Depletion of Ppp6c in hematopoietic and vascular endothelial cells causes embryonic lethality and decreased hematopoietic potential.

Protein phosphatase 6 (PP6) is a serine/threonine (Ser/Thr) protein phosphatase, and its catalytic subunit is Ppp6c. PP6 forms the PP2A subfamily with PP2A and PP4. The diverse phenotypes observed following small interfering RNA (siRNA)-based knockdown of Ppp6c in cultured mammalian cells suggest that PP6 plays roles in cell growth and DNA repair. There is also evidence that PP6 regulates nuclear factor kappa B (NF-κB) signaling and mitogen-activated protein kinases and inactivates transforming growth factor-ß-activated kinase 1 (TAK1). Loss of Ppp6c causes several abnormalities, including those of T cell and regulatory T cell function, neurogenesis, oogenesis, and spermatogenesis. PP2A has been reported to play an important role in erythropoiesis. However, the roles of PP6 in other hematopoietic cells have not been investigated. We generated Ppp6cfl/fl;Tie2-Cre (Ppp6cTKO) mice, in which Ppp6c was specifically deleted in hematopoietic and vascular endothelial cells. Ppp6cTKO mice displayed embryonic lethality. Ppp6c deficiency increased the number of dead cells and decreased the percentages of erythroid and monocytic cells during fetal hematopoiesis. By contrast, the number of Lin-Sca-1+c-Kit+ cells, which give rise to all hematopoietic cells, was slightly increased, but their colony-forming cell activity was markedly decreased. Ppp6c deficiency also increased phosphorylation of extracellular signal-regulated kinase 1/2 and c-Jun amino (N)-terminal kinase in fetal liver hematopoietic cells.

Cellular inflammatory response in the bovine uterus by Leptospira infection may be related to embryo death and subfertility.

Bovine leptospirosis is a chronic disease that causes various reproductive disorders and consequent economic losses worldwide, particularly embryo death. Although Leptospira spp. has already been detected in the genital tract of cows, little is known about the uterine cellular immune response or the intrinsic factors that could contribute to that reproductive failure. In this context, the aim of this study was to assess the uterine cellular inflammatory response after the quantification of cytokine IL-6 in bovine uteri naturally infected with leptospires compared to uninfected. Our results demonstrated that uterine tissues infected with leptospires have higher levels of IL-6 compared to uninfected tissues (p < 0.001). It suggests that the presence of leptospires in the bovine uterus can induce a cellular inflammatory response, which may be related to embryo death and consequent subfertility.

AMPK-FOXO-IP3R signaling pathway mediates neurological and developmental defects caused by mitochondrial DNA mutations.

Pathological mutations in human mitochondrial genomes (mtDNA) can cause a series of neurological, behavioral, and developmental defects, but the underlying molecular mechanisms are poorly understood. We show here that the energy-sensing adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway plays a key role in mediating similar defects caused by different mtDNA mutations in Caenorhabditis elegans, including loss or reduction of osmotic, chemical and olfactory sensing, locomotion, and associative learning and memory, as well as increased embryonic lethality. mtDNA mutations cause reduced ATP (adenosine triphosphate) levels, activation of C. elegans AMPK AAK-2, and nuclear translocation of the FOXO transcription factor DAF-16. Activated DAF-16 up-regulates the expression of inositol triphosphate receptor ITR-1, an endoplasmic reticulum calcium channel, leading to increased basal cytosolic Ca2+ levels, decreased neuronal responsiveness, compromised synapses, and increased embryonic death. Treatment of mtDNA mutants with vitamin MK-4 restores cellular ATP and cytosolic Ca2+ levels, improves synaptic development, and suppresses sensory and behavioral defects and embryonic death. Our study provides crucial mechanistic insights into neuronal and developmental defects caused by mtDNA mutations and will improve understanding and treatment of related mitochondrial diseases.

