Mid-luteal serum inhibin-A concentration as a marker of endometrial differentiation

Montserrat Creus1, Jaume Ordi2, Francisco Fábregues1, Roser Casamitjana3, Juan A. Vanrell1 and Juan Balasch1,4

1 Institut Clinic of Obstetrics and Gynaecology, 2 Department of Pathology and 3 Hormonal Laboratory, Faculty of Medicine, University of Barcelona, Hospital Clínic – Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Recent studies have indicated that the corpus luteum is a major source of circulating inhibin-A and serum concentrations of inhibin-A may reflect the human luteal function. The present prospective study was undertaken to determine the usefulness of mid-luteal serum concentrations of inhibin-A as markers of endometrial receptivity (as assessed by histological dating and {alpha}vß3 integrin expression) and whether they are better predictors of endometrial function than serum progesterone. METHODS: Consecutive infertile women (experimental group, n = 50) with regular menstrual cycles, and fertile women who were requesting contraception and had regular menstrual patterns and normal secretory endometria (control group, n = 10) were included. In all women basal body temperature, luteal serum concentrations of oestradiol, progesterone, prolactin, and inhibin-A, and endometrial biopsies were used in the same cycle to assess luteal function. RESULTS: Out-of-phase mid-secretory endometria were detected in 17 of the 50 infertile women. Lack of {alpha}vß3 integrin expression was detected in 27 of the 50 mid-luteal endometrial biopsies. Thus, hormonal concentrations were compared in the mid-luteal phase between the following eight groups of women: group 1 (n = 10), control fertile women; group 2 (n = 50), infertile women (all); subdivided into group 3 (n = 33), with in-phase biopsies; group 4 (n = 17), with out-of-phase endometria; group 5 (n = 23), expressing {alpha}vß3 integrin in endometria; group 6 (n = 27), whose endometria did not express {alpha}vß3 integrin; group 7 (n = 18), with both in-phase endometrial biopsy and {alpha}vß3 integrin expression; and finally group 8 (n = 12), whose endometria were out-of-phase and did not express {alpha}vß3 integrin. Mid-luteal serum concentrations of oestradiol, progesterone, prolactin, and inhibin-A of the seven infertile groups were similar to those of the control group of fertile women. No statistically significant difference between the infertile groups was observed for any hormonal parameter considered. CONCLUSION: Mid-luteal serum inhibin-A determination does not accurately reflect endometrial function/maturation and it is not a better indicator of endometrial luteal phase dysfunction than mid-luteal serum progesterone.

Key words: endometrium/luteal function/serum inhibin-A/serum progesterone


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The determination of luteal function is a basic goal in infertility evaluation considering that progesterone secreted by the corpus luteum is necessary to obtain adequate secretory transformation of the endometrium and it is indispensable in early pregnancy maintenance. Direct evaluation of endometrial function by premenstrual endometrial biopsy and histological dating using the criteria established by Noyes et al. (Noyes et al., 1950Go) has been the method traditionally used to assess the luteal phase (Balasch and Vanrell, 1987Go; Wentz, 1988Go). However, with the advent of radioimmunoassay for plasma progesterone, the concept that endometrial histology is sufficient to diagnose luteal insufficiency was challenged and it was postulated that mid-luteal plasma progesterone determination might offer a better assessment of luteal function (Balasch and Vanrell, 1987Go; Wentz, 1988Go). Although in-vitro studies suggest that marker molecules of human endometrial differentiation can be regulated hormonally by the corpus luteum (Classen-Linke et al., 1998Go), in the clinical setting the ovarian and endometrial cycles usually coincide in the normal state, but tend to dissociate in the abnormal or induced cycle. The lack of relationship between mid-luteal plasma progesterone concentrations and progestational transformation of the endometrium as well as between out-of-phase premenstrual endometria and infertility (Balasch and Vanrell, 1987Go) have led to multiple attempts to improve luteal phase evaluation in infertility (Somkuti et al., 1995Go).

