Genetic landscape of a large cohort of Primary Ovarian Insufficiency: New genes and pathways and implications for personalized medicine.

BACKGROUND: Primary Ovarian Insufficiency (POI), a public health problem, affects 1-3.7% of women under 40 yielding infertility and a shorter lifespan. Most causes are unknown. Recently, genetic causes were identified, mostly in single families. We studied an unprecedented large cohort of POI to unravel its molecular pathophysiology. METHODS: 375 patients with 70 families were studied using targeted (88 genes) or whole exome sequencing with pathogenic/likely-pathogenic variant selection. Mitomycin-induced chromosome breakages were studied in patients' lymphocytes if necessary. FINDINGS: A high-yield of 29.3% supports a clinical genetic diagnosis of POI. In addition, we found strong evidence of pathogenicity for nine genes not previously related to a Mendelian phenotype or POI: ELAVL2, NLRP11, CENPE, SPATA33, CCDC150, CCDC185, including DNA repair genes: C17orf53(HROB), HELQ, SWI5 yielding high chromosomal fragility. We confirmed the causal role of BRCA2, FANCM, BNC1, ERCC6, MSH4, BMPR1A, BMPR1B, BMPR2, ESR2, CAV1, SPIDR, RCBTB1 and ATG7 previously reported in isolated patients/families. In 8.5% of cases, POI is the only symptom of a multi-organ genetic disease. New pathways were identified: NF-kB, post-translational regulation, and mitophagy (mitochondrial autophagy), providing future therapeutic targets. Three new genes have been shown to affect the age of natural menopause supporting a genetic link. INTERPRETATION: We have developed high-performance genetic diagnostic of POI, dissecting the molecular pathogenesis of POI and enabling personalized medicine to i) prevent/cure comorbidities for tumour/cancer susceptibility genes that could affect life-expectancy (37.4% of cases), or for genetically-revealed syndromic POI (8.5% of cases), ii) predict residual ovarian reserve (60.5% of cases). Genetic diagnosis could help to identify patients who may benefit from the promising in vitro activation-IVA technique in the near future, greatly improving its success in treating infertility. FUNDING: Université Paris Saclay, Agence Nationale de Biomédecine.

Use of solid-phase microextraction coupled with gas chromatography for the determination of residual solvents in pharmaceutical products.

The aim of this work was to prove that solid-phase microextraction coupled with gas chromatography could be used for the determination and quantification of residual solvents in drugs. Four solvents were selected for the experiments: ethanol, cyclohexane, triethylamine and pyridine, together with a model powdered drug substance. Several kinds of fibers, together with the extraction mode, were evaluated to determine the most appropriate one for the simultaneous extraction of the four solvents. The most promising conditions were obtained with the Carboxen-polydimethylsiloxane fiber in the headspace of the aqueous solution that contained the dissolved powder. A concentrated phosphate buffer was added to the aqueous solution to set the pH at 9.6 in order to enable good extraction of triethylamine, and the optimum extraction time was experimentally determined. A multi-criteria optimization was also carried out by means of design of experiments to optimize remaining parameters: the extraction temperature was set at 40 degrees C, the ionic strength at 1.77 mol (l-1) and the volume of the aqueous solution at 7.2 ml. The method of standard additions was used for quantitative analysis. Its performance was evaluated and validated: the pooled RSD was around 15%, the limits of detection were all of the ppb level and the method was both accurate and linear.