The effect of hormone replacement therapy on the number and the proliferation index of endometrial leukocytes

M.A. Habiba1, S.C. Bell1 and F. Al-Azzawi1,2,3

1 Department of Obstetrics and Gynaecology and 2 Gynaecology Research Group, Faculty of Medicine and Biological Sciences, Leicester University, Leicester, LE2 7LX, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study aimed to determine the changes in endometrial leukocyte subpopulations under sequential hormone replacement therapy (HRT) during the late progestogenic phase. The number of leukocytes was determined using immunohistochemistry utilizing monoclonal antibodies to CD45 (total leukocytes), CD56 (endometrial granulated lymphocytes), CD3 (T-cells), and CD68 (macrophages). Leukocyte proliferation was demonstrated using in-situ hybridization with a histone probe, and the proliferation index was determined using double labelling for Ki67 (Mib1). Compared to the corresponding phase of the physiological cycle, sequential HRT-treated endometrium exhibited a 95% increase in CD45+ cells (P < 0.05), a 130% increase in CD56+ cells (P < 0.05), and a 113% increase in CD3 cells. There was a non-statistically significant drop in the number of CD68+ cells. The number of proliferating leukocytes increased in sequential HRT endometrium.

Key words: endometrium/HRT/leukocyte/menopause/proliferation


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have previously demonstrated that women receiving sequential hormone replacement therapy (HRT) can be divided according to their menstrual bleeding pattern into two groups: `early bleeders' who experience a higher frequency of short cycles where withdrawal bleeding commences before day 28 of their medication, and `late bleeders', who experience a higher frequency of long cycles where bleeding commences on or after day 28. We also demonstrated that the latter group, whose pattern of bleeding is reminiscent of the natural cycle, experience more regular bleeding compared to the former (Habiba et al., 1996Go). Despite this similarity, the endometrium obtained prior to the onset of bleeding in long cycles (>=28 days) in late bleeders exhibits marked histological differences, including an increase in round cell infiltrate, compared to the physiological cycle endometrium (Habiba et al., 1998Go).

In the physiological cycle, the leukocyte population was shown to constitute 10–15% of the total stromal cell population, and this increases premenstrually to 20–25% (Kamat and Isaacson, 1987Go). Leukocytes are either scattered individually or form aggregates of B-cells, T-cells and macrophages. The latter are more common in the basalis. In contrast to the leukocytes in the functionalis, those in aggregates do not fluctuate with the stage of the cycle.

The main populations of leukocytes during the follicular and the early-luteal phases of the physiological cycle are CD3+ T lymphocytes (mostly CD8+), macrophages, and CD56+ CD3 and CD16 lymphocytes (endometrial granulated lymphocytes, eGL). In the late-luteal phase the number of leukocytes increases, mainly due to the rise in eGL, and the number of macrophages may also rise premenstrually (Bulmer et al., 1991aGo). Increased proliferation of CD56+ cells was reported in the late-luteal phase (Pace et al., 1989Go; Bulmer et al., 1991bGo).

During the follicular phase, macrophages may comprise up to 50% of CD45+ cells (Clark and Daya, 1990Go). These have been reported to represent the most prominent cell population in the mid-luteal endometrium (Haller et al., 1993Go), and to rise before the onset of menses. The magnitude of rise in macrophages was reported in one study to be responsible for the pre-menstrual rise in CD45+ cells (Kamat and Isaacson, 1987Go), but this was not confirmed (Starkey et al., 1991Go). Another study reported that macrophages increase between day 4 and day 13 after the luteinizing hormone (LH) surge by a factor of about 49%, compared to a 62% increase in the total leukocyte count and a 92% increase in eGL (Klentzeris et al., 1992Go).

T-cells (CD3+, CD5+ and CD7+) are most often present in aggregates in the deeper endometrium (Starkey et al., 1991Go). At least 75% of the CD2+, CD3+ T-cells are CD8+, and 25% are CD4+. CD8+ cells increase from the follicular phase into the luteal phase (LP) (Bulmer et al., 1991aGo). CD3+, CD8+, CD4+ and CD2+ cells increase by 26, 27, 16 and 48% respectively from day 4 to day 13 after the LH surge, but because their rate of increase is less than that of the total number of leukocytes, T-cells are proportionately less during the late-LP compared to the early-LP (Klentzeris et al., 1992Go). B-lymphocytes and CD16+ natural killer (NK) cells are rare throughout the physiological cycle endometrium.

