CBLL1 promotes endometrial stromal cell senescence via inhibiting PTEN in recurrent spontaneous abortion.

Recurrent spontaneous abortion (RSA) is a common pregnancy-related disorder. Cbl proto-oncogene like 1 (CBLL1) is an E3 ubiquitin ligase, which has been reported to vary with the menstrual cycle in the endometrium. However, whether CBLL1 is involved in the occurrence and development of RSA remains unclear. This study aimed to investigate the effects of CBLL1 on RSA. We analyzed the expression of CBLL1 in the decidua of RSA patients, as well as its functional effects on cellular senescence, oxidative stress, and proliferation of human endometrial stromal cells (HESCs). RNA sequencing was employed to identify a key downstream target gene regulated by CBLL1. We found that CBLL1 was upregulated in the decidua of RSA patients. Additionally, overexpression of CBLL1 promoted HESC senescence, increased oxidative stress levels, and inhibited proliferation. Phosphatase and tensin homolog located on chromosome 10 (PTEN) was identified as one of the important downstream target genes of CBLL1. In vivo experiments demonstrated that CBLL1 overexpression in the endometrium caused higher embryo absorption rate in mice. Consequently, elevated CBLL1 expression is a potential cause of RSA, representing a novel therapeutic target for RSA.

ENO1 promotes trophoblast invasion regulated by E2F8 in recurrent miscarriage.

Recurrent miscarriage (RM) is related to the dysfunction of extravillous trophoblast cells (EVTs), but the comprehensive mechanisms remain largely unexplored. We analyzed single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing and microarray datasets obtained from Gene Expression Omnibus (GEO) database to explore the hub genes in the mechanisms of RM. We identified 1724 differentially expressed genes (DEGs) in EVTs from the RM, and they were all expressed along the trajectory of EVTs. These DEGs were associated with hypoxia and glucose metabolism. Single-cell Regulatory Network Inference and Clustering (SCENIC) analysis revealed that E2F transcription factor (E2F) 8 (E2F8) was a key transcription factor for these DEGs. And the expression of ENO1 can be positively regulated by E2F8 via RNA sequencing analysis. Subsequently, we performed immunofluorescence assay (IF), plasmid transfection, western blotting, chromatin immunoprecipitation (ChIP), real-time quantitative polymerase chain reaction (qRT-PCR), and transwell assays for validation experiments. We found that the expression of alpha-Enolase 1 (ENO1) was lower in the placentas of RM. Importantly, E2F8 can transcriptionally regulate the expression of ENO1 to promote the invasion of trophoblast cells by inhibiting secreted frizzled-related protein 1/4 (SFRP1/4) to activate Wnt signaling pathway. Our results suggest that ENO1 can promote trophoblast invasion via an E2F8-dependent manner, highlighting a potential novel target for the physiological mechanisms of RM.

Mechanisms of YAP1-mediated trophoblast ferroptosis in recurrent pregnancy loss.

PURPOSE: The purpose of our study is to investigate the function of YAP1 in the trophoblast ferroptosis and maternal-fetal interface communication of RPL. METHODS: We collected 25 villous tissues and detected the expression of YAP1. Cell counting kit-8 assay, scratch wound-healing assay, and Matrigel invasion assay were performed to observe the proliferation, migration, and invasion of HTR-8/SVneo and JAR cells. Subsequently, measured the levels of reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH), SLC7A11, SOD2, and GPX4. Ultimately, the use of ferroptosis activator (erastin) and inhibitor (Ferrostatin-1, fer-1) further confirmed the regulation by YAP1. In addition, established an in vitro-induced cell model to study the effect of YAP1 on the decidualization process. Finally, animal models were implemented for further confirmation. RESULTS: We found that YAP1 was downregulated in RPL patients. Overexpression of YAP1 could significantly enhance the proliferation, migration, and invasion of trophoblasts, and inhibit ferroptosis. Knocking down YAP1 exhibited the opposite effect. Rescue experiments have shown that YAP1 could upregulate the expression of SLC7A11 and GPX4, which are key molecules in the classic pathway of ferroptosis. In addition, the decidualization was impaired when hESCs were treated with conditioned medium of YAP1 knockdown trophoblasts. Moreover, we found that Yap1, Slc7a11, and Gpx4 were downregulated in the RPL mice, along with increased MDA and decreased GSH. CONCLUSION: Downregulation of YAP1 induces ferroptosis, thereby damaging the trophoblast invasion processes, which also disturbs the communication at the maternal-fetal interface. Our study identified YAP1 as a potential key molecule in the pathogenesis of RPL.

