Immunological Role of the Maternal Uterine Microbiome in Pregnancy: Pregnancies Pathologies and Alterated Microbiota.

Understanding what happens at the time of embryo implantation has been the subject of significant research. Investigators from many differing fields including maternal fetal medicine, microbiology, genetics, reproductive endocrinology and immunology have all been studying the moment the embryo interacts with the maternal endometrium. A perfect relationship between the uterus and the embryo, mediated by a tightly controlled interaction between the embryo and the endometrium, is required for successful implantation. Any factors affecting this communication, such as altered microbiome may lead to poor reproductive outcomes. Current theories suggest that altered microbiota may trigger an inflammatory response in the endometrium that affects the success of embryo implantation, as inflammatory mediators are tightly regulated during the adhesion of the blastocyst to the epithelial endometrial wall. In this review, we will highlight the various microbiome found during the periconceptual period, the microbiomes interaction with immunological responses surrounding the time of implantation, its effect on implantation, placentation and ultimately maternal and neonatal outcomes.

Risk of Chlamydia abortus transmission via embryo transfer using in vitro produced early bovine embryos.

The objectives of this study were to determine (i) whether Chlamydia (C.) abortus would adhere to the intact zona pellucida (ZP-intact) of early in vitro produced bovine embryos; (ii) whether the bacteria would adhere to the embryos (ZP-free) after in vitro infection; and (iii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol. The experimentation was made twice. For each replicate 100 (8-16-cell) bovine embryos produced in vitro were randomly divided into 10 batches. Height batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4 × 107Chlamydia/ml of AB7 strain. After incubation for 18 h at 37 °C in an atmosphere of 5% CO2, the embryos were washed in accordance with the IETS guidelines. In parallel, two batches (1 ZP-intact and 1 ZP-free) of 10 embryos were subjected to similar procedures but without exposure to C. abortus as a control group. The 10 washing fluids from each batch were collected and centrifuged for 1 h at 13,000×g. Each batch of washed embryos and each wash pellets were tested using PCR. C. abortus DNA was found in all ZP-intact and ZP-free batches of 10 embryos after 10 successive washes. For ZP-intact infected embryos, Chlamydia-DNA was also detected in all 10 wash baths for two batches (2/8) of embryos, whereas for ZP-free infected embryos, Chlamydia-DNA was detected in all 10 wash baths for 6/8 batches of embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. The bacterial load for batches of 10 embryos after the 10 wash baths was significantly higher for batches of ZP-free embryos (20.7 ±â€¯9 × 103 bacteria/mL) than for batches of ZP-intact embryos (0.47 ±â€¯0.19 × 103 bacteria/mL). These results demonstrate that C. abortus adheres to the ZP as well as the early embryonic cells of in vitro produced bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS fails to remove it.

Bartonella infections in three species of Microtus: prevalence and genetic diversity, vertical transmission and the effect of concurrent Babesia microti infection on its success.

BACKGROUND: Bartonella spp. cause persistent bacterial infections in mammals. Although these bacteria are transmitted by blood-feeding arthropods, there is also evidence for vertical transmission in their mammalian hosts. We aimed to determine: (i) the prevalence and diversity of Bartonella spp. in a Microtus spp. community; (ii) whether vertical transmission occurs from infected female voles to their offspring; (iii) the effect of concurrent Babesia microti infection on the success of vertical transmission of Bartonella; and (iv) the impact of congenital infection on pup survival. RESULTS: We sampled 124 Microtus arvalis, 76 Microtus oeconomus and 17 Microtus agrestis. In total, 115 embryos were isolated from 21 pregnant females. In the following year 11 pregnant females were kept until they had given birth and weaned their pups (n = 62). Blood smears and PCR targeting the Bartonella-specific rpoB gene fragment (333bp) were used for the detection of Bartonella. Bartonella DNA was detected in 66.8% (145/217) of the wild-caught voles. Bartonella infection was detected in 81.8% (36/44) of pregnant female voles. Bartonella-positive individuals were identified among the embryos (47.1%; 40/85) and in 54.8% (34/62) of pups. Congenitally acquired Bartonella infections and co-infection with B. microti had no impact on the survival of pups over a 3-week period post partum. Among 113 Bartonella sequences, four species were detected: Bartonella taylorii, Bartonella grahamii, Bartonella doshiae and a Bartonella rochalimae-like genotype. Bartonella taylorii clade B was the dominant species in wild-caught voles (49%), pregnant females (47%), their embryos (85%), dams (75%) and pups (95%). CONCLUSIONS: High prevalence of Bartonella spp. infection maintained in Microtus spp. community is followed by a high rate of vertical transmission of several rodent species of Bartonella in three species of naturally infected voles, M. arvalis, M. oeconomus and M. agrestis. Congenitally acquired Bartonella infection does not affect the survival of pups. Co-infection with B. microti does not affect the effectiveness of the vertical transmission of Bartonella in voles. Bartonella taylorii clade B was found to be the dominant species in wild-caught voles, including pregnant females and dams, and in their offspring, and was also found to be the most successful in vertical transmission.

