The Energy Dispersive X-ray (EDX) microanalysis is a technique of elemental analysis associated to electron microscopy based on the generation of characteristic Xrays that reveals the presence of elements present in the specimens. The EDX microanalysis is used in different biomedical fields by many researchers and clinicians. Nevertheless, most of the scientific community is not fully aware of its possible applications. The spectrum of EDX microanalysis contains both semi-qualitative and semi-quantitative information. EDX technique is made useful in the study of drugs, such as in the study of drugs delivery in which the EDX is an important tool to detect nanoparticles (generally, used to improve the therapeutic performance of some chemotherapeutic agents). EDX is also used in the study of environmental pollution and in the characterization of mineral bioaccumulated in the tissues. In conclusion, the EDX can be considered as a useful tool in all works that require element determination, endogenous or exogenous, in the tissue, cell or any other sample.

STUDY QUESTION Does LH protect mouse oocytes and female fertility from alkylating chemotherapy? SUMMARY ANSWER LH treatment before and during chemotherapy prevents detrimental effects on follicles and reproductive lifespan. WHAT IS KNOWN ALREADY Chemotherapies can damage the ovary, resulting in premature ovarian failure and reduced fertility in cancer survivors. LH was recently suggested to protect prepubertal mouse follicles from chemotoxic effects of cisplatin treatment. STUDY DESIGN, SIZE, DURATION This experimental study investigated LH effects on primordial follicles exposed to chemotherapy. Seven-week-old CD-1 female mice were randomly allocated to four experimental groups: Control (n = 13), chemotherapy (ChT, n = 15), ChT+LH-1x (n = 15), and ChT+LH-5x (n = 8). To induce primary ovarian insufficiency (POI), animals in the ChT and ChT+LH groups were intraperitoneally injected with 120 mg/kg of cyclophosphamide and 12 mg/kg of busulfan, while control mice received vehicle. For LH treatment, the ChT+LH-1x and ChT+LH-5x animals received a 1 or 5 IU LH dose, respectively, before chemotherapy, then a second LH injection administered with chemotherapy 24 h later. Then, two animals/group were euthanized at 12 and 24 h to investigate the early ovarian response to LH, while remaining mice were housed for 30 days to evaluate short- and long-term reproductive outcomes. The effects of LH and chemotherapy on growing-stage follicles were analyzed in a parallel experiment. Seven-week-old NOD-SCID female mice were allocated to control (n = 5), ChT (n = 5), and ChT+LH-1x (n = 6) groups. Animals were treated as described above, but maintained for 7 days before reproductive assessment. PARTICIPANTS/MATERIALS, SETTING, METHODS In the first experiment, follicular damage (phosphorylated H2AX histone (γH2AX) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay), apoptotic biomarkers (western blot), and DNA repair pathways (western blot and RT-qPCR) were assessed in ovaries collected at 12 and 24 h to determine early ovarian responses to LH. Thirty days after treatments, remaining mice were stimulated (10 IU of pregnant mare serum gonadotropin (PMSG) and 10 IU of hCG) and mated to collect ovaries, oocytes, and embryos. Histological analysis was performed on ovarian samples to investigate follicular populations and stromal status, and meiotic spindle and chromosome alignment was measured in oocytes by confocal microscopy. Long-term effects were monitored by assessing pregnancy rate and litter size during six consecutive breeding attempts. In the second experiment, mice were stimulated and mated 7 days after treatments and ovaries, oocytes, and embryos were collected. Follicular numbers, follicular protection (DNA damage and apoptosis by H2AX staining and TUNEL assay, respectively), and ovarian stroma were assessed. Oocyte quality was determined by confocal analysis. MAIN RESULTS AND THE ROLE OF CHANCE LH treatment was sufficient to preserve ovarian reserve and follicular development, avoid atresia, and restore ovulation and meiotic spindle configuration in mature oocytes exposed at the primordial stage. LH improved the cumulative pregnancy rate and litter size in six consecutive breeding rounds, confirming the potential of LH treatment to preserve fertility. This protective effect appeared to be mediated by an enhanced early DNA repair response, via homologous recombination, and generation of anti-apoptotic signals in the ovary a few hours after injury with chemotherapy. This response ameliorated the chemotherapy-induced increase in DNA-damaged oocytes and apoptotic granulosa cells. LH treatment also protected growing follicles from chemotherapy. LH reversed the chemotherapy-induced depletion of primordial and primary follicular subpopulations, reduced oocyte DNA damage and granulosa cell apoptosis, restored mature oocyte cohort size, and improved meiotic spindle properties. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION This was a preliminary study performed with mouse ovarian samples. Therefore, preclinical research with human samples is required for validation. WIDER IMPLICATIONS OF THE FINDINGS The current study tested if LH could protect the adult mouse ovarian reserve and reproductive lifespan from alkylating chemotherapy. These findings highlight the therapeutic potential of LH as a complementary non-surgical strategy for preserving fertility in female cancer patients. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Regional Valencian Ministry of Education (PROMETEO/2018/137), the Spanish Ministry of Science and Innovation (CP19/00141), and the Spanish Ministry of Education, Culture and Sports (FPU16/05264). The authors declare no conflict of interest.

