The endometrium versus embryonic quality in endometriosis-related ...

Human Reproduction Update, Vol.8, No.1 pp. 95±103, 2002

The endometrium versus embryonic quality in endometriosis-related infertility N.Garrido1, J.Navarro2, J.GarcõÂa-Velasco3, J.RemohõÂ1,4, A.Pellicer1,4 and C.SimoÂn1,4,5 Instituto Valenciano de Infertilidad 1Valencia, 2Sevilla, 3Madrid and 4Department of Paediatrics, Obstetrics and Gynaecology, Valencia University School of Medicine, Valencia, Spain 5

To whom correspondence should be addressed at: Instituto Valenciano de Infertilidad, C/Guardia Civil, 23, Valencia-46020, Spain. E-mail: [email protected]

In spite of a great deal of effort over many decades, the mechanisms that lead to infertility in women with endometriosis remain unknown. Moreover, controversial results in the literature add even more dif®culties in the understanding of this issue. Since the introduction of IVF, we have had the opportunity to study the effects of endometriosis at speci®c stages of the reproductive process including folliculogenesis, fertilization, embryo development and implantation. Depending on the research group, there are con¯icting data on the results of IVF in patients with endometriosis. Some researchers found impaired implantation in patients with endometriosis, but such a defect in implantation may be caused by either defective embryos or altered endometrium. The observation of a higher rate of arrested embryos in patients with endometriosis compared with disease-free women, and that women with this pathological situation undergoing ovum donation had the same chance of implantation as patients without endometriosis, initially suggested that impaired oocyte/embryo quality may be responsible for reduced implantation. Further investigations on follicular components have also found differences between women with endometriosis and those without the disease. In addition, recent advances on implantation research, mainly on markers of endometrial receptivity, also show features in the eutopic endometrium of women with endometriosis that are not found in endometrium of women without the disease, although there is no agreement on this point. In this review, we will focus on infertility-related endometriosis based on our own research and the available literature. Key words: assisted reproduction/embryo/endometriosis/endometrium/infertility

TABLE OF CONTENTS Introduction Clinical evidence of oocyte/embryo versus endometrial quality in endometriosis-associated infertility IVF in patients with endometriosis Oocyte donation model in patients with endometriosis Basic studies on follicular/oocyte/embryo quality in endometriosis Basic studies on the endometrium in endometriosis-associated infertility Molecular and morphological markers of endometrial receptivity altered in endometriosis Apoptosis and endometrium in endometriosis Endometrial contractility in women with endometriosis Concluding remarks Acknowledgements References

Introduction Endometriosis is de®ned as the attachment of endometrial cells, both stroma and epithelium, in locations other than the uterus. Ó European Society of Human Reproduction and Embryology

Within the wide spectrum of symptoms that are displayed with the disease, one of the most intriguing is the link between endometriosis and infertility in those cases where there is no mechanical alteration of the reproductive tract. This issue has been controversial for many decades, but a large body of data supported the concept of diminished fecundity in patients with endometriosis (Garrido et al., 2000). Many theories of how mild to moderate endometriosis could impair fertility have been suggested during the years, but new hypotheses and approaches to the problem have arisen with the application of assisted reproduction techniques (ART). Nevertheless, the use of ART in patients with endometriosis has yielded controversial results, and detailed discussions about this issue can be found elsewhere (GarcõÂa-Velasco and Arici, 1999). Authors who found poor success rates caused by implantation defects in women with endometriosis have described three different alterations: an oocyte/embryo impairment, endometrial defects and defective endometrial/embryonic cross-talk. Altered oocyte/embryo quality due to altered embryo development has been described (Brizek et al., 1995), or higher embryo blockage

95

N.Garrido et al. in women with endometriosis (Pellicer et al., 1995) compared with healthy controls. It seems that the impairment found in the embryos can be induced by previous defects in folliculogenesis. Moreover, clinical data regarding oocyte donation programmes seems to support the theory of a damaged oocyte condition coming from ovaries of endometriosis-affected women (Garrido et al., 2000). However, to achieve a successful implantation, pregnancy and subsequent birth, in addition to an adequate embryonic quality, an appropriate endometrium is mandatory. This endometrium should be able to accept embryo attachment and development. Although there is no general agreement, some alterations have been found in the eutopic endometrium of women with endometriosis that could be responsible, at least in part, for the subfertility shown by these women (GarcõÂa-Velasco and Arici, 1999). Also, many factors directly implicated in the cross-talk between the embryo and endometrium preceding implantation have not yet been studied in this group of women (SimoÂn, 2000). It is possible that they could play a key role against an adequate implantation. In conclusion, the mechanisms of infertility associated with minimal to mild endometriosis are poorly understood. Our aim in the present review is to describe the available information from the oocyte/embryo and the eutopic endometrium standpoint in infertility related to endometriosis.

Clinical evidence of oocyte/embryo versus endometrial quality in endometriosis-associated infertility Good quality embryos (those with the ability to implant and develop properly) surely originate from good quality oocytes, which in turn originate from follicles with an adequate environment conditioned by the follicular ¯uid (FF) and the neighbouring cells, which are able to in¯uence their progression (Canipari et al., 2000). The follicular components, oocytes and the resulting embryos can be studied either using clinical protocols (IVF or ovum donation) or by using basic studies, looking for differences between patients with and without endometriosis in molecular markers implicated in normal physiological processes within the ovary and early embryo development. IVF in patients with endometriosis

Con¯icting results are frequent among studies on IVF results in patients with endometriosis (GarcõÂa-Velasco et al., 1999; Garrido et al., 2000). These discrepancies seems to be multifactorial, since IVF outcome can be affected by patient management, stimulation, selection criteria, laboratory procedures and many other factors intrinsic to each institution. The valid option was therefore to analyse our own results in this topic to reach comparable conclusions. Our group undertook a study to investigate the origin of the subfertile status of women with different degrees of endometriosis compared with women with tubal infertility as a control group (SimoÂn et al., 1994), although the presence of hydrosalpinges within the control group was not considered. Comparable numbers of oocytes retrieved, fertilization rates and the total number of embryos transferred were seen in each group. Interestingly, a signi®cant decrease in pregnancy rate per cycle, per transfer and implantation rate were observed in the

