Endometriosis affects sperm–endosalpingeal interactions

L. Reeve1, H. Lashen1 and A. A. Pacey1,2

1 Academic Unit of Reproductive and Developmental Medicine, Level 4, The Jessop Wing, Royal Hallamshire Hospital, Sheffield S10 2SF, UK

2 To whom correspondence should be addressed. Email: a.pacey{at}sheffield.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The interaction between spermatozoa and the epithelium of the isthmic region of the uterine tube is thought to be an important part of the mechanisms of sperm transport to the site of fertilization and in preparing them for fertilization. However, it is unclear whether a dysfunction of this mechanism may contribute to subfertility in some individuals. METHODS: The sperm-binding characteristics of the epithelium from the uterine tubes of three groups of women were examined: (i) eight with pelvic endometriosis (not involving the uterine tubes); (ii) five women who had been receiving zoladex injections to control their symptoms; and (iii) as controls 10 women undergoing an elective procedure for benign gynaecological problems but with no other pathology of the reproductive tract. RESULTS: Significantly more spermatozoa bound per unit area to the ampullary epithelium of the uterine tubes taken from women with a previous diagnosis of endometriosis. CONCLUSIONS: These findings suggest that the characteristics of sperm binding to tubal epithelium may be disrupted in women with a gynaecological pathology such as endometriosis. It is suggested that this may have the potential to interfere with the availability of freely motile spermatozoa, of the appropriate physiological status, to take part in fertilization. This may be a newly described mechanism by which endometriosis can cause infertility.

Key words: endometriosis/endosalpinx/pathology/spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The importance of sperm–epithelial interaction within the mammalian oviduct is now well understood in many species (Scott, 2000Go). Numerous studies have shown that spermatozoa that bind to the endosalpinx retain their viability, motility and fertilizing capacity longer than spermatozoa incubated alone or with other cell types (Smith and Yanagimachi, 1990Go; Pollard et al., 1991Go; Smith and Nothnick, 1997Go). Epithelial-bound spermatozoa also contribute to the formation of a sperm reservoir (Suarez et al., 1991Go; Lefebvre et al., 1995Go; Suarez, 1998Go), which is thought to be important in coordinating the availability of functional spermatozoa for fertilization.

Although these events are less well described in the human uterine (Fallopian) tube, it is thought that they could have an equally important role to play in human conception. It has been shown that human spermatozoa can bind to the endosalpinx (Pacey et al., 1995aGo) and that more spermatozoa bind to cells from the isthmus region of the uterine tube compared with the ampulla (Baillie et al., 1997Go), suggestive of a sperm reservoir in the tubal isthmus. In addition, the positive effect of in vitro co-incubation with endosalpingeal cells on maintaining human sperm viability (Morales et al., 1996Go; Ellington et al 1998Go), increasing motility (Guerin et al., 1991Go; Yeung et al., 1994Go; Yao et al., 2000Go) and regulating sperm capacitation (Murray and Smith, 1997Go; Petrunkina et al., 2001Go) has been described.

Studies to date in both human and non-human species have only considered the interaction of spermatozoa with essentially ‘normal’ epithelium from individuals of either proven fertility or without obvious pathology. However, it has been suggested by both our group (Reeve et al., 2003Go) and others (Templeton et al., 1998Go) that a dysfunction in the mechanism of sperm–endosalpingeal interactions could be a reason for infertility in some couples and thus deserves further investigation. As such, we have undertaken this pilot study to investigate whether there is any evidence for altered characteristics of sperm binding to the endosalpinx from women with pelvic endometriosis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patient recruitment and surgery
The specific details regarding the recruitment of patients, surgical procedures and the isolation of endosalpingeal explants for use in experiments have been fully described elsewhere (Pacey et al., 1995aGo). Briefly, however, uterine tubes were obtained from 23 women undergoing routine (elective) abdominal hysterectomy at the Jessop Hospital for Women, Sheffield, UK for dysfunctional uterine bleeding. Eight of the women had previously been diagnosed with various degrees of endometriosis (but with no involvement of the uterine tubes) and they were not taking any drugs to control their symptoms. As controls, uterine tubes were obtained from 15 women with no known pathology of the reproductive tract, five of whom had received zoladex (goserelin acetate) injections (AstraZeneca, Macclesfield, UK) to stop their periods prior to undergoing hysterectomy (for menorrhagia) and a further 10 women taking no such drug interventions. In scheduling the surgical procedures, no attempt was made to synchronize the stage of ovarian cycle of the women in any group as it had been shown previously that the number of spermatozoa binding to the endosalpinx is unaffected by endocrine profile (Baillie et al., 1997Go). Permission to use uterine (Fallopian) tubes in this research was granted by the South Sheffield Research Ethics Committee and all patients' gave their written consent prior to surgery.

