1 Department Obstetrics and Gynaecology, Imperial College School of Science, Technology and Medicine, Hammersmith Hospital, DuCane Road, London W12 ONN, UK, 2 The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynaecology, Eastern Virginia Medical School, Norfolk, Virginia 23507-1627 USA
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Abstract |
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Key words: endometrial pinopodes/implantation/IVF/nidation window/uterine receptivity
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Introduction |
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An interesting approach to this enigma appears to be morphological studies using scanning electron microscopy (SEM). At the time of implantation, the apical membranes of the epithelial cells lining the uterine cavity lose their microvilli and develop large and smooth membrane projections (Psychoyos and Mandon, 1971a). Due to their pinocytotic function, these projections were named pinopodes (Enders and Nelson, 1973
). Their development is progesterone dependent, and in rodents coincides strictly with the implantation window (Psychoyos and Mandon, 1971b
; Martel et al., 1991
). In the human endometrium similar structures were observed around the 20th day of a normal cycle (Martel et al., 1981
), which is the presumed day of blastocyst attachment. Hormonal treatments have been shown to advance or retard the timing of pinopode formation. During ovarian stimulation with clomiphene citrate followed by human menopausal gonadotrophin (HMG)/HCG pinopodes form earlier, on days 17 or 18 (Martel et al., 1987
). In contrast, under HRT pinopodes form later, around day 22 (Psychoyos and Nikas, 1994
). Yet these days represent only mean values deriving from a group of patients. When sequential (every 23 days) midluteal samples were taken from natural or HRT cycles, the timing of pinopode appearance was found to vary up to 5 days between women. Sequential sampling also revealed that pinopode formation follows a distinct pattern allowing us to distinguish between developing, fully developed or regressing pinopodes. Fully developed pinopodes were always confined to one sample, showing a life span of less than 48 h. Finally the number of pinopodes was different between patients and there was a strong correlation between pinopode numbers and implantation after embryo transfer (Nikas et al., 1995
, 1996
, 1997
; Nikas and Psychoyos, 1997
).
Little information on pinopodes is available in ovarian stimulation cycles induced by gonadotrophins only. Conventional histology has shown advanced endometrial maturation in HMG/HCG cycles (Garcia et al., 1984). Theoretically, reduced implantation rates in IVF cycles could result from impaired or premature endometrial maturation, which could be accompanied by alterations in pinopode expression. Indeed, in a recent study pinopodes were found to form as early as 4 days after HCG administration (Kolb et al., 1997
). However, this study dealt more with the overall changes of surface morphology than with pinopode expression.
The aim of the present work was to assess the temporal expression of pinopodes as a specific marker for receptivity in IVF cycles induced by gonadotrophins. In addition, serum concentrations of steroid hormones were assessed during the preovulatory period and correlated with pinopode findings.
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Materials and methods |
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Seven women with regular menstrual periods and proven fertility served as controls. The age distribution of these women was similar to that of the donor group (29.3 ± 2.6 versus 29.8 ± 3.4 respectively). The dates in controls were assigned by detection of the LH surge using a commercially available urine kit (OvuQuick, Quidel, San Diego, CA, USA), and validated by dates of onset of the next menstrual period and histological dating according to morphological criteria (Noyes et al., 1950). All patients gave written, informed consent prior to the study, which had been approved by the Institutional Review Board of Eastern Virginia Medical School.
Endometrial tissue collection and preparation
Fifty-one endometrial samples were obtained sequentially from 17 donors. Samples were taken every 4872 h, on days 1424 (see Table I). Day of oocyte aspiration was designated day 14 of the cycle. In the natural cycle group 18 samples were taken on days 1723 (see Table II
). Sampling of uterine fundus was performed using a Pipelle (Unimar Inc., Wilton, CT, USA). The endometrial tissue was fixed in 2.5% (w/v) glutaraldehyde solution in a sodium cacodylate buffer (0.1 mol/l pH 7.3). The specimens were dehydrated in an acetone series, dried in a critical point drier using carbon dioxide, mounted on the specimen holder, coated with gold, and examined under a Stereoscan 360 SEM (Cambridge Instruments, Cambridge, UK). Many tissue pieces were viewed from each biopsy to increase the likelihood that the observations were representative, since the endometrium may show more advanced or retarded morphology from one area to another.