96

endometriosis group as compared with women with tubal infertility. No differences in the results depending on the severity of the disease were found. The quality of the embryos derived from women with endometriosis was compared with the embryos derived from women with tubal infertility in a retrospective analysis (Pellicer et al., 1995). The number of blastomeres and the degree of fragmentation were established after 48 and 72 h in culture and there was no difference between the two groups in age, number of oocytes retrieved, oocytes fertilized and mean number of blastomeres after 48 h. However, after 72 h there was a signi®cant decrease in the number of blastomeres in women with endometriosis compared with women with tubal infertility. Similarly, there was a signi®cant increase in the percentage of arrested embryos in the endometriosis compared with the tubal infertility group. Our observations have been con®rmed by other authors who have employed video cameras to record zygote formation and embryo development in vitro, showing that the percentage of abnormal embryos is higher in endometriosis patients (Brizek et al., 1995). In this study, different nuclear and cytoplasmic events were studied in 235 embryos. Day 1 preembryos were tested for darkened cytoplasm, organelle depletion, halo of anucleated fragments (HAF) and aberrant cleavage of anucleated cytoplasm. In the day 2 pre-embryos, the main measures were: the arrested presyngamy with HAF, arrested postsyngamy at the 1-cell stage with HAF, multicellular preembryos with nuclear anomalies and/or with HAF. Embryo development was signi®cantly different in women with endometriosis, as shown by the incidence of areas of organelle depletion or voids, condensation with voids, HAF and aberrant cleavage of anucleated cytoplasm on day 1, as well as arrested presyngamy with HAF on day 2. The authors concluded that altered embryo quality was affecting women with endometriosis, and given that it is a good prognostic factor of implantation rates and IVF success, these parameters could be affected in these women. Nevertheless, they were not able to demonstrate differences in implantation and pregnancy rates between endometriosis-affected women and the control group (Brizek et al., 1995). Recently, to increase knowledge on this topic and increase the power of our conclusions, we further reviewed the situation by means of a meta-analysis of eight relevant studies on this issue (Landazabal et al., 1999), including ~900 cycles in ~700 women with endometriosis and >2500 cycles of 1700 women without the disease which were used as controls. All of these studies were selected depending on their design, data presentation and inclusion criteria. Global fertilization, implantation, abortion and pregnancy rates were calculated, ®nding a statistically signi®cant impairment in pregnancy (28.6 versus 34.2% per cycle) and implantation (13.6 versus 17.6% per transferred embryo) rates in women with endometriosis versus non-endometriosis controls, while no differences were found in fertilization rates (56.6 versus 59.3% per oocyte) or in the number of oocytes retrieved (9.5 versus 9.7 per cycle). This study included only papers with analogous design. To summarize, based on the results of this research, (i) implantation was signi®cantly impaired in women with endometriosis, and (ii) the quality of the embryos replaced was poor, possibly re¯ecting poor quality oocytes. We addressed the issue

Endometriosis-related infertility further by analysing the results of our oocyte donation programme. Oocyte donation model in patients with endometriosis

Oocyte donation provides an interesting method to investigate the reproductive outcome of endometriosis-affected women, because it is possible to compare women who received fresh oocytes from non-endometriosis women with those receiving oocytes from endometriosis women. We retrospectively analysed the results of our oocyte donation programme (SimoÂn et al., 1994). Women were divided into three groups: (i) premature ovarian failure (n = 54), (ii) low responders to controlled ovarian stimulation (n = 77), and (iii) women with endometriosis who underwent oocyte donation because of low response (n = 11). A similar number of embryos were replaced in each group. There was no difference among groups in the pregnancy rate per woman, per cycle or implantation. We also analysed the outcome of oocyte donation according to the origin of the donated oocytes (SimoÂn et al., 1994). We were able to compare the ability to implant of embryos obtained from women with tubal infertility (n = 27), male infertility (n = 28), ovulation disorders (n = 58), fertile women simultaneously undergoing tubal ligation (n = 34) and patients with endometriosis (n = 11). There was no difference among groups in pregnancy rate per transfer. However, implantation rates were signi®cantly lower in recipients who received oocytes from women with endometriosis. In order to eliminate the inherent bias of the retrospective model, we designed a prospective study, in which three groups were established (Pellicer et al., 1994). Group 1 contained donors and recipients without signs of endometriosis (n = 44). Group 2 were donors with endometriosis who donated oocytes to recipients without the disease (n = 14). Group 3 were donors without endometriosis who donated oocytes to recipients with the disease (n = 16). An impairment of pregnancy rates per transfer was observed in group 2 (28.6%) as compared with the other two groups (61.4 for the ®rst and 60.0% for the second), despite a similar number of embryos replaced. A signi®cant decrease in implantation rates was also noted in group 2 as compared with groups 1 and 3, con®rming the fact that embryos derived from ovaries of women with endometriosis display a reduced ability to implant. Our observations have been con®rmed by other authors, who show that the presence of endometriomas in women undergoing ovum donation is not a factor against successful implantation and pregnancy (Sung et al., 1997). In their retrospective study, a total of 239 cycles were included, and the recipients were divided into group 1, containing women with endometriosis (n = 55; 18 with stage I±II and 37 with stage III±IV), and group 2, containing women without the disease (n = 184); the groups had a pregnancy rate of 28 and 29%, and an implantation rate of 12 and 13% respectively. The diagnoses of all women were performed by laparoscopy. Furthermore, we designed a prospective matched case±control study in order to evaluate the impact of severe endometriosis on IVF outcome in women receiving oocytes from the same donor (DõÂaz et al., 2000), thus ruling out the possibility of assigning oocytes of different quality to the different groups. Fifty-eight recipients were included in the study. Twenty-®ve patients

diagnosed by laparoscopy to have stage III±IV endometriosis (group I) and 33 women free of the disease (group II) were included (Table I). On the day of retrieval, oocytes from a single donor were donated to recipients from endometriosis and nonendometriosis groups. The results revealed no differences in pregnancy (40 versus 45%), implantation (14.8 versus 16.0%), miscarriage (30 versus 26%) and live birth rates (28 versus 27.2%) between the groups (endometriosis stage III±IV and controls respectively). The present study supports previous observations (SimoÂn et al., 1994) since oocytes provided by the same donor were allocated to different recipients, suggesting that infertility in these patients is not related to an unsuitable peritoneal and/or endometrial environment affecting endometrial receptivity, but is related to a diminished oocyte quality.

