fertilization (IVF) patients. In the present study we have evaluated the influence of hydrosalpinx fluid on normal human embryo development and implantation.
Human Reproduction vol.13 no.10 pp.2921–2925, 1998
Hydrosalpinx fluid does not adversely affect the normal development of human embryos and implantation in vitro
A.Strandell1,3, A.Sjo¨gren1, U.Bentin-Ley2, J.Thorburn1, L.Hamberger1 and M.Bra¨nnstro¨m1 1Department
of Obstetrics and Gynaecology, Go¨teborg University, Sahlgrenska University Hospital, S-413 45 Go¨teborg, Sweden and 2Department of Obstetrics and Gynaecology, Herlev Hospital, University of Copenhagen, Herlev, Denmark 3To
whom correspondence should be addressed
Several retrospectively designed studies have shown an association between the presence of hydrosalpinx and impaired implantation and pregnancy rates among in-vitro fertilization (IVF) patients. In the present study we have evaluated the influence of hydrosalpinx fluid on normal human embryo development and implantation. Surplus, donated frozen embryos (n J 183) from IVF patients were used to study the effects on blastocyst development of hydrosalpinx fluid at concentrations of 50 and 100% compared with controls in S2 medium. The fluids were analysed for concentrations of electrolytes, osmolarity, protein content, endotoxin levels, bacterial or fungal contamination, pH and haemoglobin content. There was no difference in blastocyst development in cultures under mineral oil when control cultures (15/42 J 36%) were compared with cultures in 50% hydrosalpinx fluid (32/ 96 J 33%). The only biochemical parameter which correlated with capacity for blastocyst development was pH in hydrosalpinx fluid/medium (50/50%) after equilibration in 5% CO2 in air. When embryos were cultured in 100% hydrosalpinx fluid the blastocyst development was 14% (5/ 36) in comparison to control 33% (3/9). The original experiment was repeated in an open culture system without the protection of mineral oil but still in the presence of 50% hydrosalpinx fluid. The rate of blastocyst development was within the same range in the open system. In three separate experiments, the capability of expanded blastocyst to implant on multilayer artificial endometrium was tested. In these experiments, 1/3, 4/5 and 9/9 blastocysts implanted. The present study demonstrates that hydrosalpinx fluid does not generally exert any major negative effects on in-vitro development of human embryos or on the implantation process in vitro. Key words: blastocyst/embryo/hydrosalpinx/implantation/IVF
Introduction Patients with tubal infertility constitute a large group of patients treated by in-vitro fertilization (IVF). Among these women, the presence of hydrosalpinx is a common feature. We have © European Society for Human Reproduction and Embryology
previously shown in a retrospective study that hydrosalpinx was associated with lower pregnancy and delivery rates in IVF patients (Strandell et al., 1994). Since then, several studies have confirmed these observations (Andersen et al., 1994; Kassabji et al., 1994; Vandromme et al., 1995; Fleming and Hull, 1996), although some studies have been unable to find any significant association (Sharara et al., 1996). It has been speculated that the negative influence of hydrosalpinx fluid may be due to toxic substances in the fluid. Mechanisms which have been suggested are either direct or systemic influence by inflammatory mediators on the developing embryo in utero or on the endometrium prior to or during implantation. A limited number of studies have presented results on the effects of human hydrosalpinx fluid on murine embryo