The human Ki67 antigen is a nuclear antigen (a bimolecular complex of 345 and 395 kDa) expressed in all phases of the cell cycle except G0 and early G1 (Yu et al., 1992Go). The antigen can be demonstrated in frozen (Gerdes et al., 1983Go) and formalin fixed (Gerdes et al., 1992Go) sections. Despite its usefulness and wide use, Ki67 may overestimate the proliferation fraction. This disadvantage may be overcome through quantification of the expression of histone mRNA (H3 mRNA), which because of its short half-life is a suitable marker for the S-phase. H3 mRNA is detectable in G1 and peaks during the synthetic S-phase and rapidly disappears towards the end of G2 (Chou et al., 1990Go).

The aim of the present work was to determine the leukocyte subpopulation responsible for this increased leukocytic infiltrate and the proliferation indices (PI) of the main subpopulations in the sequential HRT-treated endometrium in comparison to that of the natural cycle.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Women on sequential HRT received 2 mg of oestradiol valerate daily with the addition of 1 mg of norethisterone on days 17–28 of each treatment cycle (medication supplied by Solvay Pharma GmbH, Hannover, Germany). The inclusion and exclusion criteria, patient characteristics, and schedule have been reported previously (Habiba et al., 1998Go). Endometrial pipelle biopsies (n = 10) were obtained between days 27 and 29 and before the onset of bleeding in the sixth treatment cycle. This will be referred to as the late-pseudoluteal phase (late-PLP) endometrium.

The LH-dated natural cycle endometrial biopsies were obtained by dilatation and curettage from healthy women with regular cycles at the time of scheduled tubal sterilization. They were all given a urine ovulation detection kit (Clearplan, Unipath, Basingstoke, Hampshire, UK) and instructed in its use. Sterilization and dilatation and curettage were performed in the early (day 2–6 after the LH peak, n = 10), the mid (day 7–11 after the LH peak, n = 10), or the late (day 12–14 after the LH peak, n = 10) LP, with the day of LH peak taken as day 0.

All endometrial samples were immediately fixed in formalin and paraffin embedded within 6 h. These were then routinely processed and stained with haematoxylin and eosin to confirm normality.

Immunohistochemistry
Immunohistochemistry (IHC) was used for single labelling and for double labelling with Mib1. The antibodies used and their source are shown in Table IGo.


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Table I. The type and source of the primary antibodies used in this study, their supplier, and the method of pretreatment
 
Single labelling
IHC was performed using the avidin–biotin complex immunolabelling method (Jackson and Blythe, 1993Go) with modifications. Briefly, sections were dewaxed in xylene, and rehydrated in grades of ethyl alcohol and distilled water. Sections were pretreated using enzyme digestion or microwave according to the antibody to be used (Table IGo). Pepsin pretreatment was performed using porcine pepsin 0.4 g/100 ml (Sigma, Poole, Dorset, UK) with 5 mol/l hydrochloric acid (0.2 ml/100 ml). The duration and power used for microwave pretreatment was optimized using the method previously published (Shi et al., 1991Go) in order to enhance specific staining and to eliminate non-specific background staining. This was performed using isotype control antibody. Briefly, sections were suspended in plastic racks (maximum 20) and immersed in 6 mol/l citrate buffer and heated in a 750 W microwave at full power (Techolec Loven, T250T; Comet, Leicester, UK). A turntable was used to ensure even heating. Endogenous peroxidase was blocked by freshly prepared 6% v/v hydrogen peroxide for 10 min. Sections were washed in tap water and Tris-buffered saline/bovine serum albumin (TBS-BSA) buffer, and then covered in 100 µl of normal rabbit serum (NRS) (Dako, Ely, Cambridgeshire, UK) at 1:10 in TBS (for CD3 swine serum was used), and incubated for 20 min; these were then incubated with the primary antibody at the required dilution and time (Table IGo). Slides were washed in TBS-BSA and incubated for 30 min at 37°C with the secondary antibody (100 µl of biotinylated antibody from a heterologous species) diluted at 1:150 in TBS-BSA. They were then washed in TBS-BSA and incubated with a freshly prepared solution Vectastain ABC peroxidase® (Vector Laboratories, Bretton, Peterborough, UK) for 30 min at room temperature. Sections were washed and incubated with peroxidase substrate (DAB substrate; Vector Laboratories) for 10 min. Slides were washed in running tap water, dried, dehydrated, cleared and mounted using XAM (BDH, Poole, Dorset, UK).