Basic studies on follicular/oocyte/embryo quality in endometriosis As mentioned above, oocyte development is conditioned by the correct function of ovarian cells. Classical studies revealed an ovulatory dysfunction resulting in an increased number of follicles, but with a smaller mean size and lower estradiol levels in the pre0ovulatory phase and after the LH surge (Tummon et al., 1988). More recent studies suggested a faulty regulation of the pituitary±ovarian axis (Cahill et al., 1995), and this was reinforced by the ®nding of an altered endocrine function of granulosa cells in these patients, translated into low progesterone production and aromatase function in women with endometriosis, thus showing an impaired corpus luteum, which is unable to maintain adequate steroid production. However, in a similar experimental design our group found different results (Pellicer et al., 1998), demonstrating an overproduction of progesterone in patients with the disease. In this study, intrafollicular progesterone, estradiol, androstenedione and testosterone were determined, both in vivo (FF) and in vitro, with cultured granulosa cells from patients with and without Table I. IVF outcomes in recipients of sibling oocytes with and without stages III±IV of endometriosis (DõÂaz et al., 2000 reprinted by permission from American Society for Reproductive Medicine Fertility and Sterility, 2000, 74, 31±34) Study group

P-value

Endometriosis (III±IV) Control No. of patients No. cycles Agea (years) No. of oocytes donateda No. of embryos transferreda No. of good quality embryos transferreda Implantation (%) No. of pregnancies (%) Miscarriage (%) Live birth (%)

25 25 35.0 7.8 4.0 3.6

6 6 6 6

3.4 1.6 0.7 0.2

15/101 (14.8) 10 (40.0) 3 (30.0) 7 (28.0)

33 33 38.5 7.7 4.1 3.7

6 6 6 6

4.9 1.9 1.2 0.1

22/137 (16.0) 15 (45.5) 4 (26.0) 9 (27.2)

0.004 NS NS NS NS NS NS NS

a

Values are mean 6 SD. NS = not signi®cant

97

N.Garrido et al. endometriosis being stimulated with HCG. Only progesterone was increased in FF from patients with endometriosis. These ®ndings were further investigated by our group, with contradictory results. In a study of the expression of 3-b-hydroxysteroid dehydrogenase (the enzyme that catalyses progesterone production) and corticosteroid binding globulin (the molecule responsible for progesterone bioavailability) in granulosa cells and FF of woman with and without endometriosis (Garrido et al., 2002). No differences were found in endometriosis, and we concluded that alterations in the follicular microenvironment, if any, are not related to these molecules. Apart from the endocrine environment, multiple paracrine factors secreted by the granulosa cells and present in the FF could potentially interfere with oocyte development in inadequate amounts. For this purpose, we studied the presence of immunoreactive interleukin(IL)-6, IL-1b and vascular endothelial growth factor (VEGF) in the FF and granulosa cell cultures, both at basal and HCG-stimulated and after comparing women with and without the disease (Pellicer et al., 1998). Results indicated an increase in the production of IL-6 and a decrease in VEGF in the cultures and FF of patients with endometriosis, suggesting that these factors were implicated in an impairment of the luteal function. However, further studies conducted by our group, including only patients with ovarian endometriomas and immunopuri®cation of contaminating leukocytes in the cell cultures, revealed that IL-6 and VEGF mRNA and protein were not different in patients with and without the disease, thus discarding the implication of these factors (Garrido et al., 2001). Other authors have studied the in¯ammatory cytokine IL-6 together with other molecules, such as IL-8, IL-1b and tumour necrosis factor (TNF)-a (Carlberg et al., 2000), and concluded that only the latter is altered in women with endometriosis. In their study, they also checked for the presence of contaminating leukocytes in the granulosa cell cultures and the results were negative. The patients included in the study (n = 10) ranged across all the stages of the disease. Ovarian cell apoptosis (programmed cell death without triggering an in¯ammatory response) has also been suggested as a good predictor of oocyte quality (Nakahara et al., 1997). Moreover, in the granulosa cells of patients with endometriosis, an increased incidence of alterations in the cell cycle has been found compared with those without the disease (Toya et al., 2000), but the power of the study was low due to the small number of patients included, and there was an absence of an adequate control group with the absence of endometriotic foci con®rmed by laparoscopy. When the incidence of apoptotic bodies in these granulosa cells was checked, it was found that an increase in apoptosis correlated with the severity of the disease, but the control group showed differences in the total number of oocytes retrieved and fertilized (Nakahara et al., 1998). The in¯uence of ovarian leukocytes on oocyte maturation and growth, either directly with secretion products or mediated by interactions within the endocrine system, is beyond any doubt (Adashi, 1990). Thus, changes in number or status of these cells can directly affect gamete maturation. Flow cytometry using speci®c antibodies against concrete leukocyte subsets has been useful to evaluate these subpopulations in the FF of women with tubal factor infertility and endometriosis, demonstrating an

98

increment in the presence of natural killer cells, B lymphocytes and monocytes (Lachapelle et al., 1996). In a similar study by our group analysing percentage of stained cells (as in the study by Lachapelle et al.) and staining intensity as an approximation to the activation status of the cells, we were unable to detect any difference between groups when women with endometriosis were compared with those without the disease (Garrido et al., 1999). The main difference with the other study was our control group, which was established with the condition of absence of endometriosis con®rmed by laparoscopy. Other factors involved in the ovulation process, such as prostaglandins (speci®cally prostaglandin F2), have been determined in the FF of women with endometriosis, comparing women with visible lesions and those without, and concluding that, in accordance with previous clinical results, even if lesions disappear after treatment, fertility does not increase, and this can be mediated by these factors (Bergqvist et al., 1997). Admittedly, no comparison with a healthy group was attempted. Other intrafollicular molecules such as MuÈllerian-inhibiting substance (Fallat et al., 1997), inhibins A and B, and activin B (Akande et al., 2000) have been matter of study in a similar way, but only the presence of inhibin A is enhanced in the FF of women with endometriosis compared with the group with unexplained infertility or tubal damage; however, no relationship was found with the fertilization ability of the oocytes. Finally, endothelin-1 in the FF of women with endometriosis undergoing stimulated cycles of IVF was shown in higher levels compared with women without the disease (Abae et al., 1994). Further documentation of the implications of these molecules in the pathophysiology of endometriosis is required before conclusions can be drawn.