Cryptochrome 2 Suppresses Epithelial-Mesenchymal Transition by Promoting Trophoblastic Ferroptosis in Unexplained Recurrent Spontaneous Abortion.

Unexplained recurrent spontaneous abortion (URSA) is a serious reproductive issue that affects women of childbearing age. Studies have shown a close association between disrupted circadian rhythm and impaired epithelial-mesenchymal transition (EMT) in trophoblasts during URSA, although the underlying mechanism is not known. The current study investigated the regulatory relationship between circadian rhythm gene cryptochrome 2 (CRY2) and ferroptosis on the migratory ability of trophoblast cells. Cell proliferation experiments, wound-healing assays, and expression of related markers were conducted to study EMT. Trophoblastic ferroptosis was confirmed by the expressions of malondialdehyde, glutathione, mitochondrial membrane potential, divalent iron ions, and related genes. The results showed significant increased expression of CRY2 and decreased expression of brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) in the URSA villous tissues, accompanied by iron-dependent oxidative changes and abnormal expression of ferroptosis-related proteins. CRY2 and BMAL1 were co-localized and functioned as a feedback loop, which regulated the dynamic changes of EMT-related markers in trophoblast cells. CRY2 promoted trophoblastic ferroptosis, whereas BMAL1 had the opposite effect. Particularly, the ferroptosis inhibitor (ferrostatin-1) effectively reversed the trophoblastic ferroptosis and EMT inhibition caused by CRY2 overexpression. Collectively, these results suggest that CRY2 regulates trophoblastic ferroptosis and hinders cellular EMT and migratory ability by suppressing BMAL1 expression.

Dysregulation of CREB5 Impairs Decidualization and Maternal-Fetal Interactions by Inhibiting Autophagy in Recurrent Spontaneous Abortion.

BACKGROUND: Clinically, recurrent spontaneous abortion (RSA) is a pregnancy illness that is difficult to treat. Impaired decidualization is a documented cause of RSA, but the etiology and mechanism are still unknown. cAMP-responsive element binding protein 5 (CREB5) is a member of the ATF/CREB family. CREB5 has been reported to be related to pathological pregnancy, but there are few related studies on this topic in patients with RSA, and the underlying mechanism is unclear. METHODS: We collected decidual tissues from RSA patients and healthy pregnant women to measure the expression level of CREB5, PRL, IGFBP1, ATG5, LC3B, and SQSTM/p62. Then, the changes in CREB5 expression and autophagy levels were measured in human endometrial stromal cells (hESCs) during decidualization. The expression levels of PRL and IGFBP1 were tested in sh-CREB5/ov-CREB5 hESCs after decidualization induction, and the autophagy level in sh-CREB5/ov-CREB5 hESCs was measured without decidualization induction. The decidualization ability of sh-CREB5 and ov-CREB5 hESCs treated with an autophagy inducer or inhibitor was measured. To investigate the effect of CREB5 in hESCs on the invasion and migration of HTR8/SVneo cells, we performed a coculture experiment. Finally, we examined the expression of CREB5 and autophagy key proteins in mouse decidual tissues by constructing an abortion mouse model. RESULTS: In our study, we found that the expression of CREB5 was unusually elevated in the uterine decidua of RSA patients, but the expression of PRL, IGFBP1, and autophagy were decreased. During the decidualization of hESCs, the expression of CREB5 gradually decreases in a time-dependent manner with increasing autophagy. Moreover, by knocking down or overexpressing CREB5 in hESCs, it was found that CREB5 can impair decidualization and reduce autophagy in hESCs. Furthermore, the damage caused by CREB5 in terms of decidualization can be reversed by the addition of an autophagy inducer (rapamycin). In addition, CREB5 can increase the secretion of proteins (IL-1ß and TGF-ß1) in hESCs to inhibit trophoblast invasion and migration. CONCLUSIONS: Our data support the supposition that CREB5 disturbs the decidualization of endometrial stromal cells and interactions at the maternal-fetal interface by inhibiting autophagy and that its abnormal upregulation and dysfunction may lead to RSA. It may function as a diagnostic and therapeutic target for RSA. Similarly, we found that in the spontaneous abortion mouse model, the expression of CREB5 in the decidual tissue of the abortion group was significantly increased, and autophagy was decreased.

