The role of miRNAs in male human reproduction: a systematic review.

BACKGROUND: The presence of miRNAs in human reproductive tissue is intriguing and suggests the possibility that these important regulatory molecules play a role in reproductive function. However, the regulatory role of miRNAs in reproductive tissue remains poorly understood with a significant amount of controversial and contradicting data. OBJECTIVES: To systematically review the high-quality studies published to date investigating miRNAs associated with male human reproduction in order to describe their roles and relations with infertility and update the knowledge in the field. MATERIALS AND METHODS: A comprehensive systematic review of the published literature in MEDLINE-PubMed and EMBASE databases from the earliest available online indexing year until June 2018 (complimentary search until July 2019) was performed, in accordance with the PRISMA guidelines. We have included descriptive, case-control, cross-sectional, and observational prospective and retrospective studies in which fertile/infertile men were well-defined. The primary outcome was the miRNA expression in testis, epididymis, sperm cells, seminal plasma, and extracellular vesicles (i.e., exosomes and microvesicles). RESULTS: We identified 25,204 articles, of which 42 were selected for qualitative analysis. Of the 42 articles included, 15 evaluated the miRNAs in testis, five in epididymis, 13 in spermatozoa, and 11 in seminal plasma and/or extracellular vesicles. Two studies tackled more than one sub-group. As far as miRNA presence and content, the results of this systematic review indicated that every tissue/cell contains a well-defined and stable population of miRNAs that could be potentially related to spermatogenesis and embryogenesis. DISCUSSION AND CONCLUSION: Our systematic review of descriptive and observational studies shows a consistent relationship between aberrant miRNA expression and infertility. Therefore, it seems reasonable that measuring the expression of particular miRNAs might be useful not only as infertility biomarkers, but also for developing therapeutic strategies.

Feasibility of direct venous inoculation of the radiation-attenuated Plasmodium falciparum whole sporozoite vaccine in children and infants in Siaya, western Kenya.

PfSPZ Vaccine, composed of radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum sporozoites, is administered by direct venous inoculation (DVI) for maximal efficacy against malaria. A critical issue for advancing vaccines that are administered intravenously is the ability to efficiently administer them across multiple age groups. As part of a pediatric safety, immunogenicity, and efficacy trial in western Kenya, we evaluated the feasibility and tolerability of DVI, including ease of venous access, injection time, and crying during the procedure across age groups. Part 1 was an age de-escalation, dose escalation trial in children aged 13 months-5 years and infants aged 5-12 months; part 2 was a vaccine efficacy trial including only infants, using the most skilled injectors from part 1. Injectors could use a vein viewer, if needed. A total of 1222 injections (target 0.5 mL) were initiated by DVI in 511 participants (36 were 5-9-year-olds, 65 were 13-59-month-olds, and 410 infants). The complete volume was injected in 1185/1222 (97.0%) vaccinations, 1083/1185 (91.4%) achieved with the first DVI. 474/511 (92.8%) participants received only complete injections, 27/511 (5.3%) received at least one partial injection (<0.5 mL), and in 10/511 (2.0%) venous access was not obtained. The rate of complete injections by single DVI for infants improved from 77.1% in part 1 to 92.8% in part 2. No crying occurred in 51/59 (86.4%) vaccinations in 5-9-year-olds, 25/86 (29.1%) vaccinations in 13-59-month-olds and 172/1067 (16.1%) vaccinations in infants. Mean administration time ranged from 2.6 to 4.6 minutes and was longer for younger age groups. These data show that vaccination by DVI was feasible and well tolerated in infants and children in this rural hospital in western Kenya, when performed by skilled injectors. We also report that shipping and storage in liquid nitrogen vapor phase was simple and efficient. (Clinicaltrials.gov NCT02687373).

Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: new linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters.

