Adverse effects of hydrosalpinx fluid on sperm motility and survival

Human Reproduction vol.15 no.4 pp.772 777, 2000 Adverse effects of hydrosalpinx fluid on sperm motility and survival Ernest Hung Yu Ng 1, Louis C.Ajonuma, Estella Yee to demonstrate any adverse effect. Only hydrosalpinges visible Lan Lau, William Shu Biu Yeung and on ultrasound scanning were found to be associated with Pak Chung Ho reduced implantation and pregnancy rates in IVF treatment (Andersen et al., 1994; Wit et al, 1998). These clinical data Department of Obstetrics and Gynaecology, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong Special suggest that patients with tubal infertility with hydrosalpinx is Administrative Region, People s Republic of China a heterogeneous group and may have different effects on the 1 To whom correspondence should be addressed outcome of IVF treatment. Although the exact mechanisms are not fully known, there The negative impact of hydrosalpinx on IVF outcome is are several possibilities suggested for the implantation failure well recognized but some reports indicate that tubal infertil- associated with the presence of hydrosalpinx. Fluid accumulaity with hydrosalpinx is a heterogeneous entity and may tion in the uterine cavity due to back passage of fluid have different effects on the outcome. The embryotoxic can occasionally be observed during transvaginal ultrasound effects of hydrosalpinx fluid (HF) have been documented monitoring for IVF cycles in patients with hydrosalpinx in mouse but not human embryos. This study examined (Mansour et al., 1991; Andersen et al., 1996). Hydrosalpinx the effects of HF on sperm motility and survival after fluid (HF) within the uterine cavity may act as a mechanical various periods of incubation. Fifteen infertile patients barrier to implantation as the replaced embryos would be with hydrosalpinx shown on ultrasound monitoring during flushed out or the normal apposition of the endometrial cells ovarian stimulation underwent aspiration of HF after egg is disrupted (Edwards, 1992). collection. Electrolytes, glucose and pyruvate concentra- HF may contain micro-organisms, debris, toxins or cytokines tions were within the physiological ranges found in normal or prostaglandins, which can exert a direct detrimental effect human tubal fluid. Sperm motility and velocities remained on the receptivity of the endometrium (Meyer et al., 1997) or unchanged after 5 h of incubation with various concentra- on the developing embryos inside the uterus. The embryotoxic tions of HF but the percentage of motile spermatozoa was effects of HF were documented only in mouse embryos significantly reduced after 24 h of incubation. Both 50 (Mukherjee et al., 1996; Beyler et al., 1997; Murray et al., and 100% HF were potentially cytotoxic (survival indices 1997; Rawe et al., 1997; Sachdev et al., 1997; Koong et al., <85%). The detrimental effect seemed to be dependent on 1998) but not in human embryos (Granot et al., 1998; Strandell the concentrations of HF. Low osmolarity, low lactate et al., 1998). Inter-species difference (mouse versus human) concentrations or the protein content may be responsible and small sample sizes of 2 10 HF samples in these studies may for the loss of sperm motility. A human sperm survival account for the contrasting results in relation to embryotoxicity. test using HF may be useful in selecting appropriate The human sperm survival test (Critchlow et al., 1989) is a treatment options for patients with hydrosalpinx under- simple, reliable and objective quality control procedure to test going IVF treatment or tubal surgery. the suitability of various materials and culture media for use Key words: CASA/hydrosalpinx fluid/sperm motility and velo- in many human IVF laboratories. The objective of this study cities was to examine the effects of various concentrations of HF on sperm motility and survival after various periods of incubation. This prospective study was carried out at the Assisted Reproduction Unit, Department of Obstetrics and Gynaecology, Queen Mary Hospital, Hong