The effect of gonadotrophic stimulation on integrin expression in the endometrium

K. Thomas1,2,4, A.J. Thomson1, V. Sephton1, C. Cowan1, S. Wood1, G. Vince3, C.R. Kingsland1 and D.I. Lewis-Jones1,2

1 Reproductive Medicine Unit, Liverpool Women's Hospital, Crown Street, Liverpool, L8 7SS, 2 Department of Obstetrics and Gynaecology and 3 Department of Immunology, University of Liverpool, Liverpool, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Despite many recent advances in IVF treatment implantation rates per embryo transfer rarely exceed 30%. Three integrins ({alpha}1ß1,{alpha}4ß1 and {alpha}3) have been shown to be expressed in the endometrium in a cyclically dependent manner and are thought therefore to play a vital role in the process of implantation. METHODS: The effect of gonadotrophin stimulation on the expression of these three integrins within the endometrium was investigated by examining biopsies from oocyte donation patients and comparing them with fertile controls. RESULTS: A delay in the maturation of the glandular epithelium was found in the oocyte donation patients. There was also a reduction in the expression of all three integrins in the glandular epithelium and also a reduced expression of the {alpha}3 integrin in the luminal epithelium. CONCLUSIONS: As these integrins have been shown to be important in implantation their reduced expression after IVF treatment may have an adverse effect on pregnancy rates.

Key words: endometrium/implantation/integrins/IVF


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
With advances in the treatment of male factor infertility such as ICSI (Palmers et al., 1992Go), and subsequently surgical sperm retrieval (Craft et al., 1993Go) most couples can now reach the point in an IVF cycle where embryos, usually using their own gametes, are transferred. However pregnancy rates per embryo transfer still only average around 25–30% (HFEA, 2001Go) meaning that, in the majority of cases, implantation does not occur. The process of implantation is a complex one initially requiring the interaction of the blastocyst and, subsequently, the developing embryo and placenta with the endometrium. Implantation after embryo transfer is therefore the major stumbling block in IVF treatment making it an area of intense research. From an embryological aspect new developments such as assisted hatching (Cohen et al., 1992Go) and blastocyst culture (Gardner et al., 1998Go) may improve the success rates slightly in certain groups. However a receptive endometrium is essential for implantation to take place. Any abnormality in its structure and receptivity means that the treatment cycle is more likely to fail (Paulson et al., 1990Go).

The endometrium is controlled ultimately by the combined actions of estrogen and progesterone. The abnormal levels of these hormones during IVF treatment due to ovulation induction can affect the endometrial morphology (Toner et al., 2001Go) and this may therefore impair the receptivity of the endometrium. As well as the histology of the endometrium being affected, certain integrins expressed by the endometrium are thought to be hormonally influenced (Aplin, 1997Go). Previous work has shown the expression of integrins in the endometrium changes during the menstrual cycle (Lessey et al., 1992Go; Tabibzadeh, 1992Go; Lessey, 1998Go). Three integrins in particular ({alpha}1ß1,{alpha}4ß1 and {alpha}3) are thought to play a vital role in implantation as all are expressed during the `implantation window', the period in the menstrual cycle when the endometrium is at its most receptive. The ß3 and {alpha}1 integrins have also been shown to be reduced in infertile patients using flow cytometry (Gonzalez et al., 1999Go). Aberrant patterns of integrin expression have also been associated with certain diagnoses in infertile patients, including luteal phase defects (Lessey et al., 1994aGo), endometriosis (Lessey et al., 1994bGo), hydrosalpinx (Meyer et al., 1997Go) and unexplained infertility (Lessey et al., 1995Go; Klentzeris, 1997Go).

The exact role of integrins remains controversial and results have not been duplicated in all studies. Creus and co-workers showed no difference in integrin expression in patients who became spontaneously pregnant compared with those that do not (Creus et al., 1998Go). There is also a debate about the effect of endometriosis on integrin expression with some authors showing it has little effect (Lessey and Young, 1997Go; Hii and Rogers, 1998Go).

The purpose of the present study was therefore to look at the effect of ovulation induction on glandular development in the endometrium and the effect that this had on endometrial integrin expression. It would obviously be unethical to take an endometrial biopsy from a patient who has had IVF treatment with an embryo transfer. Oocyte donation patients are an ideal substitute as they have undergone the process of ovulation induction without the replacement of embryos. The endometrium from these patients was analysed and compared with biopsies taken from a control group of fertile women attending for sterilization. One advantage of using these two groups is that both have shown proven fertility with all patients becoming pregnant naturally without the use of any infertility treatment. Minimal abnormalities in the structure of the endometrium and therefore its receptivity would be expected in this set of patients. The only difference between these two groups of patients was that the oocyte donors had undergone ovulation induction and it would thus be possible to compare this with the unstimulated endometrium.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Liverpool Research Ethics Committee granted ethical approval for the project. Women attending the Reproductive Medicine Unit (RMU) at the Liverpool Women's Hospital for oocyte donation and women attending hospital for sterilization were asked to participate in the study. An information sheet was given to all women and informed consent was obtained from those agreeing to take part. Women were only considered if they had had at least one normal full term pregnancy. Women in the control group were not using either an intrauterine contraceptive device or hormonal methods of contraception.