Basic studies on the endometrium in endometriosisassociated infertility Implantation is an extremely co-ordinated process requiring the presence of developing embryos with the ability to induce the correct changes in the endometrial epithelium, together with the presence of an endometrium ready to receive these signals and to act in consequence (SimoÂn, 2000). Subsequently, any endometrium unable to answer properly, without the correct timing in the endometrial changes, would be adversely affecting the reproductive success. To address this issue in endometriosis, the suitable design of any study should compare eutopic endometrium of women with endometriosis with healthy controls without the disease. The vast majority of studies regarding endometrium and endometriosis are focused on the differences between eutopic and ectopic endometrium in these patients and are hence concerned with molecules presumably implicated in the origin of the disease. They are mainly focused on molecules related to the attachment and growth of the ectopic tissue, but some efforts have been dedicated to molecules previously related to implantation and infertility. Accumulated data suggest that eutopic endometrium of women with endometriosis behaves different from the endometrium of women without the disease. But, to establish a cause±effect relationship between endometrial alterations in women with

Endometriosis-related infertility endometriosis and infertility, the key factors concerning endometrium and implantation should ®rst be accurately determined. Molecular and morphological markers of endometrial receptivity altered in endometriosis

Using scanning and electron microscopy, defects in the ultrastructural morphology of endometria have been described in women with endometriosis, which could be responsible for infertility (Fedele et al., 1990). Recently, pinopods have been suggested as morphological indicators of uterine receptivity. Therefore, their presence in the eutopic endometrium of women with endometriosis must be studied. We recently addressed this issue in a prospective study in order to assess pinopod formation in women with and without endometriosis undergoing our oocyte donation programme (GarcõÂa-Velasco et al., 2001). There were no differences between the two groups analysed, suggesting that the endometrium of women with endometriosis is able to develop an adequate response to hormonal replacement therapy in terms of pinopod formation. Integrins were proposed to be sensitive indicators of endometrial receptive status because their expression pattern is modi®ed around the time of implantation, co-expressing a1, a4, and b3 chains only during the embryonic `implantation window' (Lessey et al., 1994a). The study of these molecules in eutopic endometrium from women with endometriosis demonstrated a defective expression of avb3 (Lessey et al., 1994b) in a retrospective study which compared the traditional histological criteria. First, studying `in-phase' biopsies they found (retrospectively) that avb3 was less expressed in the presence of endometriosis. They then addressed a prospective and double blind study of 89 endometrial biopsies that were taken before diagnostic laparoscopy, providing evidence that the majority of women with abnormal avb3 integrin expression had endometriosis stage I or II. Based on these results, avb3 integrin expression could be a useful marker of mild endometriosis. This method was used to evaluate the possibility that endometriosis could adversely affect the endometrial environment in natural cycles by comparing women with and without endometriosis (Lessey et al., 1994b). However, other authors did not con®rm these ®ndings (Bridges et al., 1994; Creus et al., 1998; Hill and Rogers, 1998), possibly due to a slightly different study design, controls or sample size. Afterwards, the same group correlated the increase in pregnancy rates with an increase in avb3 expression after the treatment of endometriosis, making use of surgical procedures or GnRH analogues (Lessey and Young, 1997). In addition, it has been demonstrated that increased estradiol levels negatively affect implantation (ValbuenÄa et al., 1999) in clinical studies. Furthermore, it has been demonstrated that eutopic endometrium from women with endometriosis is able to express aromatase (the enzyme that catalyses the production of estradiol), and this suggests a possible mechanism by which implantation is damaged in these patients, although more efforts on this topic are needed to establish its real implication (Noble et al., 1996). Hoxa-10 and -11, are homeobox genes essential in the mouse for embryonic development, endometrial development in adults and implantation. Moreover, targeted mutations of any of these genes provoke uterine infertility with normal embryos, thus impeding

implantation. A similar role is found in human implantation (Taylor et al., 1998, 1999b) and a cycle dependent expression has been described, with a marked up-regulation in the mid-secretory phase, just when the endometrium allows embryos to implant. In the eutopic endometrium of women with endometriosis there is an absence of this expected mid-luteal rise, which could result in implantation defects (Taylor et al., 1999a,b). The presence of anti-endometrial antibodies has been studied for many years, since it is believed that endometriosis shares some features with autoimmune diseases, but conclusions were highly controversial with many con¯icting results caused by different control group characteristics and the use of different methods of detection (Wild and Shivers, 1985; Gleicher et al., 1987; Kennedy et al., 1990; Switchenko et al., 1991; FernandezShaw et al., 1993). Their presence against eutopic endometrium epitopes was initially described in a well-designed study by Odukoya et al. who detected them in about half of the sera of the patients with endometriosis, including controls with laparoscopically demonstrated absence of endometriosis, and with a detailed analysis of previous discordant works in their discussion (Odukoya et al., 1995). More recently, in patients with endometriosis these auto-antibodies have been found in higher levels than healthy controls, but their role in infertility still has to be determined (Tanaka et al., 2000). Other immunological changes found in the eutopic endometrium of women with endometriosis are those involving immune cells, but they are mainly dedicated to factors theoretically involved in the origin of the disease and not in implantation Another molecule that is expressed in the late secretory phase as well as menstrual endometria, the ebaf, has been described as a promising novel member of the transforming growth factor (TGF)-b superfamily which could be used as a receptivity marker of implantation, and a decrease in its expression has been demonstrated in patients with endometriosis (Tabibzadeh et al., 2000) in a study where there was a comparison between endometrium from healthy and endometriosis subjects. However, the main difference is observed when comparing two subsets of women with endometriosis: women who achieved pregnancy and those who did not. Another factor involved in alterations of the endometrium of women with endometriosis is glutathione peroxidase, an enzyme that eliminates free-radicals. Glutathione peroxidase exhibits a varying expression during the menstrual cycle in normal endometria, but no variation in eutopic endometrium of women with endometriosis. This lack of variation leads to defects involving protection against oxidative stress of these endometria, whose relevance in implantation, the endometrium or embryo development is unknown but it appears a promising ®eld for study (Ota et al., 2000). Finally, many factors involved in implantation have not yet been studied comparing the eutopic endometrium of women with endometriosis with the endometrium of healthy controls, as depicted in Figure 1. Apoptosis and endometrium in endometriosis

Apoptosis is a physiological process that is employed by living organisms in order to eliminate cells without triggering an in¯ammatory or immune response. This process is a previously programmed cell death, characterized by active transcription of

99

N.Garrido et al.