Sea otters in California are commonly infected with Toxoplasma gondii. A unique Type X strain is responsible for 72% of otter infections, but its prevalence in terrestrial animals and marine invertebrates inhabiting the same area was unknown. Between 2000 and 2005, 45 terrestrial carnivores (lions, bobcats, domestic cats and foxes) and 1396 invertebrates (mussels, clams and worms) were screened for T. gondii using PCR and DNA sequencing to determine the phylogeographic distribution of T. gondii archetypal I, II, III and Type X genotypes. Marine bivalves have been shown to concentrate T. gondii oocysts in the laboratory, but a comprehensive survey of wild invertebrates has not been reported. A California mussel from an estuary draining into Monterey Bay was confirmed positive for Type X T. gondii by multilocus PCR and DNA sequencing at the B1 and SAG1 loci. This mussel was collected from nearshore marine waters just after the first significant rainfall event in the fall of 2002. Of 45 carnivores tested at the B1, SAG1, and GRA6 typing loci, 15 had PCR-confirmed T. gondii infection; 11 possessed alleles consistent with infection by archetypal Type I, II or III strains and 4 possessed alleles consistent with Type X T. gondii infection. No non-canonical alleles were identified. The four T. gondii strains with Type X alleles were identified from two mountain lions, a bobcat and a fox residing in coastal watersheds adjacent to sea otter habitat near Monterey Bay and Estero Bay. Confirmation of Type X T. gondii in coastal-dwelling felids, canids, a marine bivalve and nearshore-dwelling sea otters supports the hypotheses that feline faecal contamination is flowing from land to sea through surface runoff, and that otters can be infected with T. gondii via consumption of filter-feeding marine invertebrates.

Transplacental toxoplasmosis in a wild southern sea otter (Enhydra lutris nereis).

In September 2004, a neonatal sea otter pup was found alive on the beach in northern Monterey Bay, CA. Efforts to locate the mother were unsuccessful. Due to a poor prognosis for successful rehabilitation, the pup was euthanized. Postmortem examination revealed emaciation, systemic lymphadenopathy and a malformation of the left cerebral temporal lobe. On histopathology, free tachyzoites and tissue cysts compatible with Toxoplasma gondii were observed in the brain, heart, thymus, liver, lymph nodes and peri-umbilical adipose. The presence of T. gondii within host tissues was associated with lymphoplasmacytic inflammation and tissue necrosis. Immunofluorescent antibody tests using postmortem serum were positive for anti-T. gondii IgM and IgG (at 1:320 and 1:1280 serum dilution, respectively), but were negative for IgG directed against Sarcocystis neurona and Neospora caninum (<1:40 each). Brain immunohistochemistry revealed positive staining for tachyzoites and tissue cysts using antiserum raised to T. gondii, but not S. neurona or N. caninum. T. gondii parasite DNA was obtained from extracts of brain and muscle by PCR amplification using the diagnostic B1 locus. Restriction enzyme digestion followed by gel electrophoresis and DNA sequencing confirmed the presence of Type X T. gondii, the strain identified in the majority of southern sea otter infections.

Cigarette smoking significantly alters sperm DNA methylation patterns.

Numerous health consequences of tobacco smoke exposure have been characterized, and the effects of smoking on traditional measures of male fertility are well described. However, a growing body of data indicates that pre-conception paternal smoking also confers increased risk for a number of morbidities on offspring. The mechanism for this increased risk has not been elucidated, but it is likely mediated, at least in part, through epigenetic modifications transmitted through spermatozoa. In this study, we investigated the impact of cigarette smoke exposure on sperm DNA methylation patterns in 78 men who smoke and 78 never-smokers using the Infinium Human Methylation 450 beadchip. We investigated two models of DNA methylation alterations: (i) consistently altered methylation at specific CpGs or within specific genomic regions and (ii) stochastic DNA methylation alterations manifest as increased variability in genome-wide methylation patterns in men who smoke. We identified 141 significantly differentially methylated CpGs associated with smoking. In addition, we identified a trend toward increased variance in methylation patterns genome-wide in sperm DNA from men who smoke compared with never-smokers. These findings of widespread DNA methylation alterations are consistent with the broad range of offspring heath disparities associated with pre-conception paternal smoke exposure and warrant further investigation to identify the specific mechanism by which sperm DNA methylation perturbation confers risk to offspring health and whether these changes can be transmitted to offspring and transgenerationally.