BoHV-1 affects abortion and progesterone in dairy cows Bovine alphaherpesvirus 1 (BoHV-1) seropositivity, progesterone levels and embryo loss of 30-day-old pregnant dairy cows in Zagros Industrial Dairy Farm in Shahrekord: Examination and analysis.

BACKGROUND: Bovine alphaherpesvirus 1 (BoHV-1) is a serious disease with severe negative economic effects on the global cattle sector, especially in Iran. OBJECTIVE: This cross-sectional study was carried out to examine the seroprevalence and associated risk factors of BoHV-1 infection with progesterone levels and embryo death in 30-day pregnant dairy cattle at Zagros Industrial Dairy Farm in Shahrekord, Iran. METHODS: Between December 2017 to February 2018, blood samples were obtained from 60 dairy cow herds. To determine whether BoHV-1 was present, serum samples were examined using the ELISA for serum antibodies. To find progesterone (P4) in blood, the progesterone ELISA test was used. RESULTS: 96.7 % of sera tested positive for BoHV-1 antibodies, according to the findings. Additionally, 60.34 % of blood samples that tested positive had an experience of abortion and significantly more inseminations that resulted in pregnancy, consistent with findings from other studies conducted in Iran and other nations. CONCLUSIONS: Since this study is the first to document the risk factor for BoHV-1 infection in Shahrekord, Iran, we could infer that the virus is extensively dispersed in this area.

T Cell Subsets and the Expression of Related MicroRNAs in Patients with Recurrent Early Pregnancy Loss.

This study explored the role of T cell subsets and the expression of related microRNAs in patients with recurrent early pregnancy loss (EPL). Fifty patients with EPL loss between May 2018 and May 2021 were randomly selected as the EPL group, and 50 pregnant women with normal pregnancies or normal delivery outcomes were randomly selected as the control group. The expression levels of T cell subset-related markers and T cell subset-related miRNAs, in addition to the frequencies of T cell subsets, in peripheral blood of the two groups were analyzed. In terms of T cell-related markers, the results showed that the expression levels of the transcriptional regulator TBX-21 (T-bet) and interferon regulatory factor 4 (IRF4) were significantly upregulated in peripheral blood of the patients in the EPL group (P < 0.05), whereas the expression levels of GATA binding protein 3 (GATA3) and glucocorticoid-induced tumor necrosis factor receptor (GITR) were significantly downregulated (P < 0.05). In the EPL group, the expression of mir-106b, mir-93, and mir-25 was upregulated (1.51 ± 0.129, 1.43 ± 0.132, and 1.73 ± 0.156, respectively) in regulatory T (Treg) cell-related T cell subsets, whereas the expression of miR-146a and miR-155 was downregulated (P < 0.05). The frequencies of Treg and exhausted T cells in the EPL group were significantly lower than those in the control group (P < 0.05). The cell frequencies of T helper 17 (Th17) cells and exhausted Treg cells in the EPL group were significantly higher than those in the control group (P < 0.05). In conclusion, immune cells and associated miRNA profiles can be used as prognostic biomarkers for the treatment of human reproductive disorders, such as EPL.

Metastasis-associated lung adenocarcinoma transcript 1 induces methyl-CpG-binding domain protein 4 in mice with recurrent spontaneous abortion caused by anti-phospholipid antibody positivity.

