Important Role In Early Pregnancy Research Articles

P-436 Uterine NK cells underexpress KIR2DL1/S1 and LILRB1 receptors recognizing HLA-C2 and HLA-G on extravillous trophoblasts in reproductive failure

Abstract Study question Is there a difference in frequency, receptor expression and cytokine production of uterine NK cells (uNK) subpopulations in women with reproductive failure compared to controls? Summary answer Reproductive failure is associated with global underexpression of uNK receptors interacting with extravillous trophoblasts (EVT) that may result in defective implantation and placentation. What is known already A significant proportion of infertility, recurrent miscarriage and recurrent implantation failure are unexplained. This may be caused by immunological dysfunction at the maternal-fetal interface resulting in defective implantation and placentation. uNK is thought to play an important role in early pregnancy by cross-talking with EVT via KIR/HLA-C, LILRB1/HLA-G and CD94/HLA-E interactions to regulate EVT invasion and spiral arteriole remodeling in the decidua. Dysregulation of uNK function may cause reproductive failure; however it is unclear whether NK cell over- or under-activation is the pathology. Previous studies have been conducted on uNK obtained after miscarriage, which may be confounded by inflammatory processes. Study design, size, duration Here, we conducted a cross-sectional study to assess frequency, phenotype and function of matched pNK and uNK during the window of implantation in 16 women with reproductive failure compared to 11 controls. Women with reproductive failure cohort included unexplained subfertility (9), recurrent miscarriage (4) and recurrent implantation failure (3). The women were subsequently followed up after IVF and comparison made between women who did and did not fall pregnant. Participants/materials, setting, methods Matched peripheral blood and endometrial tissue were obtained from participants during the mid-secretory phase. Samples were accurately timed for 7-9 days after ovulation by urine LH test or histological dating. Flow cytometry was used to assess the frequency, phenotype and function. Phenotype was measured by KIR, LILRB1 and CD94 and receptor expression. Function was measured by degranulation (anti-CD107a internalization) and cytokine production (TNF-α, GM-CSF, IFN-γ and IL-8), with and without stimulation by PMA and ionomycin. Main results and the role of chance The expression of KIR2DL1/S1 and LILRB1 were significantly lower in reproductive failure group for both uNK (total uNK, uNK2 and 3) and pNK. The common finding in both uNK and pNK suggests that this is not tissue specific and may have an immunogenetic aetiology. We have previously found that degranulation peaks during secretory phase and in first trimester pregnancy. TNF-α production was also found to peak in secretory phase. Here, we found that degranulation activity of total uNK, as well as TNF-α production all uNK subsets was significantly lower in reproductive failure compared to control group. However, no significant difference was found in women in the reproductive failure cohort who fell pregnant compared to women who did not fall pregnant or miscarried after IVF. Contrary to findings from previous studies, we have not found any difference in uNK or pNK frequencies in any of the groups that were examined. Taken together, our findings suggest that reproductive failure is associated with global underexpression of receptors important for interaction with HLA-C and HLA-G on EVT, resulting in reduced uNK activation. This in turn may lead to dysregulation of implantation and placenta formation in early stage of pregnancy. Limitations, reasons for caution KIR2DL1/S1 antibody used does not distinguish between KIR2DL1 and KIR2DS1 which exerts inhibitory and activating effects respectively. Larger cohort studies on both pNK and uNK that consider KIR2DL1 and KIR2DS1 separately is needed to confirm which of these receptors are specifically reduced in reproductive failure. Wider implications of the findings Overall, our findings suggest that reproductive failure is associated with global underexpression of NK cell receptors leading to impaired implantation and placentation. This is the first study to examine uNK subsets during window of implantation and will serve as a platform to focus on particular uNK subsets in future studies. Trial registration number Not applicable

Long-chain acyl-CoA synthetase-4 regulates endometrial decidualization through a fatty acid β-oxidation pathway rather than lipid droplet accumulation

