1 Departments of Obstetrics and Gynaecology and 2 Histopathology, Imperial College School of Medicine at St Mary's, London W2 1NY, UK
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Abstract |
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Key words: endometrium/natural killer cells/recurrent miscarriage
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Introduction |
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The function of LGL remains unclear but their abundance at the time of implantation suggests they may be important in the establishment of pregnancy. As the LGL are in intimate contact with invading trophoblast both temporally and spatially, one possible role is that of limiting trophoblast invasion into the decidua (King and Loke, 1991). Experimental evidence from murine models supports this notion: NK cell-depleted transgenic mice have an increased incidence of fetal loss compared to immunocompetent mice and this is associated with histological abnormalities of placental vasculature (Guimond et al., 1997
). Further, when NK cells are replenished by bone marrow transplantation reproductive outcome is improved (Guimond et al., 1998
). The part played by endometrial leukocytes in the mechanism of infertility and early pregnancy failure is not yet clear. Alterations in endometrial leukocyte populations have been observed in women with unexplained infertility (Klentzeris et al., 1994
) and in women with infertility associated with autoimmune thyroid disease (Stewart-Akers et al., 1998
). A recent study reported no changes in endometrial T-cells in a group of women undergoing spontaneous early pregnancy loss (Vassiliadou and Bulmer, 1998a
). However the same authors report decreased cytotoxic capability of decidual CD56+ lymphocytes from spontaneous aborters indicating a functional deficiency of these cells associated with early pregnancy loss (Vassiliadou and Bulmer, 1998b
). Alterations in endometrial NK cell subsets have been reported in women with recurrent miscarriage (Lachapelle et al., 1996
). Increased numbers of peripheral blood CD56+ cells have been demonstrated in women with recurrent miscarriage (Kwak et al., 1995
) and in women who have experienced multiple in-vitro (IVF) failures (Beer et al., 1996
). These observations are of interest as they raise the possibility that alterations in the CD56+ NK cell population may result in early pregnancy loss. The purpose of this morphometric study was to quantify the endometrial CD56+ NK cell population in women with a history of recurrent miscarriage and therefore lend further support to this hypothesis.
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Materials and methods |
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Endometrial biopsies
The endometrial biopsies were taken in the luteal phase of the menstrual cycle between day 7 and day 10 after the mid-cycle LH surge. This was determined from daily early morning urinary LH measurements as described previously (Clifford et al., 1994). Endometrial tissue was obtained as an outpatient procedure, using an endometrial sampler (Z-sampler, BEI Medical Systems International, Gembloux, Belgium). Each specimen was placed onto OCT embedding matrix (CellPath, Hemel Hempstead, UK) on a cork disc and snap-frozen in isopentane in liquid nitrogen. The discs were then stored at 70°C until required.
Serial cryostat sections of 57 µm thick were mounted on clean glass slides and air-dried at room temperature. After fixing in acetone for 7 min the slides were wrapped in foil and stored at 20°C until immunolabelling.
Immunocytochemistry
Duplicate sections of all specimens were immunolabelled. The slides were washed in tris-buffered saline (TBS) and blocked with 3% bovine serum albumin in TBS for 15 min. The specimens were incubated with primary mouse anti-human natural killer cell, CD56, monoclonal antibody (Dako, Glostrup, Denmark) at a dilution of 1:20 for 60 min. After three washes in TBS the slides were incubated with secondary biotinylated rabbit anti-mouse antibody (Dako, Glostrup, Denmark) at a dilution of 1:600 for 30 min. The slides were then washed 3 times in TBS and incubated with avidin-biotin complex (ABC) for 30 min. The sections were counterstained with haematoxylin, dehydrated in 100% alcohol, and cleared with xylene prior to mounting. Negative controls were performed by omission of the primary antibody.
Histomorphometric analysis
Morphometry was performed using transmitted light microscopy using an eyepiece graticule. All sections were scored blindly without the observer's (K.C.) knowledge of the subject details. A random selection of slides was scored blindly by a second observer (A.M.F.) and similar numbers of positively staining cells found. The number of positively staining cells in 10 non-overlapping high-powered (x400) fields (h.p.f.) was counted. The procedure was repeated in a second specimen from the same subject and the mean of the absolute number of cells per 10 h.p.f. was calculated.
Statistics
The mean number of positively staining cells per 10 h.p.f. was compared in different groups using Student's two-sample t-test. Pearson's correlation test was used to analyse the relationship between the mean number of cells and various factors in the reproductive history. P = 0.05 level of significance was used throughout.
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Results |
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Discussion |
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The role played by NK cells in implantation remains unclear. Their presence in pre-implantation endometrium indicates that they are not merely part of an immune response to embryonic demise. Extensive trophoblast invasion of the uterine tissue is necessary for successful implantation and placentation. This process must be finely regulated: excessive invasion results in trophoblastic disease while insufficient invasion may lead to early pregnancy loss. Available evidence supports the theory that endometrial leukocytes play an important role in this process and their recruitment has been shown to be regulated by cytokines. Successful pregnancy depends on the presence of trophoblast growth-promoting cytokinesthe Th2 cytokinesincluding interleukin (IL)-4, IL-5 and IL-10 (Wegmann et al., 1993). In the presence of Th-1 cytokines that include IL-2 and interferon (IFN)-
, trophoblast cell lysis is favoured (King and Loke, 1990
). Thus pregnancy appears to be a Th-2 dominant state (Wegmann et al., 1993
). The LGL population itself, together with endometrial thymus-derived (T)-cells, is a major source of cytokines (Saito et al., 1993
; Jokhi et al., 1994
). Thus numerical changes in the LGL population could lead to alterations in the endometrial cytokine profile and hence to a disturbance of the Th-2/Th-1 equilibrium in favour of trophoblast cell lysis. Increased decidual NK cytotoxic activity has been described in women with recurrent miscarriage (Chao et al., 1995
). Furthermore intravenous immunoglobulin G (IVIG) therapy has been shown to down-regulate elevated peripheral blood CD56+ NK cells in women with recurrent miscarriage (Kwak et al., 1996
; Ruiz et al., 1996
) which has been reported to be associated with successful pregnancy outcome. Whether similar changes in CD56+ cell numbers are observed at the endometrial site is not known and thus the mechanism of action of such treatments is not clear. Due to the heterogeneous nature of the problem, it is unlikely that all women with recurrent miscarriage would benefit from such therapy, and clearly further large studies are needed.
The findings presented here support the hypothesis that endometrial NK cells are important for the establishment of pregnancy and alterations in the endometrial NK cell population are associated with early pregnancy failure. However a causal link remains to be established and further studies are needed to determine the precise role of endometrial leukocytes in early pregnancy. The relationship between NK cells and endometrial cytokine production warrants further investigation and how these processes may be defective in recurrent early pregnancy loss.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 14, 1999; accepted on July 14, 1999.