HOTAIR/miR-1277-5p/FBN2 signaling axis is involved in recurrent spontaneous abortion by regulating the growth, migration, and invasion of HTR-8/SVneo cells†.

OBJECTIVE: This study aimed to explore the specific pathways by which HOX transcript antisense intergenic RNA contributes to the pathogenesis of unexplained recurrent spontaneous abortion. METHODS: Real-time quantitative PCR was employed to assess the differential expression levels of HOX transcript antisense intergenic RNA in chorionic villi tissues from unexplained recurrent spontaneous abortion patients and women with voluntarily terminated pregnancies. HTR-8/SVneo served as a cellular model. Knockdown and overexpression of HOX transcript antisense intergenic RNA in the cells were achieved through siRNA transfection and pcDNA3.1 transfection, respectively. Cell viability, migration, and invasion were evaluated using cell counting kit-8, scratch, and Transwell assays, respectively. The interaction among the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 axis was predicted through bioinformatics analysis and confirmed through in vitro experiments. Furthermore, the regulatory effects of the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 signaling axis on cellular behaviors were validated in HTR-8/SVneo cells. RESULTS: We found that HOX transcript antisense intergenic RNA was downregulated in chorionic villi tissues from unexplained recurrent spontaneous abortion patients. Overexpression of HOX transcript antisense intergenic RNA significantly enhanced the viability, migration, and invasion of HTR-8/SVneo cells, while knockdown of HOX transcript antisense intergenic RNA had the opposite effects. We further confirmed the regulatory effect of the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 signaling axis in unexplained recurrent spontaneous abortion. Specifically, HOX transcript antisense intergenic RNA and fibrillin 2 were found to reduce the risk of unexplained recurrent spontaneous abortion by enhancing cell viability, migration, and invasion, whereas miR-1277-5p exerted the opposite effects. CONCLUSION: HOX transcript antisense intergenic RNA promotes unexplained recurrent spontaneous abortion development by targeting inhibition of miR-1277-5p/fibrillin 2 axis.

Downregulation of FHL2 suppressed trophoblast migration, invasion and epithelial-mesenchymal transition in recurrent miscarriage.

RESEARCH QUESTION: Is four and a half LIM domain 2 (FHL2) involved in trophoblast migration, invasion and epithelial-mesenchymal transition (EMT) in recurrent miscarriage? DESIGN: Villus tissue was collected from 24 patients who had experienced recurrent miscarriage and 24 healthy controls. FHL2 mRNA and protein expression in villus specimens were observed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Small interfering RNA and overexpression plasmid were used to change the FHL2 expression. JAR and HTR8/SVneo cell lines were used to conduct scratch-wound assay and transwell assay to detect trophoblast migration and invasion of FHL2. Downstream molecule expression of mRNA and protein and EMT markers were verified by qRT-PCR and Western blot. RESULTS: Significantly lower FHL2 mRNA (P = 0.019) and protein (P = 0.0014) expression was found in trophoblasts from the recurrent miscarriage group compared with healthy controls. FHL2 knockdown repressed migration (P = 0.0046), invasion (P < 0.001) and EMT, as shown by significant differences in mRNA and protein expression of the EMT markers N-cadherin, E-cadherin, Vimentin and Snail (all P < 0.05) of extravillus trophoblasts. FHL2 overexpression enhanced migration (P = 0.025), invasion (P < 0.001) and EMT of extravillus trophoblasts (all EMT markers P < 0.05). The positive upstream factor FHL2 in the extracellular signal-related kinase pathway induced JunD expression, thereby promoting trophoblast migration and invasion via matrix metalloproteinase 2. CONCLUSIONS: FHL2 is involved in a regulatory pathway of trophoblast migration, invasion and EMT during early pregnancy, and may have a role in recurrent miscarriage pathogenesis, which can serve as a possible target for novel therapeutic development.

Endometrial protein expression and phosphorylation landscape decipher aberrant insulin and mTOR signalling in patients with recurrent pregnancy loss.

