Peptidoglycan disrupts early embryo-maternal crosstalk via suppression of ISGs expression induced by interferon-tau in the bovine endometrium.

Uterine infection with bacteria and the release of peptidoglycan (PGN), antigenic cell wall components of both Gram-negative and Gram-positive bacteria, can cause early pregnancy losses in ruminants, but the associated mechanisms remain unsolved. Day 7 blastocyst starts to secrete a minute amount of interferon-tau (IFNT) in the uterine horn which is required for early stage of maternal recognition of pregnancy (MRP) in ruminants, and it induces interferon stimulated genes (ISGs) for driving uterine receptivity in cows. This study investigated if PGN disrupts IFNT response through modulation of endometrial ISGs expressions. Cultured bovine endometrial epithelial cells (BEECs) were treated with embryo culture medium (ECM) or IFNT (1 ng/ml) in the presence or absence of a low level of PGN (10 pg/ml) for 24 h. A real-time PCR analyses revealed that the presence of PGN suppressed IFNT-induced ISGs (OAS1 and ISG15) and STAT1 expressions in BEECs. To visualize the impact of PGN in an ex-vivo model that resembles the in vivo status, endometrial explants were treated by IFNT (1 ng/ml) with or without PGN (10 pg/ml) for 12 h. PGN suppressed IFNT-induced gene expressions of the above factors, but not for IFNA receptor type1 (IFNAR1) or type2 (IFNAR2) in explants. Immunofluorescence analysis illustrated that PGN completely suppressed the IFNT-triggered OAS1 protein expression in the luminal epithelium of explants. Of note, PGN did not stimulate pro-inflammatory cytokines (TNFA and IL1B) or TLR2 mRNA expression in both models. These findings indicate that the presence of low levels of PGN suppresses ISGs expression induced by IFNT secreted from early embryo, at the luminal epithelium of the bovine endometrium. This could severely interfere with early stage of MRP processes in cows, leading to pregnancy failure.

Does dysbiotic endometrium affect blastocyst implantation in IVF patients?

PURPOSE: To analyze the pregnancy outcomes of IVF patients presenting eubiotic or dysbiotic endometrium at the time of embryo transfer and to analyze what bacterial profiles are suitable for embryo implantation. METHODS: Ninety-nine IVF patients under 40 years old undergoing vitrified-warmed blastocyst transfer in HRT cycle had concurrent endometrial microbiome analysis. Samples from the endometrium were taken from the participants at the time of mock transfer; the bacterial profiles at genus level and percentage of lactobacilli in the endometrium of the patients were analyzed. RESULTS: Thirty-one cases (31.3%) had dysbiotic endometrium. The background profiles, pregnancy rates per transfer (52.9% vs 54.8%), and miscarriage rates (11.1% vs 5.9%) were comparable between patients with eubiotic or dysbiotic endometrium. Major bacterial genera other than Lactobacillus detected in the dysbiotic endometrium were Atopobium, Gardnerella, and Streptococcus. Some patients achieved ongoing pregnancies with 0% Lactobacillus in the endometrium. The endometrial bacterial profiles of pregnant cases with dysbiotic endometrium were comparable with those of non-pregnant cases. CONCLUSION: Analyzing microbiota at the species-level resolution may be necessary for identifying the true pathogenic bacteria of the endometrium and avoiding over-intervention against non-Lactobacillus microbiota. Further studies are necessary for analyzing the mechanism of how the pathogenic bacteria affect embryo implantation.

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.

Lipopolysaccharide-induced modulation in the expression of progesterone receptor and estradiol receptor leads to early pregnancy loss in mouse.

The objective of the present study was to investigate the effect of Gram-negative bacteria infection on ovarian steroid receptors, i.e. progesterone receptor (PR) and estradiol receptor (ER) during preimplantation days of pregnancy. A well established mouse model of Gram-negative bacteria infection was used to test this objective. Mice were treated with normal saline or lipopolysaccharide (LPS) on day 0.5 of pregnancy and used to collect embryos and uterine horns on day 1.5 to day 4.42 preimplantation day of pregnancy. Total RNA was extracted and reverse-transcription polymerase chain reaction (PCR) was performed to check the expression of PR and ER genes. The mRNA expression of PR and ER was altered in embryos and uterus of LPS-treated animals during preimplantation days of pregnancy studied. These results suggest that PR and ER play an important role in Gram-negative bacteria infection and induced implantation failure in mouse.

A reliable procedure for decontamination before thawing of human specimens cryostored in liquid nitrogen: three washes with sterile liquid nitrogen (SLN2).

