Effects of human hydrosalpinx fluid on in-vitro murine fertilization

Corinne de Vantéry Arrighi,1, Hervé Lucas, Diaa El-Mowafi, Aldo Campana and Didier Chardonnens

Clinique de Stérilité et d'Endocrinologie Gynécologique, Département de Gynécologie et d'Obstétrique, Maternité, Hôpitaux Universitaires de Genève, Geneva, Switzerland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients with hydrosalpinges show a decrease of both fertility and clinical outcome of IVF and embryo transfer treatment. Several reports have demonstrated the negative effects of hydrosalpinx fluid (HSF) on embryo development and implantation. The aim of this study was to determine whether human HSF, collected from infertile patients, might exhibit a deleterious effect on gametes and fertilization using a murine IVF system. Murine gametes were co-incubated during IVF until first cleavage with human HSF diluted to 50% from four patients (HSF1–4). It was demonstrated that HSF affected fertilization, as determined by the count of the 2-cell embryos. Pre-incubation of spermatozoa with HSF during capacitation significantly lowered the percentage of 2-cell embryos (P < 0.05). While HSF1–3 had no significant effect on motility and viability of spermatozoa, HSF4 almost completely affected their survival. In contrast, pre-incubation of ovulated oocytes surrounded by their cumulus cells with HSF before IVF did not impede first cleavage. Taken together, these results suggest that HSF has a cytotoxic effect on spermatozoa and/or impairs the fertilization process, probably by altering capacitation/acrosome reaction and/or ligand(s)–receptor(s) interactions. Hydrosalpinges may be partly associated with sterility through HSF inhibitory effects on fertilization.

Key words: embryo/hydrosalpinx fluid/IVF/oocyte/spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Tubal disease—an array of disorders including hydrosalpinges—is one of the major causes of female infertility. Hydrosalpinx is a Greek word that means a Fallopian tube filled with fluid (Bloechle, 1999Go). Hydrosalpinx fluids (HSF) vary in their electrolytes, glucose, proteins, haemoglobin, cytokine and endotoxin concentrations, and in osmolarity and pH (Amnon et al., 1969Go; Gott et al., 1990Go; Beyler et al., 1997Go; Strandell et al., 1998Go). Nevertheless, HSF contain electrolyte concentrations similar to those in serum, and concentrations of steroid hormones, progesterone, oestradiol and total proteins lower than in follicular fluid (Mukherjee et al., 1996Go; Granot et al., 1998Go).

Several reports have demonstrated the negative impact of hydrosalpinges on the clinical outcome of IVF and embryo transfer treatment. Patients with hydrosalpinges have diminished implantation and pregnancy rates, and higher miscarriage and ectopic pregnancies rates (Andersen et al., 1994Go; Kassabji et al., 1994Go; Strandell et al., 1994Go; Vandromme et al., 1995Go; Fleming and Hull, 1996Go; Katz et al., 1996Go; Blazar et al., 1997Go; Camus et al., 1999Go; Cohen et al., 1999Go). It seems that aspiration of HSF or surgical treatment of hydrosalpinges are of benefit prior to IVF–embryo transfer (Strandell et al., 1999Go).

Several studies were conducted on the potential negative effects of HSF on human embryos (Freeman et al., 1998Go; Strandell et al., 1998Go). It has been demonstrated that in vitro, HSF increased human embryo degeneration, growth arrest and abnormal development, and diminished blastulation (Freeman et al., 1998Go). In contrast, others (Strandell et al., 1998Go) showed that HSF did not generally exert any major negative effects on in-vitro embryo development and implantation process. Other investigators used an in-vitro murine model to document whether HSF collected from infertile patients might exhibit a deleterious effect on embryo mouse development. Murine embryo survival and/or development was significantly impaired in the presence of increasing concentrations of HSF, regardless of the stage at which embryos were exposed to HSF (Mukherjee et al., 1996Go; Beyler et al., 1997Go; Rawe et al., 1997Go; Sachdev et al., 1997Go; Spandorfer et al., 1999Go). However, others (Koong et al., 1998Go) reported that only 100% HSF has a mildly negative effect on blastocyst development rate.