A total of 15 women were recruited to each group and an endometrial biopsy was taken using an endometrial sampler (Wallace Ltd, Hythe, Kent, UK). This was taken 7 days after detection of the midcycle urinary LH surge (Clearplan; Unipath, Bedford, UK) in the control group, and 7 days after the administration of exogenous HCG, 5000 IU (Profasi; Serono, Middlesex, UK) in the oocyte donation patients. The biopsies were immediately frozen in liquid nitrogen and stored at –70°C until use.

Those women undergoing oocyte donation underwent ovarian stimulation following the standard protocol in the unit. Pituitary down-regulation was achieved using a gonadotrophin releasing hormone (GnRH) agonist spray, 200 µg twice daily (Synarel; Searle, High Wycombe, Bucks, UK) from the mid-luteal phase. Down-regulation was ascertained with transvaginal ultrasound (endometrial thickness <5 mm and no ovarian follicles >6 mm). Stimulation was then commenced using a recombinant FSH (Gonal F; Serono, Middlesex, UK), the initial dose being dependent on the baseline FSH. The ovarian response was monitored by ultrasound scan on day 5, and the dose of FSH adjusted if necessary. On day 10 of the treatment a further scan was performed and the estradiol level was checked. When at least three follicles had reached a diameter >16 mm, HCG (Profasi; Serono) 5000 IU was given and oocyte retrieval performed 36 h later by vaginal ultrasound-guided follicle aspiration.

Antibodies
Details of the primary antibodies used in the study are given in Table IGo.


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Table I. Antibodies used in this study
 
Immunohistocytochemistry
Cryostat sections 5 µm in thickness were cut and mounted on glass slides. After drying overnight at room temperature, slides were wrapped in aluminium foil and frozen at –20°C until immunostaining. Sections were then removed and allowed to reach room temperature. They were then fixed in acetone for 10 min and, after washing in Tris-buffered saline (TBS 0.05 mol/l, pH 7.6), were incubated with the appropriate diluted monoclonal antibody for 30 min in a humidified chamber. Mouse immunoglobulin (Ig) G was used in place of the first antibody as a negative control. After 2x5 min washes in TBS, bound antibodies were detected by the anti-alkaline phosphatase (APAAP) method using rabbit anti-(mouse IgG) IgG (diluted 1:25; Dako Ltd, High Wycombe, UK) for 30 min, washed in TBS and then incubated with pre-formed complex of calf intestinal alkaline phosphate and mouse monoclonal APAAP (diluted 1:50, Dako Ltd.) for a further 30 min. Staining was developed with Napthol AS-MX phosphate and Fast Red (Sigma, Poole, Dorset, UK) with the inclusion of 1 ml of levamisole to block any endogenous alkaline phosphatase. Slides were counterstained with haemalum and mounted in Aquamount (BDH, Poole, Dorset, UK).

The slides were evaluated using a Nikon microscope. Firstly the endometrium was dated according to the Noyes criteria (Noyes et al., 1950Go) and the staining was then analysed. The intensity of staining of the antibodies was analysed in both the glandular and luminal epithelium, and was assessed by using the HSCORE. This has previously been described by Lessey et al., who showed it has a low intra- and inter-observer error (Lessey et al., 2000Go). The HSCORE was calculated using the following equation: HSCORE = {Sigma} Pi(i + 1), where i = 1, 2 or 3 and is the intensity of the stained epithelium and Pi is the percentage of stained epithelial cells for each intensity varying from 0–100%. An HSCORE of <=0.7 was not clearly distinguishable from no staining and was used as the cut-off point for a negative test. Colour prints were made using Kodak 200 ASA film. Statistical analysis was performed using the Mann-Whitney U-test for continuous variables and the {chi}2 or Fisher's exact test (depending on sample size) for categorical variables using a commercial statistical package (Statistics Package for Social Sciences).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Fifteen patients were recruited to each group. Women were recruited only if they had a regular menstrual cycle, were known to be fertile and were <35 years of age. The mean age in the control group was 32.5 years (range 29–35) compared with 30.9 (range 27–33) in the oocyte donor group.