Thus, whereas endometrial biopsy has been traditionally performed late in the luteal phase in order to better detect the cumulative effects of progesterone secretion by the corpus luteum (Wentz, 1988Go), recent evidence suggests that earlier sampling during the mid-luteal phase (`window of implantation', days 20–24) is more sensitive for identifying delayed or otherwise altered patterns of endometrial maturation (Castelbaum et al., 1994Go; Somkuti et al., 1995Go). On the other hand, markers of normal endometrial development are being uncovered that, according to some authors, will allow us to go beyond merely histological criteria in the evaluation of the adequacy of the luteal phase. Thus, a number of comprehensive studies by Lessey et al. have indicated that an abnormal pattern of expression of the {alpha}vß3 integrin may be associated with states of impaired infertility (Lessey et al., 1992Go, 1994Go, 1995Go, 1996Go). These observations have led to the proposal for using the expression of {alpha}vß3 integrin in combination with histological dating in the determination of an endometrial cause of infertility (Lessey et al., 2000Go). Finally, recent work has indicated that the corpus luteum is a major source of circulating inhibin-A (Illingworth et al., 1996Go; Sehested et al., 2000Go; Treetampinich et al., 2000Go) and serum concentrations of inhibin-A may reflect the human luteal function (Yamoto et al., 1997Go; Danforth et al., 1998Go).

On the above evidence, the present study was undertaken to determine the usefulness of mid-luteal serum concentrations of inhibin-A as markers of endometrial receptivity (as assessed by histological dating and {alpha}vß3 integrin expression) and whether they are better predictors of endometrial function than serum progesterone, which, to our knowledge, has not been previously reported. {alpha}vß3 integrin was selected because, as previously reported by us (Creus et al., 1998Go), its expression is closely correlated with histological maturation of the endometrium irrespective of the latter being in- or out-of-phase. Since abnormalities in oestradiol and prolactin serum concentrations have been suggested as being potentially associated with defective endometrial maturation (Balasch and Vanrell, 1987Go), both hormones were also measured in the present study.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients and study cycle
We included an experimental group of 50 consecutive infertile women undergoing a routine work-up. All women gave informed consent to participate in the study, which was approved by the ethics committee of our hospital. The mean age of these women was 32.5 ± 1.5 years (mean ± SEM) (range 27–42). All of them had regular menstrual patterns every 26 to 32 days. The main causes of infertility in these women were male factor (n = 16), unexplained (n = 15), minimal to mild endometriosis (n = 14) and tubal factor with no hydrosalpinges (n = 5). As a control group we included 10 fertile women (mean age 34.0 ± 1.2 years, range 30–41; mean parity 1.2, range 1–3) who were requesting contraception.

As inhibin secretion decreases with advancing age (Welt et al., 1999Go) controls were matched by age (± 2 years) to infertile women. These control women had regular menstrual cycles (27–31 days), were taking no medication and had normal secretory endometria (mid-luteal in-phase biopsies with {alpha}vß3 integrin expression) in the study cycle. In all women, basal body temperature, luteal serum concentrations of oestradiol, progesterone, prolactin, and inhibin-A, and endometrial biopsies were used in the same cycle to assess luteal function according to a scheme of evaluation previously reported (Creus et al., 1998Go).

Starting on day 8–10 of the study cycle (depending on the cycle length of the woman) all women underwent daily transvaginal ultrasonographic evaluation of the follicular growth using a 5 MHz vaginal transducer attached to an Aloka scanner (Model SSD-620®; Aloka Co. Ltd, Tokyo, Japan). The maximum follicular diameter was measured in all women. Both ovaries were identified, and the largest diameter was measured in both the longitudinal and transverse dimensions in all follicles. The day of ovulation was designated as the day of maximum follicular enlargement, which was followed the next day by sudden disappearance or filling in of this follicle showing loss of clear demarcation of its walls and intrafollicular echoes (Shoupe et al., 1989Go; Peters et al., 1992Go).

