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.

Localization and variable expression of G alpha(i2) in human endometrium and Fallopian tubes.

BACKGROUND: Heterotrimeric G proteins take part in membrane-mediated cell signalling and have a role in hormonal regulation. This study clarifies the expression and localization of the G protein subunit G alpha(i2) in the human endometrium and Fallopian tube and changes in G alpha(i2) expression in human endometrium during the menstrual cycle. METHODS: The expression of G alpha(i2) was identified by Polymerase chain reaction (PCR), and localization confirmed by immunostaining. Cyclic changes in G alpha(i2) expression during the menstrual cycle were evaluated by quantitative real-time PCR. RESULTS: We found G alpha(i2) to be expressed in human endometrium, Fallopian tube tissue and in primary cultures of Fallopian tube epithelial cells. Our studies revealed enriched localization of G alpha(i2) in Fallopian tube cilia and in endometrial glands. We showed that G alpha(i2) expression in human endometrium changes significantly during the menstrual cycle, with a higher level in the secretory versus proliferative and menstrual phases (P < 0.05). CONCLUSIONS: G alpha(i2) is specifically localized in human Fallopian tube epithelial cells, particularly in the cilia, and is likely to have a cilia-specific role in reproduction. Significantly variable expression of G alpha(i2) during the menstrual cycle suggests G alpha(i2) might be under hormonal regulation in the female reproductive tract in vivo.

Expression of integrins in the endometrium of women with recurrent miscarriage.

Immunostaining intensity for alpha(1), alpha(4), alpha(v)beta(3), and beta(3) was assessed by H score in timed peri-implantation endometrium from 21 women with unexplained recurrent miscarriage and 16 healthy fertile women. No significant difference in H scores in gland epithelium, luminal epithelium, stroma, or blood vessels was observed between the two groups, suggesting that alpha(1), alpha(4), alpha(v)beta(3) and beta(3) integrins are expressed normally in the endometrium of women with unexplained recurrent miscarriage.

A study of luteal phase expression of inhibin, activin, and follistatin subunits in the endometrium of women with recurrent miscarriage.

OBJECTIVE: To compare the luteal phase endometrial expression of inhibin, activin, and follistatin subunits in women with recurrent miscarriage compared to a control group. Other parameters of luteal function assessed included Doppler blood flow, serum biochemical markers, and histopathology. DESIGN: This was a prospective case control study. SETTING: The study was conducted in a tertiary care hospital of Sheffield, United Kingdom. PATIENT(S): Thirty-three women with recurrent miscarriage and 10 women with no previous history of miscarriage (control group) were recruited. INTERVENTION(S): Histologic assessment of the luteal phase endometrium was done using Noyes criteria followed by immunohistochemical analysis for expression of inhibin, activin, and follistatin subunits. Doppler analysis for perifollicular and endometrial blood flow was also done. Simultaneously serum concentrations of E(2), LH, FSH, P, and inhibin B at the time of LH surge and at LH +7 days were measured. MAIN OUTCOME MEASURES: Difference in endometrial expression of inhibin, activin, and follistatin subunit in the two groups. RESULT(S): Endometrial expression of follistatin and beta A in the endometrial stromal cells of women with recurrent miscarriage was significantly lower than in control women. There were no differences in results of the Doppler studies or the hormonal profiles between cases and controls. CONCLUSION(S): The lower expression of follistatin and beta A subunit in women with recurrent miscarriage may imply an altered activity of activin A at the time of decidualization, which may lead to poor pregnancy outcome in the form of miscarriage.

Inhibin A and activin A may be used to predict pregnancy outcome in women with recurrent miscarriage.

OBJECTIVE: To look at the role of inhibin and activin in predicting pregnancy outcome in patients with history of recurrent miscarriage. DESIGN: Observational clinical study. SETTING: Recurrent miscarriage clinic of a tertiary care teaching hospital. PATIENT(S): Patients with history of recurrent miscarriage. INTERVENTION(S): Serial serum inhibin A and activin A concentrations were measured in weeks 5 though 8 of pregnancy. MAIN OUTCOME MEASURE(S): Serum concentrations of inhibin A and activin A levels. RESULT(S): Mean inhibin A concentration at 5 to 6 weeks for women who miscarried and those who had live births was 33 and 51 pg/mL, respectively; activin A at same gestation for the two groups was 534 and 643 pg/mL, respectively. After 2 weeks, mean inhibin A concentration for women who miscarried and those who had live births was 66 and 145 pg/mL, respectively, and activin A was 747 and 1,123 pg/mL, respectively. CONCLUSION(S): It is possible that inhibin A and activin A may be used as markers to predict pregnancies that are likely to miscarry.