INTRODUCTION: Antiphospholipid syndrome is an autoimmune disease characterized by pregnancy-related morbidity, related to persistent positivity of antiphospholipid antibodies (APL). One of the characteristics of pregnancy-related morbidity in patients with antiphospholipid syndrome is recurrent spontaneous abortion (RSA). This study aimed to examine the mechanism through which metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) regulates methyl-CpG-binding domain protein 4 (MBD4) expression in APL-positive RSA. METHODS: Clinical samples were subjected to microarray analysis to filter differentially expressed genes. RSA mice with APL positivity were generated, followed by adenoviral vector injection to artificially upregulate MALAT1. The effects of MALAT1 on the biological behavior of trophoblast cells were assessed. The downstream mechanism of MALAT1 was analyzed using subcellular fractionation and bioinformatics prediction, and the relationship between MALAT1 and CREB binding protein (CREBBP) or MBD4 was investigated in trophoblast cells. RESULTS: MALAT1 was downregulated in APL-positive RSA patients. MALAT1 was predominantly localized in the nucleus and recruited CREBBP to mediate the MBD4 transcription. In the APL-positive RSA mice overexpressing MALAT1, the expression of soluble Fms-related tyrosine kinase 1 and anticardiolipin antibody and the embryonic resorption rate were decreased, indicating that MALAT1 reduced the occurrence of RSA in mice. Moreover, MALAT1 enhanced proliferation, migration, and invasion of trophoblast cells through recruiting CREBBP to promote MBD4 expression. Silencing of CREBBP or MBD4 increased embryonic resorption rate in RSA mice overexpressing MALAT1. DISCUSSION: MALAT1 suppresses APL-positive RSA by promoting MBD4 transcription through recruitment of CREBBP to the MBD4 promoter region.

Investigating protein C and S levels in pregnant women with recurrent early pregnancy loss versus normal pregnancy.

Miscarriage in the first and second trimesters of pregnancy is very common, and coagulopathy can be a contributing factor. Protein C and S deficiency are rare, inherited disorders that can increase the risk of thrombophilia. Women with these deficiencies have a higher risk of developing blood clots in the placenta, which can lead to placental insufficiency and, ultimately, to a miscarriage. We aimed to compare the levels of protein C and protein S in pregnant females with recurrent first and second-trimester pregnancy loss and normal pregnant females. We performed a detailed history, examination, and various lab tests on a cohort of 40 females with a history of recurrent first and second-trimester abortions visiting an outpatient clinic at a multi-specialty hospital in Kashmir, India. All the findings were compared with 40 women with normal pregnancies. 10% of the participants had low protein C and S levels (P=0.277), out of whom 75% (p<0.001) had intrauterine growth retardation (IUGR) on ultrasound with 67% (p<0.001) having reduced doppler flow in the umbilical artery. 0.05% of participants had isolated protein S deficiency with no concomitant IUGR seen. Patients with protein C and S deficiencies were treated with heparin and progesterone and followed up for pregnancy outcomes. Screening for protein C and S deficiency is mandatory in all cases of recurrent pregnancy loss. Treatment with low molecular weight heparin and progesterone should be initiated to ensure good fetal outcomes and prevent post-partum/postoperative catastrophic venous thromboembolism events.

Late embryo mortality in Holstein cows inseminated with Holstein or Limousine frozen semen.

The objective of this study was to compare the late embryo mortality (LEM) rate (losses approximately between 32 and 53 days of gestation) and Pregnancy Specific Protein B (PSPB) and progesterone (P4) concentrations on day 32 post AI in Holstein cows bred with either Holstein or Limousine semen. A sample size of 1082 cows per group diagnosed pregnant between 28- and 35-days post breeding was calculated. The study consisted of evaluating LEM (%) in a cohort of Holstein cows bred with Holstein semen (HO × HO) or Limousine semen (HO × LM), to compare pregnancy loss from 28 to 35 days post breeding to 50-57 days post breeding. A logistic regression model to compare embryo losses was developed considering as main explanatory variable the cohort (HO × HO embryo vs. HO × LM embryo), correcting by lactation number, breeding season, days to breeding and AI technician. HO × HO embryos had greater LEM (15.16%) than HO × LM embryos (9.79%). Cows bred in summertime had higher LEM (15.23%) than cows bred in no-summertime (9.88%). There were no differences among AI technicians. Within summertime there was no difference in LEM (%) between groups within each lactation number; yet, within no-summertime, LEM (%) was higher in HO × HO than HO × LM within each lactation number. Pregnancy SPB optical densities were significantly greater in the HO × HO than in the HO × LM (p = .023) group; yet, the concentration of P4 was not different between groups (p > .05).

