The significance of cytokines, chemical composition, and murine embryo development in hydrosalpinx fluid for predicting the IVF outcome in women with hydrosalpinx

Chin-Der Chen, Jehn-Hsiahn Yang, Ko-Chen Lin, Kuang-Han Chao, Hong-Nerng Ho and Yu-Shih Yang,1

Department of Obstetrics and Gynecology, National Taiwan University College of Medicine and Hospital, Taipei, Taiwan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The aim of this study was to determine whether differences in cytokine concentrations, chemical composition, and murine embryogenesis in hydrosalpinx fluid could be observed between women with hydrosalpinx who achieved and did not achieve pregnancy. METHODS: Hydrosalpinx fluids were collected from 39 infertile women participating in an IVF–embryo transfer programme at a university teaching hospital. The fluids were analysed for concentrations of cytokine concentration (epidermal growth factor, interferon-{gamma}, leukaemia inhibitory factor, and tumour necrosis factor-{alpha}) and chemical composition. Mouse embryos were cultured in medium with various concentrations (0, 10, 50, 100%) of hydrosalpinx fluid. RESULTS: Logistic regression demonstrated that a murine blastocyst formation rate 53.3% in 50% hydrosalpinx fluid (OR = 16.6, 95% CI 2.4–116.1, P = 0.005) and patient age (OR = 0.778, 95% CI 0.61–0.99, P = 0.045) were independent predictors of IVF outcome. The diagnostic accuracy did not significantly improve when age and number of good quality embryos transferred was taken into account. The chemical composition and cytokine concentrations of hydrosalpinx fluid were not predictive of subsequent IVF outcome. CONCLUSIONS: The mouse embryo assay of hydrosalpinx fluid might potentially serve as a predictor of subsequent IVF outcome in women with hydrosalpinx. This technique may be useful in selecting appropriate treatment options for patients with hydrosalpinx undergoing IVF treatment.

Key words: cytokine/hydrosalpinx fluid/IVF outcome/mouse embryo assay


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Many retrospective studies have shown that hydrosalpinx is associated with poor IVF outcome (Camus et al., 1999Go). Recently, a large prospective multicentre Scandinavian study also showed that salpingectomy prior to IVF significantly increased implantation rates in patients with sonographically visible hydrosalpinx, particularly in those affected bilaterally (Strandell et al., 1999Go). However, indiscriminate salpingectomies prior to IVF treatment on all women with hydrosalpinx are clearly not recommended (Puttemans and Brosens, 1996Go; Strandell et al., 1999Go; Dechaud and Hedon, 2000Go). In addition, the impact of salpingectomy on ovarian function is an important concern. It has been reported that laparoscopic salpingectomy does not abate ovarian response during IVF treatment (Dar et al., 2000Go), but an adverse effect on the ipsilateral ovary has been proposed (Lass et al., 1998Go).

Recent data suggest that patients with hydrosalpinx constitute a heterogeneous population with potentially different outcomes (Sharara et al., 1996Go). It would be valuable to identify a subset of patients who would benefit most from elective salpingectomy.

No studies have been published involving the composition analyses of hydrosalpinx fluid on subsequent IVF outcome. One study (Barmat et al., 1999Go) recently reported on the absence of interferon-{gamma} (INF-{gamma}) and transforming growth factor-ß2 (TGF-ß2), and the presence of epidermal growth factor (EGF) and tumour necrosis factor-{alpha} (TNF-{alpha}) in human hydrosalpinx fluid obtained at the time of laparoscopic examination. The clinical significance of these cytokines/growth factors in hydrosalpinx fluid is not clear at present.

Cytokines and growth factors have been associated with inflammatory processes and are involved in embryotoxic and embryotrophic effects (Giudice, 1994Go). We hypothesized that their balance in hydrosalpinx fluid might influence the outcome of IVF–embryo transfer in women with hydrosalpinx. Thus, analyses of their composition as well as their effects on murine embryogenesis might allow us to classify women with hydrosalpinx more specifically into separate categories with possibly markedly different outcomes.