OBJECTIVE: To report a washing procedure, to be performed as frozen specimens are taken out of cryobanks, to minimize the risk of hypothetical culture contamination during thawing. DESIGN: Basic research. SETTING: Private assisted reproduction center. INTERVENTION(S): Two batches of liquid nitrogen (LN(2)) were experimentally contaminated, one with bacteria (Pseudomonas aeruginosa, Escherichia coli, Stenotrophomonas maltophilia) and the other with fungi (Aspergillus niger). Two hundred thirty-two of the most common human gamete/embryo vitrification carriers (Cryotop, Cryoleaf, Cryopette) were immersed in the contaminated LN(2) (117 in the bacteria and 25 in the fungi-contaminated LN(2)). The carriers were tested microbiologically, one group without washing (control) and the other after three subsequent washings in certified ultraviolet sterile liquid nitrogen (SLN(2)). The carriers were randomly allocated to the "three-wash procedure" (three-wash group, 142 carriers) or "no-wash" (control group, 90 carriers) using a specific software tool. MEAN OUTCOME MEASURE(S): Assessment of microorganism growth. RESULT(S): In the no-wash control group, 78.6% of the carriers were contaminated by the bacteria and 100% by the fungi. No carriers were found to be contaminated, either by bacteria or fungi, after the three-wash procedure. CONCLUSION(S): The three-wash procedure with SLN(2) produced an efficient decontamination of carriers in extreme experimental conditions. For this reason, this procedure could be routinely performed in IVF laboratories for safe thawing of human specimens that are cryostored in nonhermetical cryocontainers, particularly in the case of open or single-straw closed vitrification systems.

Mycoplasma contamination of murine embryonic stem cells affects cell parameters, germline transmission and chimeric progeny.

Murine embryonic stem cells (mESCs) inoculated at passage P13 with the mycoplasma species M. hominis, M. fermentans and M. orale and cultured over 20 passages showed reduced growth rate and viability (P < 0.0001) compared to control mESCs. Spectral karyotypic analysis of mycoplasma-infected mESCs showed a number of non-clonal chromosomal aberrations which increased with the duration of infection. The differentiation status of the infected mESCs was most affected at passage P13+6 where the infection was strongest and 46.3% of the mESCs expressed both POU5F1 and SSEA-1 markers whereas 84.8% of control mESCs expressed both markers. The percentage of germline chimeras from mycoplasma-infected mESCs was examined after blastocyst injection and embryo transfer to suitable recipients at different passages and, compared to the respective control group, was most affected at passage P13+5 (50% vs. 90%; P < 0.07). Further reductions were obtained at the same passage in the percentage of litters born (50% vs. 100%; P < 0.07) and in the percentage of pups born (22% vs. 45%; P < 0.001). Thirty three chimeras (39.8%) obtained from blastocyst injection with mycoplasma-infected mESCs showed reduced body weight (P < 0.0001), nasal discharge, osteoarthropathia, and cachexia. Flow cytometric analysis of plasma from chimeras produced with mycoplasma-infected mESCs revealed statistically significant differences in the proportions of T-cells and increased levels of IgG1 (P < 0.001), IgG2a (P < 0.05) and IgM (P < 0.05), anti-DNA antibodies (P < 0.05) and rheumatoid factor (P < 0.01). The present data indicate that mycoplasma contamination of mESCs affects various cell parameters, germline transmission, and postnatal development of the resulting chimeras.

West Nile virus infection induces susceptibility of in vitro outgrown murine blastocysts to specific lysis by paternally directed allo-immune and virus-immune cytotoxic T cells.

Day 3 post-coitum BALB/c and (BALB/c x CBA/H)F1 blastocysts were isolated and hatched in replicate wells. Some were treated with interferon-gamma (IFN-gamma). Whilst others were infected with West Nile Virus (WNV) at 100 plaque-forming units per cell, for 18 h. Controls were mock-treated. Gamma-irradiated (2000 rads) CBA/H, (paternal) WNV-specific and allo(CBA/H)-specific cytotoxic T (Tc) cells were then added to replicates of infected, mock-infected or IFN-gamma-treated cultures for 20 h. [3H]Thymidine was then added for a further 8 h. [3H]Thymidine incorporation was inhibited by 40-50% in WNV-infected cultures exposed to WNV-paternal-specific Tc cells and by 30-40% in WNV-infected cultures exposed to allo-paternal-specific Tc cells compared to similarly exposed, uninfected, or unexposed, WNV-infected, or unexposed, uninfected cultures. No significant differences in [3H]thymidine incorporation were found between these controls and IFN-gamma-treated cultures exposed to allo-paternal-specific Tc cells or IFN-gamma-treated cultures not exposed to Tc cells. Parallel exposure of L929 fibroblasts to the same Tc cells irradiated with 500-8000 rads in doubling doses, showed that irradiation did not alter the efficacy or specificity of the Tc cells. Relevance to maternal anti-viral immune responses during implantation is discussed.

