Number Of uNK Cells Research Articles

P-427 Do endometrial leucocytes and angiogenesis in the peri-implantation period predict hypertensive disorders in the subsequent pregnancy?

Abstract Study question Do endometrial leucocytes and angiogenesis in the peri-implantation period from women with a history of recurrent reproductive failure predict hypertensive disorders in the subsequent pregnancy? Summary answer uNK cells, T cells, B cells and macrophages in the peri-implantation endometrium did not correlate with hypertensive disease of pregnancy in the subsequent pregnancy. What is known already Women with a history of recurrent reproductive failure are thought to have a higher risk of hypertensive disorders of pregnancy (HDP) subsequently. Immunological interactions and vascular factors in the placental bed are considered to be two important pathways for the initiation and development of HDP. However, the prognostic value of endometrial leucocytes and angiogenesis around the time of implantation in a cycle prior to conception and HDP in the subsequent pregnancy is still unclear. Study design, size, duration This is a prospective cohort study. A total of 694 women were included in this study from Jan 2019 to Jun 2023. Participants/materials, setting, methods Immunohistochemistry for vWF and multiplex immunofluorescence for endometrial leukocytes, including CD56+ uNK cells, CD3+ T cells, CD20+ B cells and CD68+ macrophages, were performed in the precisely timed LH + 7 endometrium. The subsequent pregnancy outcomes of the subjects were followed up as well. Main results and the role of chance Endometrial leukocytes including CD56+ uNK cells, CD3+ T cells, CD20+ B cells and CD68+ macrophages were found in the peri-implantation endometrium. There was no significant difference in the CD56+ uNK cells, CD3+ T cells, CD20+ B cells and CD68+ macrophages numbers percentage between women with and without gestational hypertension in the subsequent pregnancy. The median micro vessel density in women who developed gestational hypertension in the subsequent pregnancy (9.4 ± 3.1 /mm2 ) was significantly (P=0.038) lower than the MVD in those who did not (13.5 ± 4.9/mm2 ). Limitations, reasons for caution 1. It is not clear whether the uNK cell number during the peri-implantation period is reflective of those in the first trimester when spiral artery remodelling occurs. 2. This study was carried out in women with reproductive failure and the results may not apply to the general populations. Wider implications of the findings Vascularization features in the endometrium may provide prognostic value of subsequent hypertensive disease of pregnancy. Trial registration number not applicable

The Association of NKp46-Positive uNK Cells With a Higher Risk of Recurrent Miscarriage and IVF Failure

Background: Uterine natural killer (uNK) cells have a significant impact on pregnancy and related complications. Given the importance of receptors in the activity of uNK cells, the present study aimed to determine the number of uNK cells and NKp46 (one of the most important NK cell-activating receptors) expression in the endometrium of women with recurrent miscarriage (RM) or a history of in vitro fertilization (IVF) failure. Materials and Methods: This case-control study was performed on 48 participants, including 16 healthy controls, 27 cases with RM, and 5 cases with repeated implantation failure (RIF) during the mid-luteal phase according to a standardized diagnostic protocol. All participants were assessed using transvaginal ultrasound to determine embryo survival rate and confirm gestational age. Endometrial specimens were collected and subjected to immunohistochemistry (IHC) staining using an anti-human NKp46 antibody expressed by uNK cells. Results: A significantly higher number of cells positive for NKp46 was obtained among two groups of cases versus healthy subjects (patients: 1.46±0.78, controls: 0.82±62, P=0.006), and the number of CD56+cells was significantly higher in patients than in controls (patients: 18.14±7.14, controls: 11.71±6.17, P=0.003). Additionally, there was not a significant difference in the frequency ratio of NKp46+NK cell subset to CD56+uNK cells between the patients (P=0.59) and control healthy group. Conclusion: The increase in the number of uterine NK cells and their cytotoxic activity during implantation and early pregnancy, possibly resulting from an excessive expression of inflammatory cytokines, confirms a significant association between uNK cell activity and a higher risk of RM and RIF. Therefore, immunomodulatory treatments may benefit these patients.

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

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. Naive T cells were stimulated in vitro with 17β-oestradiol (E2), progesterone (P4) and TGF-β1 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. 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-β1-, 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-β1-, E2- and P4-iTregs than in the PBS treated group (P<0.05, P<0.05 and P<0.01, respectively). 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.