Double labelling
For double labelling with CD45 and Mib1, the same steps as above were followed but the secondary antibody was substituted with alkaline phosphatase linked rabbit anti-mouse antibody (Dako) at a dilution of 1:100 in TBS. Sections were incubated for 30 min at 37°C, and developed using alkaline phosphatase substrate (Fast Red TR/Naphthol AS-MX®, Sigma). Sections were washed in running tap water and heated in 6 mol/l citrate buffer for 30 min in a microwave oven. After cooling, sections were transferred to tap water and then washed in TBS-BSA. NRS 100 µl (Dako), diluted 1:10 in TBS, was applied to cover the sections for 10 min. Mib1 antibody was applied to the sections and incubated overnight at room temperature. Slides were then washed in TBS-BSA and 100 µl biotinylated rabbit anti-mouse antibody (Dako) applied at 1:150 in TBS for 30 min. Colour detection was performed using Vectastain ABCelite® peroxidase solution (Vector Laboratories) and Vector SG®, blue/grey peroxidase substrate (Vector Laboratories). Sections were then washed and mounted using Apathy's aqueous mountant (Raymond A.Lamb, London, UK). The same procedure was followed for double labelling for CD3 and Mib1, substituting normal swine serum (100 µl) at 1:10 dilution, and biotinylated F(ab)2 fragmented swine anti-rabbit antibody (Dako) at a concentration of 1:150 (for 30 min at RT). This was detected using Vectastatin ABC® alkaline phosphatase (Vector Laboratories). For double labelling for CD56 and Mib1 microwave pretreatment was used prior to CD56, and enzyme digestion was omitted.

In-situ hybridization (ISH)
This was performed as previously described (Alison et al., 1994Go); we used a probe that hybridizes to the mRNA transcripts of human histone genes H2b, H3, and H4 (NCL-Histone-U; Novo Castra Laboratories, Newcastle upon Tyne, UK). Sections were dewaxed and rehydrated in DEPC (diethyl pyrocarbonate) water, and then immersed in 2xstandard saline citrate solution (SSC), at 70°C for 10 min, and washed in DEPC water. Proteinase K (Boehringer Mannheim, Mannheim, Germany) was applied to sections at a concentration of 5 µg/ml, (diluted in 0.05 mol/l Tris–HCl, pH 7.6, DEPC), and incubated at 37°C for 1 h. Slides were washed and transferred to 0.4% paraformaldehyde solution at 4°C for 20 min and washed. They were covered in 100 µl of pre-hybridization solution and incubated at 37°C for 1 h. A 50 µl aliquot of the probe was added and left overnight at 37°C. Sections were then washed twice in SSC/30% formamide at 37°C, and transferred to blocking solution. Anti-digoxigenin alkaline phosphatase 100 µl, Fab fragments antibody (Boehringer Mannheim) was applied (1/600 dilution) and incubated for 30 min at room temperature. Sections were washed in TBS and rinsed in distilled H2O. They were then transferred to Tris–HCl buffer pH 9.5 for 5 min. The alkaline phosphatase substrate kit (BCIP/NBT, Vector Laboratories) was added and incubated in the dark overnight. Sections were washed in running water for 5 min and mounted using aqua mount (BDH).

3' Labelling of oligonucleotide probes with digoxigenin
This method was used to label the Histone probe. A 2.5 µg sample of the dried probe was reconstituted in 50 µl of sterile ultra-pure water, ensuring thorough contact with the probe and left to stand for 1 h. To the tube containing the reconstituted probe, the following were added in order (reagents from Boehringer Mannheim): reconstituted probe (50 µl), sterile pure water to a final volume of 100 µl, 10 mmol/l manganese chloride (10 µl), digoxigenin-11-dUTP (10 nmol/l), terminal deoxynucleotidyl-transferase (Tdt enzyme) 50 U (2 µl), TdT buffer (20 µl). The solutions were mixed and spun in a microfuge and incubated for 2 h at 37°C. The enzyme activity was stopped by adding 5 µl of 0.5 mol/l EDTA, and the remaining reactants were removed by passing the solution through a slurry of Sephadex G50 (Sigma) spun column, and label incorporation was demonstrated by staining salmon sperm DNA test strips.