Susceptibility of mouse embryo to murine cytomegalovirus infection in early and mid-gestation stages.

The susceptibility of mouse embryonic cells to murine cytomegalovirus (MCMV) infection was studied by injecting the virus in the early and mid-gestation stages. For the early stage, blastocysts from BDF1 mice were injected with MCMV or minimal essential medium (MEM) by micromanipulator and returned to the uteri of pseudopregnant ICR mice. On day 11 of gestation, the embryos were examined immunohistochemically, using antibody specific to the early antigen of MCMV, and the placentae were examined by plaque assay. No infection was detected by either method. Furthermore, no infection was detected in MCMV-infected blastocysts that were cultured and examined for infection by immunofluorescence. For mid-gestation embryos, the conceptus was injected with MCMV on day 8.5 of gestation and was subjected to immunohistochemical analysis from day 10.5 to 12.5 of gestation. Viral antigen-positive cells were first observed in the placentae, then antigen-positive cells appeared among the blood cells, endothelial and mesodermal cells of the embryos. On day 12.5 of gestation, clusters of viral antigen-positive cells were sometimes observed in the hearts and livers. Although the incidence was lower, viral antigen-positive cells were also observed in the neuroectoderm and the eyes. These results suggest that MCMV does not infect early embryos and that infection first occurs in the placenta of postimplantation embryos, whence it extends through the blood cells to the endothelial and mesodermal cells of different embryonic regions, eventually extending to the neuroectoderm.

Host range specificity of polyomavirus EC mutants in mouse embryonal carcinoma and embryonal stem cells and preimplantation embryos.

New polyomavirus mutants (PyEC-C) selected on LT1 cells and exhibiting a strong cytopathic effect in all embryonal carcinoma (EC) cell lines tested have been isolated. They were derived by a sequence duplication event from a new multiadapted mutant isolated in PCC4 cells. A quantitative analysis of viral DNA replication and transcription in 3T6 and EC cell lines was performed to compare PyEC-C mutants and PyEC mutants previously isolated on F9 or PCC4 cell lines. Analysis of the results indicated that PyEC-C mutants were more efficient in all EC cell lines tested than all other PyEC mutants; on the contrary, they were less adapted to 3T6 cells than wild-type polyomavirus. In both 3T6 and EC cells, uncoupling between early transcription and viral DNA replication was observed; different viruses were shown to replicate with the same efficiency, while their levels of early transcripts differed by two orders of magnitude. Attempts to correlate the genome structure of the mutants with their biological properties indicate that duplication of protein-binding sequences is not the only event responsible for their phenotype. PyEC mutants were also analyzed with respect to their interactions with early mouse embryos and embryonal stem (ES) cell lines derived from the inner cell mass of blastocysts. They showed different degrees of expression in ES cells and preimplantation embryos. ES cells were most efficiently infected and lysed by mutants which exhibit both a multiadapted and a lytic phenotype in EC cells. Preimplantation embryos were not permissive to any PyEC mutants. However, EC-multiadapted mutants were infectious in blastocysts after two days of in vitro culture.

Active ras and myc oncogenes can be compatible, but Sv40 large T antigen is specifically suppressed with normal differentiation of mouse embryonic stem cells.

The pathobiological effects of oncogenes on normal differentiation of mouse embryonic stem cells from 4-day embryos were examined by introducing active ras, myc, and SV40 large T genes, all driven by mouse metallothionein I enhancer and promoter. Stem cell clones R5, M3, and T2 for ras, myc, and SV40 T genes, respectively, were particularly chosen for analyses because of their higher levels of transgene expression and their diploid chromosomal constitutions. These stem cells were then introduced into host 4-day embryos and the embryos were allowed to develop in the uterus of foster mothers. The stem cells colonized the tissues as extensively as the parent cells and gave rise to adult chimera with no apparent loss or abnormality of the embryos. The active ras and myc oncogenes introduced were expressed not only in the stem cells, but also in the developing embryos and in a variety of tissues of adult chimeras. However, although T antigen was originally expressed in the stem cells, it was not expressed in either developing embryos or tissues of adult chimeras. Induced by retinoic acid treatment in vitro or by subcutaneous grafting, this suppression of T-gene expression was also confirmed in differentiated progeny cells from several stem cell clones expressing T antigen. Permanent lines of fibroblast-like cells could be established at higher frequency from primary cultures of tissues of chimera, subcutaneous differentiated cells, and in vitro differentiated cells derived from T2 cells, and all these clones reexpressed T antigen. The results suggest that active myc and ras genes can be compatible with normal differentiation of the stem cells, but the expression of T antigen is specifically suppressed with recognition of its coding domain.

Presence of the adenovirus E1A-like activity in preimplantation stage mouse embryos.

