The effect of prednisolone on endometrial uterine NK cell concentrations and pregnancy outcome in women with reproductive failure. A retrospective cohort study.

This retrospective study of prospectively collected data examines the effect of prednisolone therapy on raised uterine Natural Killer cell (uNK) concentrations and pregnancy outcomes in women with recurrent miscarriage (RM) and recurrent implantation failure (RIF) after IVF/ICSI treatment. 136 women diagnosed with RRF who had a timed midluteal endometrial biopsy taken for uNK cell analysis were included. Women with high uNK cell concentrations (n = 45) were treated with prednisolone (10 mg/day) for one month, after which a second biopsy was taken for repeat uNK cell analysis. Women for whom prednisolone caused a decrease in uNK cell concentrations continued on prednisolone until 12 weeks of pregnancy. Pregnancy outcomes (live birth, miscarriage and implantation rates) and pregnancy complications were compared for women who received prednisolone and those who did not. Results showed that the prevalence of high uNK cells was 33.1%. Prednisolone significantly decreased the uNK cell concentration (P < 0.001), however reduction to normal limits was achieved in only 48.3% of patients. There was no difference in any of the pregnancy outcomes or complications between women who had received prednisolone and those who had not. In conclusion, this study showed a relatively high prevalence of raised uNK cells in women with recurrent reproductive failure and confirmed the effect of prednisolone on reducing uNK cell concentrations. We found however no evidence for a significant beneficial effect for prednisolone therapy on pregnancy outcomes. Until the results of an adequately powered RCT become available however, these findings should be considered preliminary.

Measurement of uterine natural killer cell percentage in the periimplantation endometrium from fertile women and women with recurrent reproductive failure: establishment of a reference range.

BACKGROUND: Uterine natural killer cells are the major leukocytes present in the periimplantation endometrium. Previous studies have found controversial differences in uterine natural killer cell percentage in women with recurrent reproductive failure compared with fertile controls. OBJECTIVE: We sought to compare the uterine natural killer cell percentage in women with recurrent reproductive failure and fertile controls. STUDY DESIGN: This was a retrospective study carried out in university hospitals. A total of 215 women from 3 university centers participated in the study, including 97 women with recurrent miscarriage, 34 women with recurrent implantation failure, and 84 fertile controls. Endometrial biopsy samples were obtained precisely 7 days after luteinization hormone surge in a natural cycle. Endometrial sections were immunostained for CD56 and cell counting was performed by a standardized protocol. Results were expressed as percentage of positive uterine natural killer cell/total stromal cells. RESULTS: The median uterine natural killer cell percentage in Chinese ovulatory fertile controls in natural cycles was 2.5% (range 0.9-5.3%). Using 5th and 95th percentile to define the lower and upper limits of uterine natural killer cell percentage, the reference range was 1.2-4.5%. Overall, the groups with recurrent reproductive failure had significantly higher uterine natural killer cell percentage than the controls (recurrent miscarriage: median 3.2%, range 0.6-8.8%; recurrent implantation failure: median 3.1%, range 0.8-8.3%). However, there was a subset of both groups (recurrent miscarriage: 16/97; recurrent implantation failure: 6/34) that had lower uterine natural killer cell percentage compared to fertile controls. CONCLUSION: A reference range for uterine natural killer cell percentage in fertile women was established. Women with recurrent reproductive failure had uterine natural killer cell percentages both above and below the reference range.

Standardisation of uterine natural killer (uNK) cell measurements in the endometrium of women with recurrent reproductive failure.