Figure 1. Factors and processes recently revealed as relevant in implantation not studied yet in women with endometriosis.

speci®c genes, and is composed of successively established phenomena resulting in cell disintegration. In the human endometrium, proliferation and apoptosis are the homeostatic mechanism of the menstrual cycle. The proliferative phase has a low number of apoptotic cells, which increase and peak in the menstrual phase (Tao et al., 1997; Toki et al., 1998), and are subsequently regulated by hormones. Some processes where apoptosis plays an important role in the endometrium are decidualization (Akcali et al., 1997) and blastocyst implantation in animals (Schlafke and Enders, 1975). Endometrial stroma is not as susceptible to apoptotic events as the epithelium (Rango et al., 1998), and our group has demonstrated an extremely coordinated apoptosis in the concrete zone where the embryo must implant, closely relating this process to human implantation (GalaÂn et al., 2000). Apoptosis is crucial for the embryo implantation, rescuing the endometrium from apoptosis in the apposition phase, and then inducing apoptosis locally by contacting endometrial surface. This process is mediated by Fas `death receptors'. This normal cascade of events could be blocked or disturbed by any alteration in the endometrium of women with endometriosis, or even the endometrial environment, which could affect the sensitivity of the endometrium to the presence of the embryo. Some alterations of factors involved in apoptotic processes have been detailed in women with endometriosis, but their in¯uence on implantation still remains unclear. Bcl-2 is a proto-oncogene that encodes an intracellular protein that can protect certain cellular types from apoptosis, thus promoting cell survival (Vaux et al., 1988). This gene is expressed in the endometrium and its level changes during the

100

menstrual cycle (Otsuki et al., 1994). On the other side, the bax gene encodes the pro-apoptotic protein related to Bcl-2 (Tao et al., 1997). The decision of cell death or cell life depends on the ratios between these two factors. No differences in the expression of endometrial Bcl-2/bax have been described between patients with or without endometriosis using immunohistochemical techniques, suggesting that these factors are not deeply involved in infertility due to endometriosis (McLaren et al., 1997). Other authors found similar results comparing DNA fragmentation and presence of bcl-2 protein in endometria from patients with and without the disease (Jones et al., 1998) Another factor potentially involved is Fas-L, which is a ligand of Fas, a surface antigen belonging to the TNF receptors superfamily, which is also localized in endometrial glands (Watanabe et al., 1997). Many components present in the peritoneal ¯uid of women with endometriosis, such as TGF-b1 and platelet-derived growth factor (PDGF) are able to induce FasL, thus deregulating the endometrium in these patients (GarcõÂaVelasco et al., 1999). Finally, when determining spontaneous apoptosis in eutopic endometrium of women with and without endometriosis, lower apoptotic rates are found in the presence of the disease (Gebel et al., 1998) and also a low apoptosis and sensitivity to macrophagemediated cytolysis is found in these women (Dmowski et al., 1998). No data are available about the reaction of eutopic endometrial cells in the presence of the embryo, depending on the source of the tissue. In conclusion, endometrial apoptosis appears to be a hopeful research ®eld to study the causes of infertility status, but a direct

Endometriosis-related infertility effect of this process on implantational status in these patients still remains unclear. Endometrial contractility in women with endometriosis

Altered endometrial contractility has been described in the uterus of women with endometriosis. These ®ndings could affect normal sperm transport and contribute to the sub-fertility status of these patients (De Vries et al., 1990). This effect could be important in the implantation process, as it has been previously described by the results of ultrasound measures on women undergoing IVF (Fanchin et al., 1998). However, this observation is merely anecdotal until further prospective studies are designed to assess the real impact of this altered endometrial contractibility in women with endometriosis.

Concluding remarks Although contradictory results have been reported, different clinical studies have demonstrated worse success rates for pregnancy in women with endometriosis compared with healthy women or those with tubal factor. Among those that found lower pregnancy rates in women with endometriosis compared with controls without the disease, some found alterations in implantation rates in women with endometriosis when they were compared with controls. Several studies suggest that an altered follicular microenvironment or an intrinsic ovarian problem could be responsible for a defective oogenesis, and subsequently low quality oocytes resulting in impaired fertilization rates or poor quality embryos, with diminished ability to implant. Many molecules associated with different physiological processes of the oocyte development and maturation such as apoptosis, steroidogenesis and angiogenesis have been investigated, but again controversial results were obtained. Endometrial aspects have also been discussed, and molecular studies on factors involved in the receptivity status of the eutopic endometrium in women with endometriosis resulted again in con¯icting data, although some differences with normal controls have been described. Mixed causes (defects both the endometrium and embryo) cannot be ruled out, and in the ovum donation programme, good embryos could by-pass an affected endometrium. The debate continues, and other approaches should be made before we can determine the authentic origin of the defects that make conception more dif®cult in these patients. These new studies must include an adequate design (prospective, randomised and controlled) and controls, in order to avoid misleading data that can complicate the interpretation of the pathophysiology of this enigmatic disease.

Acknowledgements The authors wish to acknowledge Dr A.LandazaÂbal, Instituto Valenciano de Infertilidad, Madrid, Spain, for her kind assistance in the preparation of this manuscript. N.G. was supported by grant MIT-F2 from Ministerio de EducacioÂn, Cultura y Deporte (Spain). This study was supported by grants FISss 00/0478.

