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
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Abstract |
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Key words: endometriosis/endosalpinx/pathology/spermatozoa
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Introduction |
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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., 1995a) and that more spermatozoa bind to cells from the isthmus region of the uterine tube compared with the ampulla (Baillie et al., 1997
), 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., 1996
; Ellington et al 1998
), increasing motility (Guerin et al., 1991
; Yeung et al., 1994
; Yao et al., 2000
) and regulating sperm capacitation (Murray and Smith, 1997
; Petrunkina et al., 2001
) 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., 2003) and others (Templeton et al., 1998
) that a dysfunction in the mechanism of spermendosalpingeal 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.
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Materials and methods |
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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, 12 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% penicillinstreptomycin 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, 1999). Spermatozoa were isolated from seminal plasma using a direct swim-up technique (Mortimer, 1990
). 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.
Spermendosalpingeal bioassay
To determine the sperm-binding characteristics of endosalpingeal tissue from each patient, a simple bioassay described in Baillie et al. (1997) 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 spermendosalpingeal interactions (Reeve et al., 2003
).
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). 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, 1991
) 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, 1977
). 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 MannWhitney 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.
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Results |
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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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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).
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Discussion |
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The rationale for studying the effect of endometriosis on spermepithelial 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, 2002). 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, 2000). This includes maintaining sperm viability and fertilizing ability (Smith and Yanagimachi, 1990
; Pollard et al., 1991
; Smith and Nothnick, 1997
) in the pre-ovulatory period as well as delaying the process of capacitation (Murray and Smith, 1997
; Petrunkina et al., 2001
). In addition, it has been suggested from SDSPAGE (Ellington et al., 1995
; Thomas et al., 1995
) and now micro-array (Fazeli et al., 2004
) 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 spermepithelial 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, 1992; Pacey et al., 1995b
) to assist in the detachment of spermatozoa from epithelial contact; (ii) local factors such as temperature (Bahat et al., 2003
) or chemical (Eisenbach, 1999
) 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., 1997
) as well as loss of sperm-binding properties of spermatozoa as they become more capacitated (Lefebvre and Suarez, 1996
; Fazeli et al., 1999
). 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 spermendosalpingeal 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, 2001). However, a recent study by Reeve et al. (2003)
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., 1994
; Ota and Tanaka, 1997
; Puy et al., 2002
). 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 spermendosalpingeal 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.
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Acknowledgements |
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References |
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Submitted on August 13, 2004; accepted on October 15, 2004.