development in vitro. It was demonstrated that hydrosalpinx fluid negatively affected the in-vitro development of mouse embryos (Mukherjee et al., 1996), an observation which was later confirmed to a certain extent by Rawe et al. (1997). In the latter study, it was also demonstrated that implantation rates of mice blastocysts were not affected. The effect of hydrosalpinx fluid on the process occurring after implantation was tested in a study where hydrosalpinx fluid increased the viability of trophoblastic cells and the production of trophoblastic markers such as β-human chorionic gonadotrophin (β-HCG) and trophoblast oncofetal fibronectin (Sawin et al., 1997). In the present study we have extended these investigations by testing the effects of hydrosalpinx fluid on human blastocyst development as well as on implantation capability in vitro. Materials and methods Patients Hydrosalpinx fluid was collected from 12 tubes in eight patients (mean age 35.1 years, range 32–38) undergoing laparoscopic salpingectomy for presence of hydrosalpinx. Ethical permission for this procedure had been obtained from the Human Research Ethical Committee at Go¨teborg University as part of a randomized study, examining the effect of salpingectomy prior to IVF. Aspiration of hydrosalpinx fluid was performed as the first intra-abdominal procedure during surgery. Great care was taken to avoid blood contamination in the aspirates. The hydrosalpinx fluids were immediately divided into aliquots and stored at –70°C. IVF patients were down-regulated by intranasal administration of 1.2 mg buserelin acetate per day and were subsequently stimulated with 150–225 IU per day of human menopausal gonadotrophin (Pergonal®; Serono, Rome, Italy) or urofollitrophin (Fertinorm HP®; Serono). Oocyte maturation was induced by 10 000 IU HCG (Profasi®; Serono). After follicular aspiration and conventional IVF, embryos not transferred were frozen according to our routine protocol (Testart
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Table I. Characteristics of 12 hydrosalpinx fluids in eight patients Hydrosalpinx Sodium fluid mmol/l
Potassium mmol/l
Calcium mmol/l
Osmolarity mosm/kg
Protein g/l
Haemoglobin Glucose g/l mmol/l
1A 1B 2A 2B 3 4A 4B 5 6 7A 7B 8
4.40 5.30 3.80 4.00 4.00 2.60 2.40 3.80 4.40 4.60 4.40 4.90
1.61 2.11 1.28 0.01 0.72 1.13 1.00 2.18 1.57 0.88 1.76 2.38
235.00 283.00 275.00 283.00 277.00 187.00 166.00 288.00 281.00 281.00 295.00 275.00
4.33 85.35 3.55 2.82 4.43 4.21 5.00 43.65 4.89 6.17 20.25 18.08
0.0 27.0 0.0 0.0 0.0 2.0 1.0 6.0 0.0 3.0 9.0 1.0
144.00 141.00 138.00 144.00 153.00 92.00 84.00 134.00 138.00 144.00 138.00 136.00
,0.6 0.6 ,0.6 ,0.6 ,0.6 ,0.6 ,0.6 2.4 ,0.6 ,0.6 ,0.6 ,0.6
Endotoxins IU/ml
pH before pH after equilibration equilibration
0.008 1.710 0.008 0.038 0.017 0.045 0.030 0.050 0.017 0.009 0.011 0.030
7.75 7.42 8.18 7.91 7.57 7.82 7.55 8.90 7.92 7.66 7.68 7.51
7.35 7.29 7.24 7.38 7.40 7.29 7.41 7.28 7.63 7.55 7.54 7.44
Table II. Development of blastocysts in cultures with 50% or 100% hydrosalpinx fluid from 12 different hydrosalpinx fluids and controls
Hydrosalpinx 50%
Hydrosalpinx 100%
Controls
aP bP
Hydrosalpinx fluid
Number of embryos
Number of blastocysts
% blastocyst development
pooled 1A 1B 2A 2B 3 4A 4B 5 6 7A 7B 8 Total 2B 3 4A Total 1 (versus 2 (versus 3 (versus 4 (versus 5 (versus 6 (versus Total
16 5 6 5 5 8 8 8 8 9 6 6 6 96 10 17 9 36 14 5 8 9 6 9 51
6 2 2 2 2 2 1 2 1 5 2 3 2 32 1 4 0 5 3 2 2 5 3 3 18
38 40 33 40 40 25 13 25 13 56 33 50 33 33.3a 10 24 0 13.9a,b 21 40 25 56 50 33 35.3b
50%, pooled) 50%, 1A-2A) 50%, 2B-4A) 50%, 4B-6) 50%, 7A-8) 100%)
5 0.027. 5 0.026.
