1 Department of Obstetrics and Gynaecology, 6/ F Professorial Block, Queen Mary Hospital, PokfulamRoad, Hong Kong, and 2 Department of Anatomy, The University of Hong-Kong
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Abstract |
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Key words: endometrial morphometry/high oestradiol/OHSS/secretory phase endometrium
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Introduction |
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The aim of the present study was to evaluate the effect of very high oestradiol concentrations (20 000 pmol/l) on endometrial structural parameters around the time of implantation in women at risk of OHSS with the intent of elucidating the reasons for the diminished endometrial receptivity seen in these women in our IVF programme.
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Materials and methods |
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Endometrial biopsies were obtained during the period from January 1997 to December 1998 from women who did not have embryo transfer during IVF treatment. These included women with failure of fertilization due to subnormal semen, and women at high risk of OHSS. The age range of the study population was 2739 years (mean ± SD, 33 ± 2.7). All women in stimulation cycles were stimulated with the standard departmental protocol of ovarian stimulation (Ng et al., 2000) for IVF and embryo transfer. These women were pre-treated with a gonadotrophin-releasing hormone (GnRH) analogue, buserelin (Suprecur; Hoechst, Frankfurt, Germany) nasal spray 150 µg four times a day from the mid-luteal phase of the cycle preceding the treatment cycle. Baseline transvaginal ultrasound scanning was performed on the second day of the cycle to assess uterine and ovarian morphology and to confirm pituitary down-regulation. Assay of serum oestradiol was also performed on the second day of the cycle. Human menopausal gonadotrophin (HMG, 75 IU FSH and LH; Pergonal; Serono, Aubonne, Switzerland or Humegon; Organon, Oss, The Netherlands) injections were then started. The women underwent monitoring of ovarian response by assay of serum oestradiol concentrations and serial transvaginal ultrasonographic scans during the follicular phase. Human chorionic gonadotrophin (HCG, Pregnyl; Organon) 10 000 IU was given i.m. when the mean diameter of the leading follicle was >18 mm and there were at least three follicles with a mean diameter of
16 mm. The day of HCG administration (day 0) was used as the reference for determining the cycle day. All endometrial biopsies were taken 7 (±1) days after HCG administration corresponding to the anticipated `window of implantation'.
Women with infertility due to male factor were also recruited for endometrial biopsies in natural cycles while they were on the waiting list for IVF. These were healthy women with regular ovulatory menstrual cycles with no abnormality detected during pretreatment investigations. These women were asked to use condoms for contraception during the cycle of evaluation. These women had not received any steroid hormones or used a contraceptive device for at least 2 months prior to entering the study. All endometrial biopsies in the natural cycle were timed by reference to the LH surge. During the study cycle, blood was taken daily (starting 18 days before the next expected menstruation) for assay of serum oestradiol and LH until the LH surge. The day of the LH surge (designated as LH + 0) was the day when the serum LH was found to be more than double the mean of the preceding values. The endometrial biopsies were performed on day LH+7.
At the time of the biopsy, blood samples were also collected in order to measure the serum oestradiol concentration. Oestradiol was measured using a commercially available radioimmunoassay kit (Diagnostic Products Corporation, Los Angeles, CA, USA). The inter-assay and intra-assay variability was 4.2 and 4.0% respectively. All biopsies were obtained as an outpatient procedure from the fundal and upper part of the body of the uterus by Pipelle suction curette (CCD Laboratories, France). The endometrial biopsy samples were processed for light microscopy. Each biopsy specimen was fixed immediately in 2% glutaraldehyde in sodium phosphate buffer (pH 7.4) for 46 h. It was then washed in buffer, dehydrated, and embedded in JB4 (TAAB, Berks, UK), a plastic polymer. Serial semi-thin sections of 2 µm were obtained to ensure accurate morphometric measurements. Staining was performed using haematoxylin and eosin. Multiple fields were examined in an effort to minimize the effects of endometrial variation. Two blocks of each biopsy specimen were randomly selected. Seven successive slides from each block were chosen, and two non-overlapping fields of views from each section were studied. An image analysis system (Image-1 Metamorph Imaging System, Universal Imaging Corporation) was used for morphometric analysis. All morphometric evaluation was done by one investigator (G.B.) who was blinded to the clinical data at the time of examination.