The aim of this study was to determine if differences of cytokines, chemical composition, and murine embryogenesis in hydrosalpinx fluid could be observed in women with hydrosalpinx between those who did and did not become pregnant.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
IVF protocols and embryo grading
The ovarian stimulation protocols for IVF have already been described (Chen et al., 1997Go). Embryos were transferred between 48–72 h after oocyte collection. Before embryo transfer, all embryos were graded and given a rating according to blastomere quality as follows: grade I, equal sized symmetrical blastomeres with <10% fragmentation were given a rating of 4; grade II, uneven blastomeres with <10% fragmentation were given a rating of 3; grade III, with 10–50% fragmentation were given a rating of 2, and grade IV, >50% fragmentation, were given 1. An embryo score per individual embryo was calculated by multiplying the rating of the embryo by the number of blastomeres. The scores of all embryos transferred per patient were summed to give the cumulative embryo score (CES) (Steer et al., 1992Go). Grade I and grade II embryos were considered as good quality embryos.

Patients and collection of hydrosalpinx fluids
Hydrosalpinx fluids were collected from 41 infertile women with unilateral (n = 35) or bilateral hydrosalpinx (n = 6) participating in an IVF-embryo transfer programme. Fluids were obtained after oocyte retrieval from the hydrosalpinx. Fluid from each patient with bilateral hydrosalpinx was individually pooled. Two of the 41 women with fertilization failure were excluded from this study. Overall, the remaining 39 women were divided into those who became pregnant (n = 11) and those who did not become pregnant (n = 28) according to the IVF outcome.

The medium volume of hydrosalpinx fluid aspirated was 14 ml (range 8–520 ml). An aliquot of 1 ml hydrosalpinx fluid was sent for chemical composition analysis immediately after aspiration. Fluid was centrifuged at 1000 g for 15 min to remove cellular debris and then was frozen at –80°C until mouse and cytokine assays were performed.

Chemical composition analysis and cytokine assays
Protein from these fluids was measured using a Hitachi 7250 Special Automatic analyser (Tokyo, Japan). The chemical composition (sodium, potassium, chloride, calcium, glucose, lactate, and bicarbonate) of hydrosalpinx fluid was determined using a blood analyser (Stat Profile Ultra; NOVA Biomedical, Waltham, MA, USA).

Concentrations of cytokines [EGF, leukaemia inhibitory factor (LIF), INF-{gamma}, and TNF-{alpha}] were measured by solid-phase enzyme-linked immunosorbent assay (ELISA) using commercially available kits (Quantikine; R&D Systems, Minneapolis, MN, USA). The sensitivities of these cytokine assays were 0.7 pg/ml for EGF, 8 pg/ml for LIF and INF-{gamma}, and 5 pg/ml for TNF-{alpha}. All samples were run in duplicate and were assayed at the same time to avoid interassay variations and possible alterations due to freezing and thawing.

Preparation of culture media
We used the same human tubal fluid (HTF) containing medium utilized routinely in our daily IVF programme. Fresh medium was prepared each week from stock solutions of the various components using water that had been processed through a Milli-Q water purification system (Millipore, Bedford, MA, USA). Formulation of the medium based on HTF has been previously described (Quinn et al., 1985Go).

Hydrosalpinx fluid was sterilized by filtration (Millex-GX; Millipore) before use. For each patient, HTF medium was prepared as the control culture medium, and HTF containing various concentrations of hydrosalpinx fluid was prepared as test medium. All four solutions (10, 50 and 100% of hydrosalpinx fluid) were put in a 4-well culture dish (Nunclon Delta Multidish; Nalge Nunc International, Rochester, NY, USA). Prior to addition of the collected mouse embryos, the culture dishes were equilibrated overnight in an atmosphere of air/5% CO2 at 37°C. Both pH and osmolality were monitored before and after equilibration of hydrosalpinx fluid.

