The cyto-protective effects of LH on ovarian reserve and female fertility during exposure to gonadotoxic alkylating agents in an adult mouse model.

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.

Differential effects of the immunosuppressive calcineurin inhibitors cyclosporine-A and tacrolimus on ovulation in a murine model.

STUDY QUESTION: Do therapeutic levels of cyclosporine-A and tacrolimus affect ovulation in a rat gonadotrophin-induced ovulation model? SUMMARY ANSWER: Cyclosporine-A, but not tacrolimus, decreases ovulation rate when administered for 5 days before induced ovulation. WHAT IS KNOWN ALREADY: The mainstays of immunosuppression in solid organ transplantation, to prevent rejection, are the calcineurin inhibitors cyclosporine-A or tacrolimus. These drugs could potentially affect fertility in transplanted patients. Since ovulation is an inflammation-like process with pivotal roles for several immune cells and modulators, it is possible that the calcineurin inhibitors, with broad effects on the immune system, could interfere with this sensitive, biological process. STUDY DESIGN SIZE DURATION: Experimental design at university-based animal facilities. A total of 45 immature Sprague-Dawley rats were used. The study was carried out over 3 months. PARTICIPANTS/MATERIALS SETTING METHODS: Immature Sprague-Dawley rats (n = 45) were randomly assigned to receive equivalent doses of tacrolimus (0.5 mg/kg/day; TAC), cyclosporine-A (10 mg/kg/day; CyA) or vehicle (Control). Ovarian hyperstimulation was induced with 10 IU of equine chorionic gonadotrophin, and ovulation was triggered with 10 IU of hCG. Oocytes were retrieved from the oviducts and ovulation rates were calculated. Various subpopulations of white blood cells were counted in peripheral blood and ovarian tissue samples. MAIN RESULTS AND THE ROLE OF CHANCE: Animals in the CyA group showed a lower ovulation rate when compared to the TAC and Control groups (CyA: mean 9 oocytes (range 0-22); TAC: 21 oocytes (8-41); Control: 22 oocytes (6-39); P = 0.03). Regarding counts of the white blood cell subpopulations and resident neutrophils in the ovary, no significant differences were observed between the groups. LIMITATIONS REASONS FOR CAUTION: Although the ovulation process is highly conserved within species, the differences between rodents and humans may limit the external translatability of the study. WIDER IMPLICATIONS OF THE FINDINGS: These findings suggest that tacrolimus should be the preferred calcineurin inhibitor of choice in transplanted patients who are aiming for pregnancy. STUDY FUNDING/COMPETING INTERESTS: Swedish Research Council and ALF of Sahlgrenska Academy, Sweden. Rio Hortega Grant from the Instituto de Salud Carlos III, Spain (CM09/00063). There are no conflicts of interest.

Renal Function Impairment in Kidney Transplantation: Importance of Early BK Virus Detection.

BACKGROUND: BK virus allograft nephropathy is a major complication of kidney transplantation that markedly reduces graft survival (50% graft failure 24 months after being diagnosed). BK virus replication can occur at any time posttransplantation. Viruria detection is a signal of virus reactivation and precedes viremia. Only viremia has been related to BK nephropathy. Early detection appears to be important in the prevention of BK nephropathy. METHODS: Using serial follow-up of BK infection, we sought to determine the association of BK virus infection with kidney function impairment. We included all solitary kidney recipients transplanted between February 1, 2010 and December 31, 2014 and followed for at least 1 year. Viruria at >107 copies/mL, viremia at >104 copies/mL, or biopsy-proven BK nephropathy were indicative of positivity. Various recipient, donor, and transplant characteristics were registered. Creatinine level was measured at 3, 6, 9, and 12 months and while virus replication was detected. P < .05 was considered statistically significant. RESULTS: Two hundred fifty-four kidney recipients were included. Viruria was detected in 73 of them (28.74%), of whom 32 (12.6%) also had viremia. Of the 32 recipients with viremia, 7 had biopsy-proven nephropathy. Only viremia positivity had a negative effect on kidney function (P < .01). One of 32 viremia-positive recipients had graft loss (3.1%). CONCLUSION: Serial monitoring for BK virus replication is important for detection of BK infection. Early BK detection appears crucial to prevent impairment of kidney function and subsequent graft loss.

Daily exposure to summer temperatures affects the motile subpopulation structure of epididymal sperm cells but not male fertility in an in vivo rabbit model.