Image analysis
The positive cells were counted using an image analysis system comprising a single chip colour video camera: Sony DXC-151P connected to the Sony CMA-151P camera adapter. This transmitted the image to an Apple Macintosh® computer (Centris 650) via a RasterOps 24STV graphics display board (Rasterops Corporation, Santa Clara, CA, USA). This was analysed using the Colour Vision 1.7.4a programme (Improvision, University of Warwick, Coventry, UK).

Statistical analysis
As the standard deviation of the test parameters was unknown, the number of fields randomly selected from each section (n = 17) was calculated in order to satisfy a one-sample t-test, with a power 1-b = 0.90, and a two-sided {alpha} = 0.01 to detect the population mean with a standardized difference dt = 1 (Machin and Campbell, 1987Go). The number of sections to be examined was calculated in order to satisfy a two-sided t-test with a power 1-b = 0.90, a two-sided {alpha} = 0.05 to detect a 20% difference between the means. A pilot calculation gave the estimated dt = 1. The results were analysed using the two-sided unpaired t-test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the physiological cycle, CD45+ cells were found either scattered throughout the stroma with a higher number in the more superficial functionalis, or in aggregates near the base of the glands; only a few were inter-epithelial. CD45+ cells increased by ~35–40% during the mid-LP, and by 125% during the late-LP compared to the early-LP. The distribution of CD45+ cells during the late-PLP was similar, but the total number of CD45+ cells was almost doubled compared to the late-LP (Table IIGo).


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Table II. The number of CD20+, CD3+, CD56+, CD68+, CD45+, and Mib1+ during the different stages of the LP and during the late-PLP, expressed as number (and SE)/17 random high performance fields (x400)
 
In the early-LP, CD56+ cells were scattered throughout the stroma, but formed denser aggregates near blood vessels and glands during the late-LP. The number of cells doubled from the early to the mid-LP, with almost a three-fold increase during the late-LP. CD56+ cells were the most abundant type during the late-LP and the late-PLP. During the late-PLP, CD56+ cells exceeded by more than double (230%) those in the late-LP (Table IIGo).

In the physiological cycle, CD3+ cells were found in aggregates near the gland bases as well as throughout the stroma. Although their number increased during the late-LP, the differences between the stages of the physiological cycle were not statistically significant. The cellular distribution during the late-PLP was similar to that during the physiological cycle. There was no statistically significant difference between the number of CD3+ cells in the late-PLP compared to the late-LP, but the number of CD3+ cells was higher when compared to the early, and mid-LP (P < 0.05) (Table IIGo). CD68+ cells were scattered evenly throughout the stroma. The increase in the number of these cells during the mid-LP and late-LP was not statistically significant, and there was no increase during the late-PLP. CD20+ cells were only occasionally present and their numbers and distribution did not vary in the phases examined.

Stromal Mib1+ cells were scattered or formed clusters in the basalis. Scattered stromal Mib1+ cells were more abundant in the late compared to the early or mid-LP (P < 0.05), but the highest expression was during the late-PLP, where there was a severalfold increase. Only a few isolated glandular epithelial cells were Mib1+ during any of the phases examined (Table IIGo). The expression of nuclear histone mRNA in stromal cells was highest during the late-PLP (89 ± 22), compared to the early (22 ± 9), mid (32 ± 15), and late-LP (30 ± 10) (mean ± SE positive cells, per 17 random HPF), and exhibited a moderate correlation (Pearson's correlation coefficient, r = 0.64, r2 = 0.42) with Mib1 expression. Overall pattern of expression was similar to that of Mib1.

Double labelling using Mib-1 and CD45 (Figure 1A, GoB) demonstrated that the majority (88–94%) of stromal Mib1+ cells were of haematopoietic origin as they expressed CD45. The small proportion that was CD45 was morphologically similar to lymphocytes, with large, round nuclei and sparse cytoplasm. During the physiological cycle between 8–37% of CD45+ cells were Mib1+. The proportion of CD45+ cells that were Mib1+ was high during the mid-LP and dropped during the late-LP, and was highest during the late-PLP (Table IIIGo). The proportion of Mib1+ cells that were CD56+ was relatively constant throughout the phases of the physiological cycle and the late-PLP (Figure 1c, God), but the proportion of CD56+ cells that were proliferating was higher during the mid-LP and the late-PLP. Double labelling demonstrated that although the overall proportions were small, the proportion of CD3+ cells that were Mib1+ in the late-PLP was twice that in the late-LP. The same relation was demonstrated for the proportion of Mib1+ cells that were CD3+. The proportion of CD3+ cells that were Mib1+ was lower than the proportion of CD56+ cells that were Mib1+ during any phase of the cycle (Table IIIGo).