Two endometrial biopsies were performed during a single menstrual cycle in each subject. The patient's chronological day was determined by counting forward from the ovulation day as detected by ultrasonographic scans. The early biopsy (mid-luteal) was performed on ovulation day +7.3 ± 0.2 (range, +6 to +8) whereas the late biopsy was performed on ovulation day +11.3 ± 0.3 (range, +10 to +12). The second biopsy (late luteal) was always performed 4 days after the first biopsy.

Hormones in serum were quantified on the same days as endometrial sampling. A single, well-timed determination of hormones in the mid-luteal phase was used because several hormonal measurements obtained between the 5th and 10th post-ovulatory days were found to be no better indicators of histological maturation of the endometrium (Balasch et al., 1996Go).

All samples were obtained in the fasted state between 0800 and 1000 h which corresponds to the period of minimal progesterone and prolactin variability, and adds to the accuracy of the measurement (Filicori et al., 1984Go; Zacur and Foster, 1993Go).

Endometrial samples
Endometrial samples were divided in two parts. One of them was fixed in 10% formalin and embedded in paraffin. The second portion of the tissue was snap frozen on methylbutane (Merck, Darmstadt, Germany) immersed in liquid nitrogen and stored at –70°C until immunolabelling.

Endometrial dating
For endometrial dating 4 µm sections stained with haematoxylin and eosin and periodic acid–Schiff (PAS) stain were evaluated. All endometrial biopsies were evaluated by the same expert gynaecological pathologist according to the histopathological criteria of Noyes et al. (Noyes et al., 1950Go). The pathologist was blinded with regard to ultrasonographically detected ovulatory day and clinical situation. Endometrial biopsy interpretation was performed using a single-day evaluation whenever possible and when the traditional 2 day spread evaluation method (i.e., day 20–21) was provided, the later day was used for comparison with immunohistochemical assays. An out-of-phase biopsy was defined as >=3 day lag between the chronological and the histological day.

Immunohistochemistry
{alpha}vß3 integrin was detected in frozen sections using the EnVision® system (Dako Co., Carpinteria, CA, USA). Briefly, 4 µm sections were fixed for 10 min in acetone at 4°C and dried. After washing in phosphate-buffered saline (PBS) for 5 min the peroxidase was blocked for 5 min in 0.03% H2O2 containing sodium azide. Then the slides were incubated with the primary antibody for 40 min and washed in Tris-buffered saline (TBS; Dako). The monoclonal antibody LM603 (Chemicon, Temecula, CA, USA, dilution 1:200), which recognizes the complete {alpha}vß3 heterodimer (Cheresh and Spiro, 1987Go) was used. The peroxidase labelled polymer was then applied for 40 min. After washing in TBS, the slides were incubated with the diaminobenzidine substrate chromogen solution, washed in distilled water, counterstained with haematoxylin, washed, dehydrated and mounted. In every case a negative control was performed by omission of incubation with the primary specific antibody. The reactivity in endometrial glands and surface epithelium, stromal cells and vessels was assessed. The intensity of staining of the endometrial components was evaluated by a semi-quantitative scoring system as follows: absent (–), weak or focal (+), moderate (++), and strong (+++). As in previous work it was found that the expression of {alpha}vß3 in the luminal epithelium starts abruptly on day 19–20 of the cycle, thus opening the window of implantation, and only staining in the glands seems to be clinically relevant (Lessey et al., 1992Go; Somkuti et al., 1995Go; Acosta et al., 2000Go), for the specific purpose of this study, endometrial samples were considered as expressing {alpha}vß3 integrin when this integrin was detected in both endometrial glands and surface epithelium with any intensity of the reaction ranging from weak/focal to strong.

