Immune profiling and RNA-seq uncover the cause of partial unexplained recurrent implantation failure.

BACKGROUND: Detailed knowledge of the changes in endometrial immune cells during the window of implantation in unexplained recurrent implantation failure (RIF) patients, the functions performed by immune cells, and the interactions between them is largely lacking. This study aimed to classify RIF patients and explore the mechanism through endometrial immune profiling and RNA-seq analysis. METHODS: This study enrolled a total of 172 patients, comprising 144 women with unexplained RIF and 28 fertile women. Endometrial samples were collected using endometrial scratching at the mid-luteal phase before in vitro fertilization treatment or pregnancy. Transcriptome sequencing and immunohistochemical staining of endometrial immune cells including natural killer (NK) cells, macrophages, T cells, and B cells were performed. MAIN OUTCOME MEASURE(S): Comparison of the percentage of endometrial immune cells and the RNA-seq information between RIF patients and fertile control patients. RESULT(S): The proportions of uterine CD56+ uNK cells, CD57+ NKT cells, CD68+ macrophages, and CD19+ B cells were significantly elevated in RIF patients. In addition, the number of positive CD68 glandular lumens was significantly higher in RIF patients than in the fertile group. In addition, based on this result, we classified RIF patients into three categories. CONCLUSION(S): Hyperactivation of endometrial immune cells may be associated with reduced endometrial tolerance and recurrent implantation failure, affecting pregnancy outcomes in RIF patients.

Histological endometrial dating: a reliable tool for personalized frozen-thawed embryo transfer in patients with repeated implantation failure in natural cycles.

OBJECTIVE: To evaluate the clinical availability and stability of histological endometrial dating as a tool for personalized frozen-thawed embryo transfer (pFET) in patients with repeated implantation failure (RIF) in natural cycles. METHODS: A total of 1245 RIF patients were recruited to the present study. All of the patients received an endometrial dating evaluation on day 7 post-ovulation (PO + 7) to guide their first pFET. The second and third pFETs were executed according to histological examination (again employing biopsy) or by reference to previous results. Subsequent pregnancy outcomes for all of the cycles were ultimately tracked. RESULTS: The out-of-phase rate for RIF patients was 32.4% (404/1245) and the expected dating rate (the probability of the expected endometrial dating aligning with repeat biopsy) for endometrial dating reevaluation was as high as 94.3% (50/53). The clinical pregnancy rates of first, second, and third pFETs were 65.3%, 50.0%, and 44.4%, respectively; and the cumulative clinical pregnancy rate attained 74.9% after three transfers. Endometrial dating reevaluations met expectations with more than a 2-year duration in three cases and elicited favorable clinical outcomes. CONCLUSION: We validated the relatively high stability of the histological endometrial dating platform-including the out-of-phase rate and the expected dating rate of reevaluation in patients with RIF-by expanding the sample size. The pFET, based on histological endometrial dating, was of acceptable clinical value and was worthy of promotion in patients with unexplained RIF.

Blood CD4+CD25+ T regulatory cells constitute a potential predictive marker of subsequent miscarriage in unexplained recurrent pregnancy loss.

The aim of this study was to investigate the relationship between pre-pregnancy blood immune status and unexplained recurrent pregnancy loss (URPL), and to evaluate the predictive value of pre-pregnancy blood Treg levels for subsequent miscarriage in patients with URPL. We retrospectively analyzed 76 women who had experienced two or more miscarriages before 24 weeks of gestation for no obvious reason, and 74 women who had achieved live births as controls. Flow-cytometric analysis of peripheral blood CD4 + T cells, CD8 + T cells, NK cells, NKT cells, B cells, NK cell subpopulations (including CD56bright NK cells, CD56dim NK cells, CD56dimCD16+ NK cells, and CD56brightCD16- NK cells) was executed in the luteal phase of women in the URPL and control groups. When we reviewed and analyzed reproductive outcomes in URPL patients, we found that blood Tregs were significantly lower in the URPL group than in the controls (1.89% ± 0.61% vs. 2.15% ± 0.58%, P < 0.01) during the luteal phase pre-pregnancy. However, we discerned no differences among blood CD4+T cells, CD8+T cells, B cells, NKT cells, or NK cells, NK subpopulations (CD56bright NKs, CD56dim NKs, CD56dimCD16+ NKs, or CD56brightCD16- NKs) between the two groups. By implementing receiver operating characteristic (ROC) curve analysis to determine whether Treg levels predicted subsequent miscarriages, we found that the area under the ROC curves was 0.714, and that the cutoff value was 1.35, with a sensitivity of 0.556 and specificity of 0.923. Based on the cutoff value, we divided pregnant URPL patients into two groups, demonstrating that the subsequent miscarriage rates in the low-Treg level group (<1.35%) were significantly higher than those in the normal-Treg level group (>1.35%) (71.43% vs. 14.29%, P < 0.01). CONCLUSION: The pre-pregnancy blood Treg level was a potential marker that predicted subsequent miscarriage in women with URPL.