High temperatures have negative effects on sperm quality leading to temporary or permanent sterility. The aim of the study was to assess the effect of long exposure to summer circadian heat stress cycles on sperm parameters and the motile subpopulation structure of epididymal sperm cells from rabbit bucks. Twelve White New Zealand rabbit bucks were exposed to a daily constant temperature of the thermoneutral zone (from 18 °C to 22 °C; control group) or exposed to a summer circadian heat stress cycles (30 °C, 3 h/day; heat stress group). Spermatozoa were flushed from the epididymis and assessed for sperm quality parameters at recovery. Sperm total motility and progressivity were negatively affected by high temperatures (P < 0.05), as were also specific motility parameters (curvilinear velocity, linear velocity, mean velocity, straightness coefficient, linearity coefficient, wobble coefficient, and frequency of head displacement; P < 0.05, but not the mean amplitude of lateral head displacement). Heat stress significantly increased the percentage of less-motile sperm subpopulations, although the percentage of the high-motile subpopulation was maintained, which is consistent with the fact that no effect was detected on fertility rates. However, prolificacy was reduced in females submitted to heat stress when inseminated by control bucks. In conclusion, our results suggest that environmental high temperatures are linked to changes in the proportion of motile sperm subpopulations of the epididymis, although fertility is still preserved despite the detrimental effects of heat stress. On the other hand, prolificacy seems to be affected by the negative effects of high temperatures, especially by altering female reproduction.

Absence of beneficial effects on rabbit sperm cell cryopreservation by several antioxidant agents.

The generation of reactive oxygen species associated with cryopreservation could be responsible for mammalian sperm damage and the limitable value of stored semen in artificial insemination. The aim of this study was to assess several antioxidant agents supplemented in a commercial freezing extender (Gent B®) in order to improve post-thaw rabbit sperm quality. Ejaculates of 26 New Zealand White rabbit bucks were collected, evaluated and frozen using a conventional protocol. Antioxidant agents were tested at different concentrations: bovine serum albumin (BSA; 5, 30 or 60 mg/ml), retinol (RO; 50, 100 or 200 µM) and retinyl (RI; 0.282 or 2.82 µg/ml). Per cent viability, morphological abnormalities and intact acrosomes were determined using eosin-nigrosin staining. Motility and progressivity were analyzed by computer-assisted sperm analysis (CASA). In general, all sperm quality parameters were negatively affected by the cryopreservation process, the largest effect seen was for total motility. The addition of antioxidant agents did not improve thaw sperm quality. Furthermore, for RI groups a significant decrease in sperm quality parameters was recorded. In conclusion, rabbit sperm quality is negatively affected by the cryopreservation process. To our knowledge this report is the first using these antioxidants to supplement rabbit freezing extender. BSA and RO at concentrations used in the study did not improve sperm quality parameters after thawing, whereas RI supplementation appeared to be toxic. More studies are required to find the appropriate antioxidants necessary and their most effective concentrations to improve rabbit post-thaw sperm quality.

Retinol improves in vitro oocyte nuclear maturation under heat stress in heifers.

Heat stress (HS) is especially harmful for bovine ovarian follicle development and oocyte competence. Furthermore, HS causes premature aging in oocytes due to high levels of reactive oxygen species (ROS), involved in the harmful effects over the oocyte maturation and the steroidogenic activity of follicular cells. In this study, the presumptive protective effects of antioxidant agents on heat-stressed oocytes were evaluated. Heifer oocytes were matured for 22 h under control (38°C) and HS conditions (41.5°C at 18-21 h of maturation). For each oocyte, nuclear stage and cortical granule (CG) distribution were evaluated. Steroidogenic activity of cumulus cells was also recorded. The antioxidant agents used in the study were: retinol (1.43 µg/ml), retinyl (0.28 µg/ml) and oleic acid (0.05 mg/ml). Based on a chi-squared test (P < 0.05), HS affected negatively the metaphase II (MII) progression and produced a premature CG exocytosis. Retinol improved the oocyte MII progression. However, retinyl and oleic acid, at the concentrations used in this study, could not counteract adverse effects of HS. A decrease in progesterone and increase in estradiol availability were observed when retinyl and oleic acid were supplemented to the maturation medium, respectively. In conclusion, retinol proved to be valuable in heat-stressed oocytes protecting nuclear maturation.

Bovine oocytes show a higher tolerance to heat shock in the warm compared with the cold season of the year.

Heat stress is especially harmful for bovine ovarian follicle development and oocyte competence. In this study, we assessed the effects of heat shock on oocyte maturation in oocytes collected during the cold (February-March; n = 114) or warm (May-June; n = 116) periods of the year. In both cases, cumulus-oocyte complexes were matured under control (38 °C) and heat shock conditions (41.5 °C, 18-21 h of maturation). For each oocyte, nuclear stage, cortical granule distribution and steroidogenic activity of cumulus cells were evaluated. Based on the odds ratio, heat-shocked oocytes were 26.83 times more likely to show an anomalous metaphase II morphology. When matured under heat shock conditions, oocytes obtained in both seasons were similarly affected in terms of nuclear maturation, whereas a seasonal effect was observed on cytoplasmic maturation. For oocytes collected during the cold season, the likelihood to show an anomalous maturation was 25.96 times higher when exposed to the heat treatment than when matured under control conditions. By contrast, oocytes collected during the warm season matured under control or heat shock did not show significant risk of showing an anomalous cytoplasmic maturation. Our findings indicate an increased rate of premature oocytes in response to heat shock as well as a higher tolerance to this stress of oocytes harvested in the warm season compared with those collected in the colder period.

