1 Division of Obstetrics and Gynaecology, City Hospital, Hucknall Road, Nottingham, 2 Department of Immunology and 3 Department of Obstetrics and Gynaecology, University of Liverpool, Liverpool, L69 3BX, UK
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Abstract |
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Key words: endometrial leukocytes/immunohistochemistry/recurrent miscarriage
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Introduction |
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It has been suggested that women with recurrent early pregnancy loss (RPL) may have an impaired CD56+ leukocyte response in the decidua (Hill et al., 1995a), whilst women with spontaneous losses (not RPL) have increased numbers of CD57 `classical' NK cells (Vassiliadou and Bulmer, 1996a
). However, it is difficult to characterize leukocyte populations after recurrent miscarriage, as it is difficult to distinguish causes of miscarriage from post-miscarriage inflammatory responses (Bulmer, 1996
). Lachapelle and co-workers used flow cytometry of dispersed non-pregnant endometrium to study leukocytes in women with recurrent miscarriage compared with normal obstetric histories (Lachapelle et al., 1996
). They found an increased CD4:CD8 ratio and an increase in CD20+ B cells. The proportion of NK cells was identical in both groups; however, those women with recurrent miscarriage had an increased proportion of CD16+ CD56dim LGL compared to the normal population where CD16 CD56bright LGL were more prevalent. Here, we have used immunohistochemistry to investigate the immunophenotypic profile of the endometrium of women suffering recurrent pregnancy loss.
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Materials and methods |
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Biopsies were obtained from 25 women. One biopsy was excluded because the patient subsequently had two positive tests for lupus anticoagulant. All biopsies were examined histologically by two observers to date the endometrium according to Noyes criteria (Noyes et al., 1950); patients were included in the study if their biopsy was consistent with days 1922 of the cycle. The biopsies of two women were excluded from the study; one woman did not ovulate in the cycle studied, as her progesterone level was <10 nmol/l, and the biopsy of the other woman showed endometrial differentiation consistent with early secretory phase (<day 18), perhaps because of luteal phase insufficiency [biopsies accurately timed from the luteinizing hormone (LH) surge in two consecutive cycles would be necessary to make this diagnosis]. The remaining 22 biopsies comprised the patient group (Table I
). These women had had at least three consecutive miscarriages; one had second trimester miscarriages as well and one had very early biochemical losses [raised serum human chorionic gonadotrophin (HCG) concentrations at 4 weeks gestation as the only evidence of pregnancy]. Six patients had a previous live birth and one had a history of an ectopic pregnancy. The outcomes of the pregnancies following endometrial biopsy were: 11 patients had live births at term, four patients had first trimester miscarriages and one patient had an ectopic pregnancy. Of the remaining patients, one patient was lost to follow-up and five patients have yet to conceive.
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The endometrial samples were immediately frozen in liquid nitrogen and stored at 70°C. Cryostat sections 5 µm in thickness were cut and mounted on glass slides. Sections were stained with haemalum to check that the biopsies contained endometrial tissue. After drying overnight at room temperature, slides were wrapped in aluminium foil and frozen at 20°C until immunostaining. Sections were stained using the alkaline phosphatase anti-(alkaline phosphatase) (APAAP) system using a panel of 14 monoclonal antibodies (Table II).
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The number of positive cells for each monoclonal antibody used was counted in 10 random fields at x400 magnification. The observer was blinded to the identity of the slide. On sections stained for CD45+ cells, a count was made of both positive and negative cells to allow calculation of the total number of cells per high-power field. Glandular epithelial cells were not included in this count. The numbers of positive cells for each antibody were expressed as a percentage of the total cells for that patient to allow for inter-patient variability.
The statistics were calculated on the Arcus (Cambridge, UK) software package for personal computers. The data was tested with the ShapiroWilks test and found to be non-normally distributed. Therefore the non-parametric MannWhitney U-test was used to assess differences between the control and recurrent miscarriage endometrium using two-sided P-values. The difference between the endometrium of women that had a live birth subsequent to the biopsy was compared to that of those who miscarried following the biopsy, using the MannWhitney U-test. 95% confidence intervals were calculated and the conventional level of P < 0.05 was taken as the limit of significance.
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Results |
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Discussion |
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CD3, a marker for all T lymphocytes (CD4 and CD8), labelled a similar proportion of leukocytes in the pre-implantation endometrium of patients and controls. Other studies have shown reduced numbers of T cells in normal first trimester decidua compared to normal endometrium (Vassiliadou and Bulmer, 1996b), although similar numbers of T cells were found in normal first trimester decidua compared to decidua obtained after spontaneous abortion (Vassiliadou and Bulmer, 1998
). However, the majority of spontaneous abortions are due to fetal chromosomal abnormalities and hence extrapolation of data from spontaneous abortions may not be applicable to cases of recurrent miscarriage.
In the present study, more CD4+ than CD3+ cells were observed in the endometrium. Macrophages can also express the CD4 antigen (Woods et al., 1983) and macrophages (labelled with CD14) were significantly more numerous in the patients than the controls (Table III
). Therefore, the greater proportions of CD4+ cells may be due to an increase in macrophages (co-expressing CD14 and CD4) in the patients. Further experiments with double labelling are necessary to confirm this. Women who miscarried after the biopsy had more CD4+ and CD14+ leukocytes than women who had live births following the biopsy (Table IV
), suggesting that these cells have a role in miscarriage. The exact function of these endometrial macrophages is unknown, although several roles have been suggested. Endometrial macrophages increase in number during the luteal phase of the cycle and increase further in early pregnancy decidua, suggesting that they may have a role in placentation (Bulmer et al., 1991
; Hunt and Robertson, 1996
). Macrophages strongly express MHC class II antigens necessary for antigen presentation (Bulmer, 1996
) and are therefore able to activate T cells. Decidual macrophages may also have a phagocytic role, as trophoblast invasion into the decidua in normal pregnancy could produce debris requiring removal by phagocytosis (Bulmer, 1996
). There is also evidence of a role for macrophages in miscarriage from a murine model of miscarriage, where embryo loss was found to be associated with activated macrophages (Gendron et al., 1990
). In humans, a small increase in the number of CD68 labelled macrophages was found in the decidua from spontaneous abortions compared to normal controls (Vassiliadou and Bulmer, 1996a
); however, this could have been a post-abortion inflammatory response.