From each patient, uterine tubes were transported to the laboratory within 20 min of surgery. Explants from the ampulla were taken from a region in the distal portion of the uterine tube, 1–2 cm from the cut fimbrial end and isthmic tissue from the region closest to the utero-tubal junction in the proximal portion of the tube. Epithelial mucosa was isolated from the underlying musculature by grasping the folds of mucosa with watch-maker's forceps (Fisher Scientific, Leicester, UK) and cutting them away with fine-pointed scissors (Fisher Scientific). Sections of mucosa were then carefully cut to produce explants of ~1 mm3. The explants were placed into separate wells (three explants per well, four wells per tubal region) of an untreated, flat-bottomed tissue culture plate (Gibco BRL, Paisley, UK) containing 500 µl of tissue culture medium composed of a 50/50 mixture of Ham's F-12/Dulbecco's modified Eagle's medium (DMEM) containing 1% L-glutamine (200 mmol/l), 5% of a 50/50 mixture of Nu-serum/fetal bovine serum, 1% penicillin–streptomycin solution (containing 5000 IU/ml penicillin and 5000 µg/ml streptomycin), 2% HEPES buffer (1 mol/l) and 3% sodium bicarbonate (7.5%) solution. Explants were then incubated overnight in a humid atmosphere of 5% CO2 in air at 37°C, before being used in experiments on the following day. Only explants obtained from a single patient were used in each experiment.

Preparation of spermatozoa
Motile spermatozoa were obtained from the semen of sperm donors attending the Andrology Laboratory in the Jessop Hospital for Women. All donors adhered to a 2 day period of sexual abstinence prior to sample production and were all of proven fertility, producing ejaculates containing at least 60 x 106 spermatozoa/ml and 15% ideal morphological forms (World Health Organization, 1999Go). Spermatozoa were isolated from seminal plasma using a direct swim-up technique (Mortimer, 1990Go). Briefly, aliquots of 300 µl of semen were overlaid with 600 µl of Earle's balanced salt solution (EBSS) containing 0.3% (w/v) human serum albumin (HSA) (Sigma Chemicals, Poole, Dorset, UK) and incubated in a humid atmosphere of 5% CO2 in air at 37°C for 1 h. After 1 h, the top 50% of medium was removed from each tube, the samples pooled and the concentration manually checked using a Neubauer haemocytometer. The preparation was adjusted to 5 x 106 spermatozoa/ml with EBSS containing 0.3% (w/v) HSA, and the concentration manually checked as before using a Neubauer haemocytometer.

Sperm–endosalpingeal bioassay
To determine the sperm-binding characteristics of endosalpingeal tissue from each patient, a simple bioassay described in Baillie et al. (1997)Go was used. This was developed initially to investigate the variation in spermatozoa binding to the endosalpinx both between tubal regions and also across the ovarian cycle. However, in addition, it has since been used to investigate the role of the Arg-Gly-Asp (RGD) adhesion sequence in sperm–endosalpingeal interactions (Reeve et al., 2003Go).

Following overnight incubation, the medium overlying the endosalpingeal explants was removed and 500 µl of the prepared spermatozoa suspension was added, exposing each well of explants to 2.5x106 spermatozoa. These were then incubated for 1 h at 37°C in a humid atmosphere of 5% CO2 in air. At the end of the incubation, medium was removed from each well and 500 µl of fresh (spermatozoa-free) warm EBSS added and the well gently agitated for 1 min (to remove unbound sperm). This procedure was repeated once more before the addition of fixative and the processing of samples as described in Reeve et al. (2003)Go. Once the explants had been mounted on a microscope slide, they were then observed using a x100 oil immersion objective and the number of spermatozoa bound to the epithelial surface in each field of view (equivalent to a surface area of 0.03 mm2) was recorded. A total of 25 microscope fields per experimental condition were examined in this way and the data used to determine a median number of bound spermatozoa per microscope field (0.03 mm2). The fields to be analysed were selected using a systematic random sampling method (Mayhew, 1991Go) and the number of replicates (fields examined) to provide a result with a sampling error of less than ±5% determined by performing a ‘progressive mean analysis’ (Williams, 1977Go). Only those fields that contained a flat area of epithelium, as identified by the presence of ciliated cells, were included for analysis.