Hormone assays
Hormones in serum were measured using commercially available kits. Oestradiol and prolactin were measured by a competitive immunoenzymatic assay (Immuno 1®; Bayer, Tarrytown, NY, USA). The sensitivity of the assay was 10 pg/ml for oestradiol and 0.1 ng/ml for prolactin, and the interassay coefficients of variation (CV) 5 and 4.4% respectively. Progesterone was determined by a competitive chemiluminiscent immunoassay (Immulite®; DPC, Los Angeles, CA, USA). The sensitivity of the method was 0.2 ng/ml and the interassay CV was 6.7%. Inhibin-A measurements were performed by a solid phase sandwich enzyme-linked immunosorbant assay (ELISA) carried out in microtitre plates (Serotec Ltd, Oxford, UK) (Groome and O'Brien, 1993Go; Groome et al., 1994Go). The assay sensitivity was 2 pg/ml and the within-plate CV was <7%. The cross-reactivity with activin A was <0.1%.

Statistics
Data were analysed by SPSS statistical software (Release 6.0; SPSS Inc., Chicago, IL, USA). The Mann–Whitney U test was used to compare the same parameters between groups. The Pearson correlation coefficient was used for correlative analyses. Results are expressed as means ± SEM. The concentration of significance was set at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In all instances, the endometrial specimens were noted to be clearly progestational fundal samples. Out-of-phase mid-secretory endometria were detected in 17 of the 50 infertile women. A late luteal endometrial biopsy could not be done in three of the 50 infertile women (one of them having out-of-phase endometrium in the mid-luteal phase) and one of the controls because menses had commenced at the time of the second endometrial sampling. The remaining 47 infertile women all had in-phase endometria in the late luteal phase irrespective of being in-phase or out-of-phase in the mid-luteal biopsy. The mean histological dating in the mid-luteal biopsies in the in- and out-of-phase infertile groups was 6.5 ± 0.6 and 3.9 ± 0.3 respectively. In controls (fertile women), it was 7 ± 0.3.

Weak or focal {alpha}vß3 integrin expression (Figure 1Go) was detected in only 23 of the 50 mid-luteal endometrial biopsies from infertile women but all 47 late luteal endometrial samples strongly expressed this integrin. Thus, hormonal concentrations were compared in the mid-luteal phase between the following eight groups of women: group 1 (n = 10): control fertile women; group 2 (n = 50): infertile women (all); group 3 (n = 33): infertile women having in-phase biopsies; group 4 (n = 17): infertile women with out-of-phase endometria; group 5 (n = 23): infertile women expressing {alpha}vß3 integrin in their endometria; group 6 (n = 27): infertile women whose endometria did not express {alpha}vß3 integrin; group 7 (n = 18): infertile women having both in-phase endometrial biopsy and {alpha}vß3 integrin expression; group 8 (n = 12): infertile women whose endometria were out-of-phase and did not express {alpha}vß3 integrin.



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Figure 1. Immunostaining for {alpha}vß3 integrin in a mid-luteal endometrial biopsy. Endometrial biopsy, post-ovulatory day 7. Note focal positive staining in endometrial glands (arrowheads) (monoclonal antibody anti-{alpha}vß3 integrin, EnVision®, counterstained with haematoxylin. Original magnification x100).

 
The mean age was similar for each of the eight groups of women (Table IGo). Hormonal concentrations of the study groups and controls are summarized in Table IGo. Serum concentrations of oestradiol, progesterone, prolactin, and inhibin-A of the seven infertile groups were similar to those of the control group. A positive correlation between mid-luteal serum progesterone and inhibin-A concentration was found both within the various groups studied (data not shown) and the whole study population (r = 0.42; P < 0.01). No statistically significant difference among the infertile groups was observed for any hormonal parameter considered. No differences between fertile and infertile women were observed when hormonal values obtained at the time of the late luteal biopsy were compared (data not shown). No differences in either hormonal parameters (inhibin-A and progesterone serum concentrations) or histological features (endometrial dating and {alpha}vß3 integrin expression) were detected among groups when stratified by the cause of infertility (data not shown). On the other hand, as all the mid-luteal endometrial biopsies showed weak/focal {alpha}vß3 integrin expression with no differences between the different groups studied, no correlative analyses with hormonal parameters could be carried out.


