Serial changes in two immune checkpoint receptors and ligands, Tim-3/Gal-9 and PD-1/PD-L1 in peripheral blood prior to miscarriage: Comparison with pregnancies resulting in a live birth.

PROBLEM: Immune checkpoints Tim-3/Gal-9 and PD-1/PL-1 are involved in the maintenance of maternal-fetal immune tolerance systematically and locally. This study aimed to compare the serial changes of Tim-3/Gal-9, and PD-1/PL-1 in peripheral blood over a 4-week period after blastocyst transfer, between women who had a live birth and those who miscarried. METHODS OF STUDY: Serial blood samples were obtained on the day of ET, and 9, 16, 23, and 30 days after ET for the measurement of Tim-3 and PD-1 expressions on various lymphocytes by flow cytometry. Concentrations of serum Gal-9 and PD-L1 were measured by ELISA. RESULTS: In pregnancies that resulted in a live birth, a significant and sustained increase in the proportion of Tim-3+ pNK cells was observed from the 9th to 30th days after ET, whilst the concentration of serum PD-L1 was significantly increased on the 23rd and 30th days after ET when compared to the day of ET. In pregnancies that later miscarried, none of the parameters were significantly changed across all the time points examined. When comparing the results between the two groups, the proportion of Tim-3+ CD56dim NK cells in the women who had a live birth was significantly higher than that in women who miscarried from the 9th to 30th day after ET. CONCLUSION: A significant and sustained increase in the proportion of Tim-3+ pNK cells was observed in pregnancies resulting in a live birth but not in pregnancies resulting in a miscarriage, suggesting the changes may be associated with successful pregnancy outcomes.

Uterine CD56+ cell density and euploid miscarriage in women with a history of recurrent miscarriage: A clinical descriptive study.

In women with a history of recurrent miscarriage, the uterine CD56+ cell density in subjects with subsequent euploid miscarriage was significantly higher than those with subsequent aneuploid miscarriage. Both endometrial and embryonic factors should be investigated when interpreting uterine CD56+ cell density results relating to recurrent miscarriage.

Association between chronic endometritis and uterine natural killer cell density in women with recurrent miscarriage: clinical implications.

AIM: This aim of this study was to determine the association between uterine natural killer (uNK) cell density and chronic endometritis (CE). METHODS: Endometrial biopsies from 135 women with recurrent miscarriage were obtained precisely 7 days after luteinizing hormone surge in natural cycles. Endometrial sections were immunostained for CD56 for uNK cells and CD138 for plasma cells, respectively. Uterine NK cell counting was performed according to a standardized protocol and results were expressed as percentage of CD56+ cells/ total stromal cells. High uNK cell density was defined as >4.5% and CE was diagnosed when the plasma cell density > 5.15 cells/ 10 mm2 . RESULTS: The uNK cells density in women with CE (median, 5.1%; range, 3.4-8.8%) was significantly (P < 0.05) higher than that of those without CE (median, 3.8%; range, 1.2%-7.3%). The prevalence of high uNK cell density in women with CE (11/29, 37.9%) was significantly (P < 0.05) higher than that of women without CE (8/106, 7.5%). CONCLUSION: To conclude, there was a significant association between high uNK cell density and CE. In women with high uNK cell density, plasma cell should be examined to determine if the underlying cause is associated with CE.

Early transient suppression of immune checkpoint proteins T-cell immunoglobulin mucin-3 and programmed cell death-1 in peripheral blood lymphocytes after blastocyst transfer is associated with successful implantation.