Kong. Ethical approval was obtained from the Ethics Committee, Faculty of Medicine, the University of Hong Kong and every patient gave written informed consent to participate in the study. Before the couples were enrolled into our IVF programme, they underwent a standard protocol of investigations including semen analyses and early follicular serum FSH concentrations. Tubal patency was assessed by a hysterosalpingogram and/or a diagnostic laparos- copy/hysteroscopy. The details of the ovarian stimulation regimen used at our centre had been previously published (Ng et al., 1997). Introduction IVF was first used to treat an infertile woman with severe tubal damage (Steptoe and Edwards, 1978). Tubal infertility still remains the most common indication for IVF treatment in many assisted reproduction units. Many recent reports (Andersen et al., 1994; Kassabji et al., 1994; Strandell et al., 1994; Vandromme et al., 1995; Fleming et al., 1996; Wainer et al., 1997; Murray et al., 1998) and two meta-analyses (Zeyneloglu et al., 1998; Camus et al., 1999) confirmed that the presence of hydrosalpinx significantly impaired the implantation and pregnancy rates after IVF treatment. Some studies (Sharara et al., 1996; Ng et al., 1997), however, failed Materials and methods 772 European Society of Human Reproduction and Embryology

Adverse effects of hydrosalpinx fluid In short, human menopausal gonadotrophin (Pergonal, Serono, fields were selected randomly during evaluation, and 500 spermatozoa Switzerland) injection was started after pituitary down-regulation were analysed in each specimen. with buserelin (Supracur, Hoechst, Frankfurt, Germany) nasal spray. The following parameters were determined: percentage of motile The transvaginal ultrasound-guided oocyte retrieval (oocyte retrieval) spermatozoa (MOT), mean curvilinear velocity (VCL, µm/s), mean was scheduled 36 38 h after the human chorionic gonadotrophin straight line velocity (VSL, µm/s), average path velocity (VAP, µm/ (HCG) injection. Routine prophylactic antibiotics, 1 g ampicillin s), mean linearity (LIN: VSL/VCL), amplitude of lateral head (Bristol-Meyers Squibb, NJ, USA) and 0.5 g metronidazole (McGaw displacement (ALH, µm), head beat cross frequency (BCF, Hz), Inc., Irvine, CA, USA), were given i.v. 30 min prior to oocyte retrieval. and percentage of spermatozoa exhibiting hyperactivation (HA). Spermatozoa satisfying the criteria LIN 65, VCL 100 µm/s, Aspiration of HF after oocyte retrieval ALH 7 µm (Burkman, 1991) were classified as hyperactivated Only those patients with hydrosalpinx visible on ultrasound scanning spermatozoa. on the day of HCG were recruited. They underwent aspiration of HF after all follicles of 10 mm in diameter were aspirated and the Human sperm survival test needle was flushed several times with culture media. Both sides were One semen sample was again used for each HF sample. Semen samples aspirated in cases of bilateral hydrosalpinges and HF was pooled for were processed by a swim-up method after complete liquefaction and analysis. The first part of HF was sent for culture of aerobic and the sperm concentration was adjusted to 5 10 6 /ml. Spermatozoa anaerobic organisms. The fluid was then centrifuged at 500 g for 20 from each sample were divided into four equal portions. A 0.5 ml min and the supernatant was frozen at 20 C prior to further analysis aliquot of the adjusted sperm suspension was centrifuged at 500 g and tests. for 5 min after adding 2 ml of EBSS/BSA. The supernatant was discarded as much as possible and the sperm pellet was overlaid with Hormonal and biochemical profile 0.5 ml of 10%, 50% and 100% of HF (test) and 0.5 ml of EBSS/ After thawing at room temperature, HF samples were checked BSA (control). for oestradiol, progesterone, sodium, potassium, chloride, calcium, The test and the control tubes were placed inside a modular phosphate, bicarbonate, glucose, protein, pyruvate and lactate. Oestra- chamber gassed with 5% CO 2 and left at room temperature for 4 diol was measured using a commercially available radioimmunoassay days. The