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Table I. Age and hormonal concentrations in infertility groups studied and controlsa. Values are given as mean ± SEM
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A great deal of new information has arisen in recent years concerning the physiology of inhibins and their clinical relevance in reproductive medicine. Inhibins are multifunctional molecules involved in the control of pituitary gonadotrophin secretion, ovarian follicular development, and luteal and placental function. Apart from their essential role in the selective control of the secretion of FSH during the menstrual cycle, inhibins are currently recognized as paracrine ovarian regulators and have multiple paracrine effects in the utero-placental unit. The presence and synthesis of inhibin subunits in human endometrium and decidua suggest the uterine mucosa as a further source of inhibin-related proteins and support a possible paracrine or autocrine action of these proteins as growth factors in modulating cell differentiation and/or proliferation (Petraglia et al., 1999Go; Teni et al., 1992Go). Inhibins, therefore, represent a promising marker for gynaecological and gestational diseases (Petraglia et al., 1999Go).

Inhibin is a glycoprotein hormone composed of an {alpha}-subunit disulphide-linked to one of two ß-subunits, the ßA-subunit to form inhibin-A or the ßB-subunit to form inhibin-B (Hayes et al., 1998Go). A variety of molecular forms are present in the circulation, including mature and partially processed {alpha}ß-dimers, as well as free {alpha}-subunit forms. The free {alpha}-subunits lack biological activity, and inhibin-A and inhibin-B are the active molecular forms in circulation (Hayes et al., 1998Go; Petraglia et al., 1999Go).

Early techniques for inhibin immunoassay had extensive cross-reaction with potentially inactive precursor forms and this has been a source of confusion for many years (Hayes et al., 1998Go). Recently, however, two-site directed assays have been developed capable of reliably and specifically measuring the inhibin dimers, inhibin-A and inhibin-B (Groome et al., 1994Go, 1996Go). Thus, it has been claimed that these new assays will become widespread in their use, enabling the identification of situations in which measurements of inhibin-A and -B are of clinical value (Hayes et al., 1998Go).

Inhibin-A and inhibin-B are gonadal hormones that are produced in a specific pattern in response to gonadotrophin stimulation in females. However, the source of inhibin-B is different from that of inhibin-A. Serum concentrations of inhibin-A peak in the mid-luteal phase and inhibin-A has recently been found to be the dominant form of inhibin secreted by cultured human granulosa cells (Vänttinen et al., 2000Go). The decrease in inhibin-A secretion after antagonist administration in the luteal phase confirms gonadotrophin-dependent secretion of dimeric inhibin-A by the corpus luteum (Welt et al., 1999Go). The luteal phase secretion of inhibin-A is integrated with the LH control of the corpus luteum (Hayes et al., 1998Go; Sehested et al., 2000Go). On the other hand, although daily luteal inhibin concentrations parallel those of progesterone, there is no clear association between individual inhibin and progesterone secretory patterns, which may be explained by the existence of a different mechanism for LH-dependent luteal inhibin production compared to progesterone (Nakajima et al., 1990Go; Sehested et al., 2000Go). This is to be stressed considering that endometrial biopsy-confirmed luteal phase defect is associated with decreased luteal LH-bio concentrations in the mid- and late luteal phase (Soules et al., 1989Go).

On the above evidence, it is not surprising that concentrations of inhibin-A, but not inhibin-B, have been reported as potential markers of luteal function (Yamoto et al., 1997Go; Danforth et al., 1998Go). Danforth et al. (1998) studied 25 women with regular menstrual cycles and reported that luteal phase inhibin-A is a marker of ovarian ageing in the perimenopausal transition, a finding that should be expected considering that corpus luteum dysfunction occurs more frequently with advancing age (Wentz, 1988Go). Yamoto et al. (1997) found that serum concentrations of inhibin-A in seven infertile women with luteal dysfunction, as defined by short luteal phase and a single mid-luteal progesterone concentration <10 ng/ml, were significantly lower than those found in seven normal women. Those previous reports, however, included a low number of women and did not investigate endometrial function.