Considerable work is being carried out on endometrial NK cells to determine whether they play a role in successful pregnancy outcome. In addition there is debate about whether measurements of uNK should be included in the clinical assessment for women with recurrent implantation failure or recurrent miscarriage. A hindrance to taking this forward is the fact that the density of uNK cells reported by different centres is very different. The aim of this study was to determine the reason for these differences and to develop a standardised method. Three centres participated in the study. Each centre exchanged five formalin fixed, wax embedded sections of endometrium from five women. Sections were immunostained for CD56. Images were taken of 10 random fields at ×400 magnification; total stromal and uNK cells were counted using Image J. Results were expressed as % positive uNK cells and the variation in counts obtained in each centre was compared. After initial analysis a standardised protocol was agreed and the process repeated. Significant variation was seen in the counts obtained after initial analysis (Centre A vs.B, mean difference=-0.72 P<0.001; A vs.C mean difference=-0.47 P<0.001; B vs.C, mean difference=0.25 P=0.085). Analysis suggested that differences may be due to duration of tissue fixation, the embedding and sectioning processes, selection of areas for assessment, definition of immunopositive cells and inclusion or exclusion of blood vessels. Adoption of a standardised protocol reduced the variation (Centre A vs.B mean difference=-0.105 P=0.744; A vs.C mean difference=0.219 P=0.150; B vs.C mean difference=0.32 P=0.031). Use of a standardised method is needed to establish a normal range for uNK cells and to develop a meaningful clinical test for uNK cell measurements.

The effect of a high progesterone concentration before oocyte retrieval on the peri-implantation endometrium.

In this single-centre, prospective cohort study, the effect of high progesterone level before oocyte retrieval on endometrial morphology and uterine natural killer cell (uKN) count in the peri-implantation period was investigated. A total of 106 women undergoing IVF treatment who did not proceed to fresh embryo transfer were included. Endometrial samples were obtained 7 days after HCG administration. Multiple regression analysis was used to identify factors affecting the results of histological staging and uNK cell count. Progesterone level on the day after HCG administration was the only significant variable associated with the results of histological staging (P = 0.004). Endometrial development in women with high progesterone level was significantly (P < 0.001) more advanced than that of women with normal progesterone; progesterone level on the day of HCG administration was the only significant variable associated with uNK cell count. The median (range) of uNK cell count of 9.6% (2.3-21.6%) in women with high progesterone was significantly (P < 0.001) higher than the median (range) of uNK cell count of 5.7% (1.4-18.7%) in women with normal progesterone. High progesterone level before oocyte retrieval was correlated with advancement in endometrial development as well as increased uNK cell count.

The prognostic value of uNK cell count and histological dating in the mid-luteal phase of women with reproductive failure.

OBJECTIVE: Histological dating has been used for decades to evaluate the histological maturation of the endometrium. Uterine natural killer cells are thought to play a significant role in pregnancy. While several studies have shown an increased number of uNK cells in women with recurrent reproductive failure, its prognostic value of pregnancy outcome remains uncertain. The aim of this study was to determine whether or not the prognostic value of uNK measurement on pregnancy outcome is improved when it is combined with histological dating of the same endometrial specimen. STUDY DESIGN: This is a retrospective study. Histological dating and uNK cell count was performed on endometrial biopsies taken from women with either recurrent miscarriage (RM, n=94) or recurrent implantation failure (RIF, n=72). Women who conceived within a year of the biopsy (n=83) were included in a further analysis to examine the prognostic value of uNK cell count and histological dating on the outcome of a subsequent pregnancy. RESULTS: There was a significant (p=0.01) association between uNK cells and histological dating; retarded endometrium was less likely to have a high uNK cell count (16/58, 28%) than normally developed endometrium (52/108, 48%). Whilst uNK cell count on its own did not have a significant correlation to the pregnancy outcome, a retarded endometrium is significantly (p=0.01) associated with a higher miscarriage rate (68%, 13/19) than normally developed endometrium (35%, 23/64) in women with reproductive failure. Regardless of the result of uNK cell count (normal or abnormal), combining the results of histological dating appeared to have significantly improved the prognostic value. CONCLUSIONS: We found that the prognostic value of uNK cell count is significantly increased when the result is combined with histological dating, primarily because histological dating is significantly correlated with pregnancy outcome.