Uterine Natural Killer Cells in the Context of Implantation: Immunohistochemical Analysis of Endometrial Samples from Women with Habitual Abortion and Recurrent Implantation Failure

Infertility affects approximately 48 million couples globally. Despite the enormous progress of the methods of reproductive medicine that has been made since the first test-tube baby was born in 1978, the implantation rate of day-3 embryos is only around 15-20 % and 30 % of day-5 embryos. Numerous strategies aim to improve implantation rates and prevent repeated implantation failure, however there is no specific general recommendation leading to satisfying results. One of the many risk factors relevant in this regard is the uterine immunological make-up, mainly the uterine Natural Killer (uNK) cells. They orchestrate the overall immune response during implantation by influencing trophoblast invasion and vascular remodeling and throughout pregnancy, uNK cells are also the main immune cells at the maternal-foetal interface. Previously, uNK count has been correlated with various fertility issues including idiopathic reccurent miscarriage. The present study used endometrial samples collected from 256 patients with recurrent implantation failure (RIF), habitual abortion (HA) and idiopathic sterility. Samples were collected between day 19 and 21 of the menstrual cycle mainly by Pipelle endometrial sampling. The samples were fixed in formalin for 24 hours and further processed for immunohistochemistry using anti-CD56 to visualize this antigen marker of uNK cells. Immunohistochemical counting was performed to assess the low, normal, or elevated count of uNK cells. According to the one-way ANOVA test, the age of our patients did not have any influence on the count of uNK cells. With Spearman correlation analysis, we found statistically significant correlation (p-value 0.05) of -0.133 between prior miscarriage and lower uNK cell count. Using the same analysis we found statistically significant correlation (correlation 0.233 with p-value 0.01) between number of uNK cells and activation status. Patients with higher uNK cells were more frequenty diagnosed with endometriosis (p-value 0.05, correlation 0.130). Patients with an immunological factor of sterility (defined by a clinical immunologist) had a lower chance of gravidity (-0.203 with p-value 0.01). Based on our results, we can confirm that there is a correlation between RIF, HA, idiopathic sterility, endometriosis, and immunological factor of sterility (uNK cell count). The true predictive value with regard to fertility outcomes needs to be addressed in future research.

Investigating Uterine Natural Killer Cells Through Next Generation Sequencing — A Pilot Gene Expression Study

Background: Uterine natural killer (uNK) cells form the major leucocyte population in the stroma in late secretory phase endometrium. Their actual function is unknown with available data suggesting a potential role in angiogenesis regulation, decidualization and placentation. Next generation sequencing (NGS) can be used to accurately define day of menstrual cycle through endometrial gene expression. Genes uniquely expressed by uNK are currently unknown. However, previous studies of first trimester decidua and blood have identified genes that are uniquely expressed by these NK cells. Aim: To identify potential molecular markers of uNK cells. Method: Endometrial samples were collected from 266 reproductive aged women across menstrual cycle. Gene expression profiles in endometrial tissues were generated using Illumina NovaSeq platforms. Gene expression data was normalized for cycle stage. Expression levels of previously known NK cell genes were examined across menstrual cycle. Results: Our analysis has shown that many NK cell genes (95% decidual, 71% peripheral) are expressed by the endometrium. We have identified 12 common genes that are expressed by both decidual and peripheral NK cells, and the endometrium. Expression levels of these common genes (except one) share a similar pattern across menstrual cycle. Their expression levels increase in secretory phase endometrium with peak expression levels at late secretory phase, as predicted by what is known about changing uNK cell numbers in endometrium. One example is the gene NCAM1 which codes for protein CD56 - a specific marker for uNK cells. Conclusion: This study has shown that it is possible to assess uNK cells through molecular approaches. Further molecular study of uNK cells is warranted and will help to provide evidence regarding uNK cells in human reproduction.