References Abae, M., Glassberg, M., Majercik, M.H., Yoshida, H., Vestal, R. and Puett, D. (1994) Immunoreactive endothelin-1 concentrations in follicular ¯uid of women with and without endometriosis undergoing in vitro fertilization±embryo transfer. Fertil. Steril., 61, 1083±1087. Adashi, E.Y. (1990) The potential relevance of cytokines to ovarian physiology; the emerging role of resident ovarian cells of white blood cell series. Endocr. Rev., 11, 454±464. Akande, A.V., Asselin, J., Keay, S.D., Cahill, D.J., Muttukrishna, S., Groome, N.P. and Wardle, P.G. (2000) Inhibin A, inhibin B and activin A in follicular ¯uid of infertile women with tubal damage, unexplained infertility and endometriosis. Am. J. Reprod. Immunol., 43, 61±69. Akcali, K.C., Kahn, S.A. and Moulton, B.C. (1997) Effect of decidualization on the expression of bax and bcl-2 in the rat uterine endometrium. Endocrinology, 137, 3123±3130. Bergqvist, A., Brunkwall, J. and Ploman, F. (1997) Concentrations of prostaglandin F2alpha in follicular ¯uid from women with endometriosis. Hum. Reprod., 12, 1789±1793. Boucher, A., Khar®, A., Al-Akoum, M., Bossu, P. and Akoum, A. (2001) Cycle-dependent expression of interleukin-1 receptor type II in the human endometrium. Biol. Reprod., 65, 890±898. Bridges, J., Prentice, A., Roche, W., Engle®eld, P. and Thomas, E.J. (1994) Expression of integrin adhesion molecules in endometrium and endometriosis. Br. J. Obstet. Gynaecol., 101, 696±700. Brizek, C.L., Schlaff, S., Pellegrini, V.A., Frank, J.B. and Worrilow, K.C. (1995) Increased incidence of aberrant morphological phenoptypes in human embryogenesisÐan association with endometriosis. J. Assist. Reprod. Genet., 12, 106±112. Cahill, D.J., Wardle, P.G., Maile, L.A., Harlow, C.R. and Hull, M.G. (1995) Pituitary±ovarian dysfunction as a cause for endometriosis-associated and unexplained infertility. Hum. Reprod., 10, 3142±3146. Canipari, R. (2000) Oocyte±granulosa cell interactions. Hum. Reprod. Update, 6, 279±289. Carlberg, M., Nejaty, J., Froysa, B., Guan, Y., Soder, O. and Bergqvist, A. (2000) Elevated expression of tumor necrosis factor alpha in cultured granulosa cells from women with endometriosis. Hum. Reprod., 15, 1250± 1255. Chervenak, J.L. and Illsley, N.P. (2000) Episialin acts as an antiadhesive factor in an in vitro model of human endometrial±blastocyst attachment. Biol. Reprod., 63, 294±300. Cork, B.A., Li, T.C., Warren, M.A., and Laird, S.M. (2001) Interleukin-11 (IL11) in human endometrium: expression throughout the menstrual cycle and the effects of cytokines on endometrial IL-11 production in vitro. J. Reprod. Immunol., 50, 3±17. Creus, M., Balasch, J., Ordi, J., Fabregues, F., Casamitjana, R., Quinto, L., Coutifaris, C. and Vanrrell, J.A. (1998) Integrin expression in normal and out-of-phase endometria. Hum. Reprod., 13, 3460±3468. De Vries, K., Lyons, E., Ballard, G., Levy, C.S. and Lindsay, D.J. (1990) Contractions of the inner third of the myometrium. Am. J. Obstet. Gynecol., 162, 679±682. DõÂaz, I., Navarro, J., Blasco, L., SimoÂn, C., Pellicer, A. and RemohõÂ, J. (2000) Impact of stages III±IV of endometriosis on recipients of sibling oocytes. Matched case±control study. Fertil. Steril., 74, 31±34. Dmowski, W.P., Gebel, H. and Braun, D.P. (1998) Decreased apoptosis and sensitivity to macrophage mediated cytolysis of endometrial cells in endometriosis. Hum. Reprod. Update, 4, 696±701. Fallat, M.E., Siow, Y., Marra, M., Cook, C. and Carrillo, A. (1997) Mullerianinhibiting substance in follicular ¯uid and serum: a comparison of patients with tubal factor infertility, polycystic ovarian syndrome, and endometriosis. Fertil. Steril., 67, 962±965. Fanchin, R., Righini, C., Olivennes, F., Taylor, F., Taylor, S., de Ziegler, D. and Frydman, R. (1998) Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization. Hum. Reprod., 13, 1968±1974. Fedele, L., Marchini, M., Bianchi, S., Dorta, M., Arcaini, L. and Fontana, P.E. (1990) Structural and ultrastructural defects in preovulatory endometrium of normo-ovulating infertile woman with minimal or mild endometriosis. Fertil. Steril., 53, 989±993. Fernandez-Shaw, S., Hicks, B.R., Yudkin, P.L., Kennedy, S., Barlow, D.H. and Starkey, P.M. (1993) Antiendometrial and antiendothelial autoantibodies in women with endometriosis. Hum. Reprod., 8, 310±315. GalaÂn, A., O'Connor, J.E., Valbuena, D., Herrer, R., RemohõÂ, J., Pampfer, S., Pellicer, A. and SimoÂn, C. (2000) The human blastocyst regulates