sperm suspensions were agitated every 24 h and a drop kit (Diagnostic Products Corporation, Los Angeles, CA, USA). The was removed from each tube for examination of sperm motility. For sensitivity of the assay was 8 pg/ml (conversion factor to SI unit, the assay to be regarded as valid, 70% of spermatozoa in the control 3.67), and the inter-assay and intra-assay coefficients of variation tube had to show progressive motility after 4 days of culture. The were 4.2 and 4.0% respectively. Progesterone was measured using a progressive motility of the test sample on day 4 was then used to commercially available radioimmunoassay kit (Chiron Diagnostics calculate a survival index: Corporation, MA, USA). The sensitivity of the assay was 0.11 ng/ Survival index % progressive motility of test/% progressive ml (conversion factor to SI unit, 3.18), and the inter-assay and intra- motility of control assay coefficients of variation were 9.4 and 8.4% respectively. The A survival index of 0.85 was considered to be potentially biochemical assays were performed in the general hospital laboratory. cytotoxic (Critchlow et al., 1989). Sperm motility analysis Statistical analysis HF samples were thawed at room temperature and diluted with Data were expressed as median (range). Correlation was assessed by Earle s balanced salt solution (EBSS: Flow Laboratories, Irvine, UK) the Pearson method and comparison was carried out using the supplemented with sodium bicarbonate, sodium pyruvate, penicillin- Kruskal Wallis test. Difference between groups, if present, was G, streptomycin sulphate and 0.3% bovine serum albumin (BSA), compared by the Mann Whitney test. Paired data were analysed with i.e. EBSS/BSA to 10 and 50% concentration. ph and osmolarity of the Friedman test. A P-value (two-tailed) of 0.05 was considered EBSS/BSA and HF at different concentrations were measured. to be significant. Semen samples were collected by masturbation from 15 men attending the assisted reproduction unit and only samples with normal semen parameters according to WHO criteria (WHO, 1992) were Results used in this study. One semen sample was used for each HF sample. After complete liquefaction at room temperature, semen samples were Between April 1997 and January 1999, 15 patients underwent processed through a two-gradient Percoll preparation, i.e. 45 and 90% aspiration of HF. The median volume of HF aspirated was 43 Percoll (Pharmacia, Uppsala, Sweden). Sperm suspensions were ml (range: 20 94 ml). Positive growth was present in eight adjusted with EBSS/BSA to a concentration of 20 10 6 /ml and (72.7%) out of 11 HF samples analysed and multiple organisms spermatozoa from each sample were divided into four equal portions. were found in three samples (Table I). All growths were The adjusted sperm suspension was centrifuged at 500 g for 5 min reported to be scanty in amount. Hormonal and biochemical and the supernatant was discarded as much as possible and the sperm parameters are presented in Table II. One HF sample had pellet was overlaid with 0.5 ml of 10, 50 and 100% of HF (test) and extremely high oestradiol (137 900 pmol/l) and progesterone with 0.5 ml of EBSS/BSA (control). ( 10 000 nmol/l) concentrations on repeated testing after serial Sperm motility and velocities were analysed by computer-aided dilution, probably because of the presence of substances that sperm analysis (CASA) at 30 min, 3 and 5 h after incubation at 37 C cross reacted with the antibodies used in the assays. The under 5% CO 2 using the Hobson Sperm Tracker System (HST; Hobson Tracking Systems Ltd, Sheffield, UK) as previously described oestradiol and progesterone concentrations of this HF sample (Yao et al., 1996). A 6 µl aliquot of the sperm suspension was were not included in Table II. transferred to a pre-warmed Cell-VU disposable semen analysis The median ph and osmolarity of HF were 8.1 (range 7.5 chamber (Fertility Technologies, Inc., MA, USA) with a chamber 8.1) and 271 mmol/kg (range 180 344) respectively. Table III depth of 20 µm placed on a warmed microscope stage at 37 C. The summarizes sperm motility and velocities measured by CASA 773