Porphyromonas gingivalis-mediated disruption in spiral artery remodeling is associated with altered uterine NK cell populations and dysregulated IL-18 and Htra1

Impaired spiral artery remodeling (IRSA) underpins the great obstetrical syndromes. We previously demonstrated that intrauterine infection with the periodontal pathogen, Porphyromonas gingivalis, induces IRSA in rats. Since our previous studies only examined the end stage of arterial remodeling, the aim of this study was to identify the impact of P. gingivalis infection on the earlier stages of remodeling. Gestation day (GD) 11 specimens, a transition point between trophoblast-independent remodeling and the start of extravillous trophoblast invasion, were compared to late stage GD18 tissues. P. gingivalis was found in decidual stroma of GD11 specimens that already had reduced spiral artery remodeling defined as smaller arterial lumen size, increased retention of vascular smooth muscle, and decreased invasion by extravillous trophoblasts. At GD11, P. gingivalis-induced IRSA coincided with altered uterine natural killer (uNK) cell populations, decreased placental bed expression of interleukin-18 (IL-18) with increased production of temperature requirement A1 (Htra1), a marker of oxidative stress. By GD18, placental bed IL-18 and Htra1 levels, and uNK cell numbers were equivalent in control and infected groups. However, infected GD18 placental bed specimens had decreased TNF + T cells. These results suggest disturbances in placental bed decidual stroma and uNK cells are involved in P. gingivalis-mediated IRSA.

O-300 Immune deficiency and endometrial dyshomeostasis in recurrent miscarriages

Abstract Study question Are dysregulated levels of immunomodulators (CXCL14, IL-15, and TIMP3) associated with aberrant decidual responses in the human endometrium? Summary answer Low transcript levels of CXCL14, IL-15, and TIMP-3 were associated with uterine natural killer (uNK) cell deficiency and pro-senescent response in the endometrium. What is known already In the mid-luteal phase, the endometrium becomes receptive to embryo implantation, while simultaneously poised for menstruation. Both events require decidualization, the differentiation of endometrial stromal cells into two cell subpopulations: progesterone-dependent decidual cells (DC) and progesterone-independent senescent decidual cells (snDC). Homeostatic balance between DC and snDC dictates endometrial fate. In non-conception cycles, snDC predominate as progesterone levels fall leading to menstruation. In conception cycles, endometrial transformation requires recruitment and activation of uNK cells, orchestrated by DC release of potent immunomodulators, CXCL14, IL-15, and TIMP3. This activation enables uNK cells to eliminate snDC, transforming the endometrium into the decidua of pregnancy. Study design, size, duration Endometrial biopsies (n = 730) were obtained from women attending the Implantation clinic at University Hospitals Coventry and Warwickshire (UHCW) NHS Trust. Collection was timed across the peri-implantation window based on the pre-ovulatory LH surge (LH + 6-11 days). Each biopsy was appropriately processed for immunohistochemistry and RT-qPCR analysis. Participants/materials, setting, methods Transcript levels of immunomodulators (CXCL14, IL-15, and TIMP3) were quantified in whole tissue by RT-qPCR, and uNK cells by immunohistochemical staining of CD56+. Due to changing expression levels across the luteal phase, results were normalized to the day of cycle using percentile charts. Expression levels of immunomodulators and uNK cells were compared to the expression of biomarkers of DC and snDC populations (SCARA5 and DIO2 respectively). Main results and the role of chance To understand the relationship between immunomodulators and decidual cell homeostasis, patient samples were separated into distinct categories of putative endometrial defects on the basis of DC and snDC biomarker expression. These included those with pro-decidual response (n = 61), pro-senescent response (n = 100), uNK cell deficiency (n = 42), and opposingly, uNK cell excess (n = 54). Expression of all tested immunomodulators (CXCL14, IL-15, and TIMP3) was significantly reduced in samples with a pro-senescent response (high DIO2, low SCARA5), compared to those displaying a pro-decidual response (high SCARA5, low DIO2; P &amp;lt; 0.0001; Kruskal-Wallis test). Additionally, patients with high numbers of uNK cells had a ∼2-fold-increase in CXCL14 (P = 0.0023), and IL-15 (P = 0.0052), and a 3-fold increase in TIMP3 (P &amp;lt; 0.0001) expression, compared to those with uNK cell deficiency. The data suggests that abnormal levels of these immunomodulators may dysregulate uNK cell activity and disrupt endometrial homeostasis, ultimately leading to miscarriage. Limitations, reasons for caution In this study, we showed the relationship between immunomodulators and decidual subpopulations. At present, it is not clear whether dysregulated levels of immunomodulators cause endometrial dyshomeostasis or are a result of the impaired decidual response. Further investigation is required to explore the “chicken-or-egg” dilemma. Wider implications of the findings Analysing these immunomodulators in a large number of samples provides a deeper understanding of the homeostatic relationship between uNK cells, DC, and snDC during the implantation window. This may be an invaluable diagnostic tool to identify women at risk of miscarriage, and in monitoring the effectiveness of pre-pregnancy interventions. Trial registration number N/A