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Figure 1. Double labelling (A) Mib1 and CD45 in the late-luteal phase (LP), (B) Mib1 and CD45 in the late-pseudoluteal phase (PLP), (C) Mib1 and CD56 in the late-LP, and (D) Mib1 and CD56 in the late-PLP. Nuclei exhibiting proliferation (Mib1) stained blue/grey, CD antigen stained red. Arrows point to doubly labelled cells, scale bar = 0.05 mm.

 

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Table III. The percentages of cells that are doubly labelled for both Mib1 and CD antigens per 17hpf (x400), expressed as the means and (SE) for each of the study groups
 

    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
In the endometrium, anti-CD45 antibody recognizes all lymphocytes. CD56+ cells are predominantly granulated lymphocytes, and of eGL, only a small fraction expresses CD3. CD68 is expressed on macrophages, while CD20 is present on the majority of B-cells. Recently, it was demonstrated (King et al., 1998Go) that some eGL exhibit CD3{varepsilon} immunoreactivity after microwave pretreatment, and although we used pepsin pretreatment, it is possible that some of the CD3{varepsilon}+ cells are eGL. Leukocytes in the endometrium were scattered or in aggregates. It is not possible to obtain an accurate calculation of the number of cells in these aggregates using image analysis, but there was no difference between the numbers or the size of these follicles in the specimens examined and no attempt was made to calculate their numbers.

The distribution of leukocyte subtypes during the LP of the natural cycle noted in this study agrees with those reported previously (Klentzeris et al., 1992Go). The large increase in leukocytes during the late-PLP phase is attributed to an increase in CD56+ and CD3+ cells, but there was no rise in the number of CD20+ possibly due to their lack of oestrogen receptors or progesterone receptors. CD68+ cells were reported to represent 30% of CD45+ cells during the early-LP, and to rise by a factor of ~49% between days LH+4 and LH+13 (Klentzeris et al., 1992Go). This is in broad agreement with the findings in the current study. However, because the number of CD45+ cells during the late-LP increased in this study by 126% compared to the 62% increase previously reported (Klentzeris et al., 1992Go), there are proportionately fewer macrophages in the current study (18 compared to 28%), these differences may be due to the method of tissue fixation.

The high PI of both the CD45+ and the CD56+ cells during the late-PLP may be attributable to continuous hormone administration during this phase, or may be a specific effect of norethisterone. Thus the late-PLP is more reminiscent of the mid-LP. CD56+ and CD3+ cells account for the majority of the noted increase in leukocytes during the late-PLP.

Using formalin-fixed tissues, we demonstrated a higher (275%) increase in the number of CD56+ cells from the early-LP to the late-LP, compared to the 152% increase reported using imprint preparation and frozen sections (Bulmer et al., 1991bGo). The increased number of leukocytes observed during the late-PLP endometrium underlies the evidence of stromal decidualization, and more advanced stromal compared to glandular development (Habiba et al., 1998Go). The function of the increased CD56+ cells is unknown, and although they may have a role in relation to tissue breakdown, it remains to be demonstrated whether these cells release their granules prior to the onset of tissue breakdown and bleeding.

During the LP, stromal proliferation becomes confined to CD45+ cells, and increases from the mid-follicular phase to peak during the late-LP. It is also more prominent in the scattered compared to the aggregated lymphoid cells. The small percentage of cells that were Mib1+ and CD45 exhibited morphological features similar to leukocytes, and it is possible that the inability to demonstrate CD45 antigen was due to technical limitations. CD3+ T-cells and CD11c+ macrophages were shown to follow the same pattern (Tabibzadeh, 1990Go). In agreement with our findings, the total number of lymphocytes and T-cells, but not of B-cells or macrophages, were shown to increase with oestrogen and progesterone treatment in women with premature ovarian failure (Booker et al., 1994Go).

