Role of Natural Killer Cells during Pregnancy and Related Complications

A high number of leucocytes reside in the human endometrium and are distributed differentially during the menstrual cycle and pregnancy. During early pregnancy, decidual natural killer (dNK) cells are the most common type of natural killer (NK) cells in the uterus. The increase in the number of uterine NK (uNK) cells during the mid-secretory phase of the menstrual cycle, followed by further increase of dNK cells in early pregnancy, has heightened interest in their involvement during pregnancy. Extensive research has revealed various roles of dNK cells during pregnancy including the formation of new blood vessels, migration of trophoblasts, and immunological tolerance. The present review article is focused on the significance of NK cells during pregnancy and their role in pregnancy-related diseases. The article will provide an in-depth review of cellular and molecular interactions during pregnancy and related disorders, with NK cells playing a pivotal role. Moreover, this study will help researchers to understand the physiology of normal pregnancy and related complications with respect to NK cells, so that future research work can be designed to alleviate the complications.

The Role of Decidual Subpopulations in Implantation, Menstruation and Miscarriage

In each menstrual cycle, the endometrium becomes receptive to embryo implantation while preparing for tissue breakdown and repair. Both pregnancy and menstruation are dependent on spontaneous decidualization of endometrial stromal cells, a progesterone-dependent process that follows rapid, oestrogen-dependent proliferation. During the implantation window, stromal cells mount an acute stress response, which leads to the emergence of functionally distinct decidual subsets, reflecting the level of replication stress incurred during the preceding proliferative phase. Progesterone-dependent, anti-inflammatory decidual cells (DeC) form a robust matrix that accommodates the conceptus whereas pro-inflammatory, progesterone-resistant stressed and senescent decidual cells (senDeC) control tissue remodelling and breakdown. To execute these functions, each decidual subset engages innate immune cells: DeC partner with uterine natural killer (uNK) cells to eliminate senDeC, while senDeC co-opt neutrophils and macrophages to assist with tissue breakdown and repair. Thus, successful transformation of cycling endometrium into the decidua of pregnancy not only requires continuous progesterone signalling but dominance of DeC over senDeC, aided by recruitment and differentiation of circulating NK cells and bone marrow-derived decidual progenitors. We discuss how the frequency of cycles resulting in imbalanced decidual subpopulations may determine the recurrence risk of miscarriage and highlight emerging therapeutic strategies.

The Role of Uterine Natural Killer Cells on Recurrent Miscarriage and Recurrent Implantation Failure: From Pathophysiology to Treatment

Uterine natural killer (uNK) cells constitute a unique uterine leucocyte subpopulation facilitating implantation and maintaining pregnancy. Herein, we critically analyze current evidence regarding the role of uNK cells in the events entailed in recurrent implantation failure (RIF) and recurrent miscarriages (RM). Data suggest an association between RIF and RM with abnormally elevated uNK cells’ numbers, as well as with a defective biological activity leading to cytotoxicity. However, other studies do not concur on these associations. Robust data suggesting a definitive causative relationship between uNK cells and RIF and RM is missing. Considering the possibility of uNK cells involvement on RIF and RM pathophysiology, possible treatments including glucocorticoids, intralipids, and intravenous immunoglobulin administration have been proposed towards addressing uNK related RIF and RM. When considering clinical routine practice, this study indicated that solid evidence is required to report on efficiency and safety of these treatments as there are recommendations that clearly advise against their employment. In conclusion, defining a causative relationship between uNK and RIF–RM pathologies certainly merits investigation. Future studies should serve as a prerequisite prior to proposing the use of uNK as a biomarker or prior to targeting uNK cells for therapeutic purposes addressing RIF and RM.