P-394 Peripheral and uterine natural killer cell cut-off levels and their correlation in fertile controls and women with unexplained recurrent implantation failure (RIF)

Abstract Study question What is cut-off value for peripheral and uterine natural killer (pNK, uNK) cells and any correlation between them in fertile and RIF women? Summary answer Cut-off values were 7% for uNK and 11.6% for pNK in fertile controls. No significant correlation observed among fertile and infertile women with RIF. What is known already Association of uNK and pNK cells with infertility is debatable. It is not clear whether measuring NK cell levels in RIF has a role. pNk cell levels however has been used in various studies to guide the need of immunotherapy in RIF women. Definition of normal range of uNK cell numbers has also not been stated clearly because of lack of standardised protocol. Literature reveals the cut off values for pNk cells to be 12% or 18% and for uNK cells between 5-12.9%. Correlation between uNK and pNK cells in fertile or RIF women has not been studied earlier. Study design, size, duration A prospective study was conducted at ART Centre, Department of Obstetrics and Gynecology at tertiary care institute during a period from January 2019 to January 2021. Thirty women with RIF and fifty fertile controls with age &amp;lt;35 years having regular menstrual cycles and no hormonal treatment in last 3 months were enrolled in the study for uNK and pNK cell testing. The permission was obtained from the Institute Ethics Committee. Participants/materials, setting, methods Subjects included women with RIF with age 20-35 years, tubal and unexplained factors, normal ovarian reserve, normal karyotype and normal uterine cavity. In both subjects and controls, midluteal endometrial biopsy sample was taken for Immunohistochemistry staining of CD 56+ cells to determine uNK cells. Peripheral venous blood was also obtained at the time of biopsy for flow cytometry in peripheral blood mononuclear cells to determine pNK cells. Cut off values were derived from fertile controls. Main results and the role of chance The mean age and BMI were comparable between fertile control and study group(29.45±3.3 vs 31.17±3.3 years, 22.97±1.89 vs 23.21±2.2 kg/m2; p-value &amp;gt;0.05). The control group had median value of 7% for uNK cell levels and 11.6% for pNK cell levels. In RIF subjects, the median value for uNK cells was 13.5% and even though higher compared to controls it was not statistically significant(p-value &amp;gt;0.05) . The median pNK values were comparable among controls and RIF group (11.6% vs. 12%). All RIF subjects did not have elevated NK cell levels. 18 (60%) subjects had elevated uNK cell levels (more than 7%) whereas 40% had below the reference value. pNK cells were also elevated in only 53.3% of subject population. There was positive correlation found between uNK and pNK cell levels in RIF subjects, however it was not statistically significant (r 0.312, p-value 0.09). In fertile controls, there was insignificant negative correlation between uNK and pNK cell levels (r -0.231, p-value 0.33). Limitations, reasons for caution The small sample size is the foremost limitation of study. Wider implications of the findings uNK cell testing may be worthwhile in women with unexplained RIF so as to guide immunotherapy to improve pregnancy outcomes. As no significant correlation has been found between pNK and uNK cells in fertile and RIF women, pNK cell testing in RIF women may be done with caution. Trial registration number Not Applicable

Uterine Natural Killer Cells: A Rising Star in Human Pregnancy Regulation.

Uterine natural killer (uNK) cells are an immune subset located in the uterus. uNK cells have distinct tissue-specific characteristics compared to their counterparts in peripheral blood and lymphoid organs. Based on their location and the pregnancy status of the host, uNK cells are classified as endometrial NK (eNK) cells or decidua NK (dNK) cells. uNK cells are important in protecting the host from pathogen invasion and contribute to a series of physiological processes that affect successful pregnancy, including uterine spiral artery remodeling, fetal development, and immunity tolerance. Abnormal alterations in uNK cell numbers and/or impaired function may cause pregnancy complications, such as recurrent miscarriage, preeclampsia, or even infertility. In this review, we introduce recent advances in human uNK cell research under normal physiological or pathological conditions, and summarize their unique influences on the process of pregnancy complications or uterine diseases. Finally, we propose the potential clinical use of uNK cells as a novel cellular immunotherapeutic approach for reproductive disorders.