The presence of the adenovirus E1A-like activity in embryonal carcinoma stem cells has been reported. We now show that preimplantation stage mouse embryonic cells allow transcription of the E1A-dependent E2A gene when infected with E1A-deleted mutant dl312, indicating the presence of the E1A-like activity in morulae and blastocysts. Moreover, such activity seems to decrease or disappear at about the time of implantation.

Expression of simian virus 40 early and late genes in mouse oocytes and embryos.

Simian virus 40 (SV40) large- and small-tumor antigens (T-Ag, t-Ag) are normally synthesized early after infection of either permissive (monkey) or nonpermissive (mouse) fibroblasts, whereas an equivalent amount of viral coat protein (V-Ag) is observed late after infection of permissive cells and only after viral DNA replication has occurred. To determine whether or not expression of these genes is regulated in the same manner during early mammalian development, SV40 DNA was injected into the nuclei of mouse oocytes and one- and two-cell embryos. In oocytes, about three times more V-Ag was produced than T-Ag, and both were synthesized concomitantly in the same cells. Viral mRNA and proteins synthesized in oocytes comigrated during gel electrophoresis with the same products synthesized in SV40-infected monkey cells. Viral gene expression required circular DNA molecules injected into the nuclei of transcriptionally and translationally active cells. Injected DNA was stable and underwent conformational changes consistent with chromatin assembly. Oocytes did not replicate either polyomavirus or SV40 DNA. Thus, the temporal order of viral gene expression is circumvented in mouse germ cells, allowing these proteins to be expressed concurrently and in equivalent amounts with no requirement for DNA replication. However, in preimplantation embryos, neither T-Ag nor V-Ag was detected by immunoprecipitation although T-Ag synthesis was demonstrated as a specific requirement for SV40 DNA replication. Thus, viral gene expression in mouse embryos as early as the one-cell stage was reduced at least 500-fold relative to that in oocytes. Similarities between SV40 gene expression in mouse oocytes and that in Xenopus oocytes suggest that germ cells in higher animals share common regulatory mechanisms.

Expression of virus-like particles in feline preimplantation embryos.

Embryos recovered from random-source domestic cats between 1 and 7 days after ovulation and unfertilized oocytes recovered within 6 hours of ovulation were examined ultrastructurally for the presence of types A and C viruses. Intracisternal type A particles were found consistently within cells of the inner cell mass in blastocysts but not within trophoblast cells or cells from earlier stages of development. Mature type C viruses were not observed. Novel virus-like particles were found within cytoplasmic cisternae in many of the embryos; these particles did not appear to be associated with a particular stage of embryonic development.

Effects of viral exposure of the two-cell mouse embryo on cleavage and blastocyst formation in vitro.

The effect of viral exposure of two-cell mouse embryos on their capacity to undergo subsequent cleavage and blastocyst formation in vitro was determined. Exposure to Coxsackie viruses B-4 and B-6, reovirus type 2, influenza virus type A, mouse cytomegalovirus, adenovirus type 5, and mouse adenovirus resulted in statistically significant inhibition of blastocyst formation. Development in vitro was unaffected by exposure to ECHO virus type 11, attenuated poliomyelitis virus type 2, parainfluenza virus type 1, mumps, rubella, and herpes simplex viruses types 1 and 2. Blastocyst formation was also unaffected by exposure of embryos to mouse interferon in a concentration 24 units/ml of culture fluid. Coxsackie virus B-4 was recovered from exposed embryos.

Infection of mouse preimplantation embryos with simian virus 40 and polyoma virus.

Mouse two-cell embryos, morulae, and blastocysts were killed when infected in vitro with simian virus 40 (SV40) at high multiplicities of infection. Polyoma virus was not deleterious for preimplantation embryos, even at a very high multiplicity of infection; however, the outgrowths of polyoma-infected blastocysts disintegrated after several days of culture. Indirect immunofluorescence tests revealed the presence of SV40 T and V antigens and polyoma virus V antigen in the nuclei of trophoblastic cells. Virus-specific antigens were not found in the nuclei of cells forming inner cell masses of blastocysts or in inner cell mass-derived cells in blastocyst out-growths. The appearance of SV40 T and V antigens in the nuclei was inhibited by alphaamanitin, a RNA polymerase II inhibitor. The amount of infectious virus recovered from cultures of morulae or blastocysts on subsequent days after infection with SV40 initially declined but later increased. These points of evidence indicate that some cells of early mouse embryos are permissive for the expression of early and late functions of SV40 genome and that susceptibility to infection with polyoma virus and/or permissiveness for the expression of polyoma virus late functions develop gradually between the two-cell and blastocyst stages. Electron microscope observations showed the presence of specific complexes of membranes and virions in the cytoplasm of trophoblastic cells. Single viral particles could be found in the nuclei and also in mitochondria.