Why does hydrosalpinx reduce fertility?

The importance of hydrosalpinx fluid

Annika Strandell1,3 and Anette Lindhard2

1 Reproductive Medicine, Department of Obstetrics and Gynecology, Sahlgrenska University Hospital, S-413 45, Göteborg, Sweden and 2 The Fertility Clinic, Copenhagen University hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark


    Abstract
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 Abstract
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The debate on hydrosalpinx and impaired IVF outcome has mainly focused on the best treatment before IVF and on functional surgery as an alternative treatment. We would like to initiate a debate on the possible reasons why the outcome is impaired. We know that salpingectomy is effective in terms of improved birth rates after IVF, but we do not know exactly why. The main focus is on embryotoxic properties of the hydrosalpinx fluid, which include micro-organisms, endotoxins, cytokines, oxidative stress and lack of nutrients. The endometrial receptivity may be reduced as an effect of disturbed expression of the cytokine cascade, which is essential for implantation. The presence of excessive fluid in the uterine cavity may also be a mechanical hindrance to implantation. We believe that the hydrosalpinx fluid is of crucial importance, but the actual mechanism of action needs to be clarified.

Key words: embryotoxicity/hydrosalpinx/IVF outcome/salpingectomy


    Introduction
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 Abstract
 Introduction
 Possible mechanisms
 Conclusions and issues for...
 References
 
During the past decade, the influence of the presence of hydrosalpinx on IVF success rates has been an issue of debate and research. Several retrospective studies have shown an impaired outcome of IVF in the presence of hydrosalpinx and the results have been compiled and presented in meta-analyses, demonstrating a reduction by half in the probability of achieving a pregnancy in the presence of hydrosalpinx and a doubled rate of spontaneous abortion (Zeyneloglu et al., 1998Go; Camus et al., 1999Go). According to the theory that the hydrosalpinx fluid plays a causative role, any surgical intervention interrupting the communication to the uterus would remove the leakage of the hydrosalpinx fluid and restore pregnancy rates. It has been shown that removal of a hydrosalpinx by laparoscopic salpingectomy improves pregnancy rates, particularly in patients with hydrosalpinges large enough to be visible on ultrasound examination (Strandell et al., 1999Go, 2001Go). This finding, resulting in clinical recommendation of salpingectomy in selected cases before IVF, supports the causative role of the fluid itself, but there is a lack of knowledge as to how the fluid exerts its negative effects. It is our belief that the fluid holds a key position in impairing implantation potential and we will discuss different theories concerning how this action is possible.


    Possible mechanisms
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 Abstract
 Introduction
 Possible mechanisms
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 References
 
The hydrosalpinx fluid may act on two different target systems: directly on the transferred embryos or on the endometrium and its receptivity for implantation, or both.

Embryotoxic properties of hydrosalpinx fluid
This has been evaluated using both mouse and human embryos in human hydrosalpinx fluid. There is a discrepancy in the results of culture systems using human and murine models, but the results from different mouse studies are also diverging. Five out of eight studies using a murine model described embryotoxicity at low concentrations of human hydrosalpinx fluid and three studies demonstrated impaired development, but in undiluted hydrosalpinx fluid only (Strandell, 2000Go). There are only two studies on human embryos, neither of which have been able to demonstrate any obvious toxic effect on embryo development, but undiluted hydrosalpinx fluid resulted in a 50% reduction in blastocyst development rate as compared with control medium or diluted hydrosalpinx fluid (Strandell et al., 1998Go). The other study on human embryos reported an unchanged blastocyst development rate in undiluted hydrosalpinx fluid (Granot et al., 1998Go). The experimental models using mouse and human embryos are obviously not comparable and conclusions from studies based on mouse models are evidently not applicable to humans. Part of the explanation might be that mouse embryos were exposed to hydrosalpinx fluid from another species.

From studies on embryo development, it may be concluded that hydrosalpinx fluid does not appear to host a common potent factor deleterious to embryo development, and the lack of essential substrates is more likely to be responsible for the impaired development of embryos in undiluted hydrosalpinx fluid.

Is hydrosalpinx fluid toxic in individual cases?
Even though there may not be a common toxic factor in all fluids, the presence of factors inhibitory to embryo development in fluids from certain individuals cannot be excluded. Most experiments are based on small numbers of hydrosalpinx fluids, and individual variations in content may reflect the differences in embryo development. In a study on the effect of hydrosalpinx fluid on gametes and fertilization, one out of four fluids was directly cytotoxic to murine sperm when incubated in 50% hydrosalpinx fluid during capacitation (de Vantéry Arrighi et al., 2001Go).

