APOPTOSIS RATE IN CUMULUS CELLS AS POSSIBLE MOLECULAR BIOMARKER FOR OOCYTE COMPETENCE.

Several lines of evidence showed that apoptosis rate of cumulus cells in oocytes derived by assisted reproductive technologies could be used as an indicator of fertilizing gamete quality. Aim of the study was to investigate the effects of three different ovarian stimulation protocols on the biological and clinical outcome in hyporesponder patients. Collected data showed a higher significant rate of DNA fragmentation index (DFI) in U group (patients treated with Highly Purified human Menopausal Gonadotrophin) than in P group (treated with recombinant human Follicle Stimulating Hormone (r-hFSH) combined with recombinant human Luteinizing Hormone (r-hLH)). Both groups R (treated with r-hFSH alone) and P showed a significant increase in collected and fertilized oocytes number, embryo quality number. This study showed that combined r-hFSH/r-hLH therapy could represent the best pharmacological strategy for controlled ovarian stimulation and suggests to use DFI as a biomarker of ovarian function in hyporesponder patients

Analytical identification of additional impurities in urinary-derived gonadotrophins

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Comparative Assessment of the Structural Features of Originator Recombinant Human Follitropin Alfa Versus Recombinant Human Follitropin Alfa Biosimilar Preparations Approved in Non-European Regions

Although the full primary structures of the alfa and beta subunits of reference r-hFSH-alfa and its biosimilars are identical, cell context-dependent differences in the expressing cell lines and manufacturing process can lead to variations in glycosylation profiles. In the present study, we compared the structural features of reference r-hFSH-alfa with those of five biosimilar preparations approved in different global regions outside Europe (Primapur®, Jin Sai Heng®, Follitrope®, Folisurge®, and Corneumon®) with respect to glycosylation, macro- and microheterogeneity, and other post-translational modifications and higher order structure. The mean proportion of N-glycosylation-site occupancy was highest in reference r-hFSH-alfa, decreasing sequentially in Primapur, Jin Sai Heng, Corneumon, Follisurge and Follitrope, respectively. The level of antennarity showed slightly higher complexity in Corneumon, Primapur and Follitrope versus reference r-hFSH-alfa, whereas Jin Sai Heng and Folisurge were aligned with reference r-hFSH-alfa across all N-glycosylation sites. Sialylation level was higher in Corneumon and Follitrope, but small differences were detected in other biosimilar preparations compared with reference r-hFSH-alfa. Jin Sai Heng showed higher levels of N-glyconeuramic acid than the other preparations. Minor differences in oxidation levels were seen among the different products. Therefore, in summary, we identified var ious differences in N-glycosylation occupancy, antennarity, sialylation and oxidation between reference r-hFSH-alfa and the biosimilar preparations analyzed

Exploring the human chorionic gonadotropin induced steroid secretion profile of mouse Leydig tumor cell line 1 by a 20 steroid LC-MS/MS panel

The canonical androgen synthesis in Leydig cells involves Delta 5 and Delta 4 steroids. Besides, the backdoor pathway, eompassing 5 alpha and 5 alpha,3 alpha steroids, is gaining interest in fetal and adult pathophysiology. Moreover, the role of androgen epimers and progesterone metabolites is still unknown. We developed a liquid chromatographytandem mass spectrometry (LC-MS/MS) method for measuring 20 steroids and used it to investigate the steroid secretion induced by human chorionic gonadotropin (hCG) in the mouse Leydig tumor cell line 1 (mLTC1). Steroids were extracted from 500 mu L supernatants from unstimulated or 100 pM hCG-exposed mLTC1 cells, separated on a Luna C8 100 x 3 mm, 3 mu m column, with 100 mu M NH4F and methanol as mobile phases, and analyzed by positive electrospray ionization and multiple reaction monitoring. Sensitivity ranged within 0.012-38.0 nmol/L. Intra-assay and inter-assay imprecision were < 9.1% and 10.0%, respectively. Trueness, recovery and matrix factor were within 93.4-122.0, 55.6-104.1 and 76.4-106.3%, respectively. Levels of 16OH-progesterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, 17OH-progesterone, androstenedione, epitestosterone, dihydrotestosterone, progesterone, androsterone and 17OH-allopregnanolone were effectively measured. Traces of 17OH-dihydroprogesterone, androstanediol and dihydroprogesterone were found, whereas androstenediol, 17OH-pregnenolone, dehydroepiandrosterone, pregnenolone and allopregnanolone showed no peak. hCG induced an increase of 80.2-102.5 folds in 16OH-progesterone, androstenedione and testosterone, 16.6 in dihydrotestosterone, 12.2-27.5 in epitestosterone, progesterone and metabolites, 8.1 in 17OH-allopregnanolone and <= 3.3 in 5 alpha and 5 alpha,3 alpha steroids