Mouse embryo study
Embryos were obtained by superovulating random-bred Swiss ICR (CD-1) female mice at 4–5 weeks of age. Mice were injected i.p. with 10 IU pregnant mare serum gonadotrophin (PMSG; Sigma, St Louis, MO, USA). Ovulation was induced 48 h later by administering 10 IU human chorionic gonadotrophin (HCG; Sigma). Animals were then placed overnight with 8-week-old males, and mating was confirmed by the presence of a vaginal plug the next morning. Females were sacrificed 24 h later, and 2-cell stage embryos were flushed from the oviduct and collected in a HEPES-buffered HTF medium. Embryos were washed three times in this medium and once in HEPES-free HTF medium before culture. Between 10 and 20 embryos were cultured in a volume of 500 µl in each well and scored for the incidence of cavitation 96 h after retrieval. In total, 2496 embryos were used for this study.

The blastocyst development rate on day 3 was used to calculate a blastulation index: Blastulation index = percentage blastocyst development rate of test/percentage blastocyst development rate of the control.

A blastulation index of <0.5 was considered to be potentially embryotoxic, while an index of >=1.0 was considered to be embryotrophic. If the blastulation index was between 0.5 and 1.0, then this was defined as having an inhibitory or adverse effect on embryonic development.

Statistical analysis
Receiver operating characteristic (ROC) curve analysis was used to estimate the predictive power of the measured variables (Zweig and Campbell, 1993Go). Predictive power in this study was defined by the area under the ROC curve. The area under the curve was calculated using SPSS software (Release 9.01; SPSS, Chicago, IL, USA). The cut-off point was chosen to maximize sensitivity as well as specificity. Diagnostic index values (sensitivity, specificity, positive predictive value, negative predictive value, and positive likelihood ratio) were then calculated for each of the biochemical parameters of hydrosalpinx fluid (pH, osmolality, sodium, potassium, chloride, calcium, lactate, bicarbonate, glucose and protein), the murine blastocyst development rates in various fractions of hydrosalpinx fluid, as well as cytokine concentrations (EGF, LIF, INF-{gamma}, and TNF-{alpha}). Logistic regression was performed to determine the independent effect of individual variables. An adjusted ROC curve was constructed based on predicted probabilities of the model.

Results are expressed as the mean ± SD. Statistical analyses were performed using Student's t-test, Fisher's exact test, the Mann–Whitney test, and Kruskal–Wallis test as appropriate. Correlation analysis was carried out using Spearman's correlation. Statistical significance was defined as P < 0.05. All statistical analyses were performed by SPSS software.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Baseline clinical characteristics of the study population
The pregnancy group included normal pregnancies (n = 7), ectopic pregnancies (n = 2), and blighted ovum (n = 2). The patients were 25–42 years old, with a mean age of 34.8 years. There were no significant differences between these two groups in any of the characteristics listed in Table IGo.


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Table I. Clinical characteristics of the study population.
 
Chemical composition and cytokine analyses of hydrosalpinx fluid
The results of the chemical analyses of the composition of hydrosalpinx fluid and HTF media are presented in Table IIGo. The hydrosalpinx fluid was generally physiologically normal with respect to pH and osmolality. However, the pH and bicarbonate concentrations were significantly higher and concentrations of potassium, calcium, glucose and lactate were significantly lower in hydrosalpinx fluid than in the control. The sodium/potassium (Na/K) ratio was significantly higher in hydrosalpinx fluid than in the control (39.3 ± 3.8 versus 35.1 ± 3.1, P = 0.009). However, reanalysis of the Na/K ratio in hydrosalpinx fluid between the pregnancy and non-pregnancy groups showed no significant difference between the two groups (40.9 ± 2.9 versus 39.2 ± 3.6).


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Table II. Comparison of the composition of hydrosalpinx fluid and HTF medium
 
Figure 1Go depicts scatter plots of various cytokine concentrations in hydrosalpinx fluid in women who became and did not become pregnant. There was a trend for a higher concentration of EGF and lower concentrations of LIF and INF-{gamma} in the pregnancy group compared to the non-pregnancy group; however, this did not reach statistical significance. Correlation analyses revealed that concentrations of LIF were positively correlated with concentrations of INF-{gamma} (r = 0.395, P = 0.013) and TNF-{alpha} (r = 0.318, P = 0.043). There was no significant correlation between levels of cytokines (EGF, LIF, INF-{gamma}, and TNF-{alpha}) in hydrosalpinx fluid and levels of serum oestradiol and progesterone, as well as volumes of hydrosalpinx fluid.