This study provides direct supportive evidence of in-situ proliferation of eGL (Pace et al., 1989Go). It was also reported, however (Pace et al., 1989Go), that the increased eGL proliferation from the follicular phase into the LP is not accompanied by an increase in number, and that the increase in eGL in the late-LP is not accompanied by increased proliferation. They also reported a low proliferation rate of one or two mitoses/10 high power fields (Pace et al., 1989Go). In the current study, we demonstrated a high PI, and the maximum PI preceded the peak cell density, which further supports local proliferation as the mechanism for the increase in the eGL population. However, it is also possible that a reduction of cell death, possibly through impairment of a Fas-ligand mediated or other mechanism (Jiang and Vacchio, 1998Go), may have a role. A recent study (Jones et al., 1998Go) reported a reduction of stromal proliferation in the endometrium of women receiving progestogen therapy for menorrhagia. Although this was not specifically reported, such a group would typically receive a progestogen dose severalfold higher than that used in our study.

The increased proliferative activity up to the immediate premenstrual phase argues against a significant role for leukocyte apoptosis in the initiation of menstruation (King et al., 1993Go). This view is supported by other findings (Jones et al., 1998Go), and by our finding of only occasional apoptotic cells and of an increased bcl-2 expression during the late-PLP (Habiba, 1998Go).

Progestogen administration increases the number of CD3+ and CD68+ leukocytes and particularly the number of eGL (Song et al., 1996Go), through an unknown mechanism. The progesterone receptor is absent from endometrial leukocytes but T-cells, particularly those localized to the lymphoid aggregates in the deeper functionalis or the basalis, may express the oestrogen receptor (Tabibzadeh and Saytaswaroop, 1989Go; King et al., 1996Go). Thus it is possible that the effect of steroids is mediated through cytokines such as interleukin-8 (IL-8), the monocyte chemoattractant protein-1 (MCP-1) and cyclooxygenase-2 (COX-2) (Jones et al., 1997Go).

MCP-1 is particularly interesting, for it is a potent attractant and activator for T-cells (Roth et al. 1995Go), and NK cells (Allavena et al., 1994Go), both of which showed a significant increase with sequential HRT. Decidual cells produce COX-2 in response to IL-1ß, an effect that is inhibited by progesterone (Ishihara et al., 1995Go). The inhibitory effect attributed to progesterone, and the observed increase in leukocytic infiltrate in the late-LP when levels of circulating progesterone are known to be low suggests that progesterone has an indirect inhibitory effect and that the rise observed is secondary to release of this inhibition (Jones et al., 1997Go). It was proposed that the progesterone receptor mediates the inhibitory effect of progesterone on MCP-1 release from perivascular cells (Jones et al., 1997Go). This, however, would not explain the excessive infiltrate observed under the influence of sequential HRT, or that observed with prolonged progestogen therapy (Song et al., 1996Go), or the increased eGL in the decidua (Pace et al., 1989Go). It is also possible that continued progestogen administration, through down regulating progesterone receptors, can induce the same effect. Low `effective' progestogen in sequential HRT may result from either a higher level compared to the physiological cycle leading to receptor inhibition, or from a lower oestrogen resulting in low progesterone receptor expression.

On the other hand, the lack of a rise in the number of macrophages with sequential HRT may be explained by the observation that granulocyte–macrophage colony stimulating factor (GmCSF), which may be responsible for macrophage migration as well as proliferation and differentiation (Wang et al., 1987Go), is oestrogen regulated (Robertson et al., 1996Go). The administration of the anti-progesterone mifepristone was associated with a rise in the number of decidual macrophages, but this may be partly attributable to mifepristone stimulating oestrogen receptor (Critchley et al., 1996Go). Thus this sequential HRT may be relatively hypo-oestrogenic.

The presence of lymphomyeloid cells in the stroma and their ability to produce proteolytic enzymes supports the view that the stroma may be active in the process of tissue shedding and menstruation, or in repair. eGL contain perforin, granzyme A and TIA-1 (King et al., 1993Go), which are capable of cytolysis, DNA fragmentation, and cytotoxicity. Activated eGL may be implicated in focal necrosis, stromal haemorrhage or normal or abnormal menstruation, and a similar role has been suggested for CD68+ macrophages (Song et al., 1996Go). The functional significance of the excessive leukocytic infiltrate, including eGL, during the late-PLP is not clear. Although leukocyte density does not appear to be critical for the initiation of bleeding, these cells are rich in cytokines and growth factors and exhibit cytotoxic activity comparable to that of CD56+ peripheral blood NK cells (Jones et al., 1997Go). A role in tissue breakdown or remodelling therefore cannot be ruled out.