Several mechanisms might explain the poor fertility prognosis of patients with hydrosalpinges and the potential toxicity exhibited by HSF on embryos and/or endometrium (Lass, 1999Go): (i) diminished ovarian function, follicular development and/or oocyte availability (Strandell et al., 1994Go; Freeman et al., 1998Go); (ii) tubal damage (Strandell et al., 1994Go); (iii) a purely dilutional effect of HSF on essential nutrients and substrates (Murray et al., 1997Go; Spandorfer et al., 1999Go); (iv) a direct cytotoxic effect on gametes and embryos (Mukherjee et al., 1996Go; Beyler et al., 1997Go; Rawe et al., 1997Go; Nackley and Muasher, 1998Go); (v) immunization against the human heat-shock proteins (Witkin et al., 1994Go, 1995Go; Neuer et al., 2000Go); (vi) an inflammatory response with production of pro-inflammatory cytokines (Simon et al., 1994Go); (vii) a simple mechanical problem for embryo implantation due to the leakage of HSF in the uterine cavity (Mansour et al., 1991Go; Andersen et al., 1994Go); and/or (viii) a reduced endometrial receptivity for embryos due to a reduced endometrial integrin expression (Andersen et al., 1994Go; Meyer et al., 1997Go).

Several studies have been carried out with regard to the effect of HSF on IVF–embryo transfer outcome, and on the development of human and murine embryos and implantation. However, to our knowledge, no investigations have been performed on mouse gametes, spermatozoa–oocyte interaction and fertilization. The aim of this study was to determine whether HSF might exhibit a deleterious effect on gametes and fertilization using an in-vitro murine system. The effect of human HSF collected from infertile patients on both murine gametes and fertilization was investigated in vitro. This methodology mimics the pathological conditions of gamete interaction in the tubal secretions containing HSF. This might be the case upon natural conception or intrauterine insemination with one patent tube and a contralateral hydrosalpinx.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Animals
All procedures involving mice were conducted in accordance with the Switzerland `Office Vétérinaire Cantonal' authorizations and regulations concerning the use and care of experimental animals.

Female B6D2F1 and male OF1 mice were obtained from RCC Ltd, Biotechnology & Animal Breeding Division (Füllinsdorf, Switzerland). Female mice aged 4–6 weeks were superovulated with i.p. injections of 5–7 IU of pregnant mare's serum gonadotrophin (PMSG; Folligon®; Intervet, Boxmeer, The Netherlands), followed 48 h later by 5–7 IU of human chorionic gonadotrophin (HCG; Choluron®; Intervet, Boxmeer, Holland).

Preparation of culture media
Media were prepared fresh weekly. All components were purchased from Sigma Chemical Co. (St Louis, MO, USA).

HEPES-buffered, modified KSOM medium (95 mmol/l NaCl, 2.5 mmol/l KCl, 0.35 mmol/l KH2PO4, 0.2 mmol/l MgSO4, 10 mmol/l sodium lactate, 0.2 mmol/l sodium pyruvate, 5.56 mmol/l glucose, 1 mmol/l glutamine, 4 mg/ml bovine serum albumin (BSA), 0.01 mmol/l EDTA tetrasodium salt, 5 mmol/l NaHCO3, 20 mmol/l HEPES, 1.71 mmol/l CaCl2.2H2O) was used for oocyte collection, IVF and embryo culture (Summers et al., 1995Go).