Macrophages have also been proposed as immunosuppressive cells in human early pregnancy decidua. This immunoregulatory activity has been reported to be mediated by the secretion of prostaglandin E2 by decidual macrophages and may block the function of lymphoid cells in the decidua, with potential lytic activity against the placental trophoblast (Parhar et al., 1989). However, the importance of local immunoregulatory cells in pregnancy has yet to be established. Macrophages are also capable of producing a range of cytokines including macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF) and tumour necrosis factor (TNF)-
. In common with T cells and LGL, decidual macrophage cytokine production in pregnancy could play a role in the control of placental growth (Bulmer, 1996
). CD56+, CD16 cells have been found to act synergistically with endometrial macrophages to enhance the release of interferon (IFN)-
(Marzusch et al., 1997
). NK cells can be activated by the cytokines interleukin (IL)-2, TNF-
and IFN-
; thus, they could act together with other activated decidual cell populations to attack trophoblast populations, resulting in subsequent pregnancy loss (Vassiliadou and Bulmer, 1996a
). Recent attention has focused on elucidating the immunobiological roles of cytokines in normal human pregnancy following the accumulated reports of complex cytokine activity with in-utero placental tissues (reviewed in Robertson et al., 1994
; Lim et al., 1996
, 1998
). It has been suggested that recurrent miscarriage may be associated with a Th1 cytokine (IFN-
IL-2, TNF-
) response, whereas successful pregnancy may be associated with a Th2 (IL-4, IL-5, IL-10) cytokine response (Hill et al., 1995b
).
In the present study, only two patients were found to have increased numbers of B cells in their endometrium; one subsequently had a live birth and one a miscarriage. This is in contrast with the study by Lachapelle et al. who found that recurrent miscarriers with increased B cells in their endometrium were more likely to miscarry (Lachapelle et al., 1996). However, these authors used flow cytometric analysis of dispersed cell populations, which may not be an accurate reflection of the in-vivo situation, since tissue digestion may result in a skewed population of cells.
The present study found more CD56+ LGL in the pre-implantation endometrium of the recurrent miscarriage patients than in the controls. Furthermore, there were more CD56+ leukocytes in the endometrium of patients that went on to have miscarriages than in those who had live births (Table IV). This is in contrast to previous findings (Lachapelle et al., 1996
), which found similar numbers of CD56+ cells in recurrent miscarriage patients and controls, but those women with recurrent miscarriage had an increased proportion of CD16+ CD56dim LGL compared to controls where CD16 CD56bright LGL were more prevalent. Although higher numbers of LGL were also found in decidua from spontaneous miscarriages than from normal pregnancy decidua (Vassiliadou and Bulmer, 1996a
), this may be due to post-miscarriage inflammatory changes. Christiansen put forward three hypothetical models for the pathogenesis of recurrent miscarriage (Christiansen, 1996
); one of these (model B), predicted that an increase in CD56+ cells might indicate a poor prognosis in recurrent miscarriage patients, and our results are in accordance with his prediction. However, unexplained infertility patients were found to have fewer CD56+ cells than fertile controls (Klentzeris et al., 1994
). Recently King et al. highlighted the importance of maternal LGL in human implantation, suggesting LGL are directly involved in maternal allogeneic recognition of the placenta via their expression of receptors for HLA-G and HLA-C, present on some fetal trophoblast populations (King et al., 1998
).
The present study demonstrated more CD16+ leukocytes in the pre-implantation endometrium of the patients than in the controls, and these were also more prevalent in those women who subsequently miscarried compared to those who had a successful pregnancy (Tables III and IV). This may be indicative of a chronic or latent infection; further studies would be needed to confirm this.
In the present study of pre-implantation endometrium, CD57+ cells were observed in only eight out of 22 miscarriers and were not seen in the controls. The CD57+ cells were also seen more frequently in women who subsequently miscarried than in those who had live births (Table IV). Vassiliadou and Bulmer (1996a) also found more CD57+ leukocytes in decidua of spontaneous miscarriages compared to normal pregnancy Therefore CD57+ or `classical' NK cells could be hostile to invading trophoblast.
The only two patients in this study with significant numbers of CD69+ cells miscarried, implying a deleterious role for activated leukocytes in implantation. Other evidence for this role comes from a study showing that CD56+ cells that were also CD69+ were found more commonly in the decidua from spontaneous abortions than from normal pregnancy (Kodama et al., 1998), but again, this may be due to post-miscarriage inflammatory changes.
In conclusion, a different population of leukocytes was found in the pre-implantation endometrium from recurrent miscarriage patients as compared to those from fertile controls. Furthermore, when the endometrium of women who subsequently miscarried was compared to those who had live births, the differences seen in the original analysis were accentuated. The endometrium of recurrent miscarriers could be hostile to the invading trophoblast. Another possible explanation for our data would be that the endometrium of recurrent miscarriers is more receptive, leading to the implantation of abnormal fertilized ova which subsequently miscarry.
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Acknowledgments |
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Notes |
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References |
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Submitted on February 5, 1999; accepted on June 9, 1999.