RESEARCH QUESTION: What are the proteomic and phosphoproteomic differences between the endometrium of women with recurrent pregnancy loss (RPL) and the endometrium of healthy control women during the proliferative and secretory phases of the menstrual cycle? DESIGN: In total, 54 endometrial samples were collected during the proliferative and secretory phases from women with RPL (n = 28) and healthy controls (n = 26). Comprehensive proteomic and phosphoproteomic analyses were conducted using label-free liquid chromatography-tandem mass spectrometry (n = 44), and verified through Western blotting (n = 10). Three comparison groups were established: total RPL endometrium versus total control endometrium; RPL proliferative endometrium versus control proliferative endometrium; and RPL secretory endometrium versus control secretory endometrium. RESULTS: Differentially expressed proteins and differentially phosphorylated proteins were identified in the three comparison groups. Combining pathway enrichment, network analysis and soft clustering analysis, the insulin/cyclic nucleotide signalling pathway and AMPK/mTOR signalling pathway were identified as the major contributors to the aberration of RPL endometrium. Western blotting verified altered expression of four proteins: cAMP-dependent protein kinase type I-ß regulatory subunit, adenylate cyclase type 3, 5'-AMP-activated protein kinase catalytic subunit α-2 and phosphatidate phosphatase LPIN2. CONCLUSIONS: This exploratory study provides insights into the differentiated protein expression and phosphorylation profiles of the endometrium of women with RPL in both the proliferative and sectretory phases of the menstrual cycle. The results highlight potential proteins associated with the pathogenesis of RPL that may serve as potential indicators for RPL. The findings contribute to the identification of potential targets for RPL treatment as well as its pathogenesis.

Impaired decidualization and relative increase of PROK1 expression in the decidua of patients with unexplained recurrent pregnancy loss showing insulin resistance.

A recent meta-analysis revealed that patients with unexplained recurrent pregnancy loss (RPL) show higher insulin resistance compared to healthy controls. However, the etiology of RPL remains unknown. Prokineticin (PROK1), a pleiotropic uterine endometrial protein, is important for implantation and decidualization and is regulated by hypoxia and insulin. In this study, we investigated the decidualization status and the role of PROK1 in the decidua of patients with unexplained RPL showing insulin resistance. Thirty-two patients with unexplained RPL were included in this study. Following the diagnosis of a miscarriage, the decidua and villi of the patient were surgically collected. Fasting blood glucose and insulin levels were measured, and HOMA-ß was calculated. Using IHC and ELISA, the expression of IGFBP-1, PRL and PROK1 in the decidua and IGF-2 in the villi were analyzed in patients with euploid miscarriage with a high HOMA-ß index (n = 8) and compared to controls (euploid miscarriage with normal HOMA-ß: n = 12, aneuploid miscarriage with normal HOMA-ß: n = 12). The co-localization of PROK1 and IGFBP-1 was observed in the decidua by IHC. In the decidua of RPL patients with high HOMA-ß, the expression levels of IGFBP-1 and PRL were significantly lower, whereas the PROK1/IGFBP-1 ratio was significantly higher compared to that of the controls. IGF-2 expression in villi was significantly lower in RPL patients with high HOMA-ß. Impaired decidualization and excessive PROK1 production may have pathological implications in patients with unexplained RPL with insulin resistance, especially under the state of hyper insulin production.

miRNAs associated with endoplasmic reticulum stress and unfolded protein response during decidualization.

RESEARCH QUESTION: Do microRNAs (miRNAs) play a role in regulating endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) in decidualized cells and endometrium associated with reproductive failures? DESIGN: Endometrial stromal cell line St-T1b was decidualized in vitro with 8-Br-cAMP over 5 days, or treated with the ERS inducer thapsigargin. Expression of ERS sensors, UPR markers and potential miRNA regulators was analysed by quantitative PCR. Endometrial biopsies from patients with recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF) were investigated for the location of miRNA expression. RESULTS: Decidualization of St-T1b cells resulted in increased expression of ERS sensors including ATF6α, PERK and IRE1α, and the UPR marker, CHOP. TXNIP, which serves as a link between the ERS pathway and inflammation, as well as inflammasome NLRP3 and interleukin 1ß expression increased in decidualized cells. An in-silico analysis identified miR-17-5p, miR-21-5p and miR-193b-3p as miRNAs potentially involved in regulation of the ERS/UPR pathways and inflammation associated with embryo implantation. Their expression decreased significantly (P ≤ 0.0391) in non-decidualized cells in the presence of thapsigargin. Finally, expression of the selected miRNAs was localized by in-situ hybridization in stromal and glandular epithelial cells in endometrial samples from patients with RPL and RIF. Expression in stroma cells from patients with RPL was lower in comparison with stroma cells from patients with RIF. CONCLUSIONS: Decidualization in St-T1b cells is accompanied by ERS/UPR processes, associated with an inflammatory response that is potentially influenced by miR-17-5p, miR-21-5p and miR-193b-3p. These miRNAs are expressed differentially in stromal cells from patients with RPL and RIF, indicating an alteration in regulation of the ERS/UPR pathways.