T6 medium (99.4 mmol/l NaCl, 1.42 mmol/l KCl, 0.47 mmol/l MgCl2.6H2O, 0.36 mmol/l Na2HPO4.12H2O, 1.78 mmol/l CaCl2.2H2O, 25 mmol/l NaHCO3, 24.9 mmol/l sodium lactate, 0.47 mmol/l sodium pyruvate, 5.56 mmol/l glucose, 100 U/ml penicillin, 0.01 g/l phenol red) containing 20 mg/ml BSA (T6-BSA medium) was used for sperm preparation and capacitation.

Collection of oocytes
Superovulated females B6D2F1 mice aged 4–6 weeks were killed by cervical dislocation 14–16 h following HCG injection, the peritoneal cavity was exposed, and the two oviducts were removed and placed in a 1 ml drop of HEPES-buffered, modified KSOM medium at 37°C. Cumulus masses (cumulus–oocyte complexes) were removed by tearing the oviducts with a needle at the site of the cumulus bulge in a drop of 200 µl HEPES-buffered, modified KSOM medium at 37°C under embryo-tested mineral oil (Sigma). Cumulus masses were separated in two groups and transferred to 50 µl drops of HEPES-buffered, modified KSOM medium at 37°C under mineral oil prior to IVF.

Sperm preparation
Male OF1 mice aged 10–14 weeks were killed by cervical dislocation. After exposure of the peritoneal cavity, both caudal epididymides were removed and placed in 1 ml T6 medium at 37°C. Fat and vessels were discarded, and each caudal epididymis was transferred to a 150–200 µl drop of T6-BSA medium under mineral oil. The epididymal contents were squeezed out by using a watchmaker's forceps. The residual caudal tissue was discarded. After 10 min at 37°C in a humidified atmosphere of 5% CO2, 100–150 µl of spermatozoa were recovered and transferred to a 5 ml tube containing 350 µl of T6-BSA medium for capacitation. Capacitation was allowed to proceed for 2 h at 37°C in a humidified atmosphere of 5% CO2. Sperm concentration and motility were determined using a Makler chamber and a slide-cover slide respectively.

IVF
IVF was carried out in 50 µl drops of HEPES-buffered modified KSOM medium at 37°C under mineral oil. A capacitated sperm suspension was gently added to the oocytes to give a final motile sperm concentration of 1–2x106/ml. Co-incubation of the gametes was allowed to proceed for 4 h. Potentially fertilized zygotes were washed free of excess spermatozoa and transferred to 50 µl drops of fresh HEPES-buffered modified KSOM medium at 37°C under mineral oil. Cleavage was determined the next day by counting the number of 2-cell embryos.

Hydrosalpinx fluids
HSF were aspirated at the time of oocyte retrieval for IVF from four infertile patients attending the Clinic for Infertility and Gynaecological Endocrinology-WHO Collaborating Centre, University Hospital of Geneva, Geneva, Switzerland. These patients were undergoing IVF–embryo transfer. Patients 1, 2, 3 and 4 had tubal factor sterilities, and patient 4 also had a severe endometriosis. HSF were frozen at –80°C immediately following aspiration. Before use, HSF were thawed, centrifuged at 1000 g for 10 min and filtered with a 0.2 µm Millex (Millipore, Millipore Corporation, Bedford, MA, USA). The pH values of pure HSF ranged from 7.5 to 9.5 (significantly higher than the physiological range) as determined by narrow-range pH indicator paper (Merck, Darmstadt, Germany). It has been reported that correction of pH to that of media did not improve cavitation rates in cultured mouse embryos (Mukherjee et al., 1996Go). Nevertheless, the pH was reduced to physiological values (7–7.5) after dilution of HSF to 50% with culture medium. The osmolarity of HSF was 270–282 mOsm (within physiological range) and similar to that of culture medium HEPES-buffered, modified KSOM medium (280 mOsm), as determined by a digital osmometer (5500, vapour pressure; Wescor Inc., Logan, UT, USA).

Experimental design
To simulate the in-vivo conditions in studying the effect of HSF on fertility, the potential toxicity of HSF on fertilization was tested using an in-vitro murine model.