Inheritance of paternal DNA damage by histone-mediated repair restriction.

How paternal exposure to ionizing radiation affects genetic inheritance and disease risk in the offspring has been a long-standing question in radiation biology. In humans, nearly 80% of transmitted mutations arise in the paternal germline1, but the transgenerational effects of ionizing radiation exposure has remained controversial and the mechanisms are unknown. Here we show that in sex-separated Caenorhabditis elegans strains, paternal, but not maternal, exposure to ionizing radiation leads to transgenerational embryonic lethality. The offspring of irradiated males displayed various genome instability phenotypes, including DNA fragmentation, chromosomal rearrangement and aneuploidy. Paternal DNA double strand breaks were repaired by maternally provided error-prone polymerase theta-mediated end joining. Mechanistically, we show that depletion of an orthologue of human histone H1.0, HIS-24, or the heterochromatin protein HPL-1, could significantly reverse the transgenerational embryonic lethality. Removal of HIS-24 or HPL-1 reduced histone 3 lysine 9 dimethylation and enabled error-free homologous recombination repair in the germline of the F1 generation from ionizing radiation-treated P0 males, consequently improving the viability of the F2 generation. This work establishes the mechanistic underpinnings of the heritable consequences of paternal radiation exposure on the health of offspring, which may lead to congenital disorders and cancer in humans.

ATG9A prevents TNF cytotoxicity by an unconventional lysosomal targeting pathway.

Cell death induced by tumor necrosis factor (TNF) can be beneficial during infection by helping to mount proper immune responses. However, TNF-induced death can also drive a variety of inflammatory pathologies. Protectives brakes, or cell-death checkpoints, normally repress TNF cytotoxicity to protect the organism from its potential detrimental consequences. Thus, although TNF can kill, this only occurs when one of the checkpoints is inactivated. Here, we describe a checkpoint that prevents apoptosis through the detoxification of the cytotoxic complex IIa that forms upon TNF sensing. We found that autophagy-related 9A (ATG9A) and 200kD FAK family kinase-interacting protein (FIP200) promote the degradation of this complex through a light chain 3 (LC3)-independent lysosomal targeting pathway. This detoxification mechanism was found to counteract TNF receptor 1 (TNFR1)-mediated embryonic lethality and inflammatory skin disease in mouse models.

Dysregulation in Multiple Transcriptomic Endometrial Pathways Is Associated with Recurrent Implantation Failure and Recurrent Early Pregnancy Loss.

Overlapping disease aetiologies associated with multiple altered biological processes have been identified that change the endometrial function leading to recurrent implantation failure (RIF) and recurrent early pregnancy loss (REPL). We aimed to provide a detailed insight into the nature of the biological malfunction and related pathways of differentially expressed genes in RIF and REPL. Endometrial biopsies were obtained from 9 women experiencing RIF, REPL and control groups. Affymetrix microarray analysis was performed to measure the gene expression level of the endometrial biopsies. Unsupervised clustering of endometrial samples shows scattered distribution of gene expression between the RIF, REPL and control groups. 2556 and 1174 genes (p value < 0.05, Fold change > 1.2) were significantly altered in the endometria of RIF and REPL patients' group, respectively compared to the control group. Downregulation in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the differentially expressed genes (DEGs) in RIF and REPL including ribosome and oxidative phosphorylation pathways. Gene Ontology (GO) analysis revealed ribosomes and mitochondria inner membrane as the most significantly downregulated cellular component (CC) affected in RIF and REPL. Determination of the dysregulated genes and related biological pathways in RIF and REPL will be key in understanding their molecular pathology and of major importance in addressing diagnosis, prognosis, and treatment issues

Differential MicroRNA Expression in Porcine Endometrium Related to Spontaneous Embryo Loss during Early Pregnancy.