ObjectiveLipid metabolism plays an important role in early pregnancy, but its effects on decidualization are poorly understood. Fatty acids (FAs) must be esterified by fatty acyl-CoA synthetases to form biologically active acyl-CoA in order to enter the anabolic and/or catabolic pathway. Long-chain acyl-CoA synthetase 4 (ACSL4) is associated with female reproduction. However, whether it is involved in decidualization is unknown. MethodsThe expression of ACSL4 in human and mouse endometrium was detected by immunohistochemistry. ACSL4 levels were regulated by the overexpression of ACSL4 plasmid or ACSL4 siRNA, and the effects of ACSL4 on decidualization markers and morphology of endometrial stromal cells (ESCs) were clarified. A pregnant mouse model was established to determine the effect of ACSL4 on the implantation efficiency of mouse embryos. Modulation of ACSL4 detects lipid anabolism and catabolism. ResultsThrough examining the expression level of ACSL4 in human endometrial tissues during proliferative and secretory phases, we found that ACSL4 was highly expressed during the secretory phase. Knockdown of ACSL4 suppressed decidualization and inhibited the mesenchymal-to-epithelial transition induced by MPA and db-cAMP in ESCs. Further, the knockdown of ACSL4 reduced the efficiency of embryo implantation in pregnant mice. Downregulation of ACSL4 inhibited FA β-oxidation and lipid droplet accumulation during decidualization. Interestingly, pharmacological and genetic inhibition of lipid droplet synthesis did not affect FA β-oxidation and decidualization, while the pharmacological and genetic inhibition of FA β-oxidation increased lipid droplet accumulation and inhibited decidualization. In addition, inhibition of β-oxidation was found to attenuate the promotion of decidualization by the upregulation of ACSL4. The decidualization damage caused by ACSL4 knockdown could be reversed by activating β-oxidation. ConclusionsOur findings suggest that ACSL4 promotes endometrial decidualization by activating the β-oxidation pathway. This study provides interesting insights into our understanding of the mechanisms regulating lipid metabolism during decidualization.

Formate and its role in amino acid metabolism.

The aim of this report is to examine critical relationships between amino acid and formate metabolism with particular reference to the production of formate, and to review novel functions of formate. In addition to well established mechanisms in one-carbon metabolism, formate may play an important role in early pregnancy by preventing the onset of neural tube defects in sensitive strains of mice, including mice with deficiencies in MTHFD1L, the glycine cleavage system and the mitochondrial folate transporter. Markedly elevated, circulating levels of formate are found in late pregnancy, including in cord blood, as well as elevated levels of amino acid precursors. These are consistent with specific roles for formate in late pregnancy. Serine metabolism may reduce NADP to NADPH and permit the use of NADPH in reductive reactions. Novel, noncanonical functions of formate include high rates of formate production from serine in cells and in cancers. Novel, noncanonical functions of formate continue to be discovered. Integrating their functions with well established elements of one-carbon metabolism remains an important future objective.

Unique and independent role of the GABAB1 subunit in embryo implantation and uterine decidualization in mice

Embryo implantation and decidualization are crucial for successful pregnancy, which include multiple genes and signaling pathways, while the precise mechanism regarding embryo implantation and decidualization has yet to be explored. The GABA which activates GABAA or GABAB receptors has been found playing an important role in early pregnancy. Here we seek to investigate whether GABAB receptors participate in embryo implantation in mice. This study first characterized the spatiotemporal expression pattern of GABAB receptors in the uterus during the peri-implantation period and found that GABAB1 expression was drastically upregulated in stromal cells on days 4–6, a period of embryo implantation and early stages of decidualization. Embryo delayed implantation and oil-induced decidualization models were further used to confirm that the GABAB1 was associated with embryo implantation and decidualization. We also found estrogen or progesterone had no directly effect on expression of GABAB1 in ovariectomized model. Because we were unable to detect significant GABAB2 which couples with GABAB1 to form whole GABAB receptors, and the agonist and antagonist of whole GABAB receptors had weak effect on the proliferation and differentiation of stromal cells as well, we excluded the possibility whole GABAB receptors function, and concluded it should be non-classical signals of GABAB1 involving in embryo implantation and decidualization. Future studies should focus on investigating the roles and mechanisms of GABAB1 during embryo implantation and decidualization.

P-295 - Prooxidant effects of synthetic cannabinoids on endometrial cells, results in a strenous rebound adaptative effect

Synthetic cannabinoids (SCs) are easily available and their use as recreational drugs has increased over the years. It is known that the endocannabinoid system plays an important role in early pregnancy. Here, we assessed whether the chronic exposure (24, 48 and 72 h) to SCs JWH-018, JWH-122, UR-144, THJ-2201, 5F-PB-22 and AB-FUBINACA (0.01–25 µM) may affect human primary decidual fibroblasts (HdF) and endometrial stromal cell line (St-T1b) viability and redox status. For cell proliferation/viability we used MTT, ATP and BrDU assays, whereas redox status was analysed through detection of reactive oxygen species (ROS) and of reduced glutathione (GSH). Exposure to SCs caused an increase in mitochondrial activity, as indicated by MTT assay and ATP production, although no changes were seen in BrDU or in the ratio between mitochondrial and nuclear DNA. In addition, these compounds induced a prompt ROS/RNS release independently of either CB1 or CB2 receptors. On the other hand, chronic exposure (48 h) with JWH -018, JWH-122 and UR-144, revealed a reduction in ROS species generation accompanied by an increase in GSH, mostly in HdF cells. Collectively, our data suggests that SCs improve the antioxidant status of the endometrial cells, only after 48 h, in reaction to SCs initial aggression.