Endometrial receptivity
All biopsies were taken during the period when the uterus should be at its most receptive, i.e. 7–8 days after LH surge or HCG administration. As this was a group of fertile women the endometrium would be expected to be `in phase' in the control patients. The dating of the glandular epithelium is summarized in Table IIGo. The biopsies were taken on day 7 or 8 and as the LH surge (or HCG administration) is 36 h prior to this then the biopsies should, if mature, be dated between days 5–7 postovulation. In the control group 11 of the 15 biopsies (93%) were dated between days 6–7 as opposed to six of 15 (40%) in the oocyte donors group. Overall there was a slight delay in the glandular maturation in the oocyte donors as seven biopsies (46%) were dated day 2–5 whilst only three of the controls (20%) fell within this range.


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Table II. Dating of glandular epithelium in the samples obtained 7 days after urinary LH surge (control) or administration of HCG after ovulation induction (oocyte donors)
 
Integrin expression
The integrin expression is shown in Figure 1Go. These pictures show the contrast seen between staining in the luminal and glandular epithelium for the {alpha}4 and {alpha}1 subunits. Measurements using the HSCORE for the three integrins in the luminal and glandular epithelium are shown in Figures 2, 3 and 4. GoGoGoFirstly, looking at the expression in the glandular epithelium, there was a statistically significant decrease in the expression of the {alpha}1ß1 (P < 0.005), the {alpha}4ß1 (P < 0.005) and the {alpha}3 integrin (P < 0.003) in women who had undergone ovulation induction treatment. There was also a significant difference in luminal expression of the {alpha}3 integrin (P < 0.001) but not the two other integrins. Expression of the {alpha}4 and {alpha}1 in the luminal epithelium have been shown to be minimal at all times during the menstrual cycle and these results would therefore be expected. (Lessey et al., 1996Go). However Klentzeris has demonstrated luminal staining of {alpha}4 showing that work remains controversial. (Klentzeris et al., 1993Go).



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Figure 1. Immunostaining for various antigens in the endometrium. The luminal epithelium is highlighted with arrowheads. (A) Staining for {alpha}3 integrin showing staining of luminal epithelium in a biopsy from a control patient. (B) Stainzing of {alpha}3 in a control patient. (C) Staining of glandular epithelium of {alpha}3 integrin in donor patient showing almost complete lack of expression. (D) {alpha}4 integrin subunit showing lack of uptake in luminal epithelium as compared to glandular epithelium. (E) Glandular staining of the {alpha}1 integrin subunit in a control patient. (F) Immunoglobulin G control. Magnifications: A, B, C and E x200; D and F x100.

 


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Figure 2. Expression of the {alpha}3 integrin in the glandular and luminal epithelium. The difference is statistically significant between the two groups for both the glandular (P < 0.003) and luminal epithelium (P < 0.001) ({varphi}= statistically significant).

 


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Figure 3. Expression of the {alpha}1ß1 integrin in the glandular and luminal epithelium. There is a statistically significant reduction in its expression in the glandular (P < 0.005) but not the luminal epithelium ({varphi}= statistically significant).

 


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Figure 4. Expression of the {alpha}4ß1 integrin in the glandular and luminal epithelium. There is a statistically significant reduction in the expression in the glandular (P < 0.005) but not the luminal epithelium ({varphi}= statistically significant).

 
The integrin expression in the oocyte donors was suppressed irrespective of the glandular maturation. There was no significant difference in the expression of the integrins in those biopsies dated day 2–5 and those dated day 6–9 post ovulation.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The endometrium undergoes a series of precisely regulated morphological changes after ovulation that is believed to allow a suitable environment for implantation of the embryo (Dockery et al., 1988Go). Its primary function is to provide a substrate for the implantation to occur and then maintain the development of the blastocyst. For this to happen during a natural cycle, the glandular epithelium matures under the influence of estrogen and progesterone. The embryo arrives in the endometrial cavity on day 18 or 19, but implantation does not occur until days 20–21 at the height of glandular secretory activity (Rosenwaks, 1987Go). A number of studies have however shown that the endometrium is altered during ovulation induction which may be detrimental to its receptivity, although the true clinical relevance of these observations remains unclear (Ben-Nun et al., 1992Go; Benadiva and Metzger, 1994Go; Macrow et al., 1994Go).

