment epithelium (RPE) originate from the optic cup of the developing forebrain. Previous studies have suggested that pigmentation genes are controlled by different regulatory networks in melanocytes and RPE. The promoter of the tyrosinase-related family gene Tyrp1 has been shown to drive detectable transgene expression only to the RPE, even though the gene is also expressed in melanocytes as evident from Tyrp1-mutant mice. This indicates that the regulatory elements responsible for Tyrp1 gene expression in the RPE are not sufficient for expression in melanocytes. We thus searched for a putative melanocyte-specific regulatory sequence and demonstrate that a bacterial artificial chromosome (BAC) containing the Tyrp1 gene and surrounding sequences is able to target transgenic expression to melanocytes and to rescue the Tyrp1b (brown) phenotype. This BAC contains several highly conserved non-coding sequences that might represent novel regulatory elements. We further focused on a sequence located at -15 kb, which we identified as a melanocyte-specific enhancer as shown by cell culture and transgenic mice experiments. In addition, we show that the transcription factor Sox10 can activate this conserved enhancer. The presence of a distal Tyrp1 regulatory element, which specifies melanocyte-specific expression, supports the idea that separate regulatory sequences can mediate differential gene expression in melanocytes and RPE.

Identification of evolutionarily conserved regulatory elements in the mouse Fgf8 locus.

The secreted signaling molecule fibroblast growth factor 8 (Fgf8) is an essential component of certain embryonic signaling centers including the mid-hindbrain (isthmic) organizer, the first branchial arch (BA1), and the apical ectodermal ridge (AER). In these signaling centers Fgf8 transcripts are expressed in a dynamic and transient fashion, but the mechanism by which this highly specific expression pattern is established remains largely unknown. We used DNA sequence comparisons coupled to transgenic approaches to obtain insight into the structure and function of regulatory elements in the Fgf8 locus. First, a bacterial artificial chromosome (BAC) containing the mouse Fgf8 gene partially rescues the embryonic lethality of Fgf8-deficient mice and controls Fgf8-specific gene expression of a coinjected lacZ reporter transgene. Second, sequence comparison of vertebrate Fgf8 loci revealed evolutionarily highly conserved noncoding sequences that were unexpectedly located mainly 3' of the Fgf8 coding region. Third, in transgenic mice some of these elements were sufficient to target expression to the AER, tail bud, and brain, including the isthmic organizer, indicating that they may represent Fgf8 cis-acting elements. Collectively, these data identify novel regulatory elements of the Fgf8 gene sufficient to drive expression to regions of known Fgf8 activity.

Melanocytes and pigmentation are affected in dopachrome tautomerase knockout mice.

The tyrosinase family comprises three members, tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), and dopachrome tautomerase (Dct). Null mutations and deletions at the Tyr and Tyrp1 loci are known and phenotypically affect coat color due to the absence of enzyme or intracellular mislocalization. At the Dct locus, three mutations are known that lead to pigmentation phenotype. However, these mutations are not null mutations, and we therefore set out to generate a null allele at the Dct gene locus by removing exon 1 of the mouse Dct gene. Mice deficient in Dct [Dct(tm1(Cre)Bee)] lack Dct mRNA and dopachrome tautomerase protein. They are viable and do not show any abnormalities in Dct-expressing sites such as skin, retinal pigment epithelium, or brain. However, the mice show a diluted coat color phenotype, which is due to reduced melanin content in hair. Primary melanocytes from Dct knockout mice are viable in culture and show a normal distribution of tyrosinase and tyrosinase-related protein 1. In comparison to the knockout, the slaty mutation (Dct(slt)/Dct(slt)) has less melanin and affects growth of primary melanocytes severely. In summary, we have generated a knockout of the Dct gene in mice with effects restricted to pigment production and coat color.