1 Department of Anatomy, Medical University of Zahedan, PO Box 98135396 Zahedan, Iran, 2 Department of Biomedical Science, University of Sheffield, Western Bank, S10 2TN, UK, 3 Department of Anatomy, University of Cork, Ireland, 4 Department of Obstetrics and Gynaecology, Jessop Hospital, Sheffield S3 7RE, UK, and 5 Reproductive Biology, Biotechnology and Infertility Research Centre, Tehran, Iran
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Abstract |
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Key words: basement membrane/human/implantation/luminal epithelium/morphometry
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Introduction |
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The uterine luminal epithelium, despite being the first contact between developing trophoblast (Sadler, 1981) and the mother, has received much less attention than either gland cells or stroma. Therefore, in the present study we have examined the cellular and ultrastructural changes which occur in luminal epithelial cells around the time when implantation would take place. We have used timed biopsies from a group of normal women of proven fertility in an effort to detect their preparation for implantation, i.e. the implantation window.
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Materials and methods |
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Normal fertile women (aged 1840 years) were defined (Li et al., 1988) as those who: (i) had regular menstrual cycles of between 25 and 35 days with no evidence of menstrual disorder; (ii) had not used any steroid hormonal contraception or intrauterine contraceptive device for at least 3 months prior to hospitalization; and (iii) had at least one successful pregnancy.
A single endometrial specimen was obtained using a Sharman's curette (Down Surgical Ltd, Sheffield, UK) from the fundus and upper part of the body of the uterus. All biopsies were timed by reference to the LH surge (designated day LH 0) which was determined by LH assays on daily samples of either morning urine or of plasma. Each endometrial specimen was fixed immediately in 3% glutaraldehyde and processed for light and electron microscopy as described previously (Li et al., 1992). From JB-4 blocks, 2 µm thick sections were cut using glass knives on an Anglia Scientific AS500 microtome (Anglia Instruments, Cambridge, UK) and stained with 1% acid fuchsin and 0.05% Toluidine Blue. Semi-thin sections (0.5 µm thick) were cut from Epon blocks using a Reichent ultramicrotome (OMU3, Vienna, Sweden) and stained using 0.05% Toluidine Blue. Ultrathin sections (~70 nm thick) were double-stained with aqueous uranyl acetate and lead citrate (Dockery et al., 1988
). The ultra-thin sections were examined on a Philips 301-transmission electron microscope at a range of magnifications, which were determined with the aid of a grating replica.
Morphometry
Estimation of volume fraction (VV)
Non-overlapping micrographs (between five and ten) of luminal epithelium were taken for each subject in a systematic random pattern at an initial magnification of x2200. Measurements were made by using a projecting microscope (Carl Zeiss, Jena, Germany) to magnify negatives to a final magnification of ~x22 000. The images were superimposed with a lattice of 20 mm squares (0.9 µm apart on the tissue) and the volume fraction of nucleus, mitochondria, `vesicular system' and rough endoplasmic reticulum to cell and of euchromatin to nucleus were obtained.
Basement membrane arithmetic and harmonic mean thickness (Hirose et al., 1982)
Using a projecting microscope the negatives were projected at a magnification of x50 000 on a square lattice, each square of which was 50 mm apart (1 µm on the tissue). Two perpendicular lines were drawn, one on the inner surface of the basement membrane and another on the outer surface of the basement membrane. These lines were orthogonal to the line of the square lattice. The length (l) between these two perpendicular lines was measured using a common ruler. By dividing the sum of all lengths by the number of observations the arithmetic (a) mean length was obtained (a). Basement membrane arithmetic mean thickness (Ta) was measured according to the formula:
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Surface ratios
Using the 20 mm square lattice and recording the intersections of horizontal and vertical lines on the lattice and the projected images, the ratios of microvilli surface and desmosomes to cell membrane were estimated. In addition an arbitrary straight line drawn on the base of microvilli of luminal epithelial cells was used to permit estimation of the amplification of cell apical surface due to microvilli.
Nuclear profile dimensions
Measurements were made on semi-thin sections with the aid of a drawing tube attached to an Olympus (BH-2) microscope and a microcomputer-based digitizer using previously written software. The major (a) and minor (b) axes, mean profile diameter ( a x b) and axial ratio (major axis/minor axis) of 50 longitudinally sectioned nuclear profiles were obtained from each subject.
Cell height
The height of 10 randomly sampled, luminal epithelial cells that were cut longitudinally were measured using a drawing tube and ruler in one randomly selected JB-4 embedded section per subject. Estimations were made at the mid-point of cells with clearly visible apical and basal borders using an oil immersion objective at a final magnification of x1050.
Linear nuclear density
The linear nuclear density (number of nuclear profiles per unit length of epithelium) was estimated under oil immersion at a magnification of x1050. The lengths of four randomly sampled segments of the luminal epithelium, where cells were cut longitudinally, were measured per subject. The number of nuclear profiles within that length of segment was counted and dividing the total number of nuclear profiles by the total length of epithelium gave the number of nuclear profiles per unit length of epithelium.