Estrogen-dependent regulation of human uterine natural killer cells promotes vascular remodelling via secretion of CCL2.

STUDY QUESTIONDoes intrauterine biosynthesis of estrogen play an important role in early pregnancy by altering the function of uterine natural killer (uNK) cells?SUMMARY ANSWEREstrogens directly regulate the function of human uNK cells by increasing uNK cell migration and secretion of uNK cell-derived chemokine (C-C motif) ligand 2 (CCL2) that critically facilitates uNK-mediated angiogenesis.WHAT IS KNOWN ALREADYuNK cells are a phenotypically distinct population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. Recently we discovered that decidualisation of human endometrial stromal cells results in the generation of an estrogen-rich microenvironment in areas of decidualised endometrium. We hypothesize that intrauterine biosynthesis of estrogens plays an important role in early pregnancy by altering the function of uNK cells.STUDY DESIGN, SIZE, DURATIONThis laboratory-based study used primary human uNK cells which were isolated from first trimester human decidua (n = 32).PARTICIPANTS/MATERIALS, SETTING, METHODSPrimary uNK cells were isolated from first trimester human decidua using magnetic cell sorting. The impact of estrogens on uNK cell function was assessed. Isolated uNK cells were treated with estrone (E1, 10−8 M) or estradiol (E2, 10−8 M) alone or in combination with the anti-estrogen ICI 182 780 (ICI, 10−6 M). uNK cell motility was assessed by transwell migration assay and time-lapse microscopy. Expression of chemokine receptors was assessed by quantitative PCR (qPCR) and immunohistochemistry, and angiogenic factors were assessed by qPCR and cytokine array. Concentrations of CCL2 in supernatants were measured by enzyme-linked immunosorbent assay. Angiogenesis was assessed in a human endometrial endothelial cell network formation assay.MAIN RESULTS AND THE ROLE OF CHANCETreatment with either E1 or E2 increased uNK cell migration (P = 0.0092 and P = 0.0063, respectively) compared with control. Co-administration of the anti-estrogen ICI blocked the effects of E1 and E2 on cell migration. Concentrations of C-X-C chemokine receptor type 4 (CXCR4) mRNA in uNK cells were increased by E2 treatment. The network formation assay revealed that conditioned media from uNK cells treated with E2 significantly increased human endometrial endothelial cell (HEEC) angiogenesis (P = 0.0029 versus control). Analysis of media from uNK cells treated with E2 using an antibody array identified CCL2 as the most abundant cytokine. Validation assays confirmed concentrations of CCL2 mRNA and protein were increased by E2 in uNK cells (P < 0.05 versus controls). Compared with the control, recombinant human CCL2 was found to increase HEEC network formation (P < 0.05) and neutralization of CCL2 in uNK conditioned media significantly decreased E2-dependent uNK-mediated network formation (P = 0.0006).LIMITATIONS, REASONS FOR CAUTIONOur results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in vivo in humans. Primary human uNK cells were isolated from first trimester decidua at a range of gestations (8–12 weeks), which may be a source of variation. Primary human uNK cells from non-pregnant endometrium were not assessed and therefore the responses of uNK cells to E2 treatment described in this study may be distinct to uNK cells from first trimester decidua.WIDER IMPLICATIONS OF THE FINDINGSE2 is an essential regulator of reproductive competence. This study demonstrates a critical role for E2 in regulating cellular cross-talk within the endometrium during early pregnancy. We provide the first evidence that E2 directly regulates the function of human uNK cells by altering uNK cell migration and the secretion of uNK-derived angiogenic factors. We describe a novel mechanism of estrogen-dependent secretion of CCL2 which critically mediates uNK-dependent endometrial angiogenesis. Dysregulation of uNK cell function has been implicated in the aetiology of early implantation disorders and disorders of pregnancy. These novel findings provide unique insight into the regulation of uNK cell activity during the establishment of pregnancy in women and highlight key processes which may be targeted in future therapeutic strategies.STUDY FUNDING/COMPETING INTEREST(S)Studies undertaken in the authors' laboratory were supported by MRC Programme Grant G1100356/1 to P.T.K.S. The authors have no conflicts of interest to disclose.