Litter size is an important indicator to measure the production capacity of commercial pigs. Spontaneous embryo loss is an essential factor in determining sow litter size. In early pregnancy, spontaneous embryo loss in porcine is as high as 20-30% during embryo implantation. However, the specific molecular mechanism underlying spontaneous embryo loss at the end of embryo implantation remains unknown. Therefore, we comprehensively used small RNA sequencing technology, bioinformatics analysis, and molecular experiments to determine the microRNA (miRNA) expression profile in the healthy and arresting embryo implantation site of porcine endometrium on day of gestation (DG) 28. A total of 464 miRNAs were identified in arresting endometrium (AE) and healthy endometrium (HE), and 139 differentially expressed miRNAs (DEMs) were screened. We combined the mRNA sequencing dataset from the SRA database to predict the target genes of these miRNAs. A quantitative real-time PCR assay identified the expression levels of miRNAs and mRNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed on differentially expressed target genes of DEMs, mainly enriched in epithelial development and amino acids metabolism-related pathways. We performed fluorescence in situ hybridization (FISH) and the dual-luciferase report gene assay to confirm miRNA and predicted target gene binding. miR-205 may inhibit its expression by combining 3'-untranslated regions (3' UTR) of tubulointerstitial nephritis antigen-like 1 (TINAGL1). The resulting inhibition of angiogenesis in the maternal endometrium ultimately leads to the formation of arresting embryos during the implantation period. This study provides a reference for the effect of miRNA on the successful implantation of pig embryos in early gestation.

Detection of nonpregnant cows and potential embryo losses by color Doppler ultrasound and interferon-stimulated gene expression in grazing dairy cows.

Many studies have been conducted to estimate pregnancy losses between 19 and 34 d after artificial insemination (AI) in dairy cows managed under confinement-based systems, but few studies have examined embryo mortality during this interval in dairy cows managed under gazing systems. The objectives of this prospective cohort study were (1) to assess the diagnostic value of the corpus luteum (CL) blood perfusion (BP) evaluation by Doppler ultrasound (US) to detect nonpregnant cows at 19 to 20 d post-AI, and (2) to assess the rate of potential embryo mortality between 19 to 34 d post-AI. The CL-BP of all cows included in the study (n = 131) was examined on farm by power and color mode of Doppler US and later using an image processing software by a second evaluator. The endometrium thickness and echotexture were evaluated by B-mode US at the same visit to assess if the nonpregnancy diagnosis could be improved at 19 to 20 d post-AI by this additional diagnostic tool. Blood samples were obtained at 19 to 20 d post-AI for progesterone (P4) measurement by chemiluminescence and to determine the mRNA expression of ISG by real-time PCR. Pregnancy diagnosis based on embryo visualization was performed at 33 to 34 d post-AI by US B-mode. In parallel interpretation, ISG15 and MX2 mRNA expression in leukocytes [sensitivity (Se), 100%] were regarded as suitable biomarkers for early pregnancy and were selected for molecular characterization of pregnancy at 19 to 20 d post-AI. At 19 to 20 d post-AI, 61.1% of the cows had positive CL-BP by Doppler US (Se, 98.0%), 62.7% had ISG mRNA expression in leukocytes over the cutoff point (Se, 95.7%), and 50.8% were positive, based on the combination of ISG mRNA expression, CL-BP by Doppler US, and P4 concentration (Se, 100%), and were considered as possible pregnant. At 33 to 34 d, the pregnancy rate was 37.4% diagnosed by the B-mode US. Based on the expression of the selected biomarkers in cows with active CL, we found that 28.1% of the cows could have potentially lost their pregnancy between 19 and 34 d post-AI. The Doppler US color mode showed similar accuracy and a higher negative predictive value than the genes selected as biomarkers. The additional B-mode ultrasound evaluation of the uterine stratum vasculare and the endometrium thickness improved the diagnostic accuracy. Therefore, assessing the CL-BP by Doppler US allowed early detection of nonpregnant cows at 19 to 20 d post-AI. The combination of early CL-BP by Doppler US (d 19 to 20) with early embryo detection by B-mode US (d 33-34) could be used to facilitate earlier rebreeding of dairy cows.