Volume weighted mean volume
Vertical semi-thin sections were cut and displayed on a Quantimet Q970 image analyser (Cambridge Instrument Ltd, Cambridge, UK) using a Polyvar microscope at a magnification of ~x2600 times (a graticule was used to determine exact magnification). The outlines of every luminal epithelial cell nucleus on 10 fields of vertical sections were drawn on acetate sheets using a fine point felt tip pen. A test system of parallel lines 10 mm apart was superimposed on the luminal epithelial cells. The outlines of nuclei were orientated so that their long axes were parallel to the vertical line of a measuring grid (Sorensen, 1991). When a point hit a nucleus, a line was drawn through this point. These lines produced point sampled intercepts whose lengths (which were measured by a metric ruler corrected for magnification) were raised to the third power, multiplied by [pi]/3 and then averaged over all intercepts to give an unbiased estimation of volume, i.e. weighted mean volume (
v) (Dockery et al., 1998
; Gundersen et al., 1988
). About 100 nuclear profiles were traced and measured from each subject.
Statistical analysis
Data were collected from each individual and the mean ± SE calculated per group (n = number of individuals). Where necessary, log transformations of ratio data were calculated for each group before statistical testing. Groups were compared by using correlation coefficients and one-way analysis of variance (ANOVA) tests. P < 0.05 was considered to be significantly different. Data were analysed using a Microsoft Excel spreadsheet and SPSS software running on a PC.
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Results |
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Discussion |
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Basement membrane is a mat consisting of extracellular matrix proteins and glycosaminoglycans typically ~70100 nm thick (Alberts et al., 1994). In the uterus the basement membrane provides a barrier to limit or regulate the reciprocal diffusion of macromolecules between luminal epithelial and stromal cells. Therefore, its thickness determines the rate of diffusion of substances across it. In the present study, `arithmetic mean thickness', which is an index of the relative thickness of basement membrane and `harmonic mean thickness', which shows the relative diffusing capacity, were determined. Blankenship and Given (1995) reported that in mouse uterine luminal epithelium basement membrane (UEBM) there is a loss of laminin and collagen type IV at 57 days of pregnancy. They suggested that this reduction of laminin and collagen IV may be related to stromal decidual cells, which are close to the basement membrane. Although the precise mechanism for this is unknown, it has been suggested that the three different cell types (uterine epithelium, trophoblast and stromal decidual cells) that are in contact with the basement membrane at this time may be involved with UEBM dissolution. In the rat, decidual cells often contain many lysosomes and proteolytic enzymes, which are essential for their remodelling and function at implantation (Welch et al., 1985
).
In vitro, uterine decidual cells from 6 and 7 day pregnant mice continue to synthesize basement membrane-like matrix (Wewer et al., 1986). Similarly murine decidual cells, which resemble their human equivalent, continue to synthesize laminin after explant in vitro (Wewer et al., 1985
). In 1987, it was reported for the first time that first trimester human decidual cells contain laminin and collagen types I, II, IV and V (Kisalus et al., 1987
). In both mouse (Blankenship and Given, 1992
) and rat (Schlafke et al., 1985
) decidual cells are instrumental in the removal of luminal epithelial cell basal lamina during early pregnancy and this facilitates the invasion of trophoblast at implantation. In the human, glandular epithelium lamina densa is intact during the proliferate phase (Roberts et al., 1988
), but there are occasionally small projections of epithelial cells through the lamina densa. These projections become more complex during the early to mid-luteal phase and often come so close to the underlying stromal cells that focal adhesions are visible at that location. In this adhesion area there is no cell membrane either in epithelial or stromal cells. As the authors suggested, these cells appear fused together and have a direct association between their cytoplasms (Roberts et al., 1988
). These epithelial and stromal projections contained many large convoluted gap junctions. The stromalepithelial cell interactions and the complexity of gap junctions decrease by the mid- to the late secretory phase. It was suggested that a complex physical interaction occurs between epithelial and stromal cells and this interaction indicates the maximal development of human endometrium during the preimplantation time (Roberts et al., 1988
).
It has also been suggested that stromal and decidual cells can modify the basement membrane matrix (Kisalus et al., 1987; Blankenship et al., 1990
). In the present study the harmonic and arithmetic mean thicknesses of luminal epithelial cell basement membranes were thinnest on day LH+6. This thinning of basement membrane may be due to the action of stromal cells, since in non-pregnant cycles it occurs independent of the trophoblast and other events triggered by trophoblast. Since the animal studies above (Wewer et al., 1986
) suggested decidual cells continued to secrete basement membrane-like matrix, it is possible that basement membrane thinning may be due to its enzymic degradation rather than reduced production. The agent that may be responsible for loss of luminal epithelial cell basement membrane in humans is still unknown. However, some members of the metalloproteinase family and plasminogen are candidates in this process (Matrisian et al., 1990
; Alexander and Werb, 1991
; Woessner 1991
; Murphy et al., 1992
).