Proteomic profiling analysis of human endometrium in women with unexplained recurrent spontaneous abortion.

Unexplained recurrent spontaneous abortion (URSA) seriously affects female reproductive health, causing a great burden to patients both physically and mentally. Endometrial decidualization plays an important role in pregnancy, and impaired decidualization is an essential cause of URSA, but the cause of the damage is still poorly understood. This study aimed to reveal the pathogenesis of URSA by analyzing the differential protein expression profiles in the decidual tissue of patients with recurrent abortion compared to those with normal pregnancy. Morphological analysis revealed abnormal decidualization of endometrial tissue in patients with URSA. Quantitative proteomics analysis showed that a total of 146 differentially expressed proteins were identified between the two groups, among which 95 proteins were downregulated and 51 proteins were upregulated. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed that the protein expression profile and signaling pathways of endometrium in patients with URSA changed significantly, and cytoskeleton remodeling and morphological transformation disorders were associated with abortion induced by incomplete decidualization. Meanwhile, transcription factors analysis showed that the 3 most affected families were zf-C2H2, MYB and HMG. Therefore, our study may provide a basis for searching for potential markers of decidualization injury. SIGNIFICANCE: At present, there are still about 50% of RSA patients with unknown causes, which brings great difficulties and blindness to clinical diagnosis and treatment.The limited proteomic studies on URSA further contribute to the lack of understanding in this field. However, in this study, the focus was on proteomic profiling analysis of the human endometrium in URSA patients compared to normal women. The findings revealed that cytoskeletal remodeling disorder is a significant contributor to the failure of decidualization in URSA patients. This insight highlights the potential role of cytoskeleton-related proteins in the pathogenesis of URSA, providing valuable information for further research and potential therapeutic interventions.

T cell immunity and the etiology and pathogenesis of preeclampsia.

Preeclampsia is more common in nulliparous women, their first pregnancies with a new partner in multiparous women, pregnant women with short duration of cohabitation, and in pregnancies with donor eggs, where the fetus is completely foreign to the mother. The epidemiological study findings strongly suggest that inadequate induction of tolerance to paternal/fetal antigens is involved in the pathogenesis of preeclampsia. This review proposes that preeclampsia may be caused by a reduction in paternal/fetal antigen-specific regulatory T (Treg) cells and decreased PD-1 expression on clonally expanded CD8+ effector memory T (TEM) cells, resulting in a breakdown of mother-to-fetus tolerance. The immune environment of preeclampsia is clearly different from that of recurrent pregnancy loss (RPL). In preeclampsia, cloned Treg cells decreases, and PD-1 expression on cloned CD8+TEM decreased. In RPL, the total number of Treg cells decreased, and the total number of clonally expanded CD8+TEM cells increases. In addition to these changes, increased differentiation of Th17 cells has also been observed in preeclampsia. This change is caused by soluble endoglin, that is increased in preeclampsia, neutralizing TGFß. These immunological changes make the fetus more susceptible to attacks from maternal T cells.

MITA Promotes Macrophage Proinflammatory Polarization and Its circRNA-Related Regulatory Mechanism in Recurrent Miscarriage.

MITA (also called STING), a master regulator of DNA-mediated innate immune activation, is a potential therapeutic target for viral infection and virus-related diseases. The circRNA-mediated ceRNA network plays important roles in gene regulation and may contribute to many human diseases. However, the relationship between MITA and recurrent miscarriage (RM) and its circRNA-related regulatory mechanisms remain unclear. In this study, we validated that the decidual M1/M2 ratio was upregulated in RM patients, suggesting the vital roles of decidual macrophages in the pathogenesis of RM. We found that MITA was highly expressed in decidual macrophages of RM patients and validated that MITA could promote apoptosis and macrophage proinflammatory polarization in THP-1-derived macrophage (TDM) cells. Using circRNA sequencing and bioinformatic analysis, we screened out a novel circRNA (circKIAA0391) that is overexpressed in decidual macrophages of RM patients. Mechanistically, we found that circKIAA0391 could promote the apoptosis and proinflammatory polarization of TDM cells by sponging the miR-512-5p/MITA axis. This study provides a theoretical basis for further understanding the impact of MITA on macrophages and its circRNA-related regulatory mechanisms, which may have a crucial immunomodulatory function in the pathophysiology of RM.

