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Over the last decade, the number of cancer survivors has increased thanks to progress in diagnosis and treatment. Cancer treatments are often accompanied by adverse side effects depending on the age of the patient, the type of cancer, the treatment regimen, and the doses. The testicular tissue is very sensitive to chemotherapy and radiotherapy. This review will summarize the epidemiological and experimental data concerning the consequences of exposure to chemotherapy during the prepubertal period or adulthood on spermatogenic progression, sperm production, sperm nuclear quality, and the health of the offspring. Studies concerning the gonadotoxicity of anticancer drugs in adult survivors of childhood cancer are still limited compared with those concerning the effects of chemotherapy exposure during adulthood. In humans, it is difficult to evaluate exactly the toxicity of chemotherapeutic agents because cancer treatments often combine chemotherapy and radiotherapy. Thus, it is important to undertake experimental studies in animal models in order to define the mechanism involved in the drug gonadotoxicity and to assess the effects of their administration alone or in combination on immature and mature testis. These data will help to better inform cancer patients after recovery about the risks of chemotherapy for their future fertility and to propose fertility preservation options.

Testicular tissue freezing before gonadotoxic treatments allows the preservation of fertility for children suffering from cancer. Recently, the testis organ culture method was presented as a relevant method to restore the fertility of these patients. However, the yield of spermatozoa production is low in the mouse model and no gamete has been obtained in vitro in the rat model. Here, we assess different cryopreservation protocols and culture conditions to improve the efficiency of in vitro maturation of rat prepubertal testes. Testes from male rats aged 5 or 8 days post-partum were cultured onto agarose gels of different percentages. After determining the best culture conditions, different cryopreservation protocols were assessed. Finally, testicular tissues were cultured with media of various compositions and analyzed at different time points. Our results show that the cryopreservation protocols allow the preservation of tissue architecture, cell proliferation, with no or moderate increase of cell death. In vitro spermatogenesis did not proceed beyond the pachytene spermatocyte stage. Only 2 of the 6 tested media allowed the survival of differentiated germ cells over the 45-day culture period. In conclusion, this study highlights the necessity to further improve the organ culture method before applying it into the clinics.

Children undergoing cancer treatments are at risk for impaired fertility. Cryopreserved prepubertal testicular biopsies could theoretically be later matured in vitro to produce spermatozoa for assisted reproductive technology. A complete in vitro spermatogenesis has been obtained from mouse prepubertal testicular tissue, although with low efficiency. Steroid hormones are essential for the progression of spermatogenesis, the aim of this study was to investigate steroidogenesis and steroid signaling in organotypic cultures. Histological, RT-qPCR, western blot analyses, and steroid hormone measurements were performed on in vitro cultured mouse prepubertal testicular tissues and age-matched in vivo controls. Despite a conserved density of Leydig cells after 30 days of culture (D30), transcript levels of adult Leydig cells and steroidogenic markers were decreased. Increased amounts of progesterone and estradiol and reduced androstenedione levels were observed at D30, together with decreased transcript levels of steroid metabolizing genes and steroid target genes. hCG was insufficient to facilitate Leydig cell differentiation, restore steroidogenesis, and improve sperm yield. In conclusion, this study reports the failure of adult Leydig cell development and altered steroid production and signaling in tissue cultures. The organotypic culture system will need to be further improved before it can be translated into clinics for childhood cancer survivors.

The assessment of the impact of chemotherapies on in vitro spermatogenesis in experimental models is required before considering the application of this fertility restoration strategy to prepubertal boys who received these treatments before testicular tissue cryopreservation. The present work investigated the effects of exposure of prepubertal mice to mono- (vincristine or cyclophosphamide) and polychemotherapy (a combination of vincristine and cyclophosphamide) on the first wave of in vitro spermatogenesis. When testicular tissue exposed to monochemotherapy was preserved, polychemotherapy led to severe alterations of the seminiferous epithelium and increased apoptosis in prepubertal testes prior in vitro maturation, suggesting a potential additive gonadotoxic effect. These alterations were also found in the testicular tissues of polychemotherapy-treated mice after 30 days of organotypic culture and were associated with a reduction in the germ cell/Sertoli cell ratio. The different treatments neither altered the ability of spermatogonia to differentiate in vitro into spermatozoa nor the yield of in vitro spermatogenesis. However, more spermatozoa with morphological abnormalities and fragmented DNA were produced after administration of polychemotherapy. This work therefore shows for the first time the possibility to achieve a complete in vitro spermatogenesis after an in vivo exposure of mice to a mono- or polychemotherapy before meiotic entry.