Transmission of Toxoplasma: clues from the study of sea otters as sentinels of Toxoplasma gondii flow into the marine environment.

Toxoplasma gondii affects a wide variety of hosts including threatened southern sea otters (Enhydra lutris nereis) which serve as sentinels for the detection of the parasite's transmission into marine ecosystems. Toxoplasmosis is a major cause of mortality and contributor to the slow rate of population recovery for southern sea otters in California. An updated seroprevalence analysis showed that 52% of 305 freshly dead, beachcast sea otters and 38% of 257 live sea otters sampled along the California coast from 1998 to 2004 were infected with T. gondii. Areas with high T. gondii exposure were predominantly sandy bays near urban centres with freshwater runoff. Genotypic characterisation of 15 new T. gondii isolates obtained from otters in 2004 identified only X alleles at B1 and SAG1. A total of 38/50 or 72% of all otter isolates so far examined have been infected with a Type X strain. Type X isolates were also obtained from a Pacific harbor seal (Phoca vitulina) and California sea lion (Zalophus californianus). Molecular analysis using the C8 RAPD marker showed that the X isolates were more genetically heterogeneous than archetypal Type I, II and III genotypes of T. gondii. The origin and transmission of the Type X T. gondii genotype are not yet clear. Sea otters do not prey on known intermediate hosts for T. gondii and vertical transmission appears to play a minor role in maintaining infection in the populations. Therefore, the most likely source of infection is by infectious, environmentally resistant oocysts that are shed in the feces of felids and transported via freshwater runoff into the marine ecosystem. As nearshore predators, otters serve as sentinels of protozoal pathogen flow into the marine environment since they share the same environment and consume some of the same foods as humans. Investigation into the processes promoting T. gondii infections in sea otters will provide a better understanding of terrestrial parasite flow and the emergence of disease at the interface between wildlife, domestic animals and humans.

Identification of the first reported splice site mutation (IVS7-1G-->A) in the aminomethyltransferase (T-protein) gene (AMT) of the glycine cleavage complex in 3 unrelated families with nonketotic hyperglycinemia.

A novel splice site mutation (IVS7-1G-->A) in the T-protein gene (aminomethyltransferase, or AMT) of the glycine cleavage enzyme complex was found in a patient with nonketotic hyperglycinemia (NKH). A PCR/restriction enzyme method to detect this mutation was used to screen 100 NKH alleles and identified the mutation in three unrelated families.

Dirofilaria immitis superoxide dismutase: purification and characterization.

Superoxide dismutase (SOD) was purified to apparent homogeneity from Dirofilaria immitis, the causative agent of Dog Heartworm disease which is prevalent in the Southeastern United States. The enzyme has a molecular weight of 18,000 under denaturing conditions with an isoelectric point of 5.6. Both values are similar to those found for previously purified helminth SODs. The amino acid analysis shows greater similarity with mammalian SODs than with the published Schistosoma mansoni SOD, probably because the S. mansoni enzyme appears to be an extracellular, not a cytosolic, SOD. Although SOD activity is easily detected in D. immitis homogenates, the hydrogen peroxide scavenging activities of catalase and glutathione peroxidase were below the limits of our assay. This suggests that D. immitis primary defense against oxidants may be SOD. We feel that this line of research may provide valuable insights into a vulnerable area of D. immitis that may be a good target for drug therapy.

Immunisation of mice with fractions derived from the intestines of Dirofilaria immitis.