Desmosomes help to bind cells together and are responsible for the maintenance of tissue integrity (Rhodin, 1974). It is reported that in the rabbit during implantation some changes in the molecular structure of desmosomes, including desmoplakin-I, occur and that components are translocated into the lateral membrane (Classen-Link and Denker, 1990). In the present study the proportion of cell membrane occupied by desmosomes was significantly reduced at day LH+6. This reduction may help luminal epithelium to facilitate blastocyst invasion. It was reported that human endometrial glandular epithelium in both normal and exogenous oestrogen-treated women and the number of tonofilaments that related to the formation of new desmosomes increased (Clyman et al., 1982
). When progesterone concentrations increased these bundles became scattered. In the present study desmosomes were smallest during the mid-luteal phase, when progesterone concentrations are high.
There are few published reports on luminal epithelial cells, but it has been reported that in glandular epithelium the percentage of cell occupied by the nucleus decreased between days LH+2 and LH+4 from 23.95 ± 0.49 to 19.42 ± 0.55 and then continued to increase (Dockery et al., 1993). In their study glandular cell nuclear volume did not show significant changes between days LH+2 to +6 of the cycle. The increase in the volume of cytoplasm was due mainly to the increase in glycogen from 30.91 ± 23.80 to 128.60 ± 34.00 µm3 between days LH+2 and +5, after which it suddenly declined. In comparison with these dramatic changes in glandular epithelium (Dockery et al., 1993
), the luminal epithelial cells in the present study showed much smaller changes. Therefore, luminal cells appear relatively unresponsive to hormonal changes in the luteal phase (Sundstrom et al., 1983
; Cornillie, 1985).
Microvilli decrease and apical protrusions or pinopodes project into the uterine lumen from epithelial cells (Martel et al., 1981). It has been suggested that pinopodes play a role in modulating the uterine environment by actively absorbing material from the uterine lumen (Nikas et al., 1995
). Similar projections were seen in luminal epithelial cells in the present study. Since these apical projections are variable in form they may have some influence on the cell height estimates made in the present study suggesting this feature may not be a very reliable indicator of cell height. Also it is known that values of cell profile height may be overestimates of true epithelial cell height owing to the angle of sectioning (Mayhew et al., 1990
). However these values were used as indicators of relative cell profile height change across the cycle and do not reflect absolute measures of average epithelial cell height (Beer et al., 1995
).
The volume fraction of mitochondria to cell in luminal epithelial cells numerically increased, but was not significant, between days LH+4 and LH+6, while the glandular cells (probably due to high-energy demands during secretion at this time) showed a significant change over the same time period (Dockery et al., 1993). The proportion of cell occupied by rough endoplasmic reticulum (RER) and the volume of RER in luminal epithelial cells were generally similar to glands (Dockery et al., 1993
). In the luminal epithelial cells the proportion of `vesicular system' to cell and the volume of `vesicular system' showed great variation with time, while in gland cells there was a continuous reduction (Li et al., 1988
). The reason for these differences between luminal and glandular epithelium remain unclear, but may well be a reflection of the high intersubject variation in luminal cells.
While the data presented here refer to human luminal endometrial cell morphology, it seems pertinent to mention some of the recently published molecular events occurring in luminal epithelium around the time of implantation. The early stages of apposition involve carbohydrates with firm attachment using adhesion molecules, in particular integrins and fibronectin (Poirier and Kimber, 1997). For example, the uterine H type 1 antigen may interact with trophectoderm cell surfaces during attachment, possibly via the Ley component of the blastocyst (Poirier and Kimber, 1997
). It has also been suggested in a comprehensive set of experiments (Thie et al., 1997
) that the actin cytoskeleton of luminal epithelial cells is related to their `adhesiveness'. These studies used a human uterine cell line (RL95-2) with the unusual property of permitting apical cell membrane adhesion to trophoblast. It has been suggested that it is the non-polarized actin cytoskeleton in these cells which permits adhesion with trophoblast as another cell line (HEC-1-A), which are polarized, do not permit attachment. In the present study, however, the changes which were seen in normal human endometrial tissue around the time of implantation were not in cell polarity (e.g. the admittedly crude estimation of profile cell height did not differ significantly across the time period studied), but rather were subtle changes in the basement membrane and desmosome components of cells. While the precise relevance of the in-vitro observations (Thie et al., 1997
) to the in-vivo situation needs careful consideration, their studies are likely to provide important insights into the cell biological mechanisms involved in attachment.
In summary it is suggested that luminal epithelial cells (except for basement membrane and desmosome changes which occur only in luminal epithelium) show similar trends of morphological change to those seen in glandular epithelial cells, but to a lesser extent. However, luminal epithelial cell basement membranes were thinnest and the proportion of cell membrane occupied by desmosomes was smallest on day LH+6. These reductions may help luminal epithelium to facilitate blastocyst invasion at the time that implantation is likely to occur and may represent morphological evidence for the `implantation window'.
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Notes |
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References |
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Submitted on May 24, 1999; accepted on September 7, 1999.