OBJECTIVE: To compare the changing peripheral levels of immune checkpoint proteins T-cell immunoglobulin mucin-3 (Tim-3)/galectin-9 (Gal-9), and programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) over a 9-day period after blastocyst transfer between women who did and did not conceive. DESIGN: Prospective observational study. SETTING: University teaching hospital. PATIENTS(S): Fifty-one infertile women undergoing day-5 blastocyst transfer. INTERVENTION(S): Serial blood samples obtained on the day of embryo transfer (ET), and 3, 6, and 9 days afterward for measurement of membranous Tim-3 and PD-1 expression on various peripheral lymphocytes by flow cytometry, and serum concentrations of ligands Gal-9 and PD-L1 by ELISA. MAIN OUTCOME MEASURE(S): Membranous Tim-3 and PD-1 expression on lymphocytes and serum Gal-9 and PD-L1 concentrations and comparison of results between pregnant and nonpregnant women. RESULT(S): In women who conceived, the measurements exhibited three different types of response: [1] a transient and statistically significant reduction of Tim-3+NK-like T cells, Tim-3+/PD-1+CD8+ T cells, and Tim-3+/PD-1+CD4+ T cells that returned back to baseline level 9 days after ET; [2] a reduction followed by steady increase to above baseline level on day 9 (Tim-3+CD56dimNK cells); [3] a steady increase in expression after ET to reach a level statistically significantly higher than that of the baseline by day 9 (Tim-3+CD56brightNK cells). Women who did not conceive showed no statistically significant fluctuation in any of the parameters measured across the four time pointswith exception of increased Tim-3 expression on NK cells on day 9. CONCLUSION(S): Successful blastocyst implantation is associated with a reduction of Tim-3 and PD-1 expression in peripheral lymphocytes on days 3 and 6 that is no longer apparent on day 9.

Endometrial microbiota in infertile women with and without chronic endometritis as diagnosed using a quantitative and reference range-based method.

OBJECTIVE: To systematically compare the endometrial microbiota in infertile women with and without chronic endometritis (CE), as diagnosed by a quantitative and reference range-based method. DESIGN: Case-control observational study. SETTING: University-affiliated hospital. PATIENT(S): One hundred and thirty infertile women. INTERVENTION(S): Endometrial biopsy and fluid (uterine lavage, UL) collected precisely 7 days after LH surge, with plasma cell density (PCD) determined based on Syndecan-1 (CD138)-positive cells in the entire biopsy section and culture-independent massively parallel sequencing of the 16S ribosomal RNA gene performed on both the CE and non-CE endometrial fluid samples. MAIN OUTCOME MEASURE(S): Relative abundance of bacterial taxa. RESULT(S): Chronic endometritis was diagnosed if the PCD was above the 95th percentile (>5.15 cells per 10 mm2) of the reference range in fertile control subjects. With this stringent diagnostic criterion, 12 women (9%) were diagnosed with CE. Sequencing was successfully performed on all endometrial samples obtained by UL) (CE, n = 12; non-CE, n = 118). The median relative abundance of Lactobacillus was 1.89% and 80.7% in the CE and non-CE microbiotas, respectively. Lactobacillus crispatus was less abundant in the CE microbiota (fold-change, range: 2.10-2.30). Eighteen non-Lactobacillus taxa including Dialister, Bifidobacterium, Prevotella, Gardnerella, and Anaerococcus were more abundant in the CE microbiota (fold-change, 2.10-18.9). Of these, Anaerococcus and Gardnerella were negatively correlated in relative abundance with Lactobacillus (SparCC correlation magnitude, range: 0.142-0.177). CONCLUSION(S): Chronic endometritis was associated with a statistically significantly higher abundance of 18 bacterial taxa in the endometrial cavity. CLINICAL TRIALS REGISTRY NUMBER: ChiCTR-IOC-16007882.

A comparison of uterine natural killer cell density in the peri-implantation period between natural cycles and hormone replacement therapy cycles.

PROBLEM: A reference range for uterine natural killer (uNK) cell density in the peri-implantation period has recently been established in natural cycles. However, it is uncertain whether the results can be applied to hormonal replacement therapy (HRT) cycles, used increasingly in frozen-thaw embryo replacement cycles and which is known to be capable of supporting implantation. METHOD OF STUDY: A total of 183 women from two IVF centers participated in this study, including 75 women in natural cycles and 108 women in HRT cycles. All endometrial biopsies were collected precisely on the putative day of embryo implantation, namely 7 days after LH surge (LH+7) of the natural cycles or 5 days after initiation of progesterone (P+5) of the HRT cycles. Endometrial sections were immunostained for CD56 for uNK cells. Cell counting was performed by a standardized protocol, and results were expressed as percentage of positive uNK cells/total stromal cells. RESULTS: There was no significant difference (P > 0.05) in uNK cell density between natural cycles (median 2.28%, range 0.99%-4.78%) and HRT cycles (median 2.55%, range 0.69%-5.02%) in women undergoing IVF-ET treatment on the putative day of blastocyst transfer. Using reference range from 1.2% to 4.5% for uNK cell density, there was no significant difference (P > 0.05) in high uNK cell density proportion between natural cycles (8%, 6/75) and HRT cycles (10.2%, 11/108). CONCLUSION: The results indicated that the reference range for uNK cell density derived from natural cycles may apply to HRT cycles.