No pathogenic micro-organisms have been detected in any of the published studies, but slightly elevated concentrations of endotoxin have been demonstrated in individual fluids as a sign of previous infection (Strandell et al., 1998Go). If a toxic substance was responsible for the negative influence, an assay of the aspirated hydrosalpingeal fluid before stimulation would be useful in selecting patients for salpingectomy.

An assay of mouse embryo culture in 50% hydrosalpinx fluid has been suggested to predict IVF outcome (Chen et al., 2002Go). In a population of 39 hydrosalpinx patients, the test had a sensitivity of 64%, a specificity of 86% and a positive likelihood ratio of 4.5, suggesting the test to be fairly good in detecting toxicity. The diagnostic performance was not improved by including important factors like age and number of good quality embryos transferred. The use of this technique requires transvaginal puncture, preferably before the start of any stimulation, where the result may be helpful in the decision concerning prophylactic salpingectomy. The technique still awaits clinical evaluation. In the same study, levels of cytokines [leukaemia inhibitory factor (LIF), epidermal growth factor, tumour necrosis factor (TNF)-{alpha}, interferon-{gamma}] in the hydrosalpinx fluid were not predictive of IVF outcome. The problem with studies of cytokines is that their role in implantation and embryo development may change during the cycle depending on the presence or absence of other cytokines and growth factors. This might explain why the same cytokine may be suggested to have both embryotoxic and implantation-promoting properties. Interleukin (IL)-1{alpha}, IL-1ß, IL-8 and TNF-{alpha} have been suggested as possible embryotoxic cytokines (Sung et al., 1997Go) while at least IL-1ß has documented importance during implantation.

Oxidative stress
The presence of oxidative and antioxidant systems in various reproductive tissues has evoked interest in the role of oxidative stress in reproductive diseases. Oxidative stress has been defined as an elevation in the steady-state concentration of various reactive oxygen species on a cellular level and has been suggested to be of importance in hydrosalpinx cases. A first report on this issue described a positive effect of low levels of reactive oxygen species in relation to blastocyst development, as compared with absence of reactive oxygen species in hydrosalpinx fluid (Bedaiwy et al., 2002Go). The low levels were suggested to be within a physiological range, and no high levels were detected to demonstrate a negative effect. This hypothesis will need further evaluation.

Do transferred embryos starve in the presence of hydrosal-pinx fluid?
During the development of an embryo to a blastocyst, the energy substrate requirements change from dependence on mainly pyruvate to glucose, which may mirror the environment in vivo when the embryo progresses down the female reproductive tract. However, the effects of the primary nutrients, glucose and pyruvate, on embryo development are incompletely understood. Studies on nutrient concentrations in culture media and their effect on embryo development to blastocyst support the use of a `low' glucose content (Conaghan et al., 1993Go). The levels of glucose are generally found to be very low in hydrosalpinx fluid, comparable with levels in normal human tubal fluid and culture media (Dickens et al., 1995Go; Tay et al., 1997Go). Studies on embryo development in hydrosalpinx fluid suggest a lack of nutrients, which explains the impaired development to blastocysts. Is it possible to refer this finding to in-vivo conditions and thus explain the reduced implantation by insufficient nutrient content available to the embryo?

Endometrial receptivity
The cross-talk between the embryo and endometrium, essential for allowing the embryo to implant, and mediated by the secretion and expression of certain cytokines and other substances during the implantation window, may be disturbed in the presence of hydrosalpinx fluid. Cytokines like IL-1, LIF, colony stimulating factor (CSF)-1 and integrin {alpha}vß3 are all factors which have been shown to be of importance to implantation, and they and some of their receptors are secreted or expressed by either the embryo or the endometrium in an increased manner during the implantation window.

Integrin {alpha}vß3 has been demonstrated immunohistochemically at the apical plasma membrane of the endometrial surface epithelium from day 20 onwards during a 28 day spontaneous cycle (Lessey et al., 1992Go, 1996Go; Aplin et al., 1996Go), and has in addition been demonstrated at the surface of the embryo (Campbell et al., 1995Go). Recent investigations indicate that integrin {alpha}vß3 binds to and activates matrix metalloproteinases and plasminogen activators in the extracellular matrix. This enables the integrin to act both as a receptor for the embryo at the endometrial surface epithelium and as a stimulator of trophoblastic penetration and invasion (Brooks et al., 1996Go). In patients with hydrosalpinges, the expression of integrin {alpha}vß3 was down-regulated during the implantation period and this was described as a possible explanation for failed conception in IVF (Meyer et al., 1997Go). Moreover, 70% of patients (14/20) demonstrated increased integrin expression after surgical correction of their hydrosalpinges. Thus, it can be hypothesized that the presence of hydrosalpinges results in lower endometrial receptivity, possibly by impairing integrin {alpha}vß3 expression in the endometrium. An additional published experimental study showed that blockage of murine endometrial integrin {alpha}vß3 resulted in impaired implantation (Illera et al., 2000Go).