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Figure 1. Various cytokine concentrations in hydrosalpinx fluid of women achieving and not achieving pregnancy. Horizontal bars represent the mean values in each group.

 
Effect of hydrosalpinx fluid on mouse embryo development
The mean pH of hydrosalpinx fluid after equilibration was 7.64 ± 0.14, and osmolality was 272.5 ± 25.8 mOsmol/kg. The post-equilibration values were considered to be physiological.

The mean blastocyst development rates in HTF medium, and in 10, 50, and 100% hydrosalpinx fluid were 33.2, 37.3, 30.7, and 18.3% respectively. Culture in 100% hydrosalpinx fluid produced a statistically significant decrease in the blastocyst development rate when compared with the other media groups. The mean blastulation indices of 10, 50, and 100% of hydrosalpinx fluid were 1.34, 1.02, and 0.54 respectively (Figure 2Go). The embryotoxic effects (a blastulation index <0.5) on mouse embryo development were evident in 17.5, 41.0, and 59.0% respectively, of the different hydrosalpinx fluids.



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Figure 2. Blastulation indices in various concentrations of hydrosalpinx fluid (10, 50, and 100%)

 
Diagnostic index values calculation
Table IIIGo compares the diagnostic performance of different modalities for predicting the IVF outcome in patients with hydrosalpinx. The best cut-off values to discriminate between the pregnancy and non-pregnancy groups were determined by using ROC curves. Only the blastocyst development rates in 50 and 100% hydrosalpinx fluid, and patient age showed asymptotic significance as measured by the area under the ROC curves. The chemical composition and cytokine concentrations of hydrosalpinx fluid were not predictive of subsequent IVF outcome in women with hydrosalpinx.


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Table III. Comparison of diagnostic performance of different modalities for predicting the IVF outcome in patients with hydrosalpinx.
 
It is well known that age, embryo quality and number of embryos transferred are the most important factors to achieve a clinical pregnancy. To analyse the impact of these variables on clinical pregnancy rate and to minimise the number of variables included in this small study population (n = 39), a logistic regression model was constructed using the following variables: 50% hydrosalpinx fluid mouse blastocyst formation rate >=53.3%, patient age (per additional year), and number of good quality embryos transferred.

Logistic regression demonstrated that a murine blastocyst formation rate >=53.3% in 50% hydrosalpinx fluid (OR 16.6, 95% CI 2.4–116.1, P = 0.005) and patient age (OR 0.778, 95% CI 0.61–0.99, P = 0.045) were independent predictors of IVF outcome, whereas number of good quality embryos transferred was not. The discriminatory performance of the one-variable model (50% hydrosalpinx fluid mouse assay) was slightly less than that of the model with three variables (areas under ROC curves 0.81 and 0.86 respectively, NS).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present study has shown the heterogeneous nature of hydrosalpinx fluid. We demonstrate that the mouse embryo assay of hydrosalpinx fluid might potentially serve as a predictor of subsequent IVF outcome in women with hydrosalpinx. This technique may be useful in selecting appropriate treatment options for patients with hydrosalpinx undergoing IVF treatment.

We noted a trend for higher mouse blastocyst development rates in HTF media containing various concentrations (10 and 50%) of hydrosalpinx fluid compared with HTF control medium. This cannot simply be explained by gross differences in biochemical composition between the hydrosalpinx fluid and the HTF medium. Compared with HTF medium, hydrosalpinx fluid was similar with respect to sodium and chloride concentrations, but lower in potassium, calcium, glucose and lactate. Decreasing the contents of potassium, glucose and lactate in hydrosalpinx fluid might have negative effects on the development of mouse embryos to the blastocyst stage in vitro (Quinn et al., 1985Go; Gardner et al., 1996Go; Murray et al., 1997Go). On the other hand, the obvious component in hydrosalpinx fluid that improves embryo development in vitro compared with the control may be the presence of protein in the hydrosalpinx fluid and its absence in the HTF control medium. A recent study (Liu et al., 1995Go) showed that human oviductal cells produce embryotrophic factor(s) for mouse embryo development. However, the presence of protein in the hydrosalpinx fluid cannot completely explain the heterogeneous effects of hydrosalpinx fluid on mouse embryonic development because dilution with culture medium resulted in an improvement in embryo growth and most of the undiluted hydrosalpinx fluids impaired the development of embryos. The lack of essential substrates is likely to be responsible for the impaired development of embryos in undiluted hydrosalpinx fluid (Murray et al., 1997Go). There might also be individual variations in the content of hydrosalpinx fluids, yielding different influences on embryo development.