101

N.Garrido et al. endometrial epithelial apoptosis in embryonic adhesion. Biol. Reprod., 63, 430±439. GarcõÂa-Velasco, J. and Arici, A. (1999) Is the endometrium or the oocyte/ embryo affected in endometriosis? Hum. Reprod., 14 (Suppl. 2), 77±89. GarcõÂa-Velasco, J., Arici, A., Zreik, T., Naftolin, F. and Mor, G. (1999) Macrophage derived growth factors modulated Fas ligand expression in culture endometrial stromal cells: a role in endometriosis. Mol. Hum. Reprod., 5, 642±650. GarcõÂa-Velasco, J., Nikas, G., RemohõÂ, J., Pellicer, A. and SimoÂn, C. (2001) Endometrial receptivity in terms of pinopode expression is not impaired in women with endometriosis in arti®cially prepared cycles. Fertil. Steril., 75, 1231±1233. Garrido, N., Albert, C., Mercader, A., RemohõÂ, J., SimoÂn, C. and Pellicer, A. (1999) Leukocyte subpopulations in ovulatory follicles in patients with endometriosis. Hum. Reprod., 14 (Abstract Book 1), 227. Garrido, N., Navarro, J., RemohõÂ, J., SimoÂn, C. and Pellicer, A. (2000) Follicular hormonal environment and embryo quality in women with endometriosis. Hum. Reprod. Update, 6, 67±74. Garrido, N., Albert, C., Krussel, J.S., O¢Connor, J.E., RemohõÂ, J., SimoÂn, C. and Pellicer, A. (2001) No differences in expression, production and secretion of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) by granulosa cells in women with endometriosis. Fertil. Steril., 76, 568±575. Garrido, N., Krussel, J.S., RemohõÂ, J., SimoÂn, C. and Pellicer, A. (2002) Expression and function of 3 beta hydroxisteroid dehydrogenase (3b HSD) Type II and corticosteroid binding globulin (CBG) in granulosa cells from ovaries of women with and without endometriosis. J. Assist. Reprod. Genet., 19. Gebel, H.M., Braun, D.P., Tambur, A., Frame, D., Rana, N. and Dmowski, W.P. (1998) Spontaneous apoptosis of endometrial tissue is impaired in women with endometriosis. Fertil. Steril., 69, 1042±1047. Gleicher, N., El-Roeiy, A., Con®no, E. and Friberg, (1987) Is endometriosis an autoimmune disease? Obstet. Gynecol., 70, 115±122. Gonzalez, R.R., Caballero-Campo, P., Jasper, M.J., Mercader, A., Devoto, L., Pellicer, A. and Simon, C. (2000) Leptin and leptin receptor are expressed in the human endometrium and endometrial leptin secretion is regulated by the human blastocyst. J. Clin. Endocrinol. Metab., 85, 4883±4888. Hill, L. and Rogers, P. (1998) Endometrial vascular and glandular expression of integrins alpha (v) beta 3 in women with and without endometriosis. Hum. Reprod., 13, 1030±1035. Jones, R.K., Searle, R.F. and Bulmer, J.N. (1998) Apoptosis and bcl-2 expression in normal human endometrium, endometriosis and adenomyosis. Hum. Reprod., 13, 3496±3502. Kennedy, S.H., Starkey, P.M., Sargent, I.L., Hicks, B.R. and Barlow, D.H. (1990) Antiendometrial antibodies in endometriosis measured by enzyme linked immmunosorbent assay before and after treatment with danazol and naferelin acetate. Obstet. Gynecol., 75, 914±918. Krussel, J.S., Casan, E.M., Raga, F., Hirchenhein, J., Wen, Y., Huang, H.Y., Bielfeld, P. and Polan, M.L. (1999) Expression of mRNA for vascular endothelial growth factor transmembraneous receptors Flt1 and KDR, and the soluble receptor s¯t in cycling human endometrium. Mol. Hum. Reprod., 5, 452±458. Kumar, S., Li, Q., Dua, A., Ying, Y.K., Bagchi, M.K. and Bagchi, I.C. (2001) Messenger ribonucleic acid encoding interferon-inducible guanylate binding protein 1 is induced in human endometrium within the putative window of implantation. J. Clin. Endocrinol. Metab., 86, 2420±2427. Lachapelle, M.H., Hemmings, R., Roy, D.C., Falcone, T. and Miron, P. (1996) Flow cytometric evaluation of leukocyte subpopulations in the follicular ¯uids of infertile patients. Fertil. Steril., 65, 1135±1140. Landazabal, A., MunÄoz, E., Valbuena, D., RemohõÂ, J., SimoÂn, C. and Pellicer, A. (1999) Endometriosis and in vitro fertilization (IVF): a meta-analysis. Hum. Reprod., 14 (Abstract Book 1), 181±182. Lessey, B.A. and Young, S. (1997) Integrins and other cell adhesion molecules. Semin. Reprod. Endocrinol., 15, 290±299. Lessey, B.A., Castelbaum, A.J., Buck, C.A., Lei, Y., Yowell, C.W. and Sun, J. (1994a) Further characterization of endometrial integrins during the menstrual cycle and in pregnancy. Fertil. Steril., 62, 497±506. Lessey, B.A., Castelbaum, A.J., Sawin, S.W., Buck, C.A., Schinnar, R., Bilker, W. and Strom, B.L. (1994b) Aberrant integrin expression in the endometrium of women with endometriosis. J. Clin. Endocrinol. Metab., 79, 643±649. Li, Q., Zhang, M., Kumar, S., Zhu, L.J., Chen, G., Bagchi, M.K. and Bagchi, I.C. (2001) Identi®cation and implantation stage-speci®c expression of an interferon-alpha-regulated gene in human and rat endometrium. Endocrinology, 142, 2390±2400.