Human preimplantation embryos express mRNA for the cytokine IL-1ß, its receptor IL-1RtI and the antagonist IL-1ra (Krussel et al., 1998Go). The number of IL-1 receptors increases significantly during the mid-luteal phase in human glandular epithelium, especially in the luminal epithelium (Simon et al., 1993Go). Secretion of embryonic IL-1ß seems to be the first response of the blastocysts to the receptive endometrium, inducing a second wave of cytokines in the endometrium such as LIF, IL-1 and others (Cross et al., 1994Go). By binding to their receptors, the cytokines induce molecular changes in the expression patterns of adhesion and anti-adhesion molecules such as integrin {alpha}vß3, essential for attachment of the blastocyst (Simon et al., 1997Go). A defective or missing IL-1 receptor or prohibited access to the receptor in the endometrium (caused by the presence of an increased amount of fluid in the cavity) may therefore exert detrimental effects on the cascade system of cytokines and glycoproteins secreted, leading to a reduced implantation rate.

The expressions of integrin ß3 subunit and LIF secretion have been found to increase simultaneously (Sawai et al., 1997Go; Simon et al., 1997Go). LIF seems to be regulated in the human in the same way as in the mouse, where endometrial LIF secretion is decisive for embryo implantation. LIF production predominantly occurs in the endometrial glands and is known to be involved in successful implantation of the blastocyst. Knockout mice with a defective LIF gene are unable to implant blastocysts (Stewart et al., 1992Go). Transferring blastocysts from LIF-deficient to normal mice results in implantation. Likewise, pregnancy can occur if LIF is infused in the uterine lumen of LIF-deficient mice, demonstrating that the primary action of LIF is in the endometrium. In human endometrium a maximal rise in LIF concentration is observed between cycle days 19 and 25, coinciding with the implantation window (Bhatt et al., 1991Go; Charnock-Jones et al., 1994Go; Nachtigall et al., 1996Go). The abundant expression of LIF in the late secretory phase can also be shown in uterine flushing fluid, where LIF concentrations rise from day LH+7 to a maximum at day LH+12 (Laird et al., 1997Go).

LIF and CSF-1 have also been shown to support embryo development and blastocyst hatching (Dunglison et al., 1996Go; Sjöblom et al., 1999Go). CSF-1 is expressed in human endometrial tissue during the normal menstrual cycle, and levels of immunoreactive CSF-1 in endometrial glands are higher during the secretory phase compared with the proliferative phase. Furthermore, normal first trimester decidual tissue expresses higher levels of CSF-1 mRNA and immunoreactive CSF-1 than proliferative endometrial tissue (Kauma et al., 1991Go). The CSF-1 receptor is expressed in all human endometrial and first trimester decidual tissues, including invasive extravillous trophoblast. Transcripts encoding the receptor for CSF-1 are expressed throughout preimplantation development of human embryos (Sharkey et al., 1995Go). Thereby, the increased production of CSF-1 may play a role in implantation, decidual function and placental growth through the expression of the CSF-1 receptor, possibly through autocrine and paracrine networks. The presence of hydrosalpinx fluid may cause a defective secretion of some or all of these cytokines, thereby impairing embryo development and implantation. This needs to be investigated.

The production of tropho-uteronectin (TUN) by human cytotrophoblast has been studied in order to describe early embryo–integrin interactions. The production of TUN was significantly increased by the presence of hydrosalpinx fluid in an in-vitro system (Sawin et al., 1997Go). The results indicated that hydrosalpinx fluid promoted the production of TUN in a dose-dependent manner. Furthermore, the study by Sawin demonstrated a significant increase in trophoblast cell viability as well as in the production of ß-HCG in the presence of hydrosalpinx fluid. These findings indicate that hydrosalpinx fluid possesses growth-promoting properties contradictory to a murine model in which no outgrowth of trophoblast in human hydrosalpinx fluid was documented (Jun et al., 1999Go). According to hitherto published reports, systems using human models seem to express less negative or even a positive influence from hydrosalpinx fluid as compared with murine models exposed to human hydrosalpinx fluid.