Antigens that are not normally seen by the host but that are nevertheless, accessible to host immune effector molecules and cells such as the native endoantigens associated with the intestinal epithelium of haematophagous tissue-dwelling parasites, could be potentially useful vaccine antigens. In this study, intestines were dissected from adult Dirofilaria immitis, homogenised, and a 105,000 x g pellet obtained and extracted with Triton X-100. The soluble 105,000 x g supernatant from this extract induced partial protection (51%) against a challenge infection of third stage larvae (L3) implanted in micropore chambers. Sera from mice immunised with this soluble detergent extract reacted with proteins ranging in size from 38 to 130 kDa. Immunolocalisation studies indicated the mouse sera reacted primarily to the lumenal surface of the intestines of adult D. immitis, though reactivity to the lateral nerve/epithelial chords, hypodermis and reproductive tracts was also noted, indicating the presence of shared antigens. Tissues of L3s were also recognised by the immunised mouse sera. These mouse sera did not react to a dog blood fraction prepared identically to the D. immitis fraction. Only those sera from D. immitis-infected dogs with heavy or long-term infections were reactive to a single 42 kDa protein. After 24 h incubation in fluorescein isothiocyanate-conjugated serum the intestinal tract of Onchocerca volvulus and D. immitis L3 and L4 fluoresced, indicating the serum had been ingested. These data suggest that filarial gut-associated antigens (apart from the single 42 kDa antigen) are not seen by normally infected hosts, that they can be accessible to antibodies and that they can induce an immune response which is partially protective.

An unusual genotype of Toxoplasma gondii is common in California sea otters (Enhydra lutris nereis) and is a cause of mortality.

Toxoplasma gondii-associated meningoencephalitis is a significant disease of California sea otters (Enhydra lutris nereis), responsible for 16% of total mortality in fresh, beachcast carcasses. Toxoplasma gondii isolates were obtained from 35 California otters necropsied between 1998 and 2002. Based on multi-locus PCR-restriction fragment length polymorphism and DNA sequencing at conserved genes (18S rDNA, ITS-1) and polymorphic genes (B1, SAG1, SAG3 and GRA6), two distinct genotypes were identified: type II and a novel genotype, here called type x, that possessed distinct alleles at three of the four polymorphic loci sequenced. The majority (60%) of sea otter T. gondii infections were of genotype x, with the remaining 40% being of genotype II. No type I or III genotypes were identified. Epidemiological methods were used to examine the relationship between isolated T. gondii genotype(s) and spatial and demographic risk factors, such as otter stranding location and sex, as well as specific outcomes related to pathogenicity, such as severity of brain inflammation on histopathology and T. gondii-associated mortality. Differences were identified with respect to T. gondii genotype and sea otter sex and stranding location along the California coast. Localised spatial clustering was detected for both type II (centred within Monterey Bay) and x (centred near Morro Bay)-infected otters. The Morro Bay cluster of type x-infected otters overlaps previously reported high-risk areas for sea otter infection and mortality due to T. gondii. Nine of the 12 otters that had T. gondii-associated meningoencephalitis as a primary cause of death were infected with type x parasites.

Cryopreservation of Trichinella spiralis muscle stage larvae.

A cryopreservation protocol for Trichinella spiralis muscle stage larvae is described. Larvae are pretreated in 10% bile at 37 degrees C for 1 hr to induce an increase in surface permeability, than incubated in 20% v/v ethanediol at 37 degrees C for 10 min, transferred to 0 degrees C for a second incubation step of 15 min in a v/v mixture of 33% ethanediol: 33% methanol: 34% saline at 0 degrees C followed by rapid cooling (approximately 5,100 degrees C min-1) of aliquots distributed onto glass coverslips. The larvae are thawed by dropping the coverslips into 2 ml saline at room temperature (20 degrees C) and immediately agitating, which simultaneously dilutes (1:100) the cryoprotectants. Groups of 6 mice were infected with muscle stage larvae by gastric intubation. Five days post-infection 14.5 +/- 3.2% of control untreated unfrozen MSL and 16.5 +/- 4.1% of unfrozen bile and cryoprotectant treated controls were recovered as adult worms from the small intestine. Of the cryopreserved larvae, 1.1 +/- 0.4% were recovered as adults, which represents 7.6% compared to the untreated unfrozen controls. When the bile pretreatment step was omitted fewer adult worms (0.09 +/- 0.04%) were recovered and no second generation muscle stage larvae were produced. Modifying this technique by omitting the first incubation step in 20% ethanediol and extending the second incubation step to 25 min yielded 72.3% recovery of T. nativa adult worms 5 days post-infection compared to unfrozen controls. The reproductive capacity index of bile treated cryopreserved T. spiralis was 2.5 +/- 0.6 compared to 51.8 +/- 18.8 for normal muscle stage controls.