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Study question Is luteinizing hormone (LH) able to protect ovarian follicles against cyclophosphamide (CPM)-induced damage in women? Summary answer LH significantly reduces primordial follicle (PMF) loss in ovarian cortical strips cultured in vitro with phosphoramide mustard (PM), the active metabolite of CPM. What is known already Cancer therapies are cause of premature ovarian insufficiency (POI) in female patients, since they induce a severe reduction of PMFs. For this reason, several compounds have been analyzed as adjuvant therapies to protect the ovarian reserve without interfering with cancer treatment on tumor cells. We recently demonstrated the protective effect of LH against ovotoxicity induced by PM and cisplatin on the ovary of prepuberal and adult mice. These results suggest the possibility to use LH prior or in concomitance with anticancer drugs in order to preserve oocytes in human patients, therefore preventing the early onset of menopause and/or infertility. Study design, size, duration Ovarian cortical tissues were collected from nine patients (age ± SD: 15.33 ± 4.50) who have cryopreserved their tissue before receiving anticancer treatment. For each patient, ovarian cortical strips were thawed and randomly assigned to the experimental conditions: Control (CTRL), PM and PM+LH. LH was added 1 hour before the treatment with PM. Samples were analyzed after 8, 16, 24 and 48 hrs of treatment. Participants/materials, setting, methods Ovarian cortial strips were placed onto culture plate insert and cultured for a maximum of 48 hours, in αMEM with 10% Serum Substitute Supplement with/out 200mIU/ml LH and/or 10μM PM. The samples were processed for: - Histology, for PMFs and primary follicles (PFs) analysis; - Immunohistochemistry, for the expression of protein involved in DNA damage, apoptosis and follicle activation; - Real-Time PCR, for the expression of apoptotic and inflammation related genes. Main results and the role of chance The follicle density in the untreated group varied from 233.03 to 3420.27 PMFs/mm3 between the nine patients analysed. Relative follicular density (%) was performed to analyse statistical differences between the groups. Relative PMFs density (%) was significantly reduced in PM vs CTRL either after 24 and 48 hrs, while this reduction was significantly counteracted by LH (24 hrs: CTRL=76.19±2.12; PM = 44.95±5.06; PM+LH=73.97±11.64. 48 hrs: CTRL=66.47±3.96; PM = 36.76±3.96; PM+LH=55.74±8.72). To investigate the mechanism underlying the observed effects, the expression of markers involved in DNA damage (gH2AX), apoptosis (Cleaved caspase 3, NOXA, PUMA), follicle activation (p-AKT, FOXO3a), cell cycle arrest (p21, Ki67), and inflammation (IL1β, TNFα) were analysed. The results showed that LH did not prevent DNA damage induced by PM, since gH2AX positive oocytes were seen both in PM and PH+LH group at 16h; at 24hrs however we observed a downregulation of the gH2AX expression in the PH+LH group (PM@ 100% vs PM+LH@35%). LH also counteracts the activation of chemotherapy-induced apoptotic processes, by reducing the levels of pro-apoptotic factors such as NOXA, PUMA and CC3, and follicles activation lowering AKT-FOXO3a signaling axis. Moreover, PM treatment creates a proinflammatory microenvironment, as shown by increased IL1β and TNFα gene expression, partially counteracted by LH pretreatment. Limitations, reasons for caution Experiments are carried out in vitro; the functionality of the cortex in xenografts should be evaluated. Careful evaluation of which patients might use this protector is needed. Wider implications of the findings These findings demonstrate that LH is able to reduce PMFs loss in human ovarian cortex exposed to PM. These results encourage thinking about the use of the hormone as a ferto-protector agent in clinical trials to prevent the premature onset of menopause and/or infertility in women undergoing anticancer treatments. Trial registration number not applicable