The insulin like growth factor axis as an early predictor of IVF outcome

The insulin like growth factor (IGF) axis is constituted of IGF-I, IGF-II and their ligand binding protein (BP), IGFBP-1. Pre-implantation exposure to high IGF-I concentrations has been linked to decreased embryo survival in mice. Cross talk between trophoblast-produced IGF-II and decidual IGFBP-1 has an important role in early pregnancy. We explored the use of IGF-I, IGF-II and IGFBP-1 as early stage biomarkers of IVF outcome.

Signal Transducer and Activator of Transcription 5a Mediates Mammary Ductal Branching and Proliferation in the Nulliparous Mouse

Signal transducer and activator of transcription (Stat)5a is a critical regulator of mammary gland development. Previous studies have focused on Stat5a's role in the late pregnant and lactating gland, and although active Stat5a is detectable in mammary epithelial cells in virgin mice, little is known about its role during early mammary gland development. In this report, we compare mammary gland morphology in pubertal and adult nulliparous wild-type and Stat5a-/- mice. The Stat5a-null mammary glands exhibited defects in secondary and side branching, providing evidence that Stat5a regulates these processes. In addition, Stat5a-/- mammary glands displayed an attenuated proliferative response to pregnancy levels of estrogen plus progesterone (E+P), suggesting that it plays an important role in early pregnancy. Finally, we examined one potential mediator of Stat5a's effects, receptor activator of nuclear factor-kappaB ligand (RANKL). Stat5a-/- mammary glands were defective in inducing RANKL in response to E+P treatment. In addition, regulation of several reported RANKL targets, including inhibitor of DNA binding 2 (Id2), cyclin D1, and the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), was altered in Stat5a-/- mammary cells, suggesting that one or more of these proteins mediate the effects of Stat5a in E+P-treated mammary epithelial cells.

Genetic imprinted gene PEG10 expression in deciduas of normal early pregnant women

Objective To investigate the role and significance of paternally expressed gene 10 (PEG10) in deciduas of normal pregnant women. Methods Sixty deciduas tissue from pregnant 6 to 11 weeks were divided into six groups and in each group ten samples were done every gestational week. The expression and distribution of PEG10 in deciduas were examined by RT-PCR, hybridization in situ, Western Blot and laser scanning confocal microscopy. Results The PEG10 mRNA and protein were expressed in deciduas tissue from pregnant 6 to 11 weeks. Among them, the expression of PEG10 showed a gradual increase as the pregnancy weeks increased. In RT-PCR, the PEG10 expression was lower at pregnant 6th week (0.6743±0.114), from pregnant 7 to 8 weeks, the expression was increased gradually (7th week 0.7349±0.0083) and reached the pinnacle at 8th week (0.7354±0.0074). And then the pinnacle began to descend (9th week 0.6340±0.0084, 10th week 0.5901±0.0089 and 11th week 0.5261±0.0112). There was a significant difference in the expression of PEG10 from pregnant 6 to 11 weeks except 7th week and 8th week. This expression characteristic was demonstrated by hybridization in situ. The similar conclusion was proved by Western Blot and laser scanning confocal microscopy. Conclusion Expression of PEG10 may play an important role in early pregnancy.

Natural killer cells and reproductive failure

The purpose of this review is to provide an update of what is known about the role of natural killer cells in reproduction. In humans, natural killer cells are present in abundance in the uterus and appear to play an important role in early pregnancy. Women with reproductive failure have been shown to have higher levels of natural killer cell numbers and activity in peripheral blood. The evidence relating to uterine natural killer cells, however, is contradictory. While earlier studies suggested an increase of uterine natural killer cells in women with recurrent pregnancy loss, more recent investigations have not confirmed this trend. Uterine natural killer cell number or activity appear to be higher in spontaneous abortions with normal chromosomes compared with chromosomally abnormal pregnancies. Furthermore, a rise of the cytotoxic CD56(dim)CD16+ natural killer cell subset is seen in the follicular fluid of patients with idiopathic infertility and it is postulated that this increase may decrease fertilization rates in this group. Natural killer cell number and activity appear to be altered in women with reproductive failure although it is unclear if the difference is a cause or effect of reproductive failure. Whether natural killer cells are increased or decreased depends on whether peripheral blood, endometrium or first-trimester decidua is analyzed. Until more is known about the role of natural killer cells in normal pregnancy, there does not appear to be any benefit in offering natural killer cell testing to women with reproductive failure outside of research protocols.