The uterine natural killer cell, cytotoxic T lymphocyte, and granulysin levels are elevated in the endometrium of women with nonstructural abnormal uterine bleeding

To examine the expression of uterine natural killer (uNK) cells and cytotoxic T lymphocytes (CTLs) in endometrial biopsies from reproductive-age women with and without nonstructural abnormal uterine bleeding (AUB) and evaluate the expression of granulysin within these cell populations and potential modulation of matrix metalloproteinase (MMP) expression. Experimental study, retrospective design. Academic research laboratory. Patients with nonstructural AUB with no other gynecological pathologies and control patients without AUB. None. Immunohistochemical analysis of granulysin, CD56 (uNK cell marker), and CD8 (CTL marker) expression as well as granulysin messenger ribonucleic acid (mRNA) expression levels in endometrial biopsy samples. Assessment of granulysin regulation of human endometrial stromal cell MMP-1 and MMP-3 mRNA expression. The numbers of uNK cells and CTLs were significantly greater in endometrial biopsy tissue from women with AUB than those from controls. In accord with the increased expression of uNK cells and CTLs, granulysin expression was significantly greater in endometrial biopsies from patients with AUB than in from controls and colocalized to both cell types but not endometrial stromal or epithelialcells. The increased granulysin protein expression was associated with the increased granulysin mRNA expression in adjacent serial sections from these same samples. The treatment of the human endometrial stromal cell line t-HESC with granulysin resulted in a significant increase in MMP-1 and MMP-3 mRNA expression. In the current study, immunohistochemistry showed an increased expression of uNK cells, CTLs, and granulysin among subjects with AUB compared with that of subjects without AUB, leading to conclusions that disturbances in the balance of immune cells and an increase in granulysin expression may have implications in the pathophysiology of AUB and include enhanced MMP-1 and MMP-3 expression.

Effects of Toxoplasma gondii infection on mouse and human uterine natural killer cells

Effects of Toxoplasma gondii infection on mouse and human uterine natural killer cells

Uterine Natural Killer Cells and Implantation

Decidual leukocytes make up approximately 30% of all decidual stromal cells in early pregnancy, of which 70% are uterine natural killer (uNK) cells. uNK cells are phenotypically distinct from peripheral blood NK cells, being CD56[Formula: see text]CD16[Formula: see text]. A recent single-cell sequencing project of the decidua identified three subsets of uNK cells, but we are yet to determine how they differ functionally. Several roles for uNK cells in implantation are starting to emerge including biosensing of poor-quality embryos, killing of bacteria infected trophoblast, spiral artery remodeling, and regulation of trophoblast invasion. Altered numbers of uNK cells have been identified in several pathological conditions, but whether this is causative of the condition is currently unclear.

P–375 Intralipid supplementation in women with unexplained recurrent implantation failure and elevated uterine natural killer cell levels - A randomized placebo controlled trial

