Interferon lambda signals in maternal tissues to exert protective and pathologic effects in a gestational-stage dependent manner

Interferon lambda (IFN-λ, type III IFN) is constitutively secreted from human placental cells in culture and reduces Zika virus (ZIKV) transplacental transmission in mice. However, the roles of IFN-λ during healthy pregnancy and in restricting congenital infection remain unclear. Here we used mice lacking the IFN-λ receptor (Ifnlr1-/-) to generate pregnancies lacking either maternal or fetal IFN-λ responsiveness and found that the antiviral effect of IFN-λ resulted from signaling exclusively in maternal tissues. This protective effect depended on gestational stage, as infection earlier in pregnancy (E7 rather than E9) resulted in enhanced transplacental transmission of ZIKV. In Ifnar1-/- dams, which sustain robust ZIKV infection, maternal IFN-λ signaling caused fetal resorption and intrauterine growth restriction. Pregnancy pathology elicited by poly(I:C) treatment also was mediated by maternal IFN-λ signaling, specifically in maternal leukocytes, and also occurred in a gestational stage-dependent manner. These findings identify an unexpected effect of IFN-λ signaling specifically in maternal (rather than placental or fetal) tissues, which is distinct from the pathogenic effects of IFN-αβ (type I IFN) during pregnancy. These results highlight the complexity of immune signaling at the maternal-fetal interface, where disparate outcomes can result from signaling at different gestational stages.

Effect of the prenatal action of fulvestrant on the ovaries of the offspring of laboratory mice

Relevance. Fulvestrant is used for the treatment of breast cancer in combination with other drugs. The aim of the study was to determine the effect of the prenatal action of fulvestrant on the ovaries of the offspring of laboratory mice. Materials and Methods. The experimental animals were divided into 4 groups: intact, control and 2 experimental, 5 animals in each group. Injections were administered to females after fertilization at the gestational stage E 11.5 once intramuscularly. In the control group (n=5), sterile castor oil was administered at a dose of 0.8 mcg/kg. In the first experimental group (n=5), an antiestrogen was introduced in the form of an oil solution of fulvestrant 0.08 ml 0.0005% at a dose of 20 mcg/kg. In the second group (n=5), an antiestrogen was introduced in the form of an oil solution of fulvestrant 0.4 ml 0.0005% at a dose of 100 mcg/kg. Results and Discussion . The study revealed that in the ovaries when the drug was administered at a dose of 20 mcg/kg (F-20), the number of primordial follicles was reduced. Accordingly, the number of follicles of subsequent generations decreased. With the introduction of the drug fulvestrant 100 mcg/kg (F-100) on the section of the ovary, sclerosis of the stromal component is observed, accompanied by a rearrangement of the vascular network with signs of atresia and cystic degeneration of the follicular epithelium in the secondary and tertiary follicles, formed cysts are observed in the ovarian parenchyma. Conclusion. The prenatal effect of the drug fulvestrant on the maternal body during pregnancy leads to persistent structural changes in the ovaries of the offspring, manifested in the late stages of ontogenesis, which, in turn, can lead to violations of reproductive function. The depth and scale of these changes are dose-dependent.

Testing olfaction in equids: Age and gestational stage affects olfactory interest

In spite of the highly developed olfactory apparatus of horses, implying a high adaptive value, research on equine olfaction is sparse. Our limited knowledge poses a risk that horse behaviour does not match human expectations. The benefit of acquiring more knowledge of equine olfaction is therefore twofold; 1) it can aid the understanding of horse behaviour and hence reduce the risk of dangerous situations, and 2) there may be unexploited potential of using odours in several practical situations where humans interact with horses. This study investigated olfactory abilities of, 35 Icelandic, horses who were presented to four odours: peppermint, orange, lavender and cedarwood. The response variables were sniffing duration per presentation and behavioural reaction (licking, biting, snorting, and backing). Results showed horses were able to detect and distinguish between all four odours and showed increased interest (significantly longer sniffing duration) for peppermint. More horses expressed licking behaviour when presented to peppermint compared to cedarwood and lavender. Young horses sniffed cedarwood for longer than old horses, and pregnant mares sniffed lavender less than non-pregnant mares. In conclusion, the test paradigm seemed meaningful for horses, and olfactory interest of horses varied with age and gestational status but not sex.