In one set of experiments, mice spermatozoa and oocytes were co-incubated with media containing 50% HSF during the IVF procedure until fertilized oocytes underwent first cleavage (16–20 h). In another set of experiments, mouse spermatozoa or oocytes were pre-incubated (2 h and 1 h respectively) prior to the IVF procedure with media containing 50% HSF. Each HSF (HSF1, 2, 3 or 4) and its respective control (C1, 2, 3 or 4) was tested on ovulated oocytes withdrawn from the same female, which were separated into two batches. For each HSF tested, four to seven females [as indicated in Results and figure legends (n)] were used in two or three independent manipulations. For each manipulation, all HSF (HSF1, 2, 3 and 4) and their respective controls (C1, 2, 3 or 4) were tested on spermatozoa obtained from the same male. The goal was to reduce the potential variations existing between oocytes and spermatozoa from different mice. For each set of experiments (co-incubation of both gametes, pre-incubation of spermatozoa, and pre-incubation of oocytes with HSF), the fertilization rate was determined by the count of the 2-cell embryos, i.e. the first cleavage rate.

Co-incubation of both gametes with hydrosalpinx fluid
Spermatozoa and oocytes were processed for IVF, as described above, in HEPES-buffered modified KSOM medium containing 50% of HSF1, 2, 3 or 4. Co-incubation of the gametes was allowed to proceed for 4 h. The potentially fertilized oocytes were then washed to remove excess spermatozoa, and transferred to either fresh HEPES-buffered modified KSOM medium containing 50% of one of HSF1, 2, 3 or 4, or to medium only as control, until the first cleavage (16–20 h co-incubation).

Pre-incubation of spermatozoa or ovulated oocytes with hydrosalpinx fluid
Spermatozoa from both caudal epididymides were transferred to a 5 ml tube containing 300–400 µl of T6-BSA medium and immediately divided into samples. Each spermatozoa sample was incubated in T6-BSA medium (control) or T6-BSA medium with 50% of HSF1, 2, 3 or 4 during the 2 h of capacitation. Following capacitation, sperm samples were washed with 10 ml of T6-BSA medium and centrifuged at 300 g for 10 min. The sperm pellets were resuspended in fresh T6-BSA medium and used for IVF, as described above.

The cumulus masses (ovulated oocytes surrounded by their cumulus cells) were incubated for 1 h in HEPES-buffered modified KSOM medium (control) or HEPES-buffered modified KSOM medium containing 50% of HSF1, 2 or 3. Following this pre-incubation, cumulus masses were washed and transferred to fresh HEPES-buffered modified KSOM medium, and used for IVF, as described above.

Statistical analysis
For statistical analysis, Student's t-test for paired samples at the 95% significance level and SEM were calculated.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Co-incubation of spermatozoa and oocytes with hydrosalpinx fluid during IVF
The potential negative effects of human HSF on mouse spermatozoon–oocyte interaction and fertilization rate were evaluated by co-incubating the gametes with 50% HSF1, 2, 3 or 4 during the 4 h of IVF until first cleavage, 2-cell embryos (16–20 h). Fertilization rates were analysed under control and HSF conditions.

Co-incubation of spermatozoa and cumulus masses with 50% HSF1, 2 or 3 significantly affected fertilization, as determined by the count of the 2-cell embryos (Figure 1Go). There were significant differences in the percentage of 2-cell embryos for HSF1, 3 and 4 (9.25 ± 10.31, 51.88 ± 16.43 and 0.15 ± 0.17 respectively) compared with their respective controls (84.81 ± 7.99, 84.72 ± 6.99 and 78.85 ± 7.60), but not between HSF2 (55.09 ± 18.35) and its control (69.98 ± 9.32). P-values calculated using Student's t-test were respectively for HSF1, 2, 3 and 4: P = 0.001 (n = 5), P = 0.155 (n = 5), P = 0.016 (n = 4) and P = 0.0002 (n = 5).