E.H.Y.Ng et al. Discussion Table I. Culture for aerobic and anaerobic organisms To the best of our knowledge, this is the first published study Patient no. Culture result Survival index (100% HF) demonstrating adverse effects of HF on sperm motility and survival after various periods of HF incubation. A small trial 1 Streptococcus agalactiae 73.6 (Schats et al., 1997) involving only three samples did not α-haemolytic streptococci reveal any toxic effect of HF on sperm survival. 2 Bacteroides 64.3 3 Not available 2.8 Despite the use of routine prophylactic antibiotics and 4 Not available 72.0 antiseptic solution to clean the vagina prior to oocyte retrieval, 5 Not available 60.0 positive cultures were present in nearly 75% of HF samples. 6 Lactobacillus 0 7 Negative growth 107.9 The majority of organisms found were commensal flora present 8 Negative growth 29.0 in the lower genital tract and of scanty amount only. The high 9 Gardnerella vaginalis 0 incidence of positive growth was probably due to contamination Coagulase negative staphylococcus of the needles after aspirating follicles over both sides through Streptococcus milleri the transvaginal route. None of the patients reported febrile 10 Negative growth 77.4 11 Bacteroides 89.0 episodes within a week after aspiration of HF. 12 Lactobacillus 95.2 The electrolyte concentrations of HF were similar to that 13 Streptococcus milleri 58.8 found in the serum (Table II) as human tubal fluid is mainly Candida 14 Bacteroides 48.8 derived from transudation of the plasma. Glucose, pyruvate 15 Not available 57.5 and lactate concentrations in human tubal fluid as assessed by fluorescence micro-analysis were 0.53 1.11, 0.14 0.17 and HF hydrosalpinx fluid. 5.4 8.58 mmol/l respectively (Dickens et al., 1995; Tay et al., 1997). In this study, glucose and pyruvate concentrations of Table II. Hormonal and biochemical profile of hydrosalpinx fluid (HF) HF were comparable but lactate concentrations were below the normal range. The ph of HF in this study ranged 7.5 8.1 Median Mean Range and this was similar to that found in human tubal fluid (Leese, 1988). A more alkaline condition (ph 8.45 8.70) of HF was Oestradiol (pmol/l) 156.5 2057.3 70 13 301 a reported by others (Mukherjee et al., 1996; Granot et al., Progesterone (nmol/l) 4.5 28.1 0.1 146.4 a Sodium (mmol/l) 137.0 122.4 80 142 1998). Although the median osmolarity of HF was 271 mmol/ Potassium (mmol/l) 3.8 3.5 1.9 4.7 kg, the osmolarity of one sample was only 180 mmol/kg, Chloride (mmol/l) 111.0 101.9 70 124 whereas that of another sample reached 344 mmol/kg. A Calcium (mmol/l) 0.61 0.78 0.18 2.15 Phosphate (mmol/l) 0.40 0.37 0.16 1.27 patient with HF of very low osmolarity was also found by Bicarbonate (mmol/l) 19.0 20.8 13 31 other workers (Strandell et al., 1998). Our findings differed Glucose (mmol/l) 1.35 1.31 0.5 4.4 from those of another study (Granot et al., 1998). In that study, Pyruvate (mmol/l) 0.10 0.11 0 0.2 Lactate (mmol/l) 2.15 2.16 0.7 4.8 all four HF samples (268 280 mmol/kg) were within the Protein (g/l) 2.15 12.4 0.2 59.0 physiological range. All the sperm motility and velocities were unchanged after a The HF sample with extremely high oestradiol and progesterone a relatively short period (5 h) of incubation with various concentrations was excluded. concentrations of HF, when compared to the control media. It at 30 min, 3 and 5 h after incubation with 10, 50 and 100% appears that even incubation with 100% HF does not lead to of HF. No differences were observed in MOT, VSL, VCL, acute cytotoxic effects on human spermatozoa. The HF sample VAP, ALH, BCF, LIN and HA among control and different with osmolarity of 180 mmol/kg was an exception: there was concentrations of HF (Kruskal Wallis test). The above para- a remarkable reduction in sperm motility and velocities after meters were also similar at 30 min, 3 and 5 h after incubation 30 min incubation and actually all spermatozoa became immot- with various concentrations of HF by the Friedman test. ile after 