Morphometric analysis of peri-implantation endometrium in patients having excessively high oestradiol concentrations after ovarian stimulation

Ghazala Sikandar Basir1, Wai-sum O2, Ernest Hung Yu Ng1 and Pak Chung Ho1,3

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study investigated whether high oestradiol concentrations after ovarian stimulation in infertile women affect endometrial development around the time of implantation. The glandular and stromal components of the endometrium were assessed by morphometric criteria. Endometrial biopsies were taken on day 7 (±1) after the ovulating dose of human chorionic gonadotrophin in stimulation cycles and on day 7 after the LH surge in natural cycles. Women (n = 38) undergoing assisted reproduction treatment were evaluated: 12 women in natural cycles, 11 women in stimulation cycles with oestradiol <20 000 pmol/l and failed fertilization after oocyte collection (moderate responders) and 15 women with an oestradiol concentration of >=20 000 pmol/l in stimulation cycles (high responders). High responders showed delayed glandular maturation and advanced stromal morphology, whereas moderate responders demonstrated synchronous development of glandular and stromal features. In natural cycles, the glands were in phase. The effect of excessively high oestradiol concentrations could be explained by quantitative evaluation of the endometrial biopsies as gland–stromal dyssynchrony, which indicates a deficient secretory transformation of the endometrium that represents a suboptimal endometrial environment for implantation. This substantiates our previous clinical observation of significantly lower pregnancy rates in IVF cycles of women with high oestradiol concentrations (>=20 000 pmol/l).

Key words: endometrial morphometry/high oestradiol/OHSS/secretory phase endometrium


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In most assisted reproduction programmes, gonadotrophins are used alone or in combination to enhance the growth and maturation of multiple oocytes. This is essential because of the need to recruit a greater number of follicles, thereby improving the chance of fertilization and allowing an increased number of embryos for transfer to give acceptable success rates. The transfer of multiple embryos is known to result in higher conception rates (Tan et al., 1994Go). However, in some patients there may be an increased risk of an exaggerated ovarian response, which is associated with ovarian hyperstimulation syndrome (OHSS) and its complications (MacDougall et al., 1993Go). Investigators (Paulson et al., 1990Go; Hadi et al., 1994Go; Simón et al., 1995Go) have also recognized the potential adverse effects of supraphysiological hormonal concentrations on endometrial development and differentiation. A recent study (Ng et al., 2000Go), showed a significant reduction in the implantation and pregnancy rates in women with a serum oestradiol concentration of >20 000 pmol/l. In the literature, there is controversy regarding the effect of stimulation on endometrial development. Studies have shown a high incidence of endometrial glandular advancement (Garcia et al., 1984Go; Forman et al., 1988Go) and retardation (Graf et al., 1988Go; Sterzik et al., 1988Go) using morphological and immunohistochemical criteria (Seif et al., 1992Go). In another study of natural versus ovarian stimulation cycles (Kolb et al., 1997Go), the investigators showed an advanced development of the ultrastructure of endometrial surface epithelium in stimulation cycles. It has been demonstrated (Macrow et al., 1994Go) that ovulation induction was not associated with abnormal endometrial development. In contrast, other investigators (Benadiva and Metzger, 1994Go) reported a high incidence of dyssynchronous endometrial growth under supraphysiological concentrations of oestradiol and progesterone. Nevertheless, the criteria by which supraphysiological hormonal concentrations are defined remain variable as do the timing of the biopsies and the drug regimens used for ovarian stimulation. There are no studies in literature on the effects of high oestradiol (>=20 000 pmol/l) concentrations on endometrial development.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 38 infertile women undergoing treatment for IVF were recruited from the assisted reproduction unit of the Department of Obstetrics and Gynaecology of Queen Mary Hospital, Hong Kong. Approval for the study was obtained from the Ethics Committee of the Faculty of Medicine, The University of Hong Kong. A written informed consent was obtained from the patients prior to the commencement of the study. The indications for IVF and embryo transfer included tubal (n = 12), male (n = 24) and unexplained (n = 2) factors of infertility. These women had regular menstrual cycles and there was no significant intra-uterine or ovarian abnormality detected in these women during pretreatment. All women had completed their investigations for the diagnosis of the underlying infertility disorder prior to referral to the assisted reproduction clinic.

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 27–39 years (mean ± SD, 33 ± 2.7). All women in stimulation cycles were stimulated with the standard departmental protocol of ovarian stimulation (Ng et al., 2000Go) 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 4–6 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., 1987Go) 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 Mann–Whitney U-test was used in the evaluation of the skewed data. Data are expressed as mean ± SD or as median values and range.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Women (n = 38) recruited to the study were categorized in the following three groups. Endometrial specimens were obtained from 12 women in natural cycles and 26 women receiving ovarian stimulation for IVF. Of these women, 11 had failure of fertilization after IVF. The serum oestradiol concentrations in all these women were <20 000 pmol/l. None of these women had signs or symptoms suggestive of OHSS. These women were classified as moderate responders. The other 15 women had oestradiol concentrations >=20 000 pmol/l. They were classified as high responders. The embryo transfer of all these women had to be deferred. These women, after ovarian stimulation either had oestradiol concentrations >30 000 pmol/l, or they developed severe signs and symptoms of OHSS requiring hospitalization. All high responders were also found to have developed >=15 follicles. Four women had to be excluded from the study. No biopsy specimens could be obtained from two women showing a high response, because of tight internal cervical os. In the other two women the endometrial biopsy specimens were inadequate for evaluation (one from moderate responder and the other from the high response group) because of insufficient endometrial tissue. After excluding these four women, morphometric analysis was performed on 34 biopsy samples.