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Figure 1. Effect of co-incubating spermatozoa and oocytes with hydrosalpinx fluid (HSF) during IVF until first cleavage on the fertilization rate (i.e. percentage of 2-cell embryos). Data are shown as mean ± SEM for HSF1, 2, 3 and 4 and their respective controls with respectively P < 0.05 (n = 5), P > 0.05 (n = 5), P < 0.05 (n = 4) and P < 0.05 (n = 5). {blacksquare}, controls; {square}, HSF added.

 
Pre-incubation of spermatozoa with hydrosalpinx fluid
The potential negative effects of human HSF on mouse spermatozoa were evaluated by pre-incubating the spermatozoa during capacitation (2 h) with 50% HSF1, 2, 3 or 4. The motility of capacitated spermatozoa in control and 50% HSF conditions was determined before they were used for IVF. Fertilization rates were analysed under control and 50% HSF conditions.

The motility of spermatozoa capacitated with 50% HSF1, 2 or 3 was comparable with that of spermatozoa capacitated in control medium. In contrast, all spermatozoa incubated with HSF4 were completely immobilized in less than 1 h. These spermatozoa were all dead, indicating that HSF4 had a potent cytotoxic (spermicidal) effect.

Pre-incubation of spermatozoa with 50% HSF1, 2 or 3 affected fertilization rates (Figure 2Go), there being significant differences in the percentage of 2-cell embryos for HSF1, 2 and 3 (59.72 ± 13.69, 58.87 ± 12.02 and 52.14 ± 11.10 respectively) compared with their respective controls (89.76 ± 7.58, 85.91 ± 4.68 and 85.08 ± 6.37). The P-values calculated using Student's t-test were respectively: P = 0.044 (n = 7), P = 0.046 (n = 7) and P = 0.013 (n = 7).



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Figure 2. Effect of pre-incubating spermatozoa with hydrosalpinx fluid (HSF) during the 2 h of capacitation before IVF on the fertilization rate (i.e. percentage of 2-cell embryos). Data are shown as mean ± SEM for HSF1, 2 and 3 and their respective controls with P < 0.05 (n = 7). {blacksquare}, controls; {square}, HSF added.

 
Pre-incubation of ovulated oocytes with hydrosalpinx fluid
The potential negative effects of human HSF on mouse oocytes were evaluated by pre-incubating, 1 h before IVF, ovulated oocytes surrounded by their cumulus cells (cumulus masses) with 50% HSF1, 2 or 3. Fertilization rates were analysed under control and 50% HSF conditions.

Pre-incubation of oocytes with 50% HSF1, 2 or 3 did not affect fertilization rates (Figure 3Go), since comparable numbers of 2-cell embryos were obtained under control and HSF conditions. There was no significant difference between HSF1, 2 and 3 (68.51 ± 14.09, 67.77 ± 13.56 and 78.38 ± 9.42 respectively) compared with their respective controls (70.72 ± 14.27, 70.76 ± 8.50 and 77.91 ± 6.46). The P-values calculated using Student's t-test were respectively for HSF1, 2 and 3: P = 0.444 (n = 4), P = 0.343 (n = 5) and P = 0.477 (n = 5).



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Figure 3. Effect of pre-incubating ovulated oocytes (cumulus masses) with hydrosalpinx fluid (HSF) 1 h before IVF on the fertilization rate (i.e. percentage of 2-cell embryos). Data are shown as mean ± SEM for HSF1, 2 and 3 and their respective controls with respectively P > 0.05 (n = 4), P > 0.05 (n = 5) and P > 0.05 (n = 5). {blacksquare}, controls; {square}, HSF added.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Apart from mechanical tubal obstruction, there is increasing evidence that hydrosalpinx fluid may have a direct negative impact on fertility. Since contact between HSF and spermatozoa and oocytes is possible in the uterus and/or Fallopian tubes, the effects of HSF on fertilization were analysed. This study showed the detrimental effect of human HSF on spermatozoa and fertilization in a murine IVF system.