Abstract Study question Does intralipid supplementation in women with unexplained recurrent implantation failure (RIF) with elevated uterine natural killer cell (uNK) levels improve pregnancy outcomes during IVF? Summary answer Intralipid supplementation appears to improve clinical pregnancy rate in women with unexplained RIF with elevated uNK cell levels. What is known already The increased numbers of uNK cells in peri-implantation endometrium have been reported in women with recurrent miscarriage (RM) and RIF after IVF. However, reports are contradictory when it comes to correlation of increased numbers of uNK cells with pregnancy outcome. Current opinion suggests there is a potential for intralipid therapy in improving reproductive outcome, although data on live birth rate is very limited. No studies have assessed the effect of intralipid on IVF outcomes in RIF women based on elevated uNK cells. Identified studies have all used pNK cell testing as preferred diagnostic tool for analysis of NK cell levels. Study design, size, duration A randomized placebo controlled trial was conducted at Division of Reproductive Medicine at tertiary care institute. Thirty women with RIF and fifty fertile controls with age &amp;lt;35 years having regular menstrual cycles and no hormonal treatment in last 3 months were enrolled in the study from January 2019 to December 2020 for uNK cell testing. Randomization was done using random numbers and sealed envelopes. Only the subjects were masked and allocation concealment was done. Participants/materials, setting, methods Subjects included RIF 20–35 years, normal ovarian reserve, unexplained and tubal factors, normal karyotype and normal uterine cavity. Cut off for uNK cells was derived from fertile controls by immunohistochemical staining of CD56+ cells from midluteal endometrial biopsy sample. Subjects with elevated uNK cell levels were randomized during IVF to group A (Intralipid) or group B ( saline ). The infusion was repeated within one week of positive pregnancy test and then every 2 weeks. Main results and the role of chance The mean age and BMI were comparable between fertile control and study group(29.45±3.3 vs 31.17±3.3 years, 22.97±1.89 vs 23.21±2.2 kg/m2 ;p&amp;gt;0.05). The median uNK cell levels was 7%(used as cut off) in fertile controls and 13.5% in RIF. 18 women (60%, 18/30) with RIF who had elevated uNK cell level (&amp;gt;7%) were randomized. Four women were lost to follow up. The median age, BMI, number of previous failed cycles and duration of infertility were comparable between Group A(n = 7) and Group B(n = 7){30(IQR:27–31) vs 33(IQR:30–34)years, 22.7(IQR:21.08–24.4) vs 22.6(IQR:21.37–24.2)kg/m2, 2(IQR:2–3) vs 2(IQR:2–3), 8(IQR:7–8) vs 8(IQR:7–10)years}. The median FSH, AMH and AFC were 5.86(IQR:5.13–7.67)mIU/l, 2.4(IQR:2.16–6.12)ng/ml, 10(IQR:8–12) in Group A which were comparable with Group B {6.2(IQR:4.78–6.5)mIU/l, 4.8(IQR:2.67–6.25)ng/ml, 12(IQR:12–16) }. All patients underwent antagonist protocol. The clinical pregnancy rate was 57.14%(4/7) in group A which was significantly higher as compared to 28.6%(2/7) in group B(p &amp;lt; 0.05). None of the patients reported any side effects due to intralipid. Limitations, reasons for caution The limitation of present study is its small sample size. However, the study is currently recruiting more RIF patients, and these are the interim results of the same. More RCTs with larger sample size are required to assess the efficacy of intralipid in this specific subset of population. Wider implications of the findings: The present study suggests the beneficial effect of intralipid in women with unexplained RIF with elevated uNK cell levels in increasing the chemical and clinical pregnancy rate. However, ongoing pregnancy rate and live birth rate should be investigated further in this subset of population. Trial registration number CTRI/2019/01/017213

Periconceptional exposure to lopinavir, but not darunavir, impairs decidualization: a potential mechanism leading to poor birth outcomes in HIV-positive pregnancies.

Periconceptional exposure to lopinavir, but not darunavir, impairs decidualization: a potential mechanism leading to poor birth outcomes in HIV-positive pregnancies.

Are uterine natural killer and plasma cells in infertility patients associated with endometriosis, repeated implantation failure, or recurrent pregnancy loss?

PurposeInfertility is a debilitating situation that millions of women around the world suffer from, but the causal relationship between infertility and endometriosis is still unclear. We hypothesize that the immune cell populations of uterine natural killer cells (uNK) and plasma cells (PC) which define chronic endometritis could differ in patients with or without endometriosis and therefore be the link to endometriosis-associated infertility.MethodsOur retrospective study includes 173 patients that underwent an endometrial scratching in the secretory phase of the menstrual cycle and subsequently immunohistochemical examination for uNK cells and PC. Sixty-seven patients were diagnosed with endometriosis, 106 served as the control cohort.ResultsThe risk for an elevated number of uNK cells in women with endometriosis is not increased as compared to the control group. Our findings suggest that patients with endometriosis are 1.3 times more likely to have chronic endometritis (CE) as compared to those without and that the treatment with doxycycline might increase pregnancy rates. Endometriosis and an increased number of uNK cells seem to be unrelated.ConclusionsIn contrast to the lately published connection between endometriosis, infertility and increased uNK cells, we could not find any evidence that patients with endometriosis are more prone to elevated uterine uNK cells. Counting of PC in endometrial biopsies might be a new approach in the search of biomarkers for the nonsurgical diagnosis of endometriosis since our findings suggest a connection.

Gestational Perfluorooctanoic Acid Exposure Inhibits Placental Development by Dysregulation of Labyrinth Vessels and uNK Cells and Apoptosis in Mice.