Table IGo 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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Table I. Comparison of the serum hormone concentration and the number of follicles on the days of ovulation and day of endometrial biopsy in different biopsy groups. All values are given as mean ± SD
 
Endometrial specimens from natural cycles (Figure 1AGo) showed largest glands and glandular luminal diameter, smaller cell height and fewer subnuclear vacuoles. Glandular secretions were abundant in natural cycles. In biopsy samples obtained from moderate responders (Figure 1BGo) morphometric analysis showed smaller gland volume, smaller luminal diameter and a greater number of subnuclear vacuoles. The glandular lumen showed variable degree of luminal secretions. The biopsies of high responders had smallest glandular volume fraction and glandular luminal diameter. There was retention of a large number of subnuclear vacuoles and the majority of glands were empty of secretions in this group.



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Figure 1. Morphology of the endometrium showing variable stages of glandular and stromal development in the natural cycles and ovarian stimulation cycles of moderate responders and high responders. (A) Natural cycle endometrial biopsy showing in-phase glandular development and lowest amounts of stromal oedema (*). (B) Moderate responder: in-phase endometrium showing co-ordinated development of glands and stroma (*) after ovarian stimulation. (C and D) High responders demonstrating gland stromal dyssynchrony: delayed glandular development and oedematous stromal features (*). The arrows show spiral arterial maturation appropriate to the late secretory phase.Bar = 100 µm.

 
Table IIGo summarizes the morphometric data of endometrial glandular and stromal profiles. The mean volume fraction (36.5 ± 11.1) of the endometrium occupied by the glands was greatest in the natural cycles and was significantly larger (P < 0.05) than those in cycles of moderate (22.0 ± 9.5) and high responders (20.8 ± 7.8). Biopsy specimens from high responders demonstrated the smallest volume fraction of endometrium occupied by the glands.


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Table II. Morphometric assessment of the endometrial glandular and stromal profiles. Values are given as either mean ± SD or as median with range in parentheses
 
The mean diameter of glands in natural cycles (112.0 ± 15.2 µm) was larger than that in moderate (111.5 ± 19.0 µm) and high responders (94.5 ± 17.3 µm). The differences were statistically significant between groups of natural and high responders and between moderate responders and high responders (P < 0.05). This demonstrated a trend of progressive decrease in the size of glandular volume and luminal diameter, with the increase in oestradiol concentrations from lower values in natural cycles to those of moderate oestradiol in moderate responders and excessive serum concentrations in high responders.

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 IIGo 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 IIGo) 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 1CGo), 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 1DGo) when compared with the natural cycles and cycles of moderate responders.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The most important physiological function of the endometrium is the implantation of a fertilized ovum (Grosser, 1910Go). During the menstrual cycle, the endometrium undergoes a series of well-defined cyclic morphological changes. These changes have been characterized with precision by morphometric methods throughout the normal cycle (Li et al., 1988Go). Furthermore, these histological changes reflect sequential functional states of the endometrium (Habiba et al., 1998Go). In the clinical context of gonadotrophin stimulation in IVF, studies have shown that stimulation impairs endometrial receptivity (Forman et al., 1988Go; Simón et al., 1995Go; Ng et al., 2000Go). In the literature, there is paucity of data that shows correlations between this reduced receptivity and changes in endometrial morphology associated with excessively high oestradiol concentrations. In the present study, the effect of variable ovarian response to hormonal stimulation was evaluated and compared with endometrial structural changes between the study groups.

The present data on the natural cycles are consistent with previous studies (Li et al., 1988Go; Bonhoff et al., 1993Go; Rogers et al., 1996Go) 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., 1990Go). Another study (Rogers et al., 1996Go) 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., 1994Go); 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., 1997Go) 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 7–8 post-ovulation (Johannisson et al., 1987Go; Li et al., 1988Go). 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., 1987Go) 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, 1997Go) reported a lack of oestradiol receptors in cells that were inadequately primed by oestradiol, while other investigators (Balasch et al., 1992Go; Hadi et al., 1994Go; Klentzeris, 1997Go; Noci et al., 1997Go) 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., 1996Go; Simón et al., 1998Go; Ng et al., 2000Go).


    Acknowledgments
 
The authors thank Ms May Cheung, Dr W.S.B.Yeung and all members of the reproduction team for their support.


    Notes
 
3 To whom correspondence should be addressed. E-mail: pcho{at}hksub.hku.hk Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 5, 2000; accepted on December 11, 2000.