Impact of regulatory T cell therapy on immune cell composition and fetal survival rate in abortion prone mice.

CONTEXT: Implantation of fertilised eggs and survival of a semi-allogenic embryo rely on the interactions between the cells and molecules preparing the uterus. We investigated the effect of regulatory T cell (Treg) therapy on the mechanism of local immune tolerance of mice prone to spontaneous abortion. METHODS: Naive T cells were stimulated in vitro with 17ß-oestradiol (E2), progesterone (P4) and TGF-ß1 for 96h to generate induced Tregs (iTreg). The iTregs were injected into DBA/2-mated pregnant CBA/J female mice (abortion prone model). On day 14 of pregnancy, mice were killed and decidual and placental tissues were collected for cellular composition analysis. RESULTS: Abortion prone mice (PBS treated) showed significantly lower survival rates (P <0.0001), increased CD3+ CD8+ (P <0.05), lower IDO+ (P <0.05) and increased natural killer cells (uNK) cell numbers (P <0.001) in the uterus, as well increased NK cells in the placenta (P <0.05) than in normal pregnant mice (CBA/J×BALB/c). Adoptive transfer of iTregs increased fetal survival in abortion-prone mice (P <0.01) and histopathological evaluation revealed a significantly decreased number of uNK cells in the uterus of TGF-ß1-, E2- and P4-iTregs (P<0.05, P<0.0001 and P<0.05, respectively) than in the PBS treated group. In the placenta, we found significantly lower numbers of uNK cells from TGF-ß1-, E2- and P4-iTregs than in the PBS treated group (P <0.05, P <0.05 and P <0.01, respectively). CONCLUSIONS: We propose that modulation of uterine NK cell activity through immunotherapy using Treg cells should be given more attention as an immunological strategy in the treatment of recurrent miscarriage.

Immunohistochemical study of MMP-2, MMP-9, EGFR and IL-8 in decidual and trophoblastic specimens of recurrent pregnancy loss cases.

BACKGROUND AND AIM: Unexplained recurrent pregnancy loss has been a a challenging research task to experts since there is no explicit pathophysiological mechanism and therefore, the treatment remains elusive. Immunological imbalance and morphological abnormalities are under investigation. This study aims to evaluate the implication of MMP-2, MMP-9, EGFR, and IL-8 in recurrent pregnancy loss cases. MATERIALS & METHODS: The study was carried out through comparison among two groups; the unexplained miscarriage group which consisted of 22 women, and the control group consisted of 18 women, who had electively terminated their pregnancies. Both groups were in the first trimester of gestation. The specimens included the trophoblast, decidua basalis, and decidua parietalis. The study was conducted via immunohistochemical methods. Antibodies were used against MMP-2, MMP-9, EGFR, and IL-8. The results were presented at a contingency table and were statistically analyzed with the Chi-Square Test (X2). RESULTS: There were remarkable disparities in some cases in the comparison of the two groups. MMP-9 was detected significantly high in recurrent pregnancy loss (RPL) cases, both on trophoblastic and decidual specimens (p-value < .00001), MMP-2 displayed no difference among the two groups (mild to moderate detection on trophoblast and almost negative on decidual tissues). EGFR was highly detected in trophoblastic tissue (p-value = .014). IL-8 detection was particularly different in both trophoblast and decidua parietalis of the two groups (p-value < .01). CONCLUSION: The study revealed both morphological and immunological dysregulations that might participate in the RPL pathogenesis.

Endogenous retrotransposons cause catastrophic deoxyribonucleic acid damage in human trophoblasts.

OBJECTIVE: To determine the mechanistic role of mobile genetic elements in causing widespread DNA damage in primary human trophoblasts. DESIGN: Experimental ex vivo study. SETTING: Hospital-affiliated University. PATIENT(S): Trophoblasts from a patient with unexplained recurrent pregnancy loss and patients with spontaneous and elective abortions (n = 10). INTERVENTION(S): Biochemical and genetic analysis and modification of primary human trophoblasts. MAIN OUTCOME MEASURE(S): To phenotype and systematically evaluate the underlying pathogenic mechanism for elevated DNA damage observed in trophoblasts derived from a patient with unexplained recurrent pregnancy loss, transcervical embryoscopy, G-band karyotyping, RNA sequencing, quantitative polymerase chain reaction, immunoblotting, biochemical and siRNA assays, and whole-genome sequencing were performed. RESULT(S): Transcervical embryoscopy revealed a severely dysmorphic embryo that was euploid on G-band karyotyping. RNA sequencing was notable for markedly elevated LINE-1 expression, confirmed with quantitative polymerase chain reaction, and that resulted in elevated expression of LINE-1-encoded proteins, as shown by immunoblotting. Immunofluorescence, biochemical and genetic approaches demonstrated that overexpression of LINE-1 caused reversible widespread genomic damage and apoptosis. CONCLUSION(S): Derepression of LINE-1 elements in early trophoblasts results in reversible but widespread DNA damage.