Perfluorooctanoic acid (PFOA) is a widely used perfluorinated compound and known to cause developmental toxicity which includes the increase of resorbed embryo, decrease of fetal survival, and fetal growth retardation. Nevertheless, whether it is associated with alteration of placental development remains unknown. Pregnant mice were gavaged with 0, 2.5, 5, 10 mg PFOA /kg/day from pregnancy day (PD) 1 to PD 13. Results showed that PFOA exposure markedly decreased the placental weight and caused interstitial edema of placenta. Laminin staining indicated that blood sinusoids area was shrunken in placenta of PFOA-exposed mice. Furthermore, PFOA treatment significantly reduced numbers of uNK cells. Western blot analysis revealed that levels of Bax and cleaved-caspase 3 proteins were markedly up-regulated in PFOA-treated groups. In addition, TEM examination showed that PFOA treatment caused rupture of nuclear membrane and nuclear pyknosis and fragmentation. Thus, our results suggested that gestational PFOA exposure significantly inhibited development of early placenta through shrinkage of labyrinth vessels and downregulation of uNK cells and apoptosis induction, which may result in adverse gestational outcomes.

Increase in FoxP3, CD56 immune cells and decrease in glands PGRMC1 expression in the endometrium are associated with recurrent miscarriages

ObjectiveRecurrent miscarriage (RM) is a multifactorial condition that involves frequent uterine anatomical abnormalities, parental karyotype abnormalities, and clotting disorders. We investigate the potential roles of endometrium FoxP3+ Tregs and CD56+ cells (uNK cells) and endometrial expression of PGRMC1 in the development of recurrent miscarriage. Study designThis prospective study included 102 out of 286 cases of SA patients. The cases were divided into groups with RM (+RM) and without RM (-RM). Immunohistochemistry staining was made using primary antibodies to FoxP3, CD56, and PGRMC1 in both groups. Morphometry analyses were carried out in 10 non-overlapping high power fields. Mann-Whitney U test, Fisher two-tail test, correlation analysis and relative risk (RR) were evaluated. A p < 0.05 was considered statistically significant. ResultsAn increased presence of CD56-positive (p < 0.001) and FoxP3+ Treg (p = 0.0005) cells was found in the endometrium, with a reduction in PGRMC1 expression compared with -RM group (p = 0.004). A positive correlation was shown between the number of CD56-positive cells and FoxP3+ cells (r = 0.55), and an inverse correlation with PGRMC1 (r = -0.35) in the + RM group. A similar observation was found in the -RM group, with a positive correlation of uNK cell number with the number of pregnancies (p < 0.001; r = 0.34). Endometrial infiltration of CD56-positive (p < 0.0001) and FoxP3+ (p < 0.0001) cells revealed an increased relative risk of RM. This increased risk was also revealed in SA with a loss of PGRMC1 expression (p < 0.0001). ConclusionOur prospective study suggests, for the first time, that increased endometrial infiltration of uNK, FoxP3+ Treg cells and a decreased PGRMC1 expression may play potential roles in the development of RM.

Endometrial uNK cell counts do not predict successful implantation in an IVF population.

Are uterine natural killer (uNK) cell numbers and their distribution relative to endometrial arterioles altered in women with recurrent implantation failure (RIF) compared to women with embryo implantation success (IS)? uNK cell numbers and their distribution relative to endometrial arterioles are not significantly different in women with RIF compared to women in whom embryo implantation occurs successfully following IVF. uNK cells are regulators of decidual angiogenesis and spiral arteriole remodelling during early pregnancy. Although some studies have shown that uNK cell numbers may be altered in women with RIF, the methods used to measure uNK cell numbers have proven inconsistent, making reproduction of these results difficult. It is unclear, therefore, whether the results reported so far are reproducible. Moreover, it is not known how uNK cell numbers may impact IVF outcomes. Despite the lack of conclusive evidence, uNK cell numbers are often evaluated as a prognostic criterion in women undergoing assisted reproductive procedures. Endometrial pipelle biopsies were collected 6-8days post-LH surge in natural cycles from women with RIF (n = 14), women with IS (n = 11) and women with potential RIF at the time of the study (PRIF; n = 9) from 2013 to 2015. uNK cells (i.e. CD56+ and/or CD16+ phenotypes) and their distribution relative to endometrial arterioles were investigated by standard immunohistochemistry protocols and quantified using Aperio ScanScopeXT images digitized by ImageJ and deconvoluted into binary images for single cell quantification using a Gaussian Blur and Yen algorithm. There was no significant difference in the cell density of CD56+ or CD16+ uNK cells in women with RIF compared to women with IS or PRIF. There was a higher proportion of uNK cells in the distal regions compared to the regions closest to the arterioles in all patient groups. Further, we identified a significant reduction in uNK cell density in women who had a previous pregnancy compared to those who had not, regardless of their current implantation status. Not applicable. Spiral arterioles could not always be accurately identified by digital image analysis; therefore, all endometrial arterioles were selected and analysed. Patient numbers for the study were low. However, as the clinical phenotypes of each patient were well defined, and endometrial dating was accurately determined by three independent pathologists, differences between patient groups with respect to the uNK numbers and distribution should have been measurable if uNK cell counts were to be useful as a prognostic marker of RIF. Our findings demonstrate that CD56+ and CD16+ uNK cell numbers are not significantly different in women with RIF in a typical cohort of women undergoing IVF. Further, prior pregnancy was associated with a significantly reduced number of uNK cells in both the RIF and IS patient groups, suggestive of a long-term pregnancy induced suppression of uNK cells. Combined, these findings do not support the clinical value of using uNK cell numbers as a prognostic indicator of implantation success with IVF treatment. Funding for this work was provided by Royal Women's Hospital Foundation. P.P. was supported by an NHMRC Early Career Fellowship [TF 11/14] and W.T.T. was supported by an NHMRC Postgraduate Scholarship [1055814]. The authors do not have any competing interests with this study.