    Notes
 
3 To whom correspondence should be addressed at: Gynaecology Research Group, Faculty of Medicine and Biological Sciences, Leicester University, Leicester, LE2 7LX, UK Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Alison, M., Chaudry, Z., Baker, J. et al. (1994) Liver regeneration: a comparison of in situ hybridization for histone mRNA with bromodeoxyuridine labelling for the detection of S-phase cells. J. Histochem. Cytochem., 42, 1603–1608.[Abstract/Free Full Text]

Allavena, P., Bianchi, G., Zhou, D. et al. (1994) Induction of natural-killer-cell migration by monocyte chemotactic protein-1, protein-2 and protein-3. Eur. J. Immunol., 24, 3233–3236.[ISI][Medline]

Booker, S.S., Jayanetti, C., Karalak, S. et al. (1994) The effect of progesterone on the accumulation of leukocytes in the human endometrium. Am. J. Obstet. Gynecol., 171, 139–142.[ISI][Medline]

Bulmer, J.N., Longfellow, M. and Ritson, A. (1991a) Leukocytes and resident blood cells in endometrium. Ann. N. Y. Acad. Sci., 622, 57–68.[Medline]

Bulmer, J.N., Morrison, L., Longfellow, M. et al. (1991b) Granulated lymphocytes in human endometrium: histochemical and immunohistochemical studies. Hum. Reprod., 6, 791–798.[Abstract]

Chou, M.Y., Chang, A.L.C., McBride, J. et al. (1990) A rapid method to determine proliferation patterns of normal and malignant tissues by H3 mRNA in situ hybridization. Am. J. Pathol., 136, 729–733.[Abstract]

Clark, D.A. and Daya, S. (1990) Macrophages and other migratory cells in endometrium: relevance to endometrial bleeding. In D'Arcangues, C. Newton, J.R. and Odlind, V. (eds), Contraception, and Mechanisms of Endometrial Bleeding. WHO Publications. Proceedings of a symposium organized by the special programme of research, development and research training in human reproduction of the WHO in Geneva on 28 November–2 December 1988. Cambridge University Press, Cambridge, pp. 363–382.

Critchley, H.O.D., Kelly, R.W., Lea, R.G. et al. (1996) Sex steroid regulation of leukocyte traffic in human decidua. Hum. Reprod., 11, 2257–2262.[Abstract]

Gerdes, J., Schwab, U., Lemke, H. and Stein, H. (1983) Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int. J. Cancer, 31, 13–20.[ISI][Medline]

Gerdes, J., Becker, M.H.G. and Key, G. (1992) Immunohistological detection of tumour growth fraction (Ki-67 antigen) in formalin fixed and routinely processed tissue. J. Pathol., 168, 85–86.[ISI][Medline]

Habiba, M. (1998) Endometrial Responses to Hormone Replacement Therapy. Ph.D. thesis, University of Leicester, pp. 149–164.

Habiba, M., Bell, S.C., Abrams, K. and Al-Azzawi, F. (1996) Endometrial responses to hormone replacement therapy: the bleeding pattern. Hum. Reprod., 11, 503–508.[Abstract]

Habiba, M., Bell, S.C. and Al-Azzawi, F. (1998) Endometrial responses to hormone replacement therapy: histological features compared with those of late luteal phase endometrium. Hum. Reprod., 13, 1674–1682.[Abstract]

Haller, H., Radillo, O., Rukavina, D. et al. (1993) An immunohistochemical study of leukocytes in human endometrium, first and third trimester basal decidua. J. Reprod. Immunol., 23, 41–49.[ISI][Medline]

Ishihara, O., Matsuoka, K., Kinoshita, K. et al. (1995) Interleukin-1ß-stimulated PGE2 production from early first trimester human decidual cells is inhibited by dexamethasone and progesterone. Prostaglandins, 49, 15–26.[Medline]

Jackson, P. and Blythe, D. (1993) Immunolabelling techniques for light microscopy. In Beesley, J.E. (ed.), Immunocytochemistry: a Practical Approach. IRL Press, Oxford, pp. 15–42.