102

Marions, L. and Danielsson, K.G. (1999) Expression of cyclo-oxygenase in human endometrium during the implantation period. Mol. Hum. Reprod., 5, 961±965. MartõÂn, J.C., Jasper, M.J., Valbuena, D., Meseguer, M., RemohõÂ, J., Pellicer, A. and SimoÂn, C. (2000) Increased adhesiveness in cultured endometrialderived cells is related to the absence of moesin expression. Biol. Reprod., 63, 1370±1376. McLaren, J., Prentice, A., Charnock-Jones, D.S., Sharkey, A.M. and Smith, S.K. (1997) Immulocalization of the apoptosis regulating proteins Bcl-2 and Bax in human endometrium and isolated peritoneal ¯uid macrophages in endometriosis. Hum. Reprod., 12, 146±152. Meseguer, M., Aplin, J.D., Caballero-Campo, P., O¢Connor, J.E., MartõÂn, J.C., RemohõÂ, J., Pellicer, A. and SimoÂn, C. (2001) Human endometrial MUC1 is up-regulated by progesterone and down-regulated in vitro by the human blastocyst. Biol. Reprod., 64, 590±601. Nakahara, K., Saito, H., Saito, T., Ito, M., Ohta, N., Takahashi, T. and Hiroi, M. (1997) The incidence of apoptotic bodies in membrana granulosa can predict prognosis of ova from patients participating in in vitro fertilization programs. Fertil. Steril., 68, 312±317. Nakahara, K., Saito, H., Saito, T., Ito, M., Ohta, N., Takahashi, T. and Hiroi, M. (1998) Ovarian fecundity in patients with endometriosis can be estimated by the incidence of apoptotic bodies. Fertil. Steril., 69, 931± 935. Noble, L., Simpson, E., Johns, A. and Bulun, S.E. (1996) Aromatase expression in endometriosis. J. Clin. Endocrinol. Metab., 81, 174±179. Odukoya, O.A., Wheatcroft, N., Weetman, A.P. and Cooke, I.D. (1995) The prevalence of endometrial immunoglobulin G antibodies in patients with endometriosis. Hum. Reprod., 10, 1214±1219. Ota, H., Igarashi, S., Kato, N. and Tanaka, T. (2000) Aberrant expression of glutathione peroxidase in eutopic and ectopic endometrium in endometriosis and adenomyosis. Fertil. Steril., 74, 313±318. Otsuki, Y., Misaki, O., Sugimoto, O., Ito, Y., Tsujimoto, Y. and Akao, Y. (1994) Cyclic bcl-2 gene expression in human uterine endometrium during menstrual cycle. Lancet, 334, 28±29. Pellicer, A., Oliveira, N., Gutierrez, A., RemohõÂ, J. and SimoÂn, C. (1994) Implantation in endometriosis: lessons learned from IVF and oocyte donation. In Spinola, P.Y. and Coutinho, E.M. (eds) Progress in Endometriosis, Parthenon Publishing Group, Casterton-Hill, pp. 177±183. Pellicer, A., Oliveira, N., Ruiz, A., RemohõÂ, J. and SimoÂn, C. (1995) Exploring the mechanism(s) of endometriosis-related infertility: an analysis of embryo development and implantation in assisted reproduction. Hum. Reprod., 10 (Suppl. 2), 91±97. Pellicer, A., ValbuenÄa, D., Bauset, C., Albert, C., Bonilla-Musoles, F., RemohõÂ, J. and SimoÂn, C. (1998) Fertil. Steril., 69, 1135±1141. Rango, U.V., Claessen-Linke, I., Krusche, C.A. and Beier, H.M. (1998) The receptive endometrium is characterized by apoptosis in the glands. Hum. Reprod., 13, 3177±3189. Schlafke, S. and Enders, A.C. (1975) Cellular basis of interaction between trophoblast and uterus at implantation. Biol. Reprod., 12, 41±75. SimoÂn, C., Martin, J.C. and Pellicer, A. (2000) Paracrine regulators of implantation. BaillieÁres Pract. Res. Clin. Obstet. Gynecol., 14, 815±826. SimoÂn, C., Gutierrez, A., Vidal, A., De los Santos, M.J., TarõÂn, J.J., RemohõÂ, J. and Pellicer, A. (1994) Outcome of patients with endometriosis in assisted reproduction: results from in-vitro fertilization and oocyte donation. Hum. Reprod., 9, 725±729. Sung, L., Mukherjee, T., Takeshige, T., Bustillo, M. and Copperman, A.B. (1997) Endometriosis is not detrimental to embryo implantation in oocyte recipients. J. Assist. Reprod. Genet., 14, 152±156. Switchenko, A.C., Kauffman, R.S. and Becker, M. (1991) Are there antiendometrial antibodies in sera of women with endometriosis? Fertil. Steril., 56, 235±241. Tabibzadeh, S., Mason, J.M., Shea, W., Cai, Y., Murray, M.J. and Lessey, B. (2000) Dysregulated expression of ebaf, a novel molecular defect in the endometria of patients with infertility. J. Clin. Endocrinol. Metab., 85, 2526±2536. Tanaka, T., Umesaki, N., Mizuno, K., Fujino, Y. and Ogita, S. (2000) Antiendometrial IgM autoantibodies in endometriotic patients: a preliminary study. Clin. Exp. Obstet. Gynecol., 27, 133±137. Tao, X.J., Tilly, K.I., Maravei, D.V., Shifren, J.L., Krajewski, S., Reed, J.C., Tilly, I.J. and Isaacson, K.B. (1997) Differential expression of members of the bcl-2 gene family in proliferative and secretory human endometrium: glandular epithelial cells apoptosis is associated with increased expression of bax. J. Clin. Endocrinol. Metab., 82, 2739±2746. Taskin, O., Giudice, L., Mangal, R., Dunn, R.C., Dsupin, B.A., Poindexter, A.N. and Wiehle, R.D. (1996) Insulin growth factor binding proteins in

Endometriosis-related infertility peritoneal ¯uid of women with minimal and mild endometriosis. Hum. Reprod., 11, 1741±1746. Taylor, H., Arici, A., Olive, D. and Igarashi, P. (1998) HOXA 10 is expressed in response to sex esteroids at the time of implantation in the human endometrium. J. Clin. Inv., 101, 1379±1384. Taylor, H.S., Bagot, C., Kardana, A., Olive, D. and Arici, A. (1999a) HOX gene expression is altered in the endometrium of women with endometriosis. Hum. Reprod., 14, 1328±1331. Taylor, H., Igarashi, P., Olive, D. and Arici, A. (1999b) Sex esteroids mediate HOXA 11 expression in the human peri-implantation endometrium. J. Clin. End. Met., 84, 1129±1135. Toki, T., Mori, A., Shimizu, M., Nikaido, T. and Fujii, S. (1998) Localization of apoptotic cells within the human endometrium and correlation between apoptosis and p21 expression. Mol. Hum. Reprod., 4, 1157±1164. Toya, M., Saito, H., Ohta, N., Saito, T., Kaneko, T. and Hiroi, M. (2000) Moderate and severe endometriosis is associated with alterations in the cell cycle of granulosa cells in patients undergoing in vitro fertilization and embryo transfer. Fertil. Steril., 73, 344±350. Tummon, I.S., Maclin, V.M., Radwanska, E., Binor, Z. and Dmowski, W.P. (1988) Occult ovulatory disfunction in women with minimal endometriosis or unexplained infertility. Fertil. Steril., 50, 716±720.

ValbuenÄa, D., Jasper, M., RemohõÂ, J., Pellicer, A. and SimoÂn, C. (1999) Ovarian stimulation and endometrial receptivity. Hum. Reprod., 14 (Suppl. 2), 107±111. Vaux, D., Cory, S. and Adams, J. (1988) Bcl-2 gen promotes haematopoietic cells survival and cooperates with c-myc to immortalize pre-B cells. Nature, 335, 440±442 Watanabe, H., Kanzaki, H., Narukawa, S., Inoue, T., Katsuragawa, H., Kaneko, Y. and Mori, T. (1997) Bcl-2 and Fas expression in eutopic and ectopic human endometrium during the menstrual cycle in relation to endometrial cell apoptosis. Am. J. Obstet. Gynecol., 176, 360±368. Wild, R.A. and Shivers, C.A. (1985) Endometrial antibodies in patients with endometriosis. Am. J. Reprod. Immunol. Microbiol., 8, 84±87. Williams, C.D., Boggess, J.F., LaMarque, L.R., Meyer, W.R., Murray, M.J., Fritz, M.A. and Lessey, B.A. (2001) A prospective, randomized study of endometrial telomerase during the menstrual cycle. J. Clin. Endocrinol. Metab., 86, 3912±3917.

Submitted on January 25, 2001; resubmitted on September 24, 2001; accepted on November 27, 2001

103