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Results |
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This sequence of changes was observed in all cycles studied. However, the cycle days when these changes occurred varied greatly between women, and some women showed accelerated and others retarded maturation (Figures 1, 2 and 3). Thus fully developed pinopodes appeared within a range of 5 days in ovarian stimulation cycles as follows: day 18 (n=2), day 19 (n=7), day 20 (n=4), day 21 (n=3), day 22 (n=1). In the majority of cycles (9 of 17), maturation was advanced with pinopodes already formed by day 19. In natural cycles fully developed pinopodes covered a three day range as follows: day 20 (n=2), day 21 (n=2), day 22 (n=3). Expression of fully developed pinopodes in ovarian stimulation cycles was significantly accelerated for an average of 12 days in comparison to natural cycles (19.6 ± 1.1; median 19 and 21.1 ± 0.9; median 21, respectively; MannWhitney U test: z = 2.604, P = 0.009). The number of pinopodes present was scored in three grades: abundant, moderate, and few, depending on the percentage of the endometrial surface occupied by pinopodes (>50%, 2050% and <20% respectively). These grades did not show any statistically significant difference between the two groups. Tables I and II
summarize these findings.
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Discussion |
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Ovarian stimulation using gonadotrophins alone appears more physiological than using a combination of clomiphene citrate and gonadotrophins, since in the latter the surface morphology was greatly advanced, with pinopodes already regressed by day 20, in 85% of cycles studied (Martel et al., 1987). This is in agreement with the fact that ovarian stimulation using gonadotrophins only leads to higher pregnancy rates (Tummon et al., 1992
). However, our findings seem to differ somewhat from those of Kolb et al. (1997), who observed pinopodes as soon as 4 days after HCG. In our material the earliest pinopode formation occurred 6 days after HCG. This discrepancy may be due to differences of the stimulation protocol used, or these investigators might have possibly included as pinopodes small apical projections which appear occasionally at the uterine folds during early luteal phase. Furthermore, their study protocol did not allow them to biopsy their patients sequentially, or even after day 19 (HCG+7), which might have obscured the progressive changes in pinopode expression.
In our study group the pinopodes occurred on days 1822, thus covering a 5 day interval. The expression of other proposed markers of receptivity, e.g. certain integrin subunits, was found to occur also during a 5 day interval (Lessey et al., 1994). Yet sequential sampling in our study revealed that in each woman fully developed pinopodes were present for less than 2 days. Whether sequential sampling could elaborate a similarly limited individual expression for other markers of uterine receptivity as well, remains an open question.
A further finding in our study included the link between elevated preovulatory progesterone concentrations and early appearance of pinopodes. The impact that early progesterone rise bears on success of IVF remains a matter of continuing dispute. An explanation for this controversy could lie in the diversity of consequences associated with this phenomenon. Early luteinization possibly results in better quality zygotes (Legro et al., 1993). Moreover, prolonged exposure to progesterone may decrease uterine contractility at the time of embryo transfer, which might be another parameter likely to affect the outcome of an IVF cycle (Fanchin et al., 1998
). On the other hand, in a study of endometrial histology Chetkowski et al. (1997) have shown premature progesterone rise to induce early secretory transformation. Furthermore, pinopode findings in the present study could imply that early progesterone rise (the threshold of which remains yet to be defined) may accelerate closure of the window of receptivity and thus compromise the chances of successful implantation.
Regarding some concerns about the reliability of microscopic findings after sequential sampling, SEM is a suitable method to address this question. SEM can view the entire surface of the specimen and very often the sites of a previous sampling are visible as gaps surrounded by regenerating epithelium. Such areas tend to localize at the edge of the biopsy pieces and the rest of the tissue looks perfectly normal, in terms of epithelial integrity and evolution, including pinopode formation (Figure 4). Therefore, sequential endometrial sampling provides reliable material for SEM studies.
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Notes |
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References |
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Submitted on May 7, 1998; accepted on November 18, 1998.