A decreased expression of interferon stimulated genes in peri-implantation endometrium of embryo transfer recipient sows could contribute to embryo death.

Pig pregnancy succeeds thanks to a well-coordinated system ruling both maternal immune activation and embryonic antigen tolerance. In physiological pregnancies, the maternal immune system should tolerate the presence of hemi-allogeneic conceptuses from the pre-implantation phase to term, while maintaining maternal defence against pathogens. Allogeneic pregnancies, as after embryo transfer (ET), depict high embryo mortality during the attachment phase, calling for studies of the dynamic modifications in immune processes occurring at the maternal-foetal interface, for instance, of interferon (IFN)-stimulated genes (ISGs). These ISGs are generally activated by IFN secreted by the conceptus during the process of maternal recognition of pregnancy (MRP) and responsible for recruiting immune cells to the site of embryo attachment, thus facilitating cell-antigen presentation and angiogenesis. We performed RNA-Seq analysis in peri-implantation (days 18 and 24) endometrial samples retrieved from artificially inseminated sows (hemi-allogeneic embryos (HAL) group) or sows subjected to ET (allogeneic embryos (AL) group) to monitor alterations of gene expression that could be jeopardising early pregnancy. Our results showed that endometrial gene expression patterns related to immune responses differed between hemi- or allogeneic embryo presence, with allogeneic embryos apparently inducing conspicuous modifications of immune-related genes and pathways. A decreased expression (P < 0.05; FC < -2) of several interferon ISGs, such as CXCL8, CXCL10, IRF1, IRF9, STAT1, and B2M, among others was detected in the endometrium of sows carrying allogeneic embryos on day 24 of pregnancy. This severe downregulation of ISGs in allogeneic pregnancies could represent a failure of ET-embryos to signal IFN to the endometrium to warrant the development of adequate immunotolerance mechanisms to facilitate embryo development, thus contributing to elevated embryo death.

Diminished miR-374c-5p negatively regulates IL (interleukin)-6 in unexplained recurrent spontaneous abortion.

Unexplained recurrent spontaneous abortion (URSA) is commonly observed, and seriously affects women's reproductive health. Excessive interleukin-6 (IL-6) production has been shown to frequently occur and relate to URSA pathogenesis. In this study, the miRNA expression profile in peripheral blood mononuclear cells (PBMCs) from URSA patients and normal pregnant (NP) women was assessed by miRNA microarray and real-time quantitative reverse-transcription polymerase chain reaction (qPCR). MiRNA target prediction tools and luciferase reporter assay were used to detect direct binding between miRNAs and IL6. Functional study of administering anti-IL-6 neutralizing antibody and miR-374c-5p mimics to an URSA animal model was performed to evaluate embryo resorption rates. In the results, compared with NP women, the expression of IL-6 increased markedly in PBMCs and decidual tissues at both mRNA and protein levels, while miR-374c-5p expression decreased significantly. Prediction software and luciferase reporter assay showed that miR-374c-5p binds with IL6 3'UTR via the complementary bases. Transfection of miR-374c-5p mimics into an in vitro HeLa cell line significantly downregulated the expression of IL-6, while transfection of the miR-374c-5p inhibitor induced an opposite result. In the URSA mouse model, miR-374c-5p overexpression reduced the embryo resorption rate significantly, accompanied with decreased expression of IL-6 in the decidua. To sum up, downregulated miR-374c-5p was involved in the pathogenesis of URSA by enhancing IL-6 expression. Modulation of miR-374c-5p expression may be used to regulate IL-6 production for the treatment of URSA.