Algorithms predicting gestational stage from the maternal steroid metabolome of mares

Hormone secretion by the maternal ovaries, trophoblast/placenta and fetus occurs sequentially, creating distinct steroid metabolomic “signatures” in systemic blood of pregnant mares that vary with gestational stage. Algorithms were developed to predict the gestational day (GD) from the maternal steroid metabolome [9 steroids; pregnenolone (P5), progesterone (P4), 5α-dihydroprogesterone (DHP), 17α-hydroxyprogesterone, allopregnanolone, 20α-hydroxy-DHP, 3β,20α-dihydroxy-DHP, dehydroepiandrosterone, androstenedione] determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) of eight thoroughbred mares bled longitudinally throughout pregnancy. A physiologically based model was developed to infer rates of steroid secretion during chorionic gonadotropin secretion, the luteo-placental shift, and by the equine feto-placenta unit, demonstrating more variability in P5 and DHP than P4. The average of four empirical models, using 9 steroids to predict GD, was calibrated (5 mares, R2 = 0.94, RMSE = 20 days) and validated (3 mares, R2 = 0.84, RMSE = 32 days). Validation performance was improved using paired samples taken 14 or 30 days apart (RMSE = 29 and 19 days, respectively). A second validation used an independent dataset (single serum samples from 56 mixed breed mares, RMSE = 79 days) and an additional longitudinal subset from the same population sampled monthly throughout gestation (7 mares, RMSE = 42 days). Again, using paired samples improved model performance (RMSE = 32.5 days). Despite less predictive performance of the mixed breed than the thoroughbred datasets, these models demonstrate the feasibility and potential for using maternal steroid metabolomic algorithms to estimate the stage of gestation in pregnant mares and perhaps monitor fetal development. (247 words)

Establishment and validation of a guinea pig model for human congenital toxoplasmosis

Background Toxoplasma gondii is an obligate intracellular parasite with a worldwide distribution. Congenital infection in humans and animals may lead to severe symptoms in the offspring, especially in the brain. A suitable animal model for human congenital toxoplasmosis is currently lacking. The aim of this study is to establish and validate the guinea pig as a model for human congenital toxoplasmosis by investigating the impact of the T. gondii infection dose, the duration of infection and the gestational stage at infection on the seroconversion, survival rate of dams, fate of the offspring, T. gondii DNA loads in various offspring tissues and organs and the integrity of the offspring brain. Methods Pregnant guinea pigs were infected with three different doses (10, 100, 500 oocysts) of T. gondii strain ME49 at three different time points during gestation (15, 30, 48 days post-conception). Serum of dams was tested for the presence of T. gondii antibodies using immunoblotting. T. gondii DNA levels in the dam and offspring were determined by qPCR. Offspring brains were examined histologically. Results We found the survival rate of dams and fate of the offspring to be highly dependent on the T. gondii infection dose with an inoculation of 500 oocysts ending lethally for all respective offspring. Moreover, both parameters differ depending on the gestational stage at infection with infection in the first and third trimester of gestation resulting in a high offspring mortality rate. The duration of infection was found to substantially impact the seroconversion rate of dams with the probability of seroconversion exceeding 50% after day 20 post-infection. Furthermore, the infection duration of dams influenced the T. gondii DNA loads in the offspring and the integrity of offspring brain. Highest DNA levels were found in the offspring brain of dams infected for ≥ 34 days. Conclusion This study contributes to establishing the guinea pig as a suitable model for human congenital toxoplasmosis and thus lays the foundation for using the guinea pig as a suitable animal model to study scientific questions of high topicality and clinical significance, which address the pathogenesis, diagnosis, therapy and prognosis of congenital toxoplasmosis. Graphical abstract

Transmission of Bluetongue Virus Serotype 8 by Artificial Insemination with Frozen–Thawed Semen from Naturally Infected Bulls

Transmission of bluetongue (BT) virus serotype 8 (BTV-8) via artificial insemination of contaminated frozen semen from naturally infected bulls was investigated in two independent experiments. Healthy, BT negative heifers were hormonally synchronized and artificially inseminated at oestrus. In total, six groups of three heifers received semen from four batches derived from three bulls naturally infected with BTV-8. Each experiment included one control heifer that was not inseminated and that remained BT negative throughout. BTV viraemia and seroconversion were determined in 8 out of 18 inseminated heifers, and BTV was isolated from five of these animals. These eight heifers only displayed mild clinical signs of BT, if any at all, but six of them experienced pregnancy loss between weeks four and eight of gestation, and five of them became BT PCR and antibody positive. The other two infected heifers gave birth at term to two healthy and BT negative calves. The BT viral load varied among the semen batches used and this had a significant impact on the infection rate, the time of onset of viraemia post artificial insemination, and the gestational stage at which pregnancy loss occurred. These results, which confirm unusual features of BTV-8 infection, should not be extrapolated to infection with other BTV strains without thorough evaluation. This study also adds weight to the hypothesis that the re-emergence of BTV-8 in France in 2015 may be attributable to the use of contaminated bovine semen.

