Survey of Long-Term Experiences of Sperm Cryopreservation in Oncological and Non-Oncological Patients: Usage and Reproductive Outcomes of a Large Monocentric Cohort

Progress in oncological treatment has led to an improved long-term survival of young male cancer patients over the last decades. However, standard cancer treatments frequently implicate fertility-damaging potential. Cryopreservation of sperm is the current standard option to preserve patient's fertility after treatment, yet long-term data on usage and reproductive experiences is still limited. Natural fertility after treatment and especially in relation to the type of treatment has been poorly analyzed so far. Therefore, we performed a retrospective survey including male patients with an indication for gonadotoxic treatment who cryopreserved reproductive material at our institution between 1994 and 2017. Study questionnaires regarding treatment, material usage, and reproductive outcomes were sent to eligible patients. Additionally, semen analyses of study participants from the time of cryopreservation were evaluated. A total of 99 patients were included in the study. Respondents' median age was 38.0 years. Most frequent diagnoses were testicular cancer (29.3%) and lymphoma (26.3%). A further 8.1% suffered from autoimmune diseases. Testicular cancer patients had a significantly lower pre-treatment median sperm concentration (18.0 million/ml) compared to non-testicular cancer patients (54.2 million/ml). Until November 2020, the determined sperm usage and cumulative live-birth rate per couple were 17.2% and 58.8%, respectively. Most sperm users received treatments with high (40.0%) or intermediate (33.3%) gonadotoxic potential. 20.7% of all patients reported to had fathered at least one naturally conceived child after treatment, this being the case especially if they had been treated with less or potentially gonadotoxic therapies. In conclusion, our findings emphasize the importance of sperm cryopreservation in the context of male fertility preservation. Furthermore, they indicate that the gonadotoxic potential of patients' treatments could represent a predictive factor for sperm usage

Adenoviral-mediated correction of methylmalonyl-CoA mutase deficiency in murine fibroblasts and human hepatocytes

<p>Abstract</p> <p>Background</p> <p>Methylmalonic acidemia (MMA), a common organic aciduria, is caused by deficiency of the mitochondrial localized, 5'deoxyadenosylcobalamin dependent enzyme, methylmalonyl-CoA mutase (MUT). Liver transplantation in the absence of gross hepatic dysfunction provides supportive therapy and metabolic stability in severely affected patients, which invites the concept of using cell and gene delivery as future treatments for this condition.</p> <p>Methods</p> <p>To assess the effectiveness of gene delivery to restore the defective metabolism in this disorder, adenoviral correction experiments were performed using murine <it>Mut </it>embryonic fibroblasts and primary human methylmalonyl-CoA mutase deficient hepatocytes derived from a patient who harbored two early truncating mutations, E224X and R228X, in the <it>MUT </it>gene. Enzymatic and expression studies were used to assess the extent of functional correction.</p> <p>Results</p> <p>Primary hepatocytes, isolated from the native liver after removal subsequent to a combined liver-kidney transplantation procedure, or <it>Mut </it>murine fibroblasts were infected with a second generation recombinant adenoviral vector that expressed the murine methylmalonyl-CoA mutase as well as eGFP from distinct promoters. After transduction, [1-¹⁴C] propionate macromolecular incorporation studies and Western analysis demonstrated complete correction of the enzymatic defect in both cell types. Viral reconstitution of enzymatic expression in the human methylmalonyl-CoA mutase deficient hepatocytes exceeded that seen in fibroblasts or control hepatocytes.</p> <p>Conclusion</p> <p>These experiments provide proof of principle for viral correction in methylmalonic acidemia and suggest that hepatocyte-directed gene delivery will be an effective therapeutic treatment strategy in both murine models and in human patients. Primary hepatocytes from a liver that was unsuitable for transplantation provided an important resource for these studies.</p

Effects of Ethanol and NAP on Cerebellar Expression of the Neural Cell Adhesion Molecule L1

The neural cell adhesion molecule L1 is critical for brain development and plays a role in learning and memory in the adult. Ethanol inhibits L1-mediated cell adhesion and neurite outgrowth in cerebellar granule neurons (CGNs), and these actions might underlie the cerebellar dysmorphology of fetal alcohol spectrum disorders. The peptide NAP potently blocks ethanol inhibition of L1 adhesion and prevents ethanol teratogenesis. We used quantitative RT-PCR and Western blotting of extracts of cerebellar slices, CGNs, and astrocytes from postnatal day 7 (PD7) rats to investigate whether ethanol and NAP act in part by regulating the expression of L1. Treatment of cerebellar slices with 20 mM ethanol, 10−12 M NAP, or both for 4 hours, 24 hours, and 10 days did not significantly affect L1 mRNA and protein levels. Similar treatment for 4 or 24 hours did not regulate L1 expression in primary cultures of CGNs and astrocytes, the predominant cerebellar cell types. Because ethanol also damages the adult cerebellum, we studied the effects of chronic ethanol exposure in adult rats. One year of binge drinking did not alter L1 gene and protein expression in extracts from whole cerebellum. Thus, ethanol does not alter L1 expression in the developing or adult cerebellum; more likely, ethanol disrupts L1 function by modifying its conformation and signaling. Likewise, NAP antagonizes the actions of ethanol without altering L1 expression

Mitogen-activated protein kinase modulates ethanol inhibition of cell adhesion mediated by the L1 neural cell adhesion molecule