The following endometrial parameters were measured: (i) volume fraction of the endometrium occupied by the glands measured as a percentage of the area of glandular tissue; (ii) maximal glandular diamete; (iii) height of the glandular cellular epithelium; (iv) number of subnuclear vacuoles per 100 gland cells; (v) amount of secretions in gland lumen; (vi) amount of stromal oedema; and (vii) number of venules in the stroma.
Two different methods of morphometric analysis were used for the measurements of the above seven morphological parameters: (i) parameters 1 to 4 and 7 were measured objectively with the aid of Metamorph Imaging system; (ii) semi-quantitative measurements (Johannisson et al., 1987) were employed for parameters 5 and 6. A four-point scoring system, from 0 to 3 (score 0 = absent; 1 = mild; 2 = moderate; 3 = marked) was used for parameters 5 and 6.
For semi-quantitative analysis objects of interest (parameters 5 and 6) appearing within a defined area were assessed. The frequency with which they occurred was recorded. The assumption made was that the object of interest would appear with the same frequency in all the randomly chosen fields of views.
Statistical analysis
This was carried out using statistical package for Social Science (SPSS for Windows package release 5.0; SPSS Inc, Chicago, IL, USA). P < 0.05 was considered significant. The non-parametric MannWhitney U-test was used in the evaluation of the skewed data. Data are expressed as mean ± SD or as median values and range.
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Results |
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Table I depicts the serum oestradiol concentrations in the three groups on the day of HCG administration and the day of biopsy. The serum oestradiol concentrations on the day of HCG administration were more than three-fold higher in the high responders than in moderate responders (P < 0.05). Comparison between the women in natural cycles and high responders showed an increase of more than 37-fold in the serum oestradiol concentrations.
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The mean cell height of glandular epithelium was greatest in moderate responders (20.1 ± 2.0 µm). The cells in moderate responders were significantly higher (P < 0.05) than those in cycles of high responders (17.5 ± 2.2 µm). The number of subnuclear vacuoles were significantly higher in the group of high responders than in the natural cycle group (P < 0.02). Although there was retention of subnuclear vacuoles in moderate responders there were no statistically significant differences found when compared with the other two groups.
In the natural cycles, 64% of glands had abundant luminal secretions while 36% of glands were moderately filled. The majority of glands (53%) in the group of high responders were empty, or were mild to moderately (38%) filled with secretions, only 9% showed abundant secretions. Biopsy specimens obtained from moderate responders, showed moderate to abundant filling in 56 and 33% respectively with only a few (10%) empty glands. Table II shows the mean score of glandular secretions. The difference was statistically significant (P < 0.001) between the natural cycles and cycles of high responders. Similarly, moderate responders showed a significantly larger amount of secretion than the high responders (P = 0.005). The stroma featured small round or spindle cells. In specimens demonstrating stromal oedema or break down, the stroma was less cellular and presented larger intercellular spaces per unit area of endometrium, while in dense stroma the stromal cells appeared closely packed. Morphometric assessment (Table II
) of the different biopsy groups revealed consistently advanced stromal endometrial histological features in high responders when compared with those of natural cycles and cycles of moderate responders. Eight of the 12 biopsy specimens of high responders showed marked stromal oedema (Figure 1C
), whereas the remaining four showed moderate oedema. Dense stroma was observed in seven of 12 natural cycle biopsies. In the group of moderate responders, the majority of samples showed moderate to marked oedema with only one biopsy specimen demonstrating stromal density.
In addition, in the stroma, the vascular differentiation was advanced in high responders. The number of blood vessels/mm2 were markedly higher in the group of high responders (Figure 1D) when compared with the natural cycles and cycles of moderate responders.
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Discussion |
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The present data on the natural cycles are consistent with previous studies (Li et al., 1988; Bonhoff et al., 1993
; Rogers et al., 1996
) which reported a greater endometrial glandular volume in natural cycles compared with those in endometrium after different ovarian stimulation regimens in IVF cycles. The glands in natural cycles were either more tortuous or numerous and thereby they occupied a greater area of the endometrium at the presumed time of implantation. There appears a trend towards a decline in the glandular volume fraction and glandular diameter with the rise in the serum oestradiol concentrations. The increase in the glandular diameter, and the relative decrease in cell height and subnuclear vacuolations in the natural cycles is explained by the presence of abundant secretions in the glandular lumen. Varying degree of luminal filling in moderate responders represented glands at different stages of secretory development. In high responders with impairment of secretory activity there is prolonged retention and retarded emptying of the secretory materials. This may reflect a defect in the transport of secretions from the intra-cellular to the luminal compartment and therefore the persistence of subnuclear vacuoles. As a result of the decreased secretions, the glandular lumen is less distended and occupies a smaller volume fraction of the endometrium.