Mobility trends of Patients across Italian Regions: implications for planning and evaluation of hospital services

The study of patients' mobility is useful for health planning and identifying deficiencies in care supply. The Italian Health Service, with 21 different regional realities, can be considered as a macrocosmic test bench. Our study aims: (1) to describe the trend of patients' hospital mobility across the Italian Regions; (2) to offer an immediate visual approach for decision making; (3) to identify some factors involved in patient's mobility

Myoimaging in the NGS era: The discovery of a novel mutation in MYH7 in a family with distal myopathy and core-like features -a case report

Background: Myosin heavy chain 7 related myopathies are rare disorders characterized by a wide phenotypic spectrum and heterogeneous pathological features. In the present study, we performed clinical, morphological, genetic and imaging investigations in three relatives affected by autosomal dominant distal myopathy. Whilst earlier traditional Sanger investigations had pointed to the wrong gene as disease causative, next-generation sequencing allowed us to obtain the definitive molecular genetic diagnosis in the family.Case presentation: The proposita, being found to harbor a novel heterozygous mutation in the RYR1 gene (p. Glu294Lys), was initially diagnosed with core myopathy. Subsequently, consideration of muscle magnetic resonance imaging (MRI) features and extension of family study led this diagnosis to be questioned. Use of next-generation sequencing analysis identified a novel mutation in the MYH7gene (p.Ser1435Pro) that segregated in the affected family members.Conclusions: This study identified a novel mutation in MYH7 in a family where the conclusive molecular diagnosis was reached through a complicated path. This case report might raise awareness, among clinicians, of the need to interpret NGS data in combination with muscle MRI patterns so as to facilitate the pinpointing of the main molecular etiology in inherited muscle disorders

LH prevents cisplatin-induced apoptosis in oocytes and preserves female fertility in mouse

Premature ovarian failure and female infertility are frequent side effects of anticancer therapies, owing to the extreme sensitivity of the ovarian reserve oocytes to the damaging effects of irradiation and chemotherapy on DNA. We report here a robust protective effect of luteinizing hormone (LH) on the primordial follicle pool of prepubertal ovaries against the cisplatin (Cs)-induced apoptosis. In vitro LH treatment of prepubertal ovarian fragments generated anti-apoptotic signals by a subset of ovarian somatic cells expressing LH receptor (LHR) through cAMP/PKA and Akt pathways. Such signals, reducing the oocyte level of pro-apoptotic TAp63 protein and favoring the repair of the Cs-damaged DNA in the oocytes, prevented their apoptosis. Noteworthy, in vivo administration to prepubertal female mice of a single dose of LH together with Cs inhibited the depletion of the primordial follicle reserve caused by the drug and preserved their fertility in reproductive age, preventing significant alteration in the number of pregnancy and of delivered pups. In conclusion, these findings establish a novel ovoprotective role for LH and further support the very attracting prospective to use physiological 'fertoprotective' approaches for preventing premature infertility and risks linked to precocious menopause in young patients who survived cancer after chemotherapy

Longitudinal neurophysiological assessment of intramuscular type-A botulin toxin in healthy humans

The aim of this study is to assess the neurophysiological abnormalities of type A botulin toxin-infiltrated human muscle, and their evolution over time. Seried cMAP measurements, 3 and 20 Hz repetitive nerve stimulation, EMG, SFEMG over 3 months from toxin injection. Our findings consist in lack of decrement with 3 Hz repetitive nerve stimulation and facilitation with 20 Hz repetitive nerve stimulation; progressive increasing of jitter; early appearance of fibrillations; small and short motor unit action potential in the first 3 weeks, followed by increasing of MUAP amplitude and duration, with polyphasic morphology. Although claimed as highly specific and sensible, neuromuscular junction facilitation is an inconstant finding in human botulism. Therefore, lack of neuromuscular junction facilitation cannot exclude a diagnosis of botulism. Our findings are compatible with a process of acute denervation followed by distal reinnervation, favored by terminal nerve sprouting