The first set of experiments (Figure 1Go), in which murine spermatozoa and oocytes were co-incubated with human HSF diluted to 50% during the IVF procedure, showed that HSF1, 3 and 4 each significantly affected fertilization rates, whereas the effect of HSF2 was not significant. These results showed that HSF have negative effects on fertilization, most likely by altering the spermatozoan and/or oocyte receptor(s) implicated in fertilization. Indeed, a tendency was observed that fewer spermatozoa were bound to the zona pellucida of oocytes in the presence of HSF compared with control (data not shown). To our knowledge, this is the first report showing that human HSF affects fertilization in a murine model. The only previous study investigating HSF and fertilization was clinically based, and monitored the impact of HSF on successful pregnancy in tubal factor infertility treated by IVF (Blazar et al., 1997Go). These authors reported that HSF did not decrease fertilization rates, which were comparable in women with and without HSF.

Further analysis was carried out on the effect of HSF on each gamete independently. In a second series of experiments (Figure 2Go), murine spermatozoa were pre-incubated with human HSF diluted to 50% during capacitation prior to an IVF procedure. HSF1, 2 and 3 significantly affected fertilization rates, most likely by altering capacitation and/or acrosome reaction and/or by altering spermatozoa receptor(s) involved in the fertilization process. Again, there was a tendency for fewer spermatozoa to be bound to the zona pellucida of oocytes in the presence of HSF compared with control (data not shown). These results suggest that pre-incubation of HSF with spermatozoa during capacitation is sufficient to have a negative impact on their fertilizing ability. In the presence of HSF4, spermatozoa did not survive; thus, HSF may also have a direct cytotoxic effect on spermatozoa. However, HSF4 was collected from a patient with a severe endometriosis, and this pathology might explain the cytotoxic effect of HSF4. It has been reported that peritoneal fluid from patients with endometriosis affected human sperm function in vitro (Arumugam, 1994Go; Liang et al., 1994Go; Tasdemir et al., 1995Go; Oral et al., 1996Go), and also reduced the mean number of hamster eggs penetrated by human spermatozoa (Aeby et al., 1996Go). Indeed, HSF typically results from tubal occlusion that occurs after an infectious process, which leads to the accumulation of fluid that may contain cytokines, leukotrienes, prostaglandins, or other components that potentially are detrimental to gametes and/or embryos (Ben-Rafael and Orvieto, 1992Go). Cytokines have been shown to inhibit the development of mice pre-implantation embryos in culture systems (Jakobs et al., 1992Go; Taketani et al., 1992Go). The results of the current study showed that the presence of HSF (either during IVF or capacitation of spermatozoa) lowered fertilization rates, but in differing proportions depending on the HSF tested. During IVF, HSF1, 2 and 3 induced an inhibition of fertilization of 89, 21 and 39% respectively, and during capacitation they reduced fertilization rates by 34, 32 and 39%, respectively. Thus, HSF from different patients have variable degrees of effect on the percentage of 2-cell embryos obtained by IVF. This observation is not surprising, since women with hydrosalpinges comprise a heterologous group, there being individual variations in the content of HSF with regard to concentrations of electrolytes, glucose, proteins, haemoglobin, endotoxins, cytokines, as well as in osmolarity and pH (Amnon et al., 1969Go; Gott et al., 1990Go; Beyler et al., 1997Go; Granot et al., 1998Go; Strandell et al., 1998Go). These differences might explain the variations in the effects of HSF on gametes, fertilization, embryo development, implantation and pregnancy (Strandell et al., 1994Go; Vandromme et al., 1995Go; Fleming and Hull, 1996Go; Katz et al., 1996Go; Sharara et al., 1996Go; Blazar et al., 1997Go; Ng et al., 1997Go; Freeman et al., 1998Go). In the same way, it was stated (Beyler et al., 1997Go) that mouse embryo development was significantly impaired in the presence of HSF tested, but with variation in potency. Some patients produced HSF that caused degeneration of all zygotes, whereas HSF from other patients caused increased degeneration and retardation of development compared with controls.