A significant decrease in the gland cell height of high responders could be due to a reduction in the cytoplasm and/or changes in the volume of the nucleus. However, it was not possible from this study to deduce the precise mechanism of the various morphological responses to ovarian stimulation, but more specific methods of measurement on absolute nuclear volume and glandular epithelial volume will be required to answer these questions.
Insufficient secretory transformation of the endometrium in infertile women, and impaired development of the glands after hormonal stimulation were found (Bonhoff et al., 1990). Another study (Rogers et al., 1996
) found reduced uterine receptivity in patients with small gland volume. The results presented here demonstrated that ovarian stimulation resulting in excessive response causes significantly altered endometrial morphological features. It is possible that the reduced gland volume and lack of luminal secretions observed in high responders are indicators of suboptimal endometrial environment. The secretory transformation of the endometrium, which is vital for embryo implantation, is therefore incomplete in high responders.
Unstimulated and ovarian stimulation cycles were compared (Macrow et al., 1994); they concluded that high oestradiol concentrations (8426 pmol/l) were not associated with abnormal endometrial glandular development. The present study supports these findings in the natural cycles and in the moderate responders with comparable serum oestradiol concentrations. These findings were extended in the current study to excessive responders with oestradiol concentrations of
20 000 pmol/l and significant differences in the glandular and stromal components were found. Marked stromal oedema, and significantly greater number of stromal vessels suggested advanced stromal maturation. In these cases, a direct effect on the endometrium of high oestradiol accounts for the intense biological response. In another study (Noci et al., 1997
) an asynchronous development of endometrial glands and stroma in women undergoing IVF was found. The present study supports these findings in the subjects who responded excessively after stimulation but not in moderate responders.
In the present study, stromal oedema in moderate responders corresponded to days 78 post-ovulation (Johannisson et al., 1987; Li et al., 1988
). This indicates an in-phase stromal component and is comparable to the chronological dating at this time in the normal endometrium. In natural cycles, the lower amounts of endometrial stromal oedema, compared with those of moderate and high responders, suggests that stromal density may be related to the circulating concentrations of oestradiol. In the stroma, the presence of abundant vessels in high responders indicates premature arterial maturation under the influence of high steroid concentrations or factors initiating new vessel formation. The glandular and stromal compartments of high responders did not react simultaneously to the oestradiol stimulation but rather in a discordant manner. This resulted in appearances that are unlike any of the pattern seen in the natural cycles or stimulation cycles of moderate responders.
The variation in endometrial response to different oestradiol concentrations may explain the disagreements reported in the literature, as the serum oestradiol concentrations may be different in different studies. One group (Johannisson et al., 1987) suggested that the endometrium develops synchronously with the ovarian activity and endometrial changes occurred with a high degree of regularity and precision. Another group (Thornburgh and Anderson, 1997
) reported a lack of oestradiol receptors in cells that were inadequately primed by oestradiol, while other investigators (Balasch et al., 1992
; Hadi et al., 1994
; Klentzeris, 1997
; Noci et al., 1997
) also found a decrease in endometrial oestradiol and progesterone receptors in ovarian stimulation cycles that rendered the endometrium functionally hypo-oestrogenic or hypoprogestogenic. Whether the reduction in receptors is the result of an intrinsic cellular abnormality or exogenous hormonal influence causing reduced receptor synthesis is not known. In this study, we did not examine the oestrogen and progesterone receptors in the endometrium. It would be of interest to quantitatively examine the concentration and bioactivity of these receptors.
It is concluded that excessive ovarian response leads to insufficient secretory transformation of the endometrium as well as discordant glandular and stromal development at a time that coincides with the period of maximum uterine receptivity. This may explain the findings of decreased implantation and pregnancy rates in IVF when serum oestradiol concentrations were high (Pellicer et al., 1996; Simón et al., 1998
; Ng et al., 2000
).
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Acknowledgments |
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Notes |
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References |
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Submitted on June 5, 2000; accepted on December 11, 2000.