Are embryos simply washed out?
The leakage of hydrosalpingeal fluid through the uterine cavity resulting in disposal of embryos has been suggested as a mechanism by several authors (Mansour et al., 1991Go; Andersen et al., 1996Go; Bloeche et al., 1997Go). The clinical feature of hydrorrhoea was shown to be a sign of poor prognosis among patients with hydrosalpinx undergoing IVF (Andersen et al., 1996Go). The existence of a hydrosalpingeal fluid interface on the endometrial surface, sometimes seen during assisted reproduction, has been suggested to be a hindrance to implantation (Andersen et al., 1996Go; Sharara, 1999Go). A recent study has demonstrated an association between endometrial cavity fluid and increased cancellation rates and lower clinical pregnancy rates in assisted reproduction cycles, but without any association to hydrosalpinges visible on ultrasound (Levi et al., 2001Go). These findings suggest that leakage of hydrosalpingeal fluid through the uterine cavity is only one of several possible explanations to endometrial cavity fluid. This mechanical explanation is of course difficult to study in an experimental model.

Does hydrosalpinx fluid cause an increase in endometrial peristalsis?
Ijland and co-workers have analysed endometrial wave-like activity in several investigations (Ijland et al., 1996Go). The wave-like activity was described by `the wave direction classification', by which five types of endometrial movements are described, i.e. no activity during at least 3 min of observation, waves from cervix to fundus, waves from fundus to cervix, opposing waves starting from cervix and fundus at the same time, and random waves arising from various foci. The group investigated the relationship between endometrial wave-like activity and fecundability in spontaneous cycles (Ijland et al., 1997Go). They examined 33 women with unexplained infertility in 37 spontaneous cycles. Nine women conceived within the cycle studied. Endometrial activity increased from menstruation to ovulation and decreased during the luteal phase. The conception cycles showed the lowest overall activity throughout the cycle compared with the non-pregnant women. The overall activity was highest throughout the cycle in the never pregnant group. No waves from fundus to cervix occurred after ovulation in the pregnant group.

A switch in endometrial wave direction (WDS) is described in IVF cycles (Ijland et al., 1999Go). In 22 IVF cycles with embryo transfers a switch in predominant wave direction was seen from the direction fundus–cervix to the direction cervix–fundus in 73% of the cycles. This switch always occurred before ovum retrieval. The later the WDS occurred, the higher the likelihood of pregnancy in that particular cycle. The group concluded that lowering endometrial activity and tuning the residual activity into peristaltic cervix to fundus waves at mid-cycle may be prerequisites for implantation in the human.

In one recent report, uterine dynamics of five patients with hydrosalpinx were analysed by image-processing techniques and compared with healthy volunteers (Eytan et al., 2001Go). The authors describe, from a mathematical simulation model, a reflux phenomenon (opposing the cervix-to-fundus intrauterine peristalsis) generated by a pressure gradient from tubal fluid accumulation. It was suggested that this reflux phenomenon could explain the reduced implantation rate associated with hydrosalpinx.


    Conclusions and issues for debate
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 Abstract
 Introduction
 Possible mechanisms
 Conclusions and issues for...
 References
 
There is certainly impairment of embryo development in the presence of pure hydrosalpinx fluid in vitro. If there is no direct toxic factor present, is the low energy content in the hydrosalpinx fluid meeting the embryo after transfer low enough to impair the cleavage rate and implantation ability? The positive effect of salpingectomy supports this theory and the studies on embryotoxicity and endometrial effects may also be in agreement. Studies showing obvious embryotoxicity at low concentrations of hydrosalpinx fluid (<=10%) were all conducted on five or less samples of hydrosalpinx fluid, and this small sample size may not be representative of hydrosalpinx fluids in general. If fluids from certain individuals host potent embryotoxic factor(s), how do we find these individuals? Would a mouse assay on aspirated hydrosalpinx fluid be reliable and clinically feasible?

The influence of hydrosalpinx fluid in the uterine cavity on the secretion and expression of certain cytokines and integrins, essential for early embryo–endometrial interactions and implantation, certainly needs to be explored.

It is intriguing to have an effective treatment (salpingectomy) without knowing why it works. It is not only of academic interest to know, it is also of clinical value. If only some hydrosalpinges have embryotoxic or anti-implantation properties, it would be most valuable to be able to select the patients who would benefit from salpingectomy.

The pure mechanical explanation, i.e. the fluid creates a mechanical barrier and/or causes an increased peristalsis in the uterine cavity, also needs to be investigated.

Overall, the most likely possibility is that the low implantation rate is caused by a mixture of the above-mentioned factors.


    Notes
 
3 To whom correspondence should be addressed. E-mail: annika.strandell{at}medfak.gu.se Back


    References
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 Introduction
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 Conclusions and issues for...
 References
 
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