Therefore understanding how the endometrium is different during IVF treatment, compared with natural conception, is vital if improvements to implantation rates are to be made. Since its publication half a century ago the only test of endometrial function has been histological data according to the Noyes criteria (Noyes et al., 1950Go). This has led to a search for other potential markers of endometrial receptivity. Morphologically the formation of pinopodes may be a specific marker for uterine receptivity. The number of pinopodes differs between patients and there is a strong correlation between pinopode number and implantation after embryo transfer (Nikas and Psychoyos, 1997Go). The lifespan of pinopodes never exceeds 48 h and they have been shown to appear 1–2 days earlier after ovarian stimulation (Nikas et al., 1999Go). Molecular control involves the interaction of a variety of molecules including lectins, mucins, glycoproteins and other endometrial secretory products (Ilesanmi et al., 1993Go). The endometrial expression of the three integrins ({alpha}1ß1, {alpha}4ß1 and {alpha}3) has been shown to be cyclical across the menstrual cycle and in pregnancy. These three integrins are only present at the same time during the so-called `implantation window', and are therefore regarded by many as useful markers for endometrial receptivity (Tabibzadeh, 1992Go; Klentzeris et al., 1993Go; Lessey et al., 1994aGo; Aplin, 1996Go). Their exact role remains controversial with some papers casting some doubt on their relevance (Creus et al., 1998Go).

The stage of glandular development and the expression of these integrins in the endometrium was examined during the implantation phase of fertile controls and compared with post-ovulation induction in oocyte donation patients. First, it was shown that the development of the endometrial glandular epithelium was delayed in the oocyte donors compared with the controls. Also, irrespective of the stage of glandular development, the expression of the integrins was reduced. If they are important in implantation, and are therefore useful markers of the receptivity of the endometrium, then a reduction in their level may be detrimental to IVF outcome. Meyer and co-workers showed a reduction in the expression of the {alpha}3 integrin in women who underwent ovulation induction but assigned this to a delay in the development of the glandular epithelium (Meyer et al., 1999Go). They found that in 80% (16/20) of biopsies the most advanced elements of the glandular epithelium did not exceed postovulatory day 6 which is the time that {alpha}3 expression is visible. In the four remaining only one showed expression of the {alpha}3 integrin. In the current study 53% (8/15) of the biopsies were dated between day 6–10 postovulation when all three integrins should be seen in the glandular epithelium. However it was found that despite being in phase the expression of the integrins was still reduced. Glandular histology was dated between day 6–10 postovulation in 12/15 (80%) of the control group. The HSCORE values in this group were similar to those quoted in previous studies looking at fertile women (Lessey et al., 2000Go).

The fact that integrin expression is reduced in women undergoing IVF points to a hormonal regulation of their levels. Implantation may be an event whereby the endometrium becomes receptive after a cascade of steroidally triggered events. In the review by Aplin two hypotheses are stated for the role of adhesion molecules (Aplin, 1996Go). One is that the control of attachment is by the steroidal induction of one or more of the adhesion molecules at the luminal epithelial cell surface. The other is that the loss of anti-adhesion molecules may facilitate attachment. In either case an alteration in estrogen levels may cause a failure in these processes. It is already known that the supraphysiological levels of estrogen seen in IVF can lead to the endometrium taking on bizarre appearances with quite marked dysynchrony between the glandular and stromal elements (Meyer et al., 1999Go). It is possible, therefore, that the endometrium may also be affected at a molecular level such that integrin expression is decreased. This would explain the findings presented here and would also raise the question of whether natural cycle IVF is in the long term more effective. The cumulative probability of pregnancy has been quoted as 46%, with an associated live birth rate of 32%, after four cycles (Nargund et al., 2001Go). Natural cycle IVF is safer and avoids the use of expensive drugs with a subsequent reduction in the intensity of monitoring required. It also has a less detrimental effect on the endometrium and receptivity could therefore be considered greater.

In summary, the results of the current study demonstrate that integrin expression seems to be reduced in the glandular epithelium in the endometrium after ovulation induction, irrespective of the dating. Previous work has shown the relationship between these integrins and infertility and one can only assume that if their expression is reduced then this can only be detrimental to the process of implantation. There may be an ideal estradiol level that should be reached during IVF treatment. To have low estrogen levels might reduce the yield of oocytes, but high levels might impair the receptivity of the endometrium reducing integrin expression and leading to reduced implantation rates. Due to ethical constraints patients in the current study could not take progesterone as luteal phase support that is routinely used in IVF treatment. The use of these pessaries does not seem to alter the endometrial histology (Meyer et al., 1999Go). All the ovum donors responded well and all had supraphysiological levels on the day of HCG administration and this is probably the main reason for the altered histology after IVF treatment. Ultimately a better understanding of how the endometrium does become receptive is needed in order to improve treatments for infertile women and also to enable the development of new directions in contraceptive research.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank Dr Martin Hemler for donating the {alpha}4 antibody.


    Notes
 
4 To whom correspondence should be addressed. E-mail: Kthomas{at}liverpool.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on July 6, 2001; accepted on September 28, 2001.