The development of experimental models treated by chemotherapy is needed for elucidating the side effects of cancer treatments administered prior to puberty on male gonad function and the feasibility of restoring fertility from exposed testicular tissues. This study investigated for the first time the effects of cytarabine and daunorubicin administered before meiotic initiation on the first wave of mouse spermatogenesis under both in vivo or in vitro conditions. Prepubertal exposure to cytarabine did not exhibit immediate detrimental effects on testicular tissues, whereas daunorubicin administration resulted in a decreased spermatogonia-to-Sertoli cell ratio and diminished intratubular cell proliferation within three days post-treatment. While the completion of in vivo spermatogenesis was not hindered by chemotherapy exposure, a significant increase in the proportion of spermatozoa with fragmented DNA was observed in mice more than one month after treatment. In vitro spermatogenesis was also accomplished using prepubertal testicular tissues exposed to chemotherapy, indicating that neither cytarabine nor daunorubicin impeded the differentiation potential of spermatogonia into spermatozoa. However, in vitro conditions revealed an arrest in meiotic progression in a substantial proportion of seminiferous tubules and an elevated incidence of DNA double-strand breaks in intratubular cells compared to in vivo controls, irrespective of the treatment administered.

De novo mutations (DNMs) have a significant impact on human health, notably through their contribution to developmental disorders. DNMs occur in both paternal and maternal germlines via diverse mechanisms, including parental early embryonic mosaicism, at high recurrence risk for subsequent pregnancies through germline mosaicism. This phenomenon has been studied mostly on isolated pathogenic variants, but its contribution to genome-wide phased variants in individual genomes is underexplored. We aimed to categorize DNMs and their recurrence risk by detecting and phasing a large set of DNMs via short- and long-read genome sequencing followed by systematic deep sequencing of parental blood and sperm DNA. We detected an average of 85.6 DNM per trio (n=5 trios), with an expected paternal bias of 80%. Targeted resequencing of parental blood and sperm (depth>5000x) revealed 20/334 parental germline mosaics (2–5 per trio) with variant allele fractions (VAFs) ranging from 0.24% to 14.7%, including 7 that were detected in paternal sperm exclusively (1–2 per trio). Owing to paternal bias, maternally phased variants were 3.4x more likely to be mosaic in blood. VAF in sperm samples was used as an indicator for the risk of recurrence of paternally phased DNM. Fourteen variants (out of 244, 5.7%) exhibited detectable sperm mosaicism, while the remaining 230 showed no evidence of mosaicism. Sperm sequencing therefore enabled a precise quantification of the recurrence risk of most individual DNMs. We predict that the use of long-read genome sequencing in genomic medicine will enable the critical step of variant phasing, improving the genetic counselling of rare diseases mediated by DNMs.

Testicular tissue freezing has been proposed for fertility preservation in pre-pubertal boys. Thawed frozen testicular tissue must undergo a maturation process to restore sperm production. The purpose of the current study was to evaluate the ability of retinol to improve the in vitro differentiation of pre-pubertal mouse spermatogonial stem cells into sperm. Testes from pre-pubertal mice, aged 2.5 and 6.5 days post-partum, were cultured on agarose gel at a gas-liquid interphase for 34, 38 and 60 days (D) and for 16, 30 and 36 D respectively. Assessment of basal medium (BM) supplemented with retinol (RE) alone, FSH/LH alone or a combination of both, was performed. Stereological analyses and tissue lesion scoring were performed at the culture time points indicated above. Sperm production was quantified at D30 and D34 after mechanical dissection of the testicular tissues. FSH/LH significantly increased the percentage of round spermatids at D30 and D38, when compared to BM alone. However, RE significantly increased the percentages of round but also elongated spermatids at D30 and D34. Moreover, RE significantly increased the number of spermatozoa per milligram of tissue at D30 and D34 when compared to BM. Therefore, RE improved the in vitro production of spermatids and spermatozoa from pre-pubertal SSCs during the first wave of spermatogenesis. The use of RE could be a useful tool for in vitro spermatogenesis from pre-pubertal human testicular tissue.