A Robotic System With Embedded Open Microfluidic Chip for Automatic Embryo Vitrification.

Embryo vitrification is a fundamental technology utilized in assisted reproduction and fertility preservation. Vitrification involves sequential loading and unloading of cryoprotectants (CPAs) with strict time control, and transferring the embryo in a minimum CPA droplet to the vitrification straw. However, manual operation still cannot effectively avoid embryo loss, and the existing automatic vitrification systems have insufficient system reliability, and operate differently from clinical vitrification protocol. Through collaboration with in vitro fertilization (IVF) clinics, we are in the process realizing a robotic system that can automatically conduct the embryo vitrification process, including the pretreatment with CPAs, transfer of embryo to the vitrification straw, and cryopreservation with liquid nitrogen ( LN2). An open microfluidic chip (OMC) was designed to accommodate the embryo during the automatic CPAs pretreatment process. The design of two chambers connected by a capillary gap facilitated solution exchange around the embryo, and simultaneously reduced the risk of embryo loss in the flow field. In accordance to the well-accepted procedure and medical devices in manual operation, we designed the entire vitrification protocol, as well as the robotic prototype. In a practical experiment using mouse embryos, our robotic system showed a 100 % success rate in transferring and vitrifying the embryos, achieved comparable embryo survival rates (90.9 % versus 94.4 %) and development rates (90.0 % versus 94.1 %), when compared with the manual group conducted by the senior embryologist. With this study, we aim to facilitate the standardization of clinical vitrification from manual operation to a more efficient and reliable automated process.

Polystyrene microplastics disturb maternal-fetal immune balance and cause reproductive toxicity in pregnant mice.

Microplastics (MPs), which are emerging as a new type of environmental pollutants, have raised great concerns regarding their threats to human health. A successful pregnancy depends on the sophisticated regulation of the maternal-fetal immune balance, but the risks of polystyrene MP (PS-MP) exposure in early pregnancy remain unclear. In this study, we exposed the C57BL/6-mated BALB/c mice to PS-MP particles and used the flow cytometry to explore threats towards the immune system. Herein, the allogeneic mating murine model showed an elevated embryo resorption rate with a 10 µm PS-MP particle exposure during the peri-implantation period. Both the number and diameter of uterine arterioles decreased, which might reduce the uterine blood supply. Moreover, the percentage of decidual natural killer cells was reduced, whereas the helper T cells in the placenta increased. In addition, the M1/M2 ratio in macrophages reversed significantly to a dominant M2-subtype. Lastly, the cytokine secretion shifted towards an immunosuppressive state. Overall, our results demonstrated that PS-MPs have the potential to cause adverse effects on pregnancy outcomes via immune disturbance, providing new insights into the study of reproductive toxicity of MP particles in the human body.

N-3 polyunsaturated fatty acids effectively protect against neural tube defects in diabetic mice induced by streptozotocin.