One study investigating the pathogenesis of luteal phase deficiency, as determined by an out-of-phase endometrial biopsy in the studied cycle and where daily serum concentrations of reproductive hormones were determined in a group of 10 luteal phase deficiency women, showed a significant decrease compared with normal values in the luteal phase integrated concentrations of inhibin and progesterone in the luteal defect group (Soules et al., 1989Go). However, the cost and inconvenience of obtaining daily serum samples makes this approach clinically impractical. The recommended test for the determination of luteal phase defect in the clinical setting is a single blood sample for plasma progesterone (Jordan et al., 1994Go). However, in previous studies (Balasch and Vanrell, 1987Go; Balasch et al., 1996Go) we found that neither mid-luteal progesterone nor immunoreactive {alpha}-inhibin accurately reflect histological maturation of the endometrium according to the histopathological criteria of Noyes et al. (Noyes et al., 1950Go). From that previous work it was concluded that further studies using a new assay specific for dimeric inhibin-A as well as other alternatives to evaluating endometrial function were desirable (Balasch et al., 1996Go).

The present study where concentrations of inhibin-A were measured, and endometrial function was assessed by both histological dating and {alpha}vß3 integrin expression, shows that mid-luteal serum inhibin-A determination does not accurately reflect endometrial function/maturation and it is not a better indicator of endometrial luteal phase dysfunction than mid-luteal serum progesterone. Thus, a feature of this report is that it provides evidence indicating that, unexpectedly, the predictive ability of inhibin-A is not better than that afforded by immunoreactive {alpha}-inhibin measurement in previous studies (Balasch et al., 1996Go). In our previous report an immunoenzymatic method was used which utilized antibodies directed at the {alpha}-subunit of inhibin, thus measuring inhibin-A, inhibin-B and the non-biologically active {alpha}-subunit (Balasch et al., 1996Go). Recent studies (Burger et al., 1998Go; Hayes et al., 1998Go), however, have shown that only inhibin-A is measured in significant quantity in the luteal phase of the menstrual cycle, and thus they were probably the only inhibin species measured at this time in the menstrual cycle in those earlier studies. In fact, when immunoreactive inhibin concentrations across the menstrual cycle have been compared with those obtained using the two-site directed assays, the overall pattern of inhibin secretion was similar except that the magnitude of the changes observed was greater with the new assays (Hayes et al., 1998Go). On the other hand, although immunohistochemical analysis of the pattern of endometrial integrin expression provides information beyond that derived from histological dating alone and may reflect endometrial function or receptivity, such markers cannot yet replace traditional methods of endometrial assessment (Creus et al., 1998Go; Lessey et al., 2000Go). Therefore, in the clinical setting, useful markers of luteal function and dysfunction beyond progesterone, oestradiol, prolactin and inhibin-A remain to be elucidated. Human endometrial proteins (such as placental protein 14 — now referred to as glycodelin — and placental protein 12) with cyclic changes in expression during the normal menstrual cycle (Hustin et al., 1994Go; Klentzeris et al., 1994Go; Byrjalsen et al., 1995Go) should not be overlooked in this regard.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported in part by grants FIS 98/1193 and 00/0399 from the Fondo de Investigaciones Sanitarias to J.B. The authors are grateful to Dr Antonio Palacín, Ms Margarita Mainar and Ms Montserrat Tortosa for their help with the immunohistochemical studies, and Ms Paquita Antonell for her technical assistance.


    Notes
 
4 To whom correspondence should be addressed at: Institut Clinic of Obstetrics and Gynaecology, Hospital Clínic i Provincial,c/Casanova 143, 08036-Barcelona, Spain. E-mail: jbalasch{at}medicina.ub.es Back


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 Materials and methods
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 Discussion
 Acknowledgements
 References
 
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Submitted on November 20, 2000; accepted on March 15, 2001.