Attachment of Coxiella burnetii to the zona pellucida of in vitro produced goat embryos.

Previous work demonstrated that after infection of in vivo derived caprine embryos, Coxiella burnetti (C. burnetii) showed a strong tendency to adhere to the zona pellicida (ZP). To investigate the risk of C. burnetii transmission via embryo transfer of in vitro-produced goat embryos the aim of this study was, (i) to evaluate the ability of C. burnetii to adhere to the intact zona pellicida of in vitro-produced goat embryos and to determine by confocal microscopy the location of the bacteria, (ii) to test the efficacy of IETS recommended rules for the washing of bovine embryos to eliminate C. burnetii. One hundred ZP-intact caprine embryos, produced in vitro, at the 8 to 16 cell stage, were randomly divided into 11 batches of eight to nine embryos. Nine batches were incubated for 18 h with 109Coxiella/ml of CbB1 strain (IASP, INRA Tours). The embryos then were recovered and washed in batches in 10 successive baths following the IETS guidelines. In parallel, two batches of embryos were subjected to similar procedures but without exposure to C. burnetii, to serve as the control group. One of the nine batches of infected embryos and one of the two non-infected control batches were separated to perform immunolabeling to locate the bacteria. C. burnetii DNA was detected by C-PCR in all eight batches of infected embryos after 10 successive washings. However, bacterial DNA was not detected in the embryo control batch. The first five washing media of the infected group were consistently found to be positive and Coxiella DNA was detected in the wash bath up to the 10th wash for two batches. After immunolabeling, the observation of embryos under confocal microscopy allowed C. burnetti to be found on the external part of the zona pellucida without deep penetration. This study clearly demonstrates that C. burnetii, after in vitro infection at 109Coxiella/ml, stick strongly to the external part of the zona pellucida of in vitro produced caprine embryos without deap penetration and that the 10 washings protocol recommended by IETS to eliminate the pathogenic agents of bovine embryos is unable to eliminate these bacteria from in vitro-produced goat embryo.

Whole Genome Amplification of Day 3 or Day 5 Human Embryos Biopsies Provides a Suitable DNA Template for PCR-Based Techniques for Genotyping, a Complement of Preimplantation Genetic Testing.

Our objective was to determine if whole genome amplification (WGA) provides suitable DNA for qPCR-based genotyping for human embryos. Single blastomeres (Day 3) or trophoblastic cells (Day 5) were isolated from 342 embryos for WGA. Comparative Genomic Hybridization determined embryo sex as well as Trisomy 18 or Trisomy 21. To determine the embryo's sex, qPCR melting curve analysis for SRY and DYS14 was used. Logistic regression indicated a 4.4%, 57.1%, or 98.8% probability of a male embryo when neither gene, SRY only, or both genes were detected, respectively (accuracy = 94.1%, kappa = 0.882, and p < 0.001). Fluorescent Capillary Electrophoresis for the amelogenin genes (AMEL) was also used to determine sex. AMELY peak's height was higher and this peak's presence was highly predictive of male embryos (AUC = 0.93, accuracy = 81.7%, kappa = 0.974, and p < 0.001). Trisomy 18 and Trisomy 21 were determined using the threshold cycle difference for RPL17 and TTC3, respectively, which were significantly lower in the corresponding embryos. The Ct difference for TTC3 specifically determined Trisomy 21 (AUC = 0.89) and RPL17 for Trisomy 18 (AUC = 0.94). Here, WGA provides adequate DNA for PCR-based techniques for preimplantation genotyping.