Jiang, S.-P. and Vacchio, M.S. (1998) Cutting edge: multiple mechanisms of peripheral T cell tolerance to the fetal allograft. J. Immunol., 160, 3086–3090.[Abstract/Free Full Text]

Jones, R.K., Searle, R.F., Stewart, J.A. et al. (1998) Apoptosis, bcl-2 expression, and proliferative activity in human endometrial stroma and endometrial granulated lymphocytes. Biol. Reprod., 58, 995–1002.[Abstract]

Jones, R.L., Kelly, R.W. and Critchley, H.O.D. (1997) Chemokine and cyclooxygenase-2 expression in human endometrium coincides with leukocyte accumulation. Hum. Reprod., 12, 1300–1306.[ISI][Medline]

Kamat, B.R. and Isaacson, P.G. (1987) The immunocytochemical distribution of leukocytic sub-populations in human endometrium. Am. J. Pathol., 127, 66–73.[Abstract]

King, A., Wooding, P., Gardner, L. and Loke, Y.W. (1993) Expression of perforin, granzyme A and TIA-1 by human uterine CD56+ NK cells implies they are activated and capable of effector functions. Hum. Reprod., 8, 2061–2067.[Abstract]

King, A., Gardner, L. and Loke, Y.W. (1996) Evaluation of oestrogen and progesterone receptor expression in uterine mucosal lymphocytes. Hum. Reprod., 11, 1079–1082.[Abstract]

King, A., Gardner, L., Sharkey, A. and Loke, Y.W. (1998) Expression of CD3{varepsilon}, CD3z, and RAG-1/RAG-2 in decidual CD56+ NK cells. Cell. Immunol., 183, 99–105.[ISI][Medline]

Klentzeris, L.D., Bulmer, J.N., Warren, A. et al. (1992) Endometrial lymphoid tissue in the timed endometrial biopsy: morphometric and immunohistochemical aspects. Am. J. Obstet. Gynecol., 167, 667–674.[ISI][Medline]

Machin, D. and Campbell, M.J. (1987) Statistical Tables for the Design of Clinical Trials. Blackwell, Oxford, pp. 79–88.

Pace, D., Morrison, L. and Bulmer, N.J. (1989) Proliferative activity in endometrial stromal granulocytes throughout menstrual cycle and early human pregnancy. J. Clin. Pathol., 42, 35–39.[Abstract]

Robertson, S.A., Mayrhofer, G. and Seamark, R.F. (1996) Ovarian steroid hormones regulate granulocyte–macrophage colony-stimulating factor synthesis by uterine epithelial cells in the mouse. Biol. Reprod., 54, 183–196.[Abstract]

Roth, S.J., Woldemar Carr, M. and Springer, T.A. (1995) C-C chemokines, but not the C-X-C chemokines interleukin-8 and interferon-gamma inducible protein-10, stimulate transendothelial chemotaxis of T lymphocytes. Eur. J. Immunol., 25, 482–3488.

Shi, S-R., Key, M.E., Kalra, K.L. (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissue: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. Histochem. Cytochem., 39, 741–748.[Abstract]

Song, J.Y., Russell, P., Markham, R. et al. (1996) Effect of high dose progestogens on white cells and necrosis in human endometrium. Hum. Reprod., 11, 1713–1718.[Abstract]

Starkey, P.M., Clover, L.M. and Rees, M.P.C. (1991) Variation during the menstrual cycle of immune cell populations in human endometrium. Eur. J. Obstet. Gynecol. Reprod. Biol., 39, 203–207.[ISI][Medline]

Tabibzadeh, S. (1990) Proliferative activity of lymphoid cells in human endometrium throughout the menstrual cycle. J. Clin. Endocrinol. Metab., 70, 437–443.[Abstract]

Tabibzadeh, S. and Saytaswaroop, P.G. (1989) Sex steroid receptors in lymphoid cells of human endometrium. Am. J. Clin. Pathol., 91, 656–663.[ISI][Medline]

Wang, J.M., Colella, S., Allavena, P. et al. (1987) Chemotactic activity of human recombinant granulocyte–macrophage colony-stimulating factor. Immunology, 60, 439–444.[ISI][Medline]

Yu, C-W., Woods, A.L. and Levison, D.A. (1992) The assessment of cellular proliferation by immunohistochemistry: a review of currently available methods and their applications. Histochem. J., 24, 121–131.[ISI][Medline]

Submitted on April 12, 1999; accepted on August 13, 1999.





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