Endometrial Vascularization Characterized by Optical Coherence Tomography and Immunohistochemistry in Women Undergoing In Vitro Fertilization-Embryo Transfer Treatment.

BACKGROUND AND OBJECTIVE: Endometrial angiogenesis is a prerequisite for successful pregnancy. Optical coherence tomography (OCT) is a non-invasive physically optical imaging technique widely used in ophthalmology and cardiology. However, there is no study using OCT to evaluate endometrium. The aim of this study was to use OCT and traditionally histological methods to investigate endometrial vascularization in women undergoing in vitro fertilization-embryo transfer (IVF-ET) treatment and to determine the association with the pregnancy outcome. METHODS: A total of 47 women were included in this study. OCT was used to assess endometrial vascularization by determining the high signal areas precisely on the seventh day after luteinizing hormone surge in non-conception natural cycles. Endometrial biopsies were obtained following OCT and immunohistochemistry was used to determine micro vessel and expression of vascular endothelial growth factor-A (VEGF-A) in the luminal epithelium, glandular epithelium and stroma, separately. Micro vessel counting was performed and the result was expressed as micro vessel density (MVD). A semi-quantitative H-score was used to determine the staining intensity of VEGF-A. RESULTS: In women who successfully conceived after embryo transfer, the proportion of extensive high signal area in the uterine body detected by OCT (80%, 8/10), MVD (median number of micro vessels/mm² of 10, range 4⁻17) and stromal expression of VEGF-A (median H-score of 189, range 72⁻395) were found to be significantly higher than those of women who did not conceive after embryo transfer in the subsequent IVF-ET treatment (OCT: 30%, 3/10; MVD: median number of micro vessels/mm² of 7, range 4⁻10; VEGF-A: median H-score of 125, range 86⁻299, respectively). In addition, a significantly higher stromal expression of VEGF-A (median H-score of 196, range 84⁻395) and MVD (median number of micro vessels/mm2 of 9, range 5⁻16) was found in women with extensive high signal area in uterine body, compared to those with focal or no high signal area (stromal VEGF-A: median H-score of 135, range 92⁻302; MVD: number of micro vessels/mm2 of 6, range 4-11). CONCLUSIONS: Both immunohistochemistry and OCT demonstrated significant difference in vascularization of the peri-implantation endometrium between subjects who did and did not conceive after IVF-ET treatment. Our findings also suggest OCT appears to be a promising non-invasive or minimally invasive alternative to study endometrial vascularity in women with reproductive failure.

Increased expression of angiogenic cytokines in CD56+ uterine natural killer cells from women with recurrent miscarriage.

OBJECTIVE: To compare the expression pattern of angiogenic cytokines in CD56+ uNK cells from peri-implantation endometrium in women with a history of recurrent miscarriage and fertile controls. METHODS: 28 women were recruited, from which 18 women were diagnosed as recurrent miscarriage and 10 women were of proven fertility. Endometrial biopsy samples were obtained precisely 7 days after luteinization hormone surge in a natural cycle. The angiogenic profile of isolated CD56+ uNK cells was determined by RayBio human angiogenesis antibody array G Series 1000. Differentially expressed angiogenic cytokines between groups were validated by ELISA kits. RESULTS: CD56+ uNK cells freshly isolated from peri-implantation endometrium were determined to be >90% pure. Angiogenic cytokine array demonstrated that CD56+ uNK cells are one of the angiogenic factors producers in endometrium around the time of embryo implantation. A differential angiogenic cytokine expression profile was found between two groups, with significantly higher expressions of angiogenin, VEGF-A and bFGF in CD56+ uNK cells from women with recurrent miscarriage, compared with fertile controls. CONCLUSIONS: Differential angiogenic cytokine profile of isolated CD56+ uNK cells suggested the role of uNK cells in the altered endometrial vascularity at the time of implantation, which may account for the endometrial contribution to recurrent miscarriage.