Do uterine natural killer cell numbers in peri-implantation endometrium predict hypertensive disorder in pregnancy in women with a history of reproductive failure?

The aim of this study was to investigate whether or not increased uterine natural killer (uNK) cell numbers in the peri-implantation endometrium are associated with an increased risk of hypertensive disorders in a subsequent pregnancy. This is a retrospective study including 80 women with a history of unexplained recurrent miscarriage or recurrent implantation failure. Precisely timed endometrial biopsies were obtained from women 7-9 days after the luteinising hormone surge. uNK cells were immunostained for CD56+ and expressed as a percentage of total stromal cells. Patients were defined as having a high uNK cell count if the percentage of total stromal cells was more than 13.9%. Five out of 29 (17.2%) women in the high uNK cell count group and 5 out of 51 (9.8%) women in the normal uNK cell count group developed gestational hypertension. Pre-eclampsia was diagnosed in 2 (6.9%) patients in the high uNK cell count group and 1 (2.0%) patient from the normal uNK cell count group. There was no significant difference in the incidence of either gestational hypertension (P=0.483) and pre-eclampsia (P=0.296) between groups. The overall incidence of hypertensive disease in women with high uNK cell count (24.1%) was two times higher than women with normal uNK cell count (11.8%), but it was not statistically significant (P=0.208). An increased uNK cells count in the peri-implantation period in a cycle prior to conception did not appear to significantly increase the likelihood of hypertensive disease of pregnancy.

The observer and cycle-to-cycle variability in the measurement of uterine natural killer cells by immunohistochemistry.

The aim of the study was to investigate the various potential sources of variability of counting endometrial uNK cells by immunohistochemistry. Precisely timed endometrial biopsy samples were obtained from women suffering from recurrent miscarriage or recurrent implantation failure (RIF) after IVF on days LH+7-LH+9 of the cycle. uNK cells in wax embedded sections were immunostained for CD56+ and expressed as a percentage of total stromal cells. The number of uNK cells in the same sample were counted by two methods, by the same observer on two occasions (intra-observer variability), by three different observers (inter-observer variability) and finally on samples obtained from the same individual at the same time in two different cycles (cycle-to-cycle variability). There was excellent agreement (κ=0.935) in the numbers of uNK cells obtained using both the traditional cell counting (TCC) and image analysis (IA) methods. The intra-observer variability (TCC, κ=0.944; IA, κ=0.935) was relatively small, although the inter-observer variability (TCC, κ=0.832 and 0.497; IA, κ=0.438) was modest. There was significant variation in number of cells in samples obtained from the same women in two different cycles (κ=0.348). The measurement of uNK cells in the endometrium has a reasonable degree of precision, but the significant cycle-to-cycle variation in results is a source of concern and requires further investigation.

Uterine natural killer cells in peri-implantation endometrium from women with repeated implantation failure after IVF.

Several studies have suggested that endometrial uNK (CD56+) cells may play a role in implantation. The aim of this study was to investigate the number of CD56+, CD16+ and CD69+ cells in the unstimulated endometrium of women with recurrent implantation failure after IVF. The percentage of stromal cells positive for CD56, CD16 and CD69 was identified by immunocytochemistry in endometrial biopsies from 15 normal control women and 40 women with recurrent implantation failure. All biopsies were obtained on days LH+7 to LH+9. The density of CD56+ cells in endometrium from women with repeated implantation failure after IVF [median (range) CD56+ cell density=14.5% (1.5-71.4%)] was significantly higher (P=0.005) than in endometrium from control women [5% (2.1-19.2%)]. There was no significant difference in the densities of CD16+ and CD69+ cells in the endometrium from women in the two groups. The increased density of CD56+ cells in the endometrium of women with recurrent implantation failure suggests that these cells are directly involved in the implantation process; alternatively this may indicate a general endometrial defect in these women, which leads to the inability of the embryo to implant.