Finally, in the third set of experiments (Figure 3Go), murine cumulus masses were pre-incubated with human HSF diluted to 50% prior to IVF procedure. HSF1, 2 and 3 did not reduce fertilization rates, and comparable amounts of spermatozoa were bound to the zona pellucida of oocytes under both control and HSF conditions (data not shown). These results suggest that HSF have little or no effect on the ability of oocytes to be fertilized. However, it cannot be excluded that cumulus cells may protect oocytes from the negative effects of HSF during the 1 h pre-incubation of cumulus masses with HSF. Few studies have been carried out on the effects of HSF on gametes. An indirect study suggested that hydrosalpinges may have a permanent negative influence on human ovarian function, follicular development and oocyte quality, since blastulation and implantation were seen to remain low, while in-vitro growth arrest and degeneration remained high, despite surgical treatment of hydrosalpinges (Freeman et al., 1998Go). Only two groups studied the direct effects human HSF on human gametes (Schats et al., 1997Go; Ng et al., 2000Go). In the first of these studies (Schats et al., 1997Go), the effect on the survival of spermatozoa from two proven fertile men was tested for three HSF samples. It was found that undiluted or 50%-diluted HSF did not impair survival of the spermatozoa after incubation for 1 h or 18 h. However, taking into account the variable effects of the different HSF and the fact that fertile human spermatozoa were used, a potential cytotoxic effect of HSF on spermatozoa could not be excluded. In the second study (Ng et al., 2000Go), the effects of different concentrations of HSF on human sperm mobility and survival after various periods of incubation was investigated. These authors reported that 50% and 100% HSF were each potentially cytotoxic following 24 h of incubation.

Although the targets of HSF are not known, adhesion molecules such as integrins might represent one such target. It has been proposed that alteration in endometrial integrin production was associated with hydrosalpinges (Meyer et al., 1997Go). All mammalian spermatozoa (Klentzeris et al., 1995Go; Fusi et al., 1996Go; Reddy et al., 1998Go; Bronson et al., 1999Go) and oocytes (Tarone et al., 1993Go; Evans et al., 1995Go, 1997Go, 1998Go; de Nadai et al., 1996Go; Capmany et al., 1998Go) express integrins/disintegrins at their surface that may be putative determinants in gametic interaction, in adhesion–fusion mechanisms, triggered by fertilization (Evans, 1999Go). A positive correlation was shown between the expression of ß1 integrins and the fertilizing ability of human spermatozoa (Klentzeris et al., 1995Go). Indeed, the integrin {alpha}6ß1 serves as a spermatozoan receptor that mediates spermatozoon–oocyte binding (Almeida et al., 1995Go; Sueoka et al., 1997Go).

The present study was performed using four different HSF, which is effectively a small number. Nevertheless, the HSF tested affected sperm viability or their fertilizing ability. Although this is probably not the case for all HSF, it should be borne in mind that at least some HSF can have negative effects on gametes and fertilization. These experiments were all conducted on murine gametes and IVF, using human HSF diluted to 50%, this dilution being chosen because it may mimic the in-vivo environment while avoiding a too-high dilution effect. The dilution effect of HSF on essential nutrients and substrates as a potential mechanism explaining the toxicity has been studied by others (Murray et al., 1997Go; Spandorfer et al., 1999Go). Indeed, it was shown that at 100% concentration of HSF, toxicity is somewhat mitigated by the addition of lactate (Murray et al., 1997Go), whereas others (Spandorfer et al., 1999Go) were unable to show a significant impact on embryo development when the dilution effect of HSF was simulated with normal (0.9%) saline. The significantly high pH of pure HSF should not contribute to the negative effects of HSF on gametes and/or embryos, since pH was reduced to physiological values after dilution of HSF to 50% with culture medium. Moreover, it has been reported that correction of pH to that of the media did not improve cavitation rates in cultured mouse embryos compared with non-correction of pH (Mukherjee et al., 1996Go). The osmolarity of HSF was within physiological range, and similar to that of the culture medium.