Retinol might stabilize sperm acrosomal membrane in situations of oxidative stress because of high temperatures.

High temperatures have negative effects on sperm quality leading to temporary or permanent sterility. The study tried to confirm the harmful effects of high temperatures on epididymal sperm cells in comparison with other temperatures (scrotal, environmental, and refrigeration temperatures), the main objective was the assessment of the addition of retinol as an antioxidant agent to improve sperm quality parameters. Testes from 10 bulls were collected from a slaughterhouse. Sperm cells were flushed from the cauda epididymis and deferent duct and assessed for sperm quality parameters at recovery. Afterward, sperm cell samples were exposed to one of four different temperatures (4 °C, 22 °C, 32 °C, and 41.5 °C for 3 hours) in presence or absence of retinol in the storage extender. Percentages of viability and morphologic abnormalities were determined using eosin-nigrosin staining. Acrosome integrity and sperm plasma membrane integrity were assessed by fluorescence Pisum sativum agglutinin lectin (FITC-PSA) staining and the hypo-osmotic swelling test, respectively. Total and progressive motility were analyzed by computer-assisted sperm analysis. Sperm quality parameters were mainly affected by high temperatures (41.5 °C). The addition of all-trans-retinol to the storage extender did not show any effect on sperm quality parameters. However, the percentage of sperm cells with altered acrosome was significantly reduced when retinol was present in the extender under heat stress conditions (41.5 °C). In conclusion, retinol might stabilize sperm acrosomal membrane in situations of oxidative stress because of high temperatures.

Does heat stress provoke the loss of a continuous layer of cortical granules beneath the plasma membrane during oocyte maturation?

The objective of the present study was to evaluate the influence of heat stress on bovine oocyte maturation. Both nuclear stage and distribution of cortical granules (CG) were simultaneously evaluated in each oocyte. Oocyte overmaturation under standard conditions of culture was also evaluated. For this purpose, logistic regression procedures were used to evaluate possible effects of factors such as heat stress, overmaturation, replicate, CG distribution and metaphase II (MII) morphology on oocyte maturation. Based on the odds ratio, oocytes on heat stressed (HSO) and overmaturated (OMO) oocyte group were, respectively, 14.5 and 5.4 times more likely to show anomalous MII morphology than those matured under control conditions (CO). The likelihood for an oocyte of showing the CG distribution pattern IV (aging oocyte) was 6.3 and 9.3 times higher for HSO and OMO groups, respectively, than for the CO group. The risk of undergoing anomalous oocyte maturation, considering both nuclear stage and distribution of CG was 17.1 and 18 times greater in oocytes cultured in HSO and OMO groups, respectively, than those in the CO group. In conclusion, heat stress proved to be valuable in aging oocytes. Heat stress advanced age for nuclear and cytoplasmic processes in a similar form to that of oocyte overmaturation.

Fatal levofloxacin failure in treatment of a bacteremic patient infected with Streptococcus pneumoniae with a preexisting parC mutation.

The fatal outcome of levofloxacin treatment in a patient with bacteremic pneumonia caused by Streptococcus pneumoniae with a preexisting parC mutation is reported. Failure was due to the emergence of a gyrA mutation after 4 days of therapy. Problems encountered in detecting first-step mutation isolates are discussed.

Meningitis de Mollaret por virus herpes simple 1 / Herpes simplex 1 virus Mollaret meningitis

La meningitis de Mollaret es una enfermedad rara, caracterizada por episodios recurrentes y autolimitados de meningitis aséptica. Se considera una enfermedad de etiología benigna. Se la ha asociado con infecciones víricas: Epstein-Barr y herpes simple, más frecuente el tipo 2. El virus de herpes simple tipo 1 se ha aislado raramente en el líquido cefalorraquídeo de los casos de meningitis de Mollaret; para ello se ha utilizado cultivo, ampliación con PCR y análisis de inmunoblot. En este artículo se describe un caso de meningitis de Mollaret por virus de herpes simple tipo 1; el interés radica en el uso para apoyar al diagnóstico de la demostración del virus herpes simple tipo 1 dentro del citoplasma de las células de Mollaret mediante la técnica de inmunohistoquímica (método ABC-peroxidasa) usando anticuerpos monoclonales antivirus de herpes simple tipo 1, siendo éste el primer caso descrito en la literatura (AU)

[Herpes simplex 1 virus Mollaret meningitis]. / Meningitis de Mollaret por virus herpes simple 1.