CD19 and intraglandular CD163-positivity as prognostic indicators of pregnancy outcome in CD138-negative women with a previous fresh-embryo-transfer failure.

Many factors impede embryonic implantation, and excluding obvious known factors such as chronic endometritis, the immune status of the endometrium may be related to pregnancy. Although an abundantly large number of immune cells infiltrate the endometrium during the secretory phase, whether these immune cells can be used as a predictor of prognosis in ART has not yet been clarified. In the present study we therefore retrospectively analyzed 97 CD138-negative women with a previous fresh-embryo-transfer failure. We assessed the expression of CD56+ uNK cells, CD16+ NK cells, CD57+ NK cells, CD68+ pan-macrophages, CD163+ M2 macrophages, CD4+T cells, CD8+T cells, FOXP3+ regulatory T cells, and CD19+ B cells in the endometrium by IHC to evaluate mid-luteal endometrial immune cells as prognostic indicators of pregnancy outcome in the next frozen-embryo-transfer cycle. CD19-positive cells and the intraglandular CD163-positivity rate increased significantly in the clinically non-pregnant group (0.47 % vs. 0.20 %, P = 0.021; 61 % vs. 30 %, P = 0.017). The ratios of CD4/CD8 were also higher in the non-pregnant group (1.96 vs. 1.45, P = 0.005).The area under the ROC curve of CD19 cell number alone, the intraglandular CD163-positivity alone, and CD19 number combined with the intraglandular CD163-positivity were 0.692 (95 % CI, 0.55-0.834), 0.661 (95 % CI, 0.514-0.809), and 0.748 (95 % CI, 0.614-0.882), respectively. The optimal cut-off value of CD19 was 0.464 %, and the clinical pregnancy rate and live-birth rate diminished significantly when the CD19 level was above this cut-off value. Our study suggests that CD19-positive cells and intraglandular CD163-positivity can be used as prognostic indicators of pregnancy outcome in CD138-negative patients who experienced first-fresh-embryo transfer failure.

Endometrial CD138 count appears to be a negative prognostic indicator for patients who have experienced previous embryo transfer failure.

OBJECTIVE: To explore the predictive value of endometrial CD138 expression in the natural cycle preceding frozen embryo transfer in patients with normal endometrial dating and histopathologic features, who previously failed the transfer of two high-quality fresh embryos. DESIGN: Retrospective analysis. SETTING: University-affiliated hospital. PATIENT(S): Women with normal endometrial dating and histopathologic features who previously failed the transfer of two high-quality fresh embryos, and who then underwent an endometrial scratching operation preceding a natural cycle. INTERVENTION(S): Paraffin-embedded endometrial samples cut into sections for immunohistochemistry staining of CD138 (syndecan-1) expression, then clinical information for these patients reviewed and analyzed. MAIN OUTCOME MEASURE(S): Clinical rates of pregnancy and implantation. RESULT(S): A total of 141 women met the inclusion criteria. Of these patients, about 31.2% (44 of 141) were positive for CD138 expression, with CD138 counts ranging from 0 to 33. Receiver operating characteristic (ROC) curves were analyzed to determine whether the number of cells expressing CD138 (CD138+ cells) predicted a successful pregnancy. The areas under the ROC curves based on CD138+ cell density and CD138+ cell count were 0.660 and 0.658, respectively. The clinical pregnancy and embryo implantation rates in patients not expressing CD138 (80.04% and 64.9%, respectively) were statistically significantly higher than rates in CD138+ patients (52.7% and 46.8%, respectively). In addition, the higher the number of cells expressing CD138, the worse the outcome of the pregnancy. Finally, clinical data showed that free pelvic fluid on the day of endometrial sampling (identified using transvaginal ultrasound) might be a risk factor for CD138 expression. CONCLUSION(S): Endometrial CD138+ count might be a valuable marker predicting pregnancy outcomes after frozen embryo transfer in patients with normal endometrial dating and histopathologic features who previously failed the transfer of two high-quality fresh embryos.