Can Chlamydia abortus be transmitted by embryo transfer in goats?

The objectives of this study were to determine (i) whether Chlamydia abortus would adhere to or penetrate the intact zona pellucida (ZP-intact) of early in vivo-derived caprine embryos, after in vitro infection; and (ii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. Fifty-two ZP-intact embryos (8-16 cells), obtained from 14 donors were used in this experiment. The embryos were randomly divided into 12 batches. Nine batches (ZP-intact) of five embryos were incubated in a medium containing 4 × 10(7)Chlamydia/mL of AB7 strain. After incubation for 18 hours at 37 °C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a phosphate buffer saline and 5% fetal calf serum solution in accordance with IETS guidelines. In parallel, three batches of ZP-intact embryos were used as controls by being subjected to similar procedures but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 hour at 13,000 × g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at -20 °C before examination for evidence of C. abortus using polymerase chain reaction. C. abortus DNA was found in all of the infected batches of ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the 10th wash fluid for seven batches of embryos, whereas for the two other batches, the last positive wash bath was the eighth and the ninth, respectively. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results report that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor goats to healthy recipients and/or their offspring. Nevertheless, the detection of C. abortus DNA by polymerase chain reaction does not prove that the bacteria found was infectious. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of caprine embryos infected by C. abortus would eliminate the bacteria from the ZP.

Bacterial and fungal contamination risks in human oocyte and embryo cryopreservation: open versus closed vitrification systems.

OBJECTIVE: To study the contamination risk in open and closed vitrification devices for oocyte/embryo cryopreservation by evaluating the contaminants present (bacteria and fungi) in the thaw medium and in liquid nitrogen (LN) storage containers. DESIGN: Retrospective study. SETTING: Human reproduction unit. PATIENT(S): None. INTERVENTION(S): Retrospective study of vitrification device safety and LN sterility performed from July to October 2014. MAIN OUTCOME MEASURE(S): From each bank container, both open and closed vitrification devices, devitrification media and LN in the containers and as supplied by the company were evaluated for contaminants. An automated system and the corresponding susceptibility to antibiotics were used for bacteria identification. Fungus detection was performed by evaluating the colony morphology and their microscopic characteristics. RESULT(S): No bacteria or fungi were observed in any of the devitrification media regardless of the type of device used, nor in the LN supplied by the company. No fungi were observed in any of the LN samples tested. Stenotrophomonas maltophilia and Bacillus spp. were found in all oocyte/embryo bank LN containers. There was no relationship between the number of samples or the time that each container had been used and the presence of microbiologic contaminants in the LN. At the container's bottom, Acinetobacter lwoffii, Alcaligenes faecalis ssp. faecalis, and Sphingomonas paucimobilis were found. CONCLUSION(S): Bacteria cross-contamination may not occur in oocyte/embryo banking in either open or closed storage devices. However, microorganisms can survive in LN. The bacteria cross-contamination risk is no greater for open than for closed containers. Storage containers should be cleaned periodically owing to the risk of lost straws or small particles of contaminated material.

Invasion and persistence of Mycoplasma bovis in embryonic calf turbinate cells.