The Change of Membrane Complement Regulatory Protein in Chorion of Early Pregnancy

Research on membrane complement inhibitors (DAF, MCP, and CD59) has led to understanding of the regulation of complement system; however, their precise role and distribution remain speculative. In this study, we used an indirect fluorescent immunostaining to investigate the distribution of complements, MCP, DAF, and CD59, in the villi before the 10th week of pregnancy in 18 women. DAF and MCP were observed at the surface of syncytiotrophoblasts (ST) and extravillous trophoblast (EVT) in all subjects. They were observed in villous cytotrophoblast (VCT) in the subjects before the 8th week of pregnancy but were not observed in any subject after the 9th week. However, CD59 appeared in ST but never in VCT. MCP, DAF, and CD59 were observed in EVT. These findings indicated that these complement inhibitors played an important role in early pregnancy, and that CD59 continued to appear in early pregnancy, whereas the expression of MCP and DAF depended on the stage of pregnancy.

Lactoferrin in the mouse uterus: analyses of the preimplantation period and regulation by ovarian steroids.

Uterine expression of lactoferrin (LF) during the preimplantation period and its regulation by the ovarian steroids estradiol (E2) and/or progesterone (P4) in ovariectomized adult mice were examined. Immunoblot detection of LF in uterine cell lysates demonstrated the presence of this protein from days 1-8 of pregnancy [day 1 (D1) = day of vaginal plug]. Immunoprecipitation of 35S pulse-labeled uterine proteins showed that the relative rate of LF synthesis was high on D1, but below the level of detection by D4. Immunolocalization of LF in uterine sections showed intense luminal and glandular epithelial staining on D1 and D2, and progressively decreased staining through D4. Immunoreactive protein was also detected in cells, primarily concentrated in the stroma. The relative number of these cells was greatest on D1 and decreased progressively to a low number by D4. These cells were morphologically similar to neutrophils, which are known to contain LF protein, but little or no LF mRNA. Northern blotting showed that uterine LF mRNA levels were very high on D1 and D2 of pregnancy and decreased to low, but detectable, levels by D4. In situ hybridization to uterine sections showed that LF mRNA was highly abundant only in glandular and luminal epithelial cells, and followed the same pattern as immunolocalization on D1-D4 in epithelial cells. These results document two sources of LF in the preimplantation mouse uterus: neutrophils and epithelial cells. The synthesis of LF in the uterus reflects the abundance of epithelial LF mRNA, which is high on the first 2 days of pregnancy. Neutrophils that contain LF are also abundant in the uterine stroma during this time. E2 and/or P4 regulation of uterine LF was examined. LF mRNA was rapidly induced by E2 in ovariectomized adult mice, and this mRNA was localized exclusively to epithelial cells. P4 had little effect on uterine LF mRNA levels, but antagonized the prolonged induction of this gene by E2. E2 induced the accumulation of immunoreactive LF in uterine epithelial cells and the appearance of numerous immunopositive neutrophils distributed throughout the uterine stroma. P4 also antagonized these effects. Thus, E2 regulates LF gene expression in uterine epithelial cells and causes the recruitment of neutrophils into the uterus. These results suggest that LF may play an important role in early pregnancy and that uterine LF gene expression is regulated by a balance between estrogen and P4.

Late luteal estradiol patterns are a better prognosticator of pregnancy outcome than serial beta-human chorionic gonadotropin concentrations

Since the corpus luteum (CL) is known to play an important role in early pregnancy, its activity could possibly be a marker for pregnancy outcome.The late estradiol (E2) concentration in 48 viable pregnancies and 39 pregnancies which resulted in spontaneous abortions after in vitro fertilization and embryo transfer were used to evaluate such predictability.All patients studied were of the Center for Reproductive Medicine at Cornell University Medical College.Eighty-seven patients.None.Serum E2 and human chorionic gonadotropin (hCG) concentrations on day +11, +13, +15 (day +1 = day of ovum pick-up) were measured and studied.The late luteal CL activity after rescue had a positive correlation with the number and quality of the implanted embryos. Reduced CL activity was indicative of abortion. The late luteal E2 pattern when compared with hCG doubling time had a better abortion predictability (37.8% versus 63.9%, respectively).Corpus luteum activity demonstrated to be a better prognosticator of abortion than serial beta-hCG titers.