Folate cannot prevent all neural tube defects (NTD), indicating that other pathogeneses still exist except for the folate deficiency. Maternal diabetes mellitus during pregnancy can increase the risk of offspring NTD. Our previous study showed that polyunsaturated fatty acids (PUFA) were lower in the placenta of human NTD cases than in healthy controls, and the supplementation of fish oil (rich in long-chain (LC) n-3 PUFA, mainly C20:5n-3 and C22:6n-3) had a better prevention effect against sodium valproate induced NTD than corn oil (rich in C18:2n-6) and flaxseed oil (rich in C18:3n-3). The aim of the present study was to investigate whether PUFA could prevent diabetes-induced NTD in mice. Streptozotocin (STZ)-induced diabetic pregnant mice were fed with a normal diet (DMC), a diet containing a low dose of fish oil (DMLn-3), a diet containing a high dose of fish oil (DMHn-3) or a diet rich in corn oil (DMn-6). Healthy pregnant mice were fed with a normal diet (HC). Compared with the DMC group, the rate of NTD was significantly lower in the DMHn-3 group (4.44% vs. 12.50%), but not in the DMLn-3 (11.11%) or DMn-6 group (12.03%). The NTD rate in the DMHn-3 group was comparable with that in the HC group (1.33%) (p = 0.246), and lower than that in the DMn-6 group (p = 0.052). The NTD rate in DMLn-3 and DMn-6 groups was significantly higher than that in the HC group. No significant difference was observed in NTD rate between DMLn-3 and DMHn-3 groups, and between DMLn-3 and DMn-6 groups. Compared with the HC group, the DMC group had a significantly lower C22:6n-3 in both serum and embryos. Fish oil supplementation ameliorated neuroepithelial cell apoptosis, and the apoptotic rate was comparable between DMHn-3 and HC groups. Although the apoptotic rate was significantly lower in the DMn-6 group than the DMC group, it was still much higher than that in the HC group. The proteins P53 and Bax in embryos were higher, while the proteins Bcl-2 and Pax3 were lower in the DMC group than in the HC group. The disturbance of Pax3, P53 and Bax induced by diabetes was abolished in DMLn-3, DMHn-3 and DMn-6 groups. Importantly, Bcl-2 in embryos was restored to the normal level only in the DMHn-3 group but not in the DMLn-3 or DMn-6 group. In conclusion, LC n-3 PUFA enriched fish oil has a protective effect against NTD in diabetes induced by STZ through improving neuroepithelial cell apoptosis, and the mechanism may be by increasing the anti-apoptosis protein Bcl-2 independently of Pax3 and P53.

Uterine glands impact embryo survival and stromal cell decidualization in mice.

Uterine glands are essential for the establishment of pregnancy and have critical roles in endometrial receptivity to blastocyst implantation, stromal cell decidualization, and placentation. Uterine gland dysfunction is considered a major contributing factor to pregnancy loss, however our understanding of how glands impact embryo survival and stromal cell decidualization is incomplete. Forkhead box A2 (FOXA2) is expressed only in the glandular epithelium and regulates its development and function. Mice with a conditional deletion of FOXA2 in the uterus are infertile due to defective embryo implantation arising from a lack of leukemia inhibitory factor (LIF), a critical factor of uterine gland origin. Here, a glandless FOXA2-deficient mouse model, coupled with LIF repletion to rescue the implantation defect, was used to investigate the roles of uterine glands in embryo survival and decidualization. Studies found that embryo survival and decidualization were compromised in glandless FOXA2-deficient mice on gestational day 6.5, resulting in abrupt pregnancy loss by day 7.5. These findings strongly support the hypothesis that uterine glands secrete factors other than LIF that impact embryo survival and stromal cell decidualization for pregnancy success.

Human MLH1/3 variants causing aneuploidy, pregnancy loss, and premature reproductive aging.

Embryonic aneuploidy from mis-segregation of chromosomes during meiosis causes pregnancy loss. Proper disjunction of homologous chromosomes requires the mismatch repair (MMR) genes MLH1 and MLH3, essential in mice for fertility. Variants in these genes can increase colorectal cancer risk, yet the reproductive impacts are unclear. To determine if MLH1/3 single nucleotide polymorphisms (SNPs) in human populations could cause reproductive abnormalities, we use computational predictions, yeast two-hybrid assays, and MMR and recombination assays in yeast, selecting nine MLH1 and MLH3 variants to model in mice via genome editing. We identify seven alleles causing reproductive defects in mice including female subfertility and male infertility. Remarkably, in females these alleles cause age-dependent decreases in litter size and increased embryo resorption, likely a consequence of fewer chiasmata that increase univalents at meiotic metaphase I. Our data suggest that hypomorphic alleles of meiotic recombination genes can predispose females to increased incidence of pregnancy loss from gamete aneuploidy.