The in-vitro model used in these studies does not closely mimic the in-vivo environment of IVF–embryo transfer to study the possible toxicity of HSF. In IVF–embryo transfer, human embryos transferred to a patient with hydrosalpinges are placed within the endometrium, which is known actively to secrete a variety of growth factors that play a role in early embryonic development and probably help to detoxify HSF (Spandorfer et al., 1999Go). Indeed, these authors studied the effect of HSF on mouse embryo development using an endometrial co-culture system. They demonstrated that high concentrations of HSF are required to impede embryogenesis to a significant extent: 100% for a decrease in blastocyst formation, and 75% for hatching and outgrowth stages instead of 50% without co-culture. Although the use of a murine model can be criticised for testing of the potential negative effects of human HSF on gametes and/or embryos, it is used routinely in the quality control of medium and culture conditions in fertility centres. Moreover, if HSF-negative effects are mediated through alteration of spermatozoa disintegrins/oocyte integrins or other cell adhesion molecules implicated in gamete interaction, the murine model could likely be extrapolated to the human situation (Bronson and Fusi, 1996Go; Sueoka et al., 1997Go; Glander et al., 1998Go; Evans, 1999Go).

Taken together, the results of the present study showed that HSF may have negative effects on spermatozoa, gamete interactions and/or fertilization until the first cleavage, with effects of variable intensity among different HSF. In contrast, HSF has no effect on cumulus masses to be used for IVF. Further studies will be required to elucidate the mechanisms by which HSF impede the fertilization rate, for example on: (i) capacitation; (ii) the acrosome reaction; (iii) spermatozoa–zona pellucida and/or spermatozoa–oolemma interactions; (iv) oocyte activation upon fertilization; and/or (v) embryo first cleavage and development. In conclusion, hydrosalpinges may be partly associated with sterility through HSF inhibitory effects on fertilization.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was supported by the HUG (Hôpitaux Universitaires de Genève), Geneva, Switzerland. We are grateful to I.Wagner for technical help.


    Notes
 
1 To whom correspondence should be addressed at: Clinique de Stérilité et d'Endocrinologie Gynécologique, Département de Gynécologie et d'Obstétrique, Maternité, Hôpitaux Universitaires de Genève, 30, bd de la Cluse, 1211 Geneva 14, Switzerland.E-mail: Corrine.deVantery{at}heuge.ch Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Aeby, T.C, Huang, T. and Nakayama, R.T. (1996) The effect of peritoneal fluid from patients with endometriosis on human sperm function in vitro. Am. J. Obstet. Gynecol., 174, 1779–1783.[ISI][Medline]

Almeida, E.A., Huovila, A.P., Sutherland, A.E. et al. (1995) Mouse egg integrin {alpha}6ß1 functions as a sperm receptor. Cell, 81, 1095–1104.[ISI][Medline]

Amnon, D., Garcia, C.R. and Czernbilsky, B. (1969) Human hydrosalpinx: histologic study and chemical composition of fluid. Am. J. Obstet. Gynecol., 105, 400–411.[ISI][Medline]

Andersen, A.N., Yue, Z., Meng, F.J. et al. (1994) Low implantation rate after in-vitro fertilization in patients with hydrosalpinges diagnosed by ultrasonography. Hum. Reprod., 9, 1935–1938.[Abstract]

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Submitted on July 18, 2000; accepted on .