There is a genetic contribution to fetal alcohol spectrum disorders (FASD), but the identification of candidate genes has been elusive. Ethanol may cause FASD in part by decreasing the adhesion of the developmentally critical L1 cell adhesion molecule through interactions with an alcohol binding pocket on the extracellular domain. Pharmacologic inhibition or genetic knockdown of ERK2 did not alter L1 adhesion, but markedly decreased ethanol inhibition of L1 adhesion in NIH/3T3 cells and NG108-15 cells. Likewise, leucine replacement of S1248, an ERK2 substrate on the L1 cytoplasmic domain, did not decrease L1 adhesion, but abolished ethanol inhibition of L1 adhesion. Stable transfection of NIH/3T3 cells with human L1 resulted in clonal cell lines in which L1 adhesion was consistently sensitive or insensitive to ethanol for more than a decade. ERK2 activity and S1248 phosphorylation were greater in ethanol-sensitive NIH/3T3 clonal cell lines than in their ethanol-insensitive counterparts. Ethanol-insensitive cells became ethanol sensitive after increasing ERK2 activity by transfection with a constitutively active MAP kinase kinase 1. Finally, embryos from two substrains of C57BL mice that differ in susceptibility to ethanol teratogenesis showed corresponding differences in MAPK activity. Our data suggest that ERK2 phosphorylation of S1248 modulates ethanol inhibition of L1 adhesion by inside-out signaling and that differential regulation of ERK2 signaling might contribute to genetic susceptibility to FASD. Moreover, identification of a specific locus that regulates ethanol sensitivity, but not L1 function, might facilitate the rational design of drugs that block ethanol neurotoxicity

Nicotinamide Protects against Ethanol-Induced Apoptotic Neurodegeneration in the Developing Mouse Brain

BACKGROUND: Exposure to alcohol during brain development may cause a neurological syndrome called fetal alcohol syndrome (FAS). Ethanol induces apoptotic neuronal death at specific developmental stages, particularly during the brain-growth spurt, which occurs from the beginning of third trimester of gestation and continues for several years after birth in humans, whilst occuring in the first two postnatal weeks in mice. Administration of a single dose of ethanol in 7-d postnatal (P7) mice triggers activation of caspase-3 and widespread apoptotic neuronal death in the forebrain, providing a possible explanation for the microencephaly observed in human FAS. The present study was aimed at determining whether nicotinamide may prevent ethanol-induced neurodegeneration. METHODS AND FINDINGS: P7 mice were treated with a single dose of ethanol (5g/kg), and nicotinamide was administered from 0 h to 8 h after ethanol exposure. The effects of nicotinamide on ethanol-induced activation of caspase-3 and release of cytochrome-c from the mitochondria were analyzed by Western blot ( n = 4–7/group). Density of Fluoro-Jade B–positive cells and NeuN-positive cells was determined in the cingulated cortex, CA1 region of the hippocampus, and lateral dorsal nucleus of the thalamus ( n = 5–6/group). Open field, plus maze, and fear conditioning tests were used to study the behavior in adult mice ( n = 31–34/group). Nicotinamide reduced the activation of caspase-3 (85.14 ± 4.1%) and the release of cytochrome-c (80.78 ± 4.39%) in postnatal mouse forebrain, too. Nicotinamide prevented also the ethanol-induced increase of apoptosis. We demonstrated that ethanol-exposed mice showed impaired performance in the fear conditioning test and increased activity in the open field and in the plus maze. Administration of nicotinamide prevented all these behavioral abnormalities in ethanol-exposed mice. CONCLUSIONS: Our findings indicate that nicotinamide can prevent some of the deleterious effects of ethanol on the developing mouse brain when given shortly after ethanol exposure. These results suggest that nicotinamide, which has been used in humans for the treatment of diabetes and bullous pemphigoid, may hold promise as a preventive therapy of FAS

Alteration of gene expression by alcohol exposure at early neurulation

<p>Abstract</p> <p>Background</p> <p>We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables.</p> <p>Result</p> <p>Alcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, <it>Sox5, Bhlhe22</it>), neural growth factor genes [<it>Igf1, Efemp1</it>, <it>Klf10 </it>(<it>Tieg), and Edil3</it>], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (<it>Rbp1</it>), and <it>de novo </it>expression of aldehyde dehydrogenase 1B1 (<it>Aldh1B1</it>). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos.</p> <p>Conclusion</p> <p>This study revealed a set of genes vulnerable to alcohol exposure and genes that were associated with neural tube defects during early neurulation.</p

Diverse Modes of Axon Elaboration in the Developing Neocortex

The development of axonal arbors is a critical step in the establishment of precise neural circuits, but relatively little is known about the mechanisms of axonal elaboration in the neocortex. We used in vivo two-photon time-lapse microscopy to image axons in the neocortex of green fluorescent protein-transgenic mice over the first 3 wk of postnatal development. This period spans the elaboration of thalamocortical (TC) and Cajal-Retzius (CR) axons and cortical synaptogenesis. Layer 1 collaterals of TC and CR axons were imaged repeatedly over time scales ranging from minutes up to days, and their growth and pruning were analyzed. The structure and dynamics of TC and CR axons differed profoundly. Branches of TC axons terminated in small, bulbous growth cones, while CR axon branch tips had large growth cones with numerous long filopodia. TC axons grew rapidly in straight paths, with frequent interstitial branch additions, while CR axons grew more slowly along tortuous paths. For both types of axon, new branches appeared at interstitial sites along the axon shaft and did not involve growth cone splitting. Pruning occurred via retraction of small axon branches (tens of microns, at both CR and TC axons) or degeneration of large portions of the arbor (hundreds of microns, for TC axons only). The balance between growth and retraction favored overall growth, but only by a slight margin. Given the identical layer 1 territory upon which CR and TC axons grow, the differences in their structure and dynamics likely reflect distinct intrinsic growth programs for axons of long projection neurons versus local interneurons