Mycoplasma bovis is a wall-less bacterium causing bovine mycoplasmosis, a disease showing a broad range of clinical manifestations in cattle. It leads to enormous economic losses to the beef and dairy industries. Antibiotic treatments are not efficacious and currently no efficient vaccine is available. Moreover, mechanisms of pathogenicity of this bacterium are not clear, as few virulence attributes are known. Microscopic observations of necropsy material suggest the possibility of an intracellular stage of M. bovis. We used a combination of a gentamicin protection assay, a variety of chemical treatments to block mycoplasmas entry in eukaryotic cells, and fluorescence and transmission electron microscopy to investigate the intracellular life of M. bovis in calf turbinate cells. Our findings indicate that M. bovis invades and persists in primary embryonic calf turbinate cells. Moreover, M. bovis can multiply within these cells. The intracellular phase of M. bovis may represent a protective niche for this pathogen and contribute to its escape from the host's immune defense as well as avoidance of antimicrobial agents.

Shaping the norms that regulate international commerce of embryos.

As various embryo technologies in livestock were developed and evolved to a state of usefulness over the past 40 years, scientists with a specific interest in infectious diseases sought to determine the epidemiologic consequences of movement, especially international movement, of increasing numbers of embryos. Many of the foundational studies in this area were reported in Theriogenology, beginning in the 1970s and especially throughout the 1980s and 1990s. Unquestionably, Theriogenology has been a widely used venue for dissemination of basic information on this subject, which ultimately led to the development of the now universally accepted techniques for certification of embryo health. Today it is well-recognized that movement in commerce of embryos, especially in vivo-derived embryos, is a very low-risk method for exchange of animal germ plasm. This paper chronicles the evolution of strategies for health certification of embryos. An overview is provided of the calculated efforts of practitioners, scientists, and regulators to organize, forge necessary partnerships, stimulate needed research, provide purposeful analysis of the results, and, through these processes, guarantee the universal acceptance of efficient protocols for certifying the health of embryos intended for movement in international commerce.

In vitro development of murine embryos in presence of campylobacter fetus / Desarrollo de embriones murinos in vitro en presencia de campylobacter fetus

Bovine campylobacteriosis caused by Campylobacter fetus is associated with reproductive losses. The knowledge about the mechanisms of bacterial pathogenesis is limited, then a murine experimental model is proposed. BALB/c females and males were used. Two-cell embryos were cultured in Ham-F10 as control group (CG). Treatment groups were constituted by the addition of Cfv 1 and 3, or Cff 2 and 5. Morulae were placed in Ham-F10 (CG); treatment groups were constituted by the addition of Cfv27, CFF (cell-free filtrate) and Brucella broth (BB). Blastocysts were cultured in MEM (CG); challenge group were constituted by the addition of Cfv 27. Differentiation, hatching, hatched, adhesion and expansion were evaluated. Results were analyzed by Chi2 test. In two-cell embryo, the differentiation rate was not modified when the study strains were added (p > 0.05). The differentiation rate at 24 h for embryos at the morula stage was lower for BB, Cfv, and CFF, compared with CG (p < 0.05). After 48 h culture, no differences were observed in blastocyst formation for Cfv and BB, compared to CG (p > 0.05). However, the differentiation rate for the CFF group was lower than for CG (p < 0.05). At 48 and 72 h, the hatching rate was higher in CFF and Cfv groups than in CG (p < 0.05). Differences were not detected in blastocyst cultures. In conclusion, under these experimental conditions, Cf was not detrimental to the development of murine embryos. Efforts will be intensified to establish in vitro infection models that reproduce their pathogenicity.

La campilobacteriosis bovina caudada por Campylobacter fetus produce pérdidas reproductivas existiendo poca información de los mecanismos de patogenicidad de dicha bacteria, por lo cual se propone un modelo utilizando ratones BALC/c. Embriones de dos células fueron cultivados en Ham-F10: grupo control (GC), los grupos experimentales fueron adicionados con las cepas Cfv 1, Cfv 3, Cff 2 y Cff 5. Mórulas fueron cultivadas en Ham-F10 (GC); los grupos tratados recibieron Cfv27, CFF (filtrado libre de células) y caldo Brucella (BB). Blastocistos fueron cultivados en MEM (GC) y MEM más Cfv 27 (grupo desafiado). Se evaluó: diferenciación, "hatching", "hatched", adhesión y expansión. Los resultados fueron analizados por Chi2. En embriones de dos células, la diferenciación no fue modificada por acción de las cepas evaluadas (p > 0,05). Para embriones en estadío de mórula, la diferenciación a las 24 h de cultivo fue menor para BB, Cfv, y CFF, comparado con el GC (p < 0,05). Luego de 48 h de cultivo, no hubo diferencias entre Cfv, BB, y CG (p > 0,05), no obstante para el grupo CFF la diferenciación fue menor al CG (p < 0,05). El porcentaje de "hatching" (48 y 72 h de cultivo), fue mayor en los grupos CFF y Cfv comparado con el GC (p < 0,05). La adición de Cfv 27 no modificó el desarrollo de blastocistos. En el modelo propuesto, Cf no afectó negativamente el desarrollo embrionario. Futuros trabajos serán necesarios para establecer un modelo de infección in vitro en pos de reproducir su patogenicidad.