et al., 1986). Donated cryopreserved embryos, which had exceeded the allowed freezing time in Sweden of one year, were thawed at the four cell stage. Embryos with a normal cleavage rate, when observed the following day, were used for the experiments and cultured up to the expanded blastocyst stage. The patients had given their informed consent to use these surplus embryos for this study, which was approved by the Human Research Ethical Committee of Go¨teborg University. Laboratory procedures Individual hydrosalpinx fluid aliquots were initially thawed and centrifuged at 794 g for 10 min (Biofuge 15R, Heraeus Sepatech). Samples from these aliquots were taken for analysis of sodium, potassium, calcium ion concentrations and osmolarity according to clinical routines in the Department of Clinical Chemistry, Sahlgrenska University Hospital, Go¨teborg, Sweden. Haemoglobin concentrations
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and pH were measured (ABL system 625®; Radiometer, Copenhagen, Denmark). Protein concentrations were measured (Lowry et al., 1951) and endotoxins were analysed (Kinetic, chromogenic LAL assay, GMP-complying THERMOmax™ Microplate Reader, SOFTMAX® equipment) (European Pharmacopeia, 1997). As a pilot study, hydrosalpinx fluid from three patients were pooled and diluted 1:1 with S2-medium™ (Scandinavian IVF Science Ltd, Go¨teborg, Sweden). The pH was measured on two occasions; firstly before dilution and secondly after dilution and equilibration overnight in a 5% CO2 incubator. After incubation overnight the fluid had a cloudy appearance. To exclude micro-organism contamination as a cause, cultures for bacteria and fungi were performed but were negative. After the pilot study had shown the capacity for blastocyst development, the study was continued, performing embryo cultures in 12 separate hydrosalpinx fluids. Prolonged cultures were performed in microdrops under mineral oil. In four different experiments, each
Hydrosalpinx fluid and human embryo development
one including three separate hydrosalpinx fluids diluted to 50% and one control group with S2 medium, embryos were cultured after thawing up to 5 days. Development was followed in an inverted microscope daily. The proportion of expanded or hatched blastocysts was calculated for each group. In order to test embryo development in undiluted hydrosalpinx fluid another experiment was undertaken using 100% hydrosalpinx fluid from three different patients for embryo culture. The prolonged culture procedure using microdrops under mineral oil was compared in three additional experiments with embryo cultures in an open system. Embryos were cultured in 50% hydrosalpinx fluids from three different patients, comparing the blastocyst development in the open system with that in microdrops under mineral oil. Implantation was studied using a multilayer in-vitro culture of human endometrium according to Bentin-Ley and co-workers (1994). Three separate experiments were performed under identical conditions using a total of 17 blastocysts that had developed in 50% hydrosalpinx fluid. Statistics Correlation analysis was performed using Pearson’s correlation test. χ2 test was used to compare proportions between groups. A P value less than 0.05 was considered significant.
Results Hydrosalpinx fluid content characteristics are shown in Table I. The only detectable correlation between blastocyst development and hydrosalpinx fluid characteristics was the pH value after equilibration in the CO2 incubator. There was a positive correlation (r 5 0.601) between blastocyst development and increasing pH. There were differences between tubes within the individual, regarding electrolyte concentrations, haemoglobin and consequently protein content and endotoxin levels as shown in Table I. The development of embryos to blastocysts in 50% and 100% hydrosalpinx fluids versus controls are shown in Table II. The development to blastocysts was significantly lower in 100% hydrosalpinx fluid compared with both 50% and controls (P 5 0.027). Figure 1 shows a hatched blastocyst cultured in 50% hydrosalpinx fluid. When comparing the open culture system with that in microdrops under mineral oil, the development of blastocysts in 50% hydrosalpinx fluid differed in the individual experiments but was equal when total numbers were compared (15/ 26 in both systems) (Table III). In the three experiments testing the implantation ability, the blastocysts implanted in 33% (1/3), 80% (4/5) and 100% (9/9). Discussion This is to our knowledge the first report evaluating the developmental capacity of human embryos, related to hydrosalpinx fluid. Previous published studies using murine models have reported conflicting findings with signs of impaired embryo development. Mukherjee et al. (1996) tested embryo development in different concentrations of hydrosalpinx fluid ranging from 0–100%, describing embryo development as cavitation rate. In these experiments, there was a significant negative correlation between cavitation rate and increasing concentrations of hydrosalpinx fluid. A similar design was
Figure 1. A hatched blastocyst 96 h after thawing, cultured in 50% hydrosalpinx fluid. Original magnification 3400. Printed from Leitz DM IRB (Leica).