Comprehensive RNA expression profile analysis of γδ T cells from peripheral blood and decidual tissues in normal pregnancy (NP) donors and patients with recurrent pregnancy loss (RPL).

Recurrent pregnancy loss (RPL) is a significant adverse pregnancy complication. The loss of immune tolerance has been proposed in the pathogenesis of RPL, however, the role of γδ T cells in RPL is still controversial. In this study, the gene expression patterns of circulated and decidual tissue-resident γδ T cells from normal pregnancy donors and patients with RPL were analyzed by SMART-seq. We demonstrate that the transcriptional expression profile of different subsets of γδ T cells in peripheral blood and decidual tissue is strikingly different. Vδ2 γδ T cells, as the major cytotoxic subset, are found to be enriched considerably, and the potential cytotoxicity of this subset is further enhanced in the decidua of RPL patients may be due to detrimental ROS reduction, enhanced metabolic activity, downregulation of immunosuppressive molecules expression in resident γδ T cells. Time-series Expression Miner (STEM) analysis of transcriptome indicates complex changes in gene expression in decidual γδ T cells over time from NP and RPL patients. Taken together, our work identifies high heterogeneity of gene signature in γδ T cells from NP and RPL patients between peripheral blood and decidua, which will be a useful resource for further studies of the critical roles of γδ T cells in RPL.

Age-related changes in CD4+ T and NK cell compartments may contribute to the occurrence of pregnancy loss in advanced maternal age.

A recent study characterized novel immune cell subsets (T, NK, and γδ T cell subsets) related to recurrent pregnancy loss (RPL). This study aims to assess whether these RPL-related immune cell subsets are affected by aging. The percentages of peripheral blood immunes cells from nulligravida women (NGW), women with a history of normal pregnancy (NP), and women with a history of pregnancy loss (PL) were detected by flow cytometry. The correlations between maternal age and cell percentages were assessed. We found a significant positive correlation between PL and maternal age. The percentages of effector memory CD4+ T (CD3+ CD4+ CD45RA¯ CCR7¯), terminally differentiated CD4+ T (CD3+ CD4+ CD45RA+ CCR7¯), and mature NK cells (CD3¯ CD56+lo) significantly increased with maternal age. A significant decrease in the percentage of Naïve CD4+ T cells (CD3+ CD4+ CD45RA+ CCR7+) with age was observed in women from the NP group. Women aged 35 or older had significantly higher percentages of effector memory CD4+ T cells, terminally differentiated CD4+ T cells, and mature NK cells than younger women. Maternal age positively correlates with terminally differentiated CD4+ T, effector memory CD4+ T, and mature NK cell percentages. In contrast, an inverse correlation was observed between Naïve CD4+ T cell and age among women from the NP group. Our findings indicate that age-related CD4+ T and NK cell dysregulation might be involved in the pathogenesis of PL in women with advanced maternal age. The underlying mechanism needs further investigation.

Clinical parameters associated with altered sperm DNA fragmentation index among primary infertile men: Findings from a real-life cross-sectional study.