Listeria phospholipases subvert host autophagic defenses by stalling pre-autophagosomal structures.

Listeria can escape host autophagy defense pathways through mechanisms that remain poorly understood. We show here that in epithelial cells, Listeriolysin (LLO)-dependent cytosolic escape of Listeria triggered a transient amino-acid starvation host response characterized by GCN2 phosphorylation, ATF3 induction and mTOR inhibition, the latter favouring a pro-autophagic cellular environment. Surprisingly, rapid recovery of mTOR signalling was neither sufficient nor necessary for Listeria avoidance of autophagic targeting. Instead, we observed that Listeria phospholipases PlcA and PlcB reduced autophagic flux and phosphatidylinositol 3-phosphate (PI3P) levels, causing pre-autophagosomal structure stalling and preventing efficient targeting of cytosolic bacteria. In co-infection experiments, wild-type Listeria protected PlcA/B-deficient bacteria from autophagy-mediated clearance. Thus, our results uncover a critical role for Listeria phospholipases C in the inhibition of autophagic flux, favouring bacterial escape from host autophagic defense.

Francisella tularensis harvests nutrients derived via ATG5-independent autophagy to support intracellular growth.

Francisella tularensis is a highly virulent intracellular pathogen that invades and replicates within numerous host cell types including macrophages, hepatocytes and pneumocytes. By 24 hours post invasion, F. tularensis replicates up to 1000-fold in the cytoplasm of infected cells. To achieve such rapid intracellular proliferation, F. tularensis must scavenge large quantities of essential carbon and energy sources from the host cell while evading anti-microbial immune responses. We found that macroautophagy, a eukaryotic cell process that primarily degrades host cell proteins and organelles as well as intracellular pathogens, was induced in F. tularensis infected cells. F. tularensis not only survived macroautophagy, but optimal intracellular bacterial growth was found to require macroautophagy. Intracellular growth upon macroautophagy inhibition was rescued by supplying excess nonessential amino acids or pyruvate, demonstrating that autophagy derived nutrients provide carbon and energy sources that support F. tularensis proliferation. Furthermore, F. tularensis did not require canonical, ATG5-dependent autophagy pathway induction but instead induced an ATG5-independent autophagy pathway. ATG5-independent autophagy induction caused the degradation of cellular constituents resulting in the release of nutrients that the bacteria harvested to support bacterial replication. Canonical macroautophagy limits the growth of several different bacterial species. However, our data demonstrate that ATG5-independent macroautophagy may be beneficial to some cytoplasmic bacteria by supplying nutrients to support bacterial growth.

Structural and functional integrity of spermatozoa is compromised as a consequence of acute uropathogenic E. coli-associated epididymitis.