Adoptive transfer of vaccine-induced resistance to Leishmania donovani.

BALB/c mice were immunized with three subcutaneous injections combining killed parasites and glucan, or were untreated. Spleen cells were transferred to syngeneic recipients. Mice which received 5 X 10(8) spleen cells from vaccinated donors demonstrated significant protection against Leishmania donovani challenge as compared to untreated mice receiving immune sera, or mice which received untreated spleen cells.

Leishmania donovani: immunization against infection as a function of parasite growth phase.

C57BL/6 mice were immunized against Leishmania donovani infection with a subcutaneous vaccination protocol. Groups received 3 injections at 4-day intervals combining glucan and killed promastigotes harvested from either logarithmic or stationary phase cultures. Controls were immunized with glucan alone, stationary or log phase promastigotes alone, or were untreated. All groups were challenged intravenously with stationary phase promastigotes at day 45 post-immunization. Results revealed that animals immunized with the glucan-killed parasite vaccine, utilizing promastigotes derived from either log (GPL) or stationary phase cultures (GPS), demonstrated significant resistance against infection as compared to controls or untreated mice. Additionally, the reduction in hepatic amastigote proliferation in mice immunized with GPS was significantly greater than in mice immunized with GPL.

Recovery of infective Schistosoma mansoni schistosomula from liquid nitrogen: a step towards storage of a live schistosomiasis vaccine.

Successful recovery of infective schistosomula of Schistosoma mansoni following storage at -196 degrees C is reported. The technique involves a two-step cooling procedure--slow cooling (0.65 degrees C min-1) to an intermediate temperature of -28 degrees C, followed by rapid cooling into liquid nitrogen (10,000 degrees C min-1). Rewarming (10,000 degrees C min-1) and rapid dilution to remove the cryoprotectant (17.5% methanol) yielded motile organisms some of which developed to adult worms in mice after intramuscular injection. The percentage of schistosomula developing to adult worms was small (0.44%), but is a significant step towards storage of trematode larvae and of a live attenuated vaccine for schistosomiasis.

Successful cryopreservation of Brugia pahangi third-stage larvae in liquid nitrogen.

Experiments are described which lead to the retention of infectivity of Brugia pahangi third-stage larvae after cooling to -196 degrees C. Methanol, a well documented cryoprotectant was used at a concentration of 20% (v/v). The schedule consisted of a 5 degrees C min-1 cool to an intermediate temperature of -21 degrees C and a subsequent rapid cool into liquid nitrogen. A rapid thaw of the parasites led to approximately 34% of motile cryopreserved larvae developing in multimammate rats (Mastomys natalensis) compared to unfrozen control larvae. Cooling rate and intermediate temperature were both found to be crucial variables affecting survival levels of the larvae.

Separation of viable and non-viable Onchocerca microfilariae using an ion exchanger.

A technique has been developed for separating viable and non-viable Onchocerca lienalis microfilariae (mff) by passing a suspension of the parasites through a column of DEAE cellulose. A proportion (between 42% and 87%) of normally motile parasites, whether unfrozen or cryopreserved, passed through the cellulose columns and retained their viability. Non-motile and sluggish mff were retained by the cellulose. The proportion of viable cryopreserved mff was greater after passage through the column as assessed by motility, migration in mice and development in the insect vector. Numbers of cryopreserved mff eluted through the columns peak quickly at a relatively high level after about 20 ml of eluate had been passed through, whereas normal unfrozen parasites rose and fell in numbers more slowly. By using sodium chloride gradients and buffers of differing conductivity it was concluded that the separation of the mff is probably due to net charge changes as well as motility. It is believed that this technique offers considerable promise as a tool to provide populations of mff of more uniform viability following freezing and thawing with existing cryopreservation techniques.