used by Rawe et al. (1997) who studied mouse blastocyst development in hydrosalpinx fluids at concentrations ranging from 5–20%. A negative correlation was found between blastocyst development and increasing concentrations of hydrosalpinx fluid. In another murine model, the development to blastocysts was affected only when 100% concentration of hydrosalpinx fluid was used as culture medium, as compared with 50% concentration and controls (Murray et al., 1997). Beyler et al. (1997) recently showed impaired blastocyst development of murine embryos when cultured in hydrosalpinx fluid in an open system. They suggested that the difference in results between the open and closed system was due to the presence of embryotoxic lipophilic factors in hydrosalpinx fluid. Interestingly, we were not able to detect any suppression of human blastocyst development at a concentration of 50% hydrosalpinx fluid when comparing the closed and open systems. In our experiments using normal human embryos, we could not detect any difference between blastocyst development in cultures of 50% hydrosalpinx fluid and controls. There was, however, a significant impairment of blastocyst development if cultures were performed in 100% hydrosalpinx fluid compared with 50% or controls. This finding could be explained by the lack of necessary substrate in absence of culture medium. The presence of any embryotoxic factor would 2923
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Table III. Development of blastocysts in cultures with 50% hydrosalpinx fluid from three different patients, comparing open culture system with microdrops under mineral oil
Open system
Microdrops under mineral oil
Hydrosalpinx fluid
Number of embryos
Number of blastocysts
% blastocyst development
2B 3 4A Total 2B 3 4A Total
7 10 9 26 7 10 9 26
6 4 5 15 7 7 1 15
86 40 56 58 100 70 11 58
probably have had an effect on blastocycst development even if diluted to 50%. This theory is supported by Murray et al. (1997) who demonstrated in a murine model that the impaired blastocyst development in 100% hydrosalpinx fluid could to some extent be restored by the addition of lactate, implying that the inhibitory effect of hydrosalpingeal fluid on embryonic development is due to the absence of essential factors. Our experiment comparing cultures under mineral oil with an open culture system did not support the theory of a lipophilic factor with embryotoxic properties. Obviously, the murine model should not be used in studies aimed to gain knowledge of hydrosalpinx fluid influence on human embryos. The only detectable correlation between characteristics of hydrosalpinx fluid and embryo development in our study was pH after equilibration. This finding points out that correct pH is important and that low values are of disadvantage for embryo development. Interestingly, the wide variety in osmolarity and to a certain extent in electrolyte concentration did not seem to be of major importance. Endotoxin analysis showed in the majority of cases extremely low values, with few exceptions. This suggests that in some cases the causative reaction may recently have occurred or that a low grade persistent infection is present. There was no significant correlation between endotoxin levels and blastocyst development. When comparing two hydrosalpinx fluid characteristics within the individual, there were some obvious differences in calcium, protein and haemoglobin levels as well as in endotoxins. This finding suggests that the left and right diseased tube may carry different properties, although it did not yield an obvious difference in blastocyst development. We have not been able to detect any embryotoxic factor that relates to impaired embryo development, suggesting that the potential negative influence of hydrosalpinx on IVF outcome might be on the level of the endometrium. In a murine implantation study, where transfers were performed with hydrosalpinx fluid or culture medium, the number of implantation sites were equal (Rawe et al., 1997). Our implantation experiments were based on a model with normal endometrial cells, although the embryos were cultured in hydrosalpinx fluid. The presence of hydrosalpinx could alter endometrial receptivity, a theory which is supported by Meyer et al. (1997). It was shown that women with hydrosalpinges express significantly less of the αvβ3 integrin, which is a marker of endometrial receptivity. Another plausible explanation of the 2924
potential negative influence of hydrosalpinx is mechanical leakage of fluid from the salpinges, thereby exerting a wash out of the uterine cavity during the period of implantation. There are several case reports of patients with hydrosalpinges describing this phenomenon, which becomes more evident during ovarian hyperstimulation than in natural cycles (Mansour et al., 1991; Bloechle et al., 1997; Sharara and McClamrock, 1997). Our suggestion in a previous publication (Strandell et al., 1994), that circulatory factors and disturbed innervation may contribute negatively, still remains as another plausible explanation for the poor outcome after IVF in women with hydrosalpinges. We conclude from this study that hydrosalpinx fluid in general does not have any major embryotoxic properties on human embryo development to blastocysts. There might be individual variations in the content of hydrosalpinx fluids, yielding different influences on embryo development. The potential negative influence of hydrosalpinx on IVF outcome may have other explanations as well, that may be found among endometrial factors such as receptivity of implantation and direct leakage through the cavity.