Biology and pathology of the uterine microenvironment and its natural killer cells

Tissues are the new frontier of discoveries in immunology. Cells of the immune system are an integral part of tissue physiology and immunity. Determining how immune cells inhabit, housekeep, and defend gut, lung, brain, liver, uterus, and other organs helps revealing the intimate details of tissue physiology and may offer new therapeutic targets to treat pathologies. The uterine microenvironment modulates the development and function of innate lymphoid cells [ILC, largely represented by natural killer (NK) cells], macrophages, T cells, and dendritic cells. These immune cells, in turn, contribute to tissue homeostasis. Regulated by ovarian hormones, the human uterine mucosa (endometrium) undergoes ~400 monthly cycles of breakdown and regeneration from menarche to menopause, with its fibroblasts, glands, blood vessels, and immune cells remodeling the tissue into the transient decidua. Even more transformative changes occur upon blastocyst implantation. Before the placenta is formed, the endometrial glands feed the embryo by histiotrophic nutrition while the uterine spiral arteries are stripped of their endothelial layer and smooth muscle actin. This arterial remodeling is carried out by invading fetal trophoblast and maternal immune cells, chiefly uterine NK (uNK) cells, which also assist fetal growth. The transformed arteries no longer respond to maternal stimuli and meet the increasing demands of the growing fetus. This review focuses on how the everchanging uterine microenvironment affects uNK cells and how uNK cells regulate homeostasis of the decidua, placenta development, and fetal growth. Determining these pathways will help understand the causes of major pregnancy complications.

P–361 Endometrium optical coherence tomography (OCT) and histomorphometry in implantation window and their relationships with reproductive failure and implantation outcome

Study question How do endometrium OCT image characteristics during peri-implantation window correlate with histomorphometry and associate with implantation outcomes in women with reproductive failure? Summary answer Endometrium OCT intensity correlated with stromal cell density and gland size. Endometrium with recurrent implantation failure had low OCT intensity but reversed in successful implantation. What is known already OCT is a non-invasive imaging technique using low energy near-infrared light to capture micrometer-scale resolution images from optical scattering media. An image produced by OCT resembles tissue architecture observed in histology, so OCT imaging has been regarded as “optical biopsy”. Our previous findings demonstrated OCT is better than ultrasound to identify endometrial pathology. We also showed association of OCT signal with microvessel density in peri-implantation endometrium. However, other histomorphometry were not evaluated. It is still unclear whether endometrium OCT image characteristics are different in reproductive failure and can predict implantation outcomes. Study design, size, duration This was a prospective study conducted at teaching hospital of The Chinese University of Hong Kong from Jan 2018 to Dec 2019. 46 infertile women with or without recurrent miscarriage (RM) and implantation failure (RIF) were recruited in this study. Endometrium OCT imaging and subsequent biopsy were performed on the seventh day after luteal hormone surge (LH + 7) in natural cycle prior to the consecutive natural conception or embryo transfer (ET) cycle. Participants/materials, setting, methods At least 5 systematic random endometrium OCT images from uterine fundus, body and lower segment of each subject were included for intensity analysis by two independent observers. OCT intensity of each image was classified as low, moderate, high based on optical range and then average OCT intensity in each uterine region was calculated for group comparison. Endometrium glandular epithelial, stromal, endothelial, uNK cells were defined by standard H&E and specific immunostaining for histomorphometry and correlation. Main results and the role of chance OCT intensity significantly correlated with endometrial cell and gland parameters regardless classifications of reproductive failure and implantation outcome. Higher OCT intensity indicated higher stromal cell density, gland to stromal (G/S) ratio and average gland area, but fewer microvessel and uNK cells. None of the endometrium histomorphometry were significantly different among different reproductive failure types and implantation outcomes, suggesting it may not be sensitive enough to detect the abnormal histological features. However, OCT intensity was significantly lower in the uterine fundus and body of RIF group than in that of infertile and RM groups. There was no significant difference of OCT intensity in the lower part of the endometrium among three groups. It indicates that OCT intensity is a sensitive marker to differentiate endometrium with RIF from the endometrium with other conditions and also endometrium with RIF is characterized with less stromal cells and smaller glands. Compared with infertile group with unsuccessful implantation, OCT intensity was higher in all three parts of the uterus from the infertile group with successful implantation, but the results were not statistically different. The results further implied that endometrial cells and gland size may potentially contribute to the endometrium receptivity for implantation. Limitations, reasons for caution Current endometrium OCT imaging depth is within 3mm, change beyond this thickness is inaccessible but still the most important layer for implantation. This is a pilot and small study with lack of normal fertile control. Endometrium OCT imaging in the same natural conception or ET cycle will be more accurate. Wider implications of the findings: OCT imaging could be used as a potential noninvasive modality to evaluate peri-implantation window endometrium. It enables real-time and in-situ visualization of endometrium structure and pathology with no additional biopsy risk and examination delay. Larger clinical trials are needed to confirm its clinical applications and utilities. Trial registration number CREC 2016.160