Background First-year medical candidates encounter steep learning demands when transitioning from high school to university. Spaced repetition– a method of distributing review sessions over time– improves long-term memory retention. This study assessed its effectiveness alongside lifestyle and academic factors in students preparing for the medical school entrance examination. Methods In 2023, all 618 candidates at the University of Rouen were invited to complete a post-exam, self-administered questionnaire; 523 responded (84.6%). We collected data on revision methods (e.g., spaced repetition, reviewing archives of previous exams), participation in private preparatory classes and summer courses, lifestyle behaviors (e.g., sleep duration, physical activity or smoking), and secondary-school grades. Predictors of success were identified through univariate analyses. A multivariate logistic regression was then conducted to determine independent predictors of success. Results Of 523 respondents, 134 (25.6%) passed the entrance exam. In univariate analysis, successful candidates significantly more often used spaced repetition (44.8% vs. 20.3%; p  < 0.001), reviewed archives of previous exams, attended private preparatory classes or summer courses, and had higher secondary-school grades. In multivariate logistic regression, independent predictors of success included spaced repetition (aOR 2.09; 95% CI, 1.16–3.48), secondary-school grades (aOR 3.19; 95% CI, 2.33–4.37), private preparatory class attendance (aOR 2.02; 95% CI, 1.11–3.66), sleep duration (aOR 1.49; 95% CI, 1.12–1.99), and regular sport practice (aOR 1.81; 95% CI, 1.13–2.93). Conclusions Admission success in the medical school entrance examination appeared to be influenced by multiple factors: while spaced repetition significantly enhanced performance, academic background, private preparatory classes, and healthy lifestyle habits also contributed. These findings support integrating validated study techniques and wellness strategies into university support programs for entrance examinations. Practice points Students frequently feel underprepared for the transition from small-group secondary-school instruction to large-lecture university formats. Structured study strategies– such as spaced repetition– are associated with higher success rates in medical school entrance examinations. Additional factors– including sleep duration, regular physical activity, attendance at private preparatory classes, and strong secondary school performance– also correlate with admission success.

Telomere length can be influenced by reactive oxygen species (ROS) generated by lifestyle factors or environmental exposure. We sought to determine whether oxidative stress has an impact on sperm nuclear alterations, especially on chromatin organization and telomere interactions in the spermatozoa of infertile males. We performed an observational and prospective study including fifty-two males, allocated in the “case group” (30 infertile males presenting conventional semen parameter alterations) and the “control group” (22 males with normal conventional semen parameters). ROS detection was determined on spermatozoa using CellROX© probes. Sperm nuclear damage was assessed using quantitative fluorescence in situ hybridization (Q-FISH) for relative telomere length and telomere number, aniline blue staining for chromatin condensation, terminal deoxynucleotidyl transferase dUTP nick-end labeling for DNA fragmentation, and FISH for aneuploidy and 8-hydroxy-2′-deoxyguanosine immunostaining for oxidative DNA damages. Infertile males had significantly increased levels of cytoplasmic ROS and chromatin condensation defects as well as a higher mean number of telomere signals per spermatozoon in comparison with controls. In addition, the mean number of sperm telomere signals were positively correlated with the percentage of spermatozoa with chromatin condensation defect. In infertile males with conventional semen parameter alterations, oxidative stress is associated with telomere interaction impairment and chromatin condensation defects.

PurposePrevious preclinical and preliminary clinical data suggest an appetite-stimulating effect of propofol compared with halogenated drugs. This study compared the effects of propofol with those of sevoflurane on recovery of hunger during the postoperative period.MethodsPatients undergoing outpatient transvaginal oocyte retrieval were randomized to propofol-remifentanil (propofol group) or sevoflurane-remifentanil (sevoflurane group) anesthesia. The primary endpoint was the time before feeling hungry (≥ 50/100 mm on a visual analogue scale). Secondary endpoints included plasma levels of ghrelin, leptin, and insulin (ten minutes, one hour, and two hours after anesthesia), caloric intake at first feed, and discharge readiness time.ResultsIn the 58 patients allocated to either the propofol or sevoflurane group, there was no difference in the median [interquartile range] recovery time of hunger (97 [75–138] vs 97 [80–140] min, respectively; median difference, 1; 95% confidence interval [CI], − 15 to 14; P = 0.91); caloric intake (245 [200–343] vs 260 [171–314] kcal; P = 0.39); or discharge readiness time (125 [85–153] vs 125 [95–174] min, P = 0.29). The groups showed no difference in crude plasma levels of ghrelin, leptin, and insulin at any time-point. When peptide plasma levels were expressed as a % change from baseline, there was a higher insulin plasma level one hour after anesthesia in the sevoflurane group (median difference, 4.9%; 95% CI, − 16.2 to 43.4) compared with the propofol group (median difference, − 21.2%; 95% CI, − 35.7 to 9.1; adjusted P = 0.01).ConclusionPropofol did not accelerate the recovery of hunger compared with sevoflurane after outpatient minor surgery. Moreover, propofol did not have distinguishable effects on other clinical or biological parameters associated with food intake.Trial registrationwww.ClinicalTrials.gov (NCT02272166); registered 22 October, 2014.