Schistosoma haematobium in the baboon (Papio anubis): effect of vaccination with irradiated larvae on the subsequent infection with percutaneously applied cercariae.

Groups of five baboons were vaccinated three times at approximately six-weekly intervals at a rate of 1,000 organisms per kg of gamma-irradiated Schistosoma haematobium larvae. Five vaccines were tested: 3 and 20 Krad cercariae applied percutaneously; fresh 3 and 20 Krad mechanically transformed schistosomula injected intramuscularly; and cryopreserved 20 Krad schistosomula injected intramuscularly. These five groups and an unvaccinated control group were challenged percutaneously with 7,500 S. haematobium cercariae three months after the last vaccination. The efficacy of the vaccines was judged by faecal egg excretion, and by adult worm and tissue egg recoveries at necropsy 4.5 months after challenge. Significant protection, with 64 to 89% reductions in worm burden and parallel reductions in egg production, was achieved by all but the cryopreserved vaccine, although egg production was not significantly reduced in those female worms which did mature. Cercariae tended to give more protection than schistosomula and 20 Krad more protection than 3 Krad. No significant pathology could be detected in an additional baboon vaccinated with 20 Krad schistosomula but not challenged with cercariae. This is an encouraging result for the development of a live vaccine against S. haematobium.

A note on the efficacy of a new class of compounds, 9-acridanone-hydrazones, against Schistosoma mansoni in a primate--the baboon.

Five 9-acridanone-hydrazone compounds were tested against moderately heavy Schistosoma mansoni infections in baboons. They were administered as a single oral dose at a rate of 50 mg/kg body-weight. Compared with results from an untreated control baboon, four of the compounds showed high levels of activity judged by the reduction or elimination of faecal egg production, adult worms and tissue eggs.

Partial protection of baboons against Schistosoma mansoni using radiation-attenuated cryopreserved schistosomula.

Three groups of five baboons were vaccinated in Kenya using three doses of 10,000 viable cryopreserved schistosomula attenuated with either 10, 20 or 60 krad 60Co-irradiation prepared in England. Animals were vaccinated at four-week intervals, challenged after a further six weeks with 2,000 cercariae and perfused at 10 weeks after challenge. High antibody titres to schistosomula mediating in vitro cytoadherence with P 388D1 macrophage-like cells were demonstrated in all vaccinated animals but not in controls. Significant titres to soluble egg antigen (SEA) were also demonstrated by ELISA in the 10 and 20 krad vaccinated groups following the first vaccination. The subsequent vaccinations and the challenge boosted this response considerably. Mean anti-SEA titres were only elevated above background in the 60-krad group six weeks after the third vaccination and in the challenge controls six weeks after challenge. Peripheral eosinophil counts were slightly reduced and neutrophil counts slightly elevated before challenge while eosinophil and erythrocyte counts were elevated and neutrophil counts depressed after challenge. PCV values were erratic in all groups. Eggs appeared in the faeces from six weeks after challenge and excretion rates were higher in all three vaccinated groups than in the challenge controls by necropsy 10 weeks after challenge. Body-weights were depressed in all groups after challenge but subsequently rose in the 10 and 20 krad groups. The 60 krad and challenge control groups lost 12.4% and 7.9% of body-weight respectively after challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme polymorphism in Trichinella.

The isoenzyme profiles of five isolates of the supposed 'species' of Trichinella, T. nativa, T. spiralis and T. nelsoni were compared. Four enzymes (AK, PGM, MPI and GPI) gave good resolution and clearly differentiated T. Spiralis from the other two species. T. nativa and T. nelsoni had similar isoenzyme patterns; the two separate isolates of T. nativa and T. spiralis used gave similar results, thus indicating the validity and the reproducibility of the technique. The value of enzyme electrophoresis for specific and subspecific classification of Trichinella is discussed and compared with the more traditional methods of taxonomy which have failed to resolve the controversy surrounding speciation.