Mollaret meningitis is a rare disease characterized by recurrent and self-limited episodes of aseptic meningitis. It is considered a disease with benign etiology and it has been related to viral infections: Epstein-Barr virus and herpes simplex virus (HSV), type 2 most frequently. Herpes simplex 1 virus has been rarely isolated in the cerebrospinal fluid in patients with Mollaret meningitis; to this end culture, expansion with PCR, and inmunoblot has been utilized. In this article a case of Mollaret meningitis related to type 1 HSV is described. The interest is the demonstration of the herpes simplex virus type 1 within the cytoplasm of the Mollaret cells with a immunohistochemical technique (ABC-peroxidase method) using monoclonal antibodies anti HSV-1 in order to support the diagnosis, being this the first case described in the literature.

[Is basic the anesthetist presence in cataract surgery?]. / Es necesaria la presencia del médico anestesista en la cirugía de la catarata?

PURPOSE: To determine the need for anesthesiology in cataract surgery. METHODS: For the present study a total of 406 consecutive patients who underwent cataract surgery under regional peribulbar anesthesia were selected. RESULTS: In 109 cases (28,5%) the anesthetist was required, the causes of intervention were: high blood pressure in 68 patients (16.74%), perturbation in 20 patients (4.92%), cardiac arrhytmia in 20 patients (4.92%) and one case of myocardial infarction. CONCLUSIONS: Anesthetist intervention was requiered in one third of cataract surgery cases. This is why the anesthetist care seems to be justified in cataract surgery under local anesthesia.

Prevalence of hepatitis A virus antibodies in patients with chronic liver diseases.

OBJECTIVE: The age of persons with hepatitis A virus (HAV) infection in the general population has risen; these persons are at increased risk of clinically severe disease, especially patients with chronic liver disease. The aim of the present study was to analyze the prevalence of total antibodies against HAV in patients with chronic liver disease. METHODS: In a prospective study carried out between September 1998 and June 1999, 180 patients seen in the chronic liver disease outpatient department were studied. The prevalence of total anti-HAV antibodies was determined by age group, etiology and degree of histological damage, and according to the antecedents of risk for parenteral infection. A nonconditional logistic regression model was fitted with anti-HAV positivity as the dependent variable. RESULTS: Mean age was 44.1 years, with an anti-HAV prevalence of 77.2% (varying from 42.9% in the 21-25-year-old group to more than 83% in patients > 56-years old). Differences across groups regarding other categories (histological damage, etiology and history of parenteral or drug use) were not statistically significant, but the probability of anti-HAV positivity increased with age and a history of drug addiction. CONCLUSIONS: The prevalence of total anti-HAV antibodies is high among patients with chronic liver disease. We therefore recommend this test before vaccination against HAV, until current recommendations on universal childhood vaccination are implemented, in order to prevent hepatitis A epidemics in the general population.

Prevalencia de anticuerpos antivirus de la hepatitis A en pacientes con hepatopatías crónicas / Prevalence of hepatitis A virus antibodies in patients with chronic liver diseases

OBJETIVO: la edad de infección por el virus de la hepatitis A (VHA) ha aumentado en la población, con un mayor riesgo de gravedad clínica, sobre todo en pacientes con hepatopatía crónica. Se pretende conocer la prevalencia de anticuerpos totales frente al VHA en pacientes con hepatopatía crónica. PACIENTES Y MÉTODOS: se estudió de forma prospectiva a 180 pacientes atendidos consecutivamente en nuestra consulta de Hepatopatías Crónicas desde septiembre de 1998 hasta junio de 1999. Se determinó la prevalencia de anticuerpos totales anti-VHA por grupos de edad, etiologías, grado de afectación histológica y antecedentes de riesgo de infección por vía parenteral. Finalmente se ajustó un modelo de regresión logística no condicional con variable dependiente la presencia de anti-VHA. RESULTADOS: la edad media fue de 44,1 años, con una prevalencia de anti-VHA del 77,2 por ciento (desde el 42,9 por ciento entre 21-25 años hasta más del 83 por ciento a partir de 56 años). No existieron diferencias estadísticamente significativas según la afectación histológica, etiología o antecedentes de riesgo parenteral o de drogadicción, pero la probabilidad de tener anti-VHA aumentaba con la edad y con el antecedente UDVP. CONCLUSIONES: los pacientes con hepatopatía crónica presentan una alta prevalencia de anticuerpos totales anti-VHA, En consecuencia, en estos pacientes recomendamos su determinación previamente a la vacunación frente al VHA hasta que se introduzcan las recomendaciones actuales de vacunación generalizada de la población infantil para evitar epidemias de hepatitis A en la población general (AU)