Acknowledgements We thank Mesihan Jin for performing the endotoxin analyses. The study was conducted with grants from The Swedish Medical Research Council (2873, 11607), The Swedish Society for Medical Research, Hjalmar Svensson Foundation and the Society Ordensa¨llskapet W6. The Michelsen Foundation, Mimi and Victor Larsens Foundation, Organon Denmark and Serono Nordic are acknowledged for their support of the implantation studies.
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Hydrosalpinx fluid and human embryo development Fleming, C. and Hull, M.G.R. (1996) Impaired implantation after in vitro fertilization treatment associated with hydrosalpinx. Br. J. Obstet. Gynaecol., 103, 268–272. Kassabji, M., Sims, J.A., Butler, L. and Muasher, S.J. (1994) Reduced pregnancy outcome in patients with unilateral or bilateral hydrosalpinx after in vitro fertilization. Eur. J. Obstet. Gynecol. Reprod. Biol., 56, 129–132. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin reagent. J. Biol. Chem., 193, 265–275. Mansour, R.T., Aboulghar, M.A., Serour, G.I. and Riad, R. (1991) Fluid accumulation of the uterine cavity before embryo transfer: a possible hindrance for implantation. J. In Vitro Fert. Embryo Transfer, 8, 157–159. Meyer, W.R., Castelbaum, A.J., Somkuti, S. et al. (1997) Hydrosalpinges adversely affect markers of endometrial receptivity. Hum. Reprod., 12, 1393–1398. Mukherjee, T., Copperman, A.B., McCaffrey, C. et al. (1996) Hydrosalpinx fluid has embryotoxic effects on murine embryogenesis: a case for prophylactic salpingectomy. Fertil. Steril., 66, 851–853. Murray, C.A., Clarke, H.J., Tulandi, T. and Tan, S.L. (1996) Inhibitory effect of human hydrosalpingeal fluid on mouse preimplantation embryonic development is significantly reduced by the addition of lactate. Hum. Reprod., 12, 2504–2507. Rawe, V.J., Liu, J., Shaffer, S., Compton, M.G. et al. (1997) Effect of human hydrosalpinx fluid on murine embryo development and implantation. Fertil. Steril., 68, 668–670. Sawin, S.W., Loret de Mola, J.R., Monzon-Bordonaba, F. et al. (1997) Hydrosalpinx fluid enhances human trophoblast viability and function in vitro: implications for embryonic implantation in assisted reproduction. Fertil. Steril., 68, 65–71. Sharara, F.I. and McClamrock, H.D. (1997) Endometrial fluid collection in women with hydrosalpinx after human chorionic gonadotrophin administration: a report of two cases and implications for management. Hum. Reprod., 12, 2816–2819. Sharara, F.I., Scott Jr, R.T., Marut, E.L. and Queenan Jr, J.T. (1996) In-vitro fertilization outcome in women with hydrosalpinx. Hum. Reprod., 11, 526–530. Strandell, A., Waldenstro¨m, U., Nilsson, L. and Hamberger, L. (1994) Hydrosalpinx reduces in-vitro fertilization/embryo transfer rates. Hum. Reprod., 9, 861–863. Testart, J., Lasalle, B., Belaisch-Allart, J. et al. (1986) High pregnancy rate after early human embryo freezing. Fertil. Steril., 46, 268–272. Vandromme, J., Chasse, E., Lejeune, B. et al. (1995) Hydrosalpinges in in-vitro fertilization: an unfavourable prognostic feature. Hum. Reprod., 10, 576–579. Received on March 18, 1998; accepted on July 14, 1998
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