Uropathogenic Escherichia coli (UPEC)-associated epididymitis is commonly diagnosed in outpatient settings. Although the infection can be successfully cleared using antimicrobial medications, 40% of patients unexplainably show persistent impaired semen parameters even after treatment. Our aim was to investigate whether pathogenic UPEC and its associated virulence factor hemolysin (hlyA) perturb the structural and functional integrity of both the epididymis and sperm, actions that may be responsible for the observed impairment and possibly a reduction of fertilization capabilities. Semen collected from patients diagnosed with E. coli-only related epididymitis showed that sperm counts were low 14 days postantimicrobial treatment regardless of hlyA status. At Day 84 following treatment, hlyA production correlated with approximately 4-fold lower sperm concentrations than in men with hlyA-negative strains. In vivo experiments with the hlyA-producing UPEC CFT073 strain in a murine epididymitis model showed that just 3 days postinfection, structural damage to the epididymis (epithelial damage, leukocyte infiltration, and edema formation) was present. This was more severe in UPEC CFT073 compared to nonpathogenic E. coli (NPEC 470) infection. Moreover, pathogenic UPEC strains prematurely activated the acrosome in vivo and in vitro. Raman microspectroscopy revealed that UPEC CFT073 undermined sperm integrity by inducing nuclear DNA damage. Consistent with these observations, the in vitro fertilization capability of hlyA-treated mouse sperm was completely abolished, although sperm were motile. These findings provide new insights into understanding the possible processes underlying clinical manifestations of acute epididymitis.

Microorganisms in cryopreserved semen and culture media used in the in vitro production (IVP) of bovine embryos identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS).

Commercial cattle breeders produce their own herd offspring for the dairy and beef market using artificial insemination. The procedure involves sanitary risks associated with the collection and commercialization of the germplasm, and the in vitro production and transfer of the bovine embryos must be monitored by strict health surveillance. To avoid the spreading of infectious diseases, one must rely on using controlled and monitored germplasm, media, and reagents that are guaranteed free of pathogens. In this article, we investigated the use of a new mass spectrometric approach for fast and accurate identification of bacteria and fungi in bovine semen and in culture media employed in the embryo in vitro production process. The microorganisms isolated from samples obtained in a commercial bovine embryo IVP setting were identified in a few minutes by their conserved peptide/protein profile, obtained applying matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), matched against a commercial database. The successful microorganisms MS identification has been confirmed by DNA amplification and sequencing. Therefore, the MS technique seems to offer a powerful tool for rapid and accurate microorganism identification in semen and culture media samples.

Teratogenicity of Staphylococcus aureus L-forms using a mouse whole-embryo culture model.

Our previous studies have suggested that Staphylococcus aureus L-forms are able to pass through the placental barrier of mice from the maternal side to the fetal body and affect fetal growth and development, but little is known about the direct influence of S. aureus L-forms on embryos during the critical period of organogenesis. Mouse embryos at gestational day 8.5 were cultured in vitro for 48 h with 0, 50, 100, 200 or 400 c.f.u. S. aureus L-forms ml(-1). At the end of the culture period, the mouse embryos were assessed morphologically for viability, growth and development. Bacteriological and immunohistochemical staining were used to determine the existence of S. aureus L-forms in embryonic tissues. We found that both crown-rump length and head length of mouse embryos exposed to S. aureus L-forms at a concentration of 50 c.f.u. ml(-1) were reduced. When the mouse embryos were exposed to 100, 200 or 400 c.f.u. S. aureus L-forms ml(-1), the total morphological score, number of somites, dry embryo weight, yolk sac diameter, crown-rump length and head length were significantly lower than those of the control group. With the increased concentration of S. aureus L-forms in the culture medium, there were fewer normally developed embryos and more embryos with abnormalities or retardation in growth. S. aureus L-forms detected by Gram-staining and immunohistochemical detection of antigen were found in the tissues of embryos infected by S. aureus L-forms. These data suggest that S. aureus L-forms exert a direct teratogenic effect on cultured mouse embryos in vitro.

IFNγ inhibits the cytosolic replication of Shigella flexneri via the cytoplasmic RNA sensor RIG-I.