The literature predominantly suggests a negative effect of hydrosalpinx on IVF–embryo transfer outcome, although the mechanism of this effect is still controversial. Mukherjee et al. (1996) first reported that hydrosalpinx fluid was embryotoxic and suggested performing salpingectomy (Mukherjee et al., 1996Go). Other studies have demonstrated that the fluid composition itself has no cytotoxic or adverse effect on the normal development of human embryos and their implantation in vitro (Granot et al., 1998Go; Strandell et al., 1998Go). Two explanations may be proposed to explain the difference between our results and those of Mukherjee et al. (1996). First, embryos obtained from different strains of mice were used, and these may show different sensitivities to agents commonly present in hydrosalpinx fluid. Using the same strain of mouse as in our study, the results of another published study (Murray et al., 1997Go) do not support the concept that a potent embryotoxic agent is commonly present in hydrosalpinx fluid. Another reason for this discrepancy may be due to obtaining hydrosalpinx fluid from a different stage.

To our knowledge, the clinical significance of various cytokine concentrations in hydrosalpinx fluid on subsequent IVF outcome has not been investigated. Our study shows slight in-vitro differences of various cytokines in hydrosalpinx fluid in women with hydrosalpinx in those who became and did not become pregnant. We found a trend for higher levels of EGF and lower levels of LIF and INF-{gamma} in the pregnancy group compared with the non-pregnancy group, although the difference did not reach statistical significance. Our results may suffer from a type II statistical error (falsely accept null hypothesis) because only 39 patients were studied. The possible role of the periovulatory cytokine profile in hydrosalpinx fluid as a predictor of subsequent IVF outcome awaits further clarification.

Our results imply a pregnancy rate of 28% and a birth rate of 18%, which is quite a good result in hydrosalpinx patients. It has to be considered that all patients were treated by transvaginal aspiration at the time of oocyte retrieval. This method has been suggested to improve pregnancy outcome, but it has not been properly evaluated. However, in the present study population it might have influenced pregnancy rates positively. Recently, two retrospective studies investigating the potential benefit of hydrosalpinx drainage on IVF outcome have reached conflicting results (Sowter et al., 1997Go; Van Voorhis et al., 1998Go). The role of hydrosalpinx aspiration at oocyte retrieval still awaits evaluation in a well designed prospective trial.

These results must be interpreted with caution, however, given the small sample size and the fact that we did not control for the various confounding factors associated with the outcome of IVF–embryo transfer. It is well known that age, embryo quality and number of embryos transferred are the most important factors to achieve a clinical pregnancy. When a statistical model adjusting for these variables was used, the predictive power of the 50% hydrosalpinx fluid mouse assay became even stronger, although the diagnostic accuracy did not significantly improve when age and number of good quality embryos transferred were taken into account.

In conclusion, the present study demonstrates that the mouse embryo assay of hydrosalpinx fluid might potentially serve as a predictor of subsequent IVF outcome in women with hydrosalpinx. This technique can be applied to the current treatment cycle by draining off the fluid at the start of stimulation, or even in advance of that so that a more rational decision can be made either to go ahead with the cycle or to cancel and consider the possibility of tubal occlusion or salpingectomy.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Miss Yin-Hsueh Wang for her technical assistance. This study was supported by grant NTUH89N-024 from the National Taiwan University Hospital.


    Notes
 
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, National Taiwan University Hospital, 7, Chung-Shan South Road, Taipei, Taiwan. E-mail: yayang{at}ntumc.org Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on August 6, 2001; accepted on September 14, 2001.