Comprehensive Profiling of Secretome Formulations from Fetal- and Perinatal Human Amniotic Fluid Stem Cells

We previously reported that c-KIT+ human amniotic-fluid derived stem cells obtained from leftover samples of routine II trimester prenatal diagnosis (fetal hAFS) are endowed with regenerative paracrine potential driving pro-survival, anti-fibrotic and proliferative effects. hAFS may also be isolated from III trimester clinical waste samples during scheduled C-sections (perinatal hAFS), thus offering a more easily accessible alternative when compared to fetal hAFS. Nonetheless, little is known about the paracrine profile of perinatal hAFS. Here we provide a detailed characterization of the hAFS total secretome (i.e., the entirety of soluble paracrine factors released by cells in the conditioned medium, hAFS-CM) and the extracellular vesicles (hAFS-EVs) within it, from II trimester fetal- versus III trimester perinatal cells. Fetal- and perinatal hAFS were characterized and subject to hypoxic preconditioning to enhance their paracrine potential. hAFS-CM and hAFS-EV formulations were analyzed for protein and chemokine/cytokine content, and the EV cargo was further investigated by RNA sequencing. The phenotype of fetal- and perinatal hAFS, along with their corresponding secretome formulations, overlapped; yet, fetal hAFS showed immature oxidative phosphorylation activity when compared to perinatal ones. The profiling of their paracrine cargo revealed some differences according to gestational stage and hypoxic preconditioning. Both cell sources provided formulations enriched with neurotrophic, immunomodulatory, anti-fibrotic and endothelial stimulating factors, and the immature fetal hAFS secretome was defined by a more pronounced pro-vasculogenic, regenerative, pro-resolving and anti-aging profile. Small RNA profiling showed microRNA enrichment in both fetal- and perinatal hAFS-EV cargo, with a stably- expressed pro-resolving core as a reference molecular signature. Here we confirm that hAFS represents an appealing source of regenerative paracrine factors; the selection of either fetal or perinatal hAFS secretome formulations for future paracrine therapy should be evaluated considering the specific clinical scenario.

Kisspeptin and Hematologic Parameters as Predictive Biomarkers for First-Trimester Abortions

Objective: Spontaneous abortion is the most common complication of early pregnancy, affecting up to 20% of recognized pregnancies. Kisspeptin is predominantly released by placental syncytiotrophoblasts, and regulates their placental invasion into the uterine matrices. We aimed to establish an association of serum kisspeptin levels with pregnancy outcomes during the early gestational stage of the first trimester. Method: In this prospective study, 90 pregnant women in their 7 to 8 6/7 gestational weeks were classified into three groups: (i) The control group, consisting of healthy pregnant women (n=30), (ii) the threatened abortion group (n=30), and (iii) the spontaneous abortion group (n=30). The maternal serum samples were analyzed for complete blood count parameters and kisspeptin levels. Results: There was no statistical difference regarding body mass index (BMI) and gestational age (p=0.370). Regarding detailed obstetric notations, including gravida, parity, abortion, and living children, socioeconomic levels, and employment rates, all study groups were comparable (p>0.05, for all). No significant association was found regarding the biochemical parameters of complete blood count, including neutrophil, lymphocyte, and platelet concentrations, as well as neutrophil-to-lymphocyte ratios (NLR) and platelet-to-lymphocyte ratios (PLR) (p>0.05, for all). The median serum kisspeptin levels of the study groups did not differ between the groups (p=0.153). Correlation analysis revealed no significant relationship between serum kisspeptin levels and other study parameters in any study groups (p>0.05, for all) Conclusions: We found no statistically significant relationship between serum kisspeptin concentrations and pregnancy outcomes in the early gestational stage of the first trimester, and serum kisspeptin concentrations did not seem to be a reliable marker to distinguish abortion status from viable pregnancy

Temporal Changes in Physical Signs of Estrus and Validation of Fetal Parameters for Estimation of Gestational Stage through B-mode Ultrasonography in Beetal goats

The current study evaluates changes in the physical signs of estrus (vulvar hyperemia, edema and mucous discharge) from their onset until ovulation, and validates fetal parameters for the estimation of gestational stage in Beetal goats. In experiment 1, temporal changes in estrus signs were monitored subjectively after the administration of two injections of PGF2α at 10-days interval and a cumulative behavioural index (BI) was calculated in goats that showed estrus response (n=7). Ovulation occurred after 18.8±2.3 h relative to peak vulvar hyperemia and edema; ovulation coincided with maximum mucous discharge (0±2.4 h). Maximum BI was observed between 7 to 31 h after the onset of estrus and was highly correlated with vulvar hyperemia (r=0.94), followed by vulvar edema (r=0.90) and mucous discharge (r=0.85). In experiment 2, fetal parameters were monitored in pregnant goats (n=7) via ultrasonography for estimation of gestational stage, and for validation in a separate set of goats (n=20; experiment 3). Gestational stage correlated significantly (P<0.05) with the uterine diameter (UD), crown-rump length (CRL), trunk diameter (TD), intercostal space (ICS); diameter of amniotic vesicle (AV), biparietal diameter (BPD), placentome diameter (PD) and umbilical cord (UC) diameter. Validation revealed the difference between the actual and estimated day of gestation was less for TD than BPD (4.1±0.6 vs 6.8±1.0 days; P<0.05). In conclusion, vulvar hyperemia, edema, and mucous discharge are highly correlated with BI. Fetal parameters are strongly associated with the gestational stage, and the estimation of the actual gestational stage is less variable through TD than BPD in Beetal goats