Statistical analysis
A Mann–Whitney test was used to compare spermatozoa binding between the isthmic and ampullary regions within each experimental group, and to compare differences between the level of binding between groups within the same anatomical region (isthmus or ampulla). All tests were performed using the Minitab for Windows release 13.32 (Minitab Inc., PA). Data are presented as median (ranges). A P-value of <0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
All uterine (Fallopian) tubes obtained for study were macroscopically normal and, following dissection, the internal mucosal surface (the endosalpinx) appeared as expected and without obvious disease. Moreover, the ages of the women who agreed to take part in this study were not significantly different from each other although the mean age (±SEM) of women with a previous diagnosis of endometriosis was slightly younger (39.2±1.8 years) than women who had received zoladex injections (42.2±3.81 years) or who had no known pathology other than dysfunctional uterine bleeding (43.1±2.2 years).

Table I summarizes the median (ranges) number of spermatozoa bound per 0.03 mm2 of epithelium obtained from the isthmic and ampullary regions of the endosalpinx of the eight women with a previous diagnosis of endometriosis (but not taking any medication to manage their symptoms), in addition to the five women receiving zoladex injections, and the 10 women who had not received any drug therapy prior to surgery.


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Table I. The number of sperm bound to isthmic or ampullary epithelium of the human uterine tube from women: (i) with a prior diagnosis of endometriosis unrelated to the uterine tubes; (ii) who had received zoladex injections to control their dysfunctional uterine bleeding; or (iii) undergoing elective hysterectomy for dysfunctional uterine bleeding but with no associated gynaecological pathology

 
As shown previously using the same bioassay system (Baillie et al., 1997Go; Reeve et al., 2003Go), in each experimental group numerically more spermatozoa bound to the tubal isthmus in comparison with the tubal ampulla (Table I). However, this was only statistically significant in control tissue (P<0.05).

When the median numbers of spermatozoa bound to the epithelium from the same anatomical region of the endosalpinx were compared across the three groups of patients, a statistical difference was found only with respect to sperm binding to epithelium from the tubal ampulla. Briefly, the number of spermatozoa bound per 0.03 mm2 of ampullary epithelium from women with a previous diagnosis of endometriosis was statistically higher than that observed in women who had no drug therapy prior to surgery (P<0.01).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Previous studies that have investigated human sperm–endosalpingeal interaction (Pacey et al., 1995aGo,bGo; Baillie et al., 1997Go; Murray and Smith, 1997Go; Kervancioglu et al., 2000Go; Reeve et al., 2003Go) have specifically excluded women from tubal donation who were not of proven fertility or who had medical conditions that might influence the normal function of the uterine (Fallopian) tubes. As such, there have been no studies to date that have investigated whether gynaecological pathology, such as endometriosis, could directly or indirectly influence the sperm-binding properties of the endosalpinx. Yet the possibility that this could occur may provide an insight into as yet unidentified causes of subfertility in some individuals, such as those women with endometriosis. This study, therefore, investigated the ability of spermatozoa to bind to the endosalpinx of women who, prior to surgery, had previously been diagnosed with various degrees of endometriosis or who had been receiving injections of zoladex to control their dysfunctional uterine bleeding. To quantify the level of sperm–endosalpingeal interactions, a simple in vitro assay was used that has been employed previously to investigate the variation in sperm binding between tubal regions and also across the ovarian cycle (Baillie et al., 1997Go) as well as the role of the RGD adhesion sequence in sperm–endosalpingeal interactions (Reeve et al., 2003Go).

The rationale for studying the effect of endometriosis on sperm–epithelial interaction was 2-fold: (i) because it is a relatively common gynaecological condition; and (ii) it is known that women with only minimal endometriosis with no tubal distortion/obstruction suffer infertility for reasons that cannot be immediately explained (Mahutte and Arici, 2002Go). As such, the finding that the ampullary epithelium from women with a previous diagnosis of endometriosis can bind significantly more spermatozoa per unit area than observed in the controls may provide a tantalizing insight into a novel potential mechanism that could contribute to the subfertility of women with this condition.