BACKGROUND: Recurrent pregnancy loss and unexplained infertility are the current indications to test sperm DNA fragmentation according to the European Association of Urology Guidelines on sexual and reproductive health. OBJECTIVE: To identify a novel and better performing model to diagnose primary infertile men presenting with altered sperm DNA fragmentation and to outline its predictive ability in respect to current European Association of Urology Guidelines' recommendations. MATERIALS AND METHODS: Data from the latest 515 consecutive primary infertile men as for World Health Organization criteria were analyzed. Semen analysis, sperm DNA fragmentation (according to sperm chromatin structure assay), and serum hormones were considered in every patient. Altered sperm DNA fragmentation was defined with levels greater than 30%. Descriptive statistics was applied to compare patients with normal versus SDF > 30%. The new predicting model was identified through logistic regression analysis exploring potential predictors of SDF > 30% at first clinical presentation. Diagnostic accuracy between the two predictive models (European Association of Urology Guidelines vs. new) was assessed, and decision curve analyses tested their clinical benefit. RESULTS: Of 515, 268 (51.9%) patients had SDF > 30% at clinical presentation. Patients with SDF > 30% were older (median [interquartile range] 39 [35-43] vs. 37 [34-41] years), had lower mean testicular volume (Prader 15 [12-20] vs. 17.5 [13.5-20] and lower total motile sperm count (1.80 [0.7-13.2] vs. 11.82 [4.2-44.5] × 106 ), all p < 0.001. No other clinical differences were depicted. The two groups showed similar rates of history of recurrent pregnancy loss and unexplained infertility. At multivariable logistic regression analysis, age more than 38 years (odds ratio: 2.43) and baseline total motile sperm count less than 20 × 106 (odds ratio: 3.72) were associated with SDF > 30%, after adjusting for Prader < 15, history of miscarriages and unexplained infertility, all p < 0.0001. The newly identified model (unexplained infertility + history of poli-abortions + Prader < 15 + age ≥38 years + total motile sperm count <20 × 106 ) showed higher accuracy to identify SDF > 30% at baseline in respect to European Association of Urology Guidelines (area under the curve: 72.1 vs. 52.7), with superior clinical net benefit use. CONCLUSIONS: The application of the European Association of Urology sexual and reproductive health guidelines does not ensure proper identification of primary infertile men with pathological sperm DNA fragmentation. We propose a novel and better performing predictive model to identify the infertile men with altered sperm DNA fragmentation at first clinical assessment. DISCUSSION: As altered sperm DNA fragmentation has been widely linked with the inability to conceive, this second-level test could be further implemented over the diagnostic workup of a broader subset of patients presenting for male factor infertility. We propose a better performing model to identify this specific category of patients.

Sperm DNA fragmentation and idiopathic recurrent pregnancy loss: Results from a multicenter case-control study.

BACKGROUND: Sperm DNA fragmentation was hypothesized to have a role in the pathogenesis of recurrent pregnancy loss. Unfortunately, the quality of already published evidence is low. OBJECTIVES: To investigate the association between sperm DNA fragmentation and idiopathic recurrent pregnancy loss by limiting, as much as possible, the interference of confounding factors. MATERIALS AND METHODS: This was a retrospective multicenter case-control study conducted in two Italian University Hospitals (i.e., Policlinico Gemelli, Rome and Humanitas S. Pio X, Milan) from July 2020 to March 2022. Cases were men belonging to couples affected by first trimester idiopathic recurrent pregnancy loss, defined as the previous loss of two or more pregnancies. Two control groups were selected: (i) men belonging to couples with proven fertility (i.e., at least two previous full-term pregnancies) (control group A); (ii) men belonging to couples with proven infertility (i.e., the failure to achieve a pregnancy after 12 months or more of regular unprotected sexual intercourse) (control group B). The sperm DNA fragmentation index was measured by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. RESULTS: We included 74 cases, 37 men with proven fertility (control group A) and 100 men belonging to infertile couples (control group B). The median sperm DNA fragmentation index was significantly lower in control group A (17%, interquartile range: 14.3%-20.6%) compared to both case group (24.5%, interquartile range: 17%-32%; p < 0.0001) and control group B (24%, interquartile range: 18.9%-30%; p = 0.001). The rate of subjects with sperm DNA fragmentation index greater than 30% was significantly higher in both case groups (28%, 95% confidence interval [18%-40%]) and control group B (26%, 95% confidence interval [18%, 36%]) compared to control group A (0%, 95% confidence interval [0%-10%]) (p < 0.001). Multivariate regression models yielded a significant association between sperm DNA fragmentation index and recurrent pregnancy loss (adjusted odds ratio 1.13, 95% confidence interval [1.04-1.23], p = 0.006), but failed to show an association between sperm DNA fragmentation index and infertility (adjusted odds ratio 1.13, 95% CI [1-1.29], p = 0.05). CONCLUSIONS: Men within couples affected by recurrent pregnancy loss or infertility had a significantly higher rate of sperm DNA fragmentation compared to fertile controls. However, after adjusting for covariates, sperm DNA fragmentation index was associated only with recurrent pregnancy loss.