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

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 <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>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 (>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 < 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

SPP1 expression in the mouse uterus and placenta: Implications for implantation

Secreted phosphoprotein 1 [SPP1, also known as osteopontin (OPN)] binds integrins to mediate cell–cell and cell-extracellular matrix communication to promote cell adhesion, migration, and differentiation. Considerable evidence links SPP1 to pregnancy in several species. Current evidence suggests that SPP1 is involved in implantation and placentation in mice, but in vivo localization of SPP1 and in vivo mechanistic studies to substantiate these roles are incomplete and contradictory. We localized Spp1 mRNA and protein in the endometrium and placenta of mice throughout gestation, and utilized delayed implantation of mouse blastocysts to link SPP1 expression to the implantation chamber. Spp1 mRNA and protein localized to the endometrial luminal (LE), but not glandular epithelia (GE) in interimplantation regions of the uterus throughout gestation. Spp1 mRNA and protein also localized to uterine naturel killer (uNK) cells of the decidua. Within the implantation chamber, Spp1 mRNA localized only to intermittent LE cells, and to the inner cell mass. SPP1 protein localized to intermittent trophoblast cells, and to the parietal endoderm. These results suggest that SPP1: 1) is secreted by the LE at interimplantation sites for closure of the uterine lumen to form the implantation chamber; 2) is secreted by LE adjacent to the attaching trophoblast cells for attachment and invasion of the blastocyst; and 3) is not a component of histotroph secreted from the GE, but is secreted from uNK cells in the decidua to increase angiogenesis within the decidua to augment hemotrophic support of embryonic/fetal development of the conceptus.

Intralipid Therapy and Adverse Reproductive Outcome: Is there Any Evidence?

Controversy exists regarding the benefits of intravenous intralipid therapy in patients with poor reproductive history. It is frequently reported that there is no evidence to support the effectiveness, utility or safety for this treatment. While individual studies may be perceived as weak, a systematic review and meta-analysis was performed to determine if there is any advantage to patients. PubMed, Embase and Scopus searches were performed with the target populations being either recurrent pregnancy loss (RPL), or recurrent implantation failure (RIF) undergoing assisted reproductive technology (ART) and receiving intralipid infusions. These cohorts were compared with either placebo, no intervention or alternative treatments. The most relevant outcome measures were considered to be clinical pregnancy rate (CPR), live birth rate (LBR), implantation rate (IR) and miscarriage rate (MR). Twelve studies encompassing 2,676 participants met the criteria for selection and were included and reviewed. Treatment of the target population with intralipid led to an improvement in IR (Odds Ratio (OR) 2.97, 2.05-4.29), pregnancy rate (OR 1.64, 1.31-2.04), and LBR (OR 2.36, 1.75-3.17), with a reduction in MR (OR 0.2, 0.14-0.30). Although intravenous intralipid is not recommended as a routine treatment for recurrent miscarriage or implantation failure, there is enough data to suggest consideration in selected patients where routine testing is unremarkable, standard treatments have failed and immunological risk factors are present. The presence of abnormal uterine natural killer (uNK) cells needs more study as a target marker to determine those who could benefit.

Continuous human uterine NK cell differentiation in response to endometrial regeneration and pregnancy

Immune cell differentiation is critical for adequate tissue-specific immune responses to occur. Here, we studied differentiation of human uterine natural killer cells (uNK cells). These cells reside in a tissue undergoing constant regeneration and represent the major leukocyte population at the maternal-fetal interface. However, their physiological response during the menstrual cycle and in pregnancy remains elusive. By surface proteome and transcriptome analysis as well as using humanized mice, we identify a differentiation pathway of uNK cells in vitro and in vivo with sequential acquisition of killer cell immunoglobulin-like receptors and CD39. uNK cell differentiation occurred continuously in response to the endometrial regeneration and was driven by interleukin-15. Differentiated uNK cells displayed reduced proliferative capacity and immunomodulatory function including enhanced angiogenic capacity. By studying human uterus transplantation and monozygotic twins, we found that the uNK cell niche could be replenished from circulation and that it was under genetic control. Together, our study uncovers a continuous differentiation pathway of human NK cells in the uterus that is coupled to profound functional changes in response to local tissue regeneration and pregnancy.