The activation of host cells by interferon gamma (IFNγ) is essential for inhibiting the intracellular replication of most microbial pathogens. Although significant advances have been made in identifying IFNγ-dependent host factors that suppress intracellular bacteria, little is known about how IFNγ enables cells to recognize, or restrict, the growth of pathogens that replicate in the host cytoplasm. The replication of the cytosolic bacterial pathogen Shigella flexneri is significantly inhibited in IFNγ-stimulated cells, however the specific mechanisms that mediate this inhibition have remained elusive. We found that S. flexneri efficiently invades IFNγ-activated mouse embryonic fibroblasts (MEFs) and escapes from the vacuole, suggesting that IFNγ acts by blocking S. flexneri replication in the cytosol. This restriction on cytosolic growth was dependent on interferon regulatory factor 1 (IRF1), an IFNγ-inducible transcription factor capable of inducing IFNγ-mediated cell-autonomous immunity. To identify host factors that restrict S. flexneri growth, we used whole genome microarrays to identify mammalian genes whose expression in S. flexneri-infected cells is controlled by IFNγ and IRF1. Among the genes we identified was the pattern recognition receptor (PRR) retanoic acid-inducible gene I (RIG-I), a cytoplasmic sensor of foreign RNA that had not been previously known to play a role in S. flexneri infection. We found that RIG-I and its downstream signaling adaptor mitochondrial antiviral signaling protein (MAVS)--but not cytosolic Nod-like receptors (NLRs)--are critically important for IFNγ-mediated S. flexneri growth restriction. The recently described RNA polymerase III pathway, which transcribes foreign cytosolic DNA into the RIG-I ligand 5'-triphosphate RNA, appeared to be involved in this restriction. The finding that RIG-I responds to S. flexneri infection during the IFNγ response extends the range of PRRs that are capable of recognizing this bacterium. Additionally, these findings expand our understanding of how IFNγ recognizes, and ultimately restricts, bacterial pathogens within host cells.

A review of the risk of contamination of semen and embryos during cryopreservation and measures to limit cross-contamination during banking to prevent disease transmission in ET practices.

This review summarizes pertinent data and opinions regarding the potential hazard of disease transmission through cryopreserved and banked embryos in liquid nitrogen (LN). Special attention is given to the survival of pathogens in LN, new vitrification methods, sterility of LN, risks associated with the use of straws and cryovials, and LN dewars including dry shippers. It was experimentally demonstrated that cross-contamination between LN and embryos may occur, when infectious agents are present in LN and embryos are not protected by a sealed container. It is important, therefore, to prevent direct contact of embryos with LN during cryopreservation and their banking. This includes the usage of hermetically sealed, high-quality, shatter-proof freezing containers and/or the application of a secondary enclosure such as "double bagging or straw in straw." A periodic disinfection of cryo-dewars should be considered as an additional precaution to diminish the potential for inadvertent cross-contamination. It might be advisable to use separate LN dewars to quarantine embryos derived from infected donors of valuable genotype or from unknown health status, extinction-threatened species. Nevertheless, in summary, it has been concluded that over 25 yr with no direct evidence of disease transmission by transferred cryopreserved human and animal embryos, that the present cryopreservation technology is sanitary sound, with the stipulation that biocontainment measures recommended by the International Embryo Transfer Society (IETS) and the World Organization for Animal Health - Office International des Epizooties (OIE), are strictly followed.

Risk of contamination of germplasm during cryopreservation and cryobanking in IVF units.

Cryopreservation of sperm, embryos and, more recently, oocytes plays an important and increasing role in assisted reproduction, due to improvements of old, and introduction of new technologies. In parallel, concerns are increasing about the technical and biological safety of these procedures. However, published data regarding the confirmed or theoretical hazards of these procedures are sparse and sometimes contradictory. The purpose of this review will summarize data and opinions about one of the most disputed risks, the potential hazard of contamination and disease transmission through cryopreservation. Special attention is concentrated on the weak points of the technology including open vitrification systems, sterilization of liquid nitrogen and safety of commonly used storage tanks including straws and cryovials. Suggestions are also made for practical measures to avoid these dangers while preserving the benefits and perspectives of new cryopreservation technologies.

In vitro fertilization outcomes after transfer of embryos contaminated with yeast.

In this retrospective, matched-paired study, yeast in the embryo culture medium was associated with a trend toward decreased developmental competency that was more pronounced when observed early in culture. Because live births occurred after transfer of embryos in the yeast-contaminated group, we concluded that yeast contamination is not a reason to cancel embryo transfer (ET).