It is thought that intimate and specific contact between mammalian spermatozoa and the epithelium of the endosalpinx of the tubal isthmus is of crucial importance in regulating a number of aspects of spermatozoa function prior to fertilization (Scott, 2000Go). This includes maintaining sperm viability and fertilizing ability (Smith and Yanagimachi, 1990Go; Pollard et al., 1991Go; Smith and Nothnick, 1997Go) in the pre-ovulatory period as well as delaying the process of capacitation (Murray and Smith, 1997Go; Petrunkina et al., 2001Go). In addition, it has been suggested from SDS–PAGE (Ellington et al., 1995Go; Thomas et al., 1995Go) and now micro-array (Fazeli et al., 2004Go) techniques that spermatozoa contact can trigger gene expression in the epithelium that leads to the synthesis of new proteins. Whilst the role of these proteins remains unclear, it is further evidence to suggest that sperm–epithelial contact is both a specific and coordinated part of the reproductive process in mammals.

Following ovulation, and in order for fertilization to occur, spermatozoa must be released from the sperm reservoir in the tubal isthmus and then travel to the site of fertilization in the tubal ampulla. Whilst the mechanism by which this occurs has not been fully described, it is thought to be controlled by a number of factors including: (i) the onset of hyperactivated motility in spermatozoa (DeMott and Suarez, 1992Go; Pacey et al., 1995bGo) to assist in the detachment of spermatozoa from epithelial contact; (ii) local factors such as temperature (Bahat et al., 2003Go) or chemical (Eisenbach, 1999Go) gradients within the uterine tubes; and (iii) the fact that the epithelium from the tubal ampulla has a reduced sperm-binding ability when compared with the tubal isthmus (Baillie et al., 1997Go) as well as loss of sperm-binding properties of spermatozoa as they become more capacitated (Lefebvre and Suarez, 1996Go; Fazeli et al., 1999Go). Each of these changes play an important part in ensuring that a sufficient number of motile and capacitated spermatozoa are available in the tubal ampulla for fertilization to occur. Therefore, the observation that the ampullary epithelium from women with a previous diagnosis of endometriosis binds more spermatozoa than controls (i.e. a similar number to that usually seen binding in the tubal ampulla) could potentially hinder fertilization by reducing the number of free spermatozoa in the tubal lumen that are available to take part in fertilization.

Unfortunately, very little is known about the molecular basis for sperm–endosalpingeal interactions, and so it is impossible to determine how increased sperm binding to the epithelium of the tubal ampulla of these women is being mediated. In a number of non-human species, it has been proposed that it may involve carbohydrate recognition (reviewed by Suarez, 2001Go). However, a recent study by Reeve et al. (2003)Go has proposed that in the human, the binding of sperm to the tubal isthmus may involve the interaction between integrins on the epithelial surface with an RGD adhesion sequence of an as yet unidentified sperm protein. Interestingly, however, it has been shown that integrin expression is aberrant in the endometrium of women with endometriosis (Lessey et al., 1994Go; Ota and Tanaka, 1997Go; Puy et al., 2002Go). As such, it would be interesting to speculate whether an increased integrin expression in the ampullary epithelium of these women could lead to increased sperm binding at that location.

In conclusion, this preliminary study has provided the first evidence to support the hypothesis that gynaecological pathology, such as endometriosis, has the potential to influence the function of the endosalpinx and could subtly affect the nature of sperm–endosalpingeal interaction (binding). Given the importance of sperm binding for modulating sperm function in the period prior to fertilization, there is therefore a strong possibility that in some circumstances this may have a negative impact on the reproductive process and in some individuals contribute to their infertility. We propose that this is an area of human reproduction that has received too little attention and is worthy of greater attention by researchers.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors would like to express thanks to staff and patients of the Jessop Hospital for Women, Sheffield for their help in obtaining the tissue samples to carry out this study. Financial assistance for the project was provided by the Infertility Research Trust (Registered Charity No: 512973).


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Submitted on August 13, 2004; accepted on October 15, 2004.