Flutamide ameliorates uterine decidualization and angiogenesis in the mouse hyperandrogenemia model during mid-pregnancy.

Background/AimPatients with polycystic ovary syndrome (PCOS), characterized by anovulation, hyperandrogenemia and polycystic ovaries, are still vulnerable to undergo recurrent pregnancy loss and premature labor even though the ovulatory process is pharmacologically recovered. However, its potential mechanism remains unknown. Thus, our aim was to investigate the effect and mechanism of hyperandrogenemia and flutamide (a non-steroidal anti-androgen) on the embryo implantation and pregnancy during mid-pregnancy.MethodsWe used a mouse model in which PCOS-like hyperandrogenemia was induced by subcutaneous injection of testosterone propionate. In this model, we observed the effect of hyperandrogenemia and flutamide on the decidualization, angiogenesis and uNK cells by methods of immunohistochemistry, quantitative PCR, western blotting and Dolichos biflorus agglutinin (DBA) lectin staining.ResultsTestosterone and flutamide treatment did not significantly influence the numbers of implanted embryo compared with the control group. However, different doses of testosterone significantly increased the ratio of resorbed /implanted embryo, decreased the level of prl8a2 mRNA and cyclin D3 protein, inhibited the uterine angiogenesis and reduced the numbers of uNK cells, but combined treatment with flutamide markedly decreased the resorbed embryos, increased expressions of prl8a2 mRNA and cyclin D3 protein and angiogenesis and numbers of uNK cells.ConclusionFlutamide treatment can efficiently ameliorate the hyperandrogenemia-induced the disorders in aspects of decidualization, angiogenesis and uNK cells, which further improve the poor endometrial receptivity in PCOS patients.

Lectin histochemical analysis of uterine natural killer cells in normal, hydatidiform molar and invasive molar pregnancy.

Uterine natural killer (uNK) cells have been hypothesized to serve a role in controlling trophoblast invasion and proliferation. The aim of the present study was to identify the distribution and number of uNK cells in normal pregnancy (NP), partial mole (PM), complete mole (CM) and invasive mole (IM). uNK cells were detected using dolichos biflorus agglutinin lectin immunohistochemistry in decidual and villous tissues from early NP (n=15), late NP (n=15), PM (n=22), CM (n=20) and IM (n=10). A scaled eye piece was used for cell counting to obtain semi-quantitative results. It was revealed that uNK cells were mainly located in the uterine deciduas of early NP. As pregnancy progressed, the number of decidual uNK cells significantly decreased. Decidual uNK cells of PM, CM and IM were located near blood vessel endothelial cells. No significant differences were detected with respect to the numbers of decidual uNK between early NP and PM. However, the number of decidual uNK cells was significantly reduced in CM and IM compared with early NP. The populations of decidual uNK cells were not significantly different between CM and IM. No uNK cells were detected in the villi of PM, CM or IM. The decrease of decidual uNK cells in late NP, CP and IM, compared with early NP, suggested that uNK cells served an important role in controlling trophoblast invasion and proliferation.