3hofincubation. Sperm motility was significantly different on day 1 to day Prolonged incubation (1 4 days) of HF was clearly associ- 4 incubation between control and 100% HF and between 10 ated with a reduction of the percentage of progressive motile and 100% HF, whereas significant differences were seen spermatozoa. It appears that higher concentrations of HF would between control and 50% HF on day 2 to day 4 and between be associated with a more rapid decline in the sperm motility control and 10% HF on day 4 only (Table IV). There were no as shown by significant differences in sperm motility between differences in sperm motility over 4 days of incubation between control and 100% HF and between 10 and 100% HF from day 10% and 50% HF and between 50% and 100% HF. 1 onwards. The differences between control and 50% HF and Median survival indices of 50 and 100% HF were 85%. between control and 10% HF reached significance from day The data were re-analysed after exclusion of the HF sample 2 onwards and on day 4 respectively. The survival indices of with extremely high hormonal concentrations and the results 50 and 100% HF were 79.8 and 58.8% respectively. were essentially the same (data not shown). No significant The rate of loss of sperm motility in vitro is related in part correlation existed between survival index and various biochemical to the rate of endogenous lipid peroxidation. This results in and hormonal parameters of 100% HF. extensive damage to the plasma and acrosomal membranes, 774

Adverse effects of hydrosalpinx fluid Table III. CASA parameters at 30 min, 3 and 5 h after hydrosalpinx fluid (HF) incubation (n 15) Parameters Control 10% HF 50% HF 100% HF P value a ph 7.6 (7.2 8.1) 7.8 (7.2 8.1) 7.8 (7.5 8.1) 8.1 (7.5 8.1) Osmolarity 285 (267 295) 281 (269 301) 278 (234 320) 271 (180 344) (mmol/kg) MOT (%) 30 min 88.0 (33 95) 92.0 (56 98) 88.0 (19 98) 83.0 (10 97) 0.82 3 h 86.0 (48 97) 89.0 (37 97) 90.0 (36 99) 88.0 (0 99) 0.64 5h b 92.0 (84 99) 85.0 (56 98) 89.0 (54 99) 89.0 (0 97) 0.28 VSL (µm/s) 30 min 42.1 (24.4 57.1) 49.3 (32.7 59.0) 45.6 (18.1 60.0) 29.8 (1.6 57.1) 0.10 3 h 39.1 (5.4 58.5) 42.3 (14.3 53.1) 41.8 (15.0 50.1) 26.4 (0 64.6) 0.16 5h b 41.7 (28.6 50.5) 44.7 (17.1 55.0) 44.1 (16.5 59.1) 35.6 (0 59.6) 0.50 VCL (µm/s) 30 min 103.3 (55.3 121.9) 107.2 (43.1 127.9) 96.4 (44.6 129.4) 81.4 (16.6 121.1) 0.28 3 h 105.3 (23.7 113.9) 102.7 (41.2 126.5) 96.8 (37.0 119.7) 59.6 (0 132.3) 0.38 5h b 91.8 (59.4 121.9) 89.6 (57.7 116.5) 82.7 (46.9 133.1) 74.2 (0 121.5) 0.43 VAP (µm/s) 30 min 56.5 (33.3 72.6) 66.8 (45.9 76.8) 59.2 (29.5 72.2) 41.8 (15.2 78.1) 0.10 3 h 54.2 (17.1 70.4) 58.5 (26.4 67.1) 56.7 (24.1 63.4) 36.2 (0 75.4) 0.12 5h b 54.9 (37.7 62.0) 54.8 (30.9 68.9) 54.7 (27.5 70.5) 44.7 (0 70.4) 0.38 ALH (µm) 30 min 8.8 (4.8 66.6) 8.3 (4.1 11.0) 6.5 (2.0 15.8) 7.7 (0.3 10.5) 0.19 3 h 7.9 (0.9 13.1) 8.3 (2.0 15.3) 6.9 (11.7 14.8) 7.6 (0 11.8) 0.84 5h b 8.3 (4.5 11.6) 6.5 (5.2 10.4) 6.3 (3.2 9.6) 5.6 (0 8.9) 0.18 BCF (Hz) 30 min 7.6 (4.5 11.0) 8.8 (5.5 10.4) 7.8 (2.9 10.7) 6.2 (0.1 11.5) 0.16 3 h 7.1 (0.5 11.4) 7.5 (1.9 10.8) 8.1 (2.4 10.0) 5.4 (0 12.2) 0.11 5h b 7.1 (5.0 9.8) 7.9 (2.7 10.0) 7.4 (3.0 12.0) 7.4 (0 11.5) 0.81 LIN 30 min 42.3 (26.6 54.1) 43.3 (35.9 60.8) 43.7 (25.2 56.0) 38.2 (12.6 48.0) 0.07 3 h 37.6 (19.6 52.7) 42.1 (22.7 53.2) 43.2 (23.9 49.1) 36.2 (0 51.2) 0.41 5h b 41.1 (32.5 53.4) 46.4 (25.1 60.7) 46.2 (31.1 53.4) 42.4 (0 50.3) 0.23 HA (%) 30 min 38.0 (4.0 58.0) 48.0 (12.0 68.0) 29.0 (7.0 82.0) 30.0 (0 68.0) 0.69 3 h 31.0 (0 56.0) 34.0 (8.0 78.0) 34.0 (2.0 171.0) 12.0 (0 66.0) 0.53 5h b 26.0 (10.0 62.0) 25.0 (9.0 52.0) 18.0 (2.0 69.0) 17.0 (0 59.0) 0.71 Results are given in median (range). a Kruskal Wallis test used to compare parameters among various HF concentrations. b Only 11 HF samples subject to 5 h incubation. For parameter abbreviations, see Materials and methods. Table IV. Sperm motility over 4 days of hydrosalpinx fluid (HF) incubation and sperm survival index (n 15) Control 10% HF 50% HF 100% HF P value* Motility (%) Day 1 87.0 (72.0 93.0) 78.0 (60.0 94.0) 82.0 (45.0 92.0) 65.0 (25.0 83.0) 0.005 a,b Day 2 80.0 (70.0 91.0) 81.0 (42.0 90.0) 74.0 (5.0 89.0) 73.0 (0 91.0) 0.018 a,b,c Day 3 75.0 (71.0 91.0) 77.0 (39.0 87.0) 65.0 (1.0 86.0) 55.0 (0 79.0) 0.001 a,b,c Day 4 78.0 (70.0 93.0) 71.0 (17.0 86.0) 63.0 (3.0 79.0) 47.0 (0 80.0) 0.001 a,b,c,d Survival index 100 92.5 (24.3 101.6) 79.8 (4.3 106.3) 58.8 (0 107.9) Results are given in median (range). *Kruskal Wallis test used to compare motility among various HF concentrations. Mann Whitney test used to show the difference between two groups: a Significant difference between control and 100% HF. b Significant difference between 10% and 100% HF. c Significant difference between control and 50% HF. d Significant difference between control and 10% HF. leading to loss of permeability and leaking of pyridine nucleotides that ultimately renders the spermatozoa immotile (Alvarez et al., 1996). The mechanisms leading to the loss of sperm motility in HF remain speculative. Low osmolarity (180 mmol/ kg) in one of the HF samples certainly caused a rapid loss of sperm motility, as the effect is similar to that occurring in the 775

E.H.Y.Ng et al. hypo-osmotic swelling test. Lactate acts as an energy substrate human spermatozoa and tubal epithelium (Williams et al., for spermatozoa and was found to be low in HF. The median 1993). Based on our results, it is tempting to speculate that total protein concentration in HF was 2.15 g/l, but the concen- some of the failure after tubal surgery might be due to the trations ranged from 0.2 to 59.0 g/l. The detailed composition cytotoxic effects of HF on human spermatozoa because after of the proteins present in HF was not examined in this study the tubal surgery, the diseased tube will continue to produce and they may adversely affect the sperm motility. A protein tubal fluid, which might have a similar content to that of of 54 kda molecular weight with an isoelectric point of 4.5 hydrosalpinx. Human sperm survival test of HF from hydrosalpinx and containing carbohydrate was identified in human oviductal may be incorporated as part of the evaluation in the fluid and the protein could bind to the surface of the entire fertility outcome after surgery. Such information would be spermatozoon (Lippes and Wagh, 1989; Wagh and Lippes, useful in counselling patients prior to surgery. 1989). In summary, sperm motility and velocities remained Positive growth of aerobic and/or anaerobic organisms in unchanged after a relatively short period (5 h) of incubation HF was unlikely to be the cause of toxicity because the with various concentrations of HF. The percentage of motile proportion of positive cultures was similar in the toxic (6/8) spermatozoa was significantly reduced after 24 h of HF and non-toxic (2/3) HF samples (Table I). The electrolytes, incubation and the detrimental effect seemed to be dependent glucose and pyruvate concentrations in HF were all within the on the concentrations of HF used. Both 50 and 100% HF were physiological range found in normal human tubal fluid. It is potentially cytotoxic as the survival indices were 85%. Low difficult to assess the effects of hormonal concentrations on osmolarity, low lactate concentrations and protein content may sperm motility because of the small number of HF samples be responsible for the loss of sperm motility. A human sperm available. survival test using HF may be useful in selecting appropriate The range of survival indices of 100% HF was large and treatment options for patients with hydrosalpinx undergoing three (20%) HF samples had survival indices 85%, i.e. nontoxic. IVF treatment or tubal surgery. The larger sample size (n 15) of this study revealed the heterogeneity of HF. Our data may help to explain in part the difference in embryotoxicity observed in the literature and Acknowledgements to provide a possible explanation for the difference in clinical This work was supported by grants from the Committee on Research results among studies. There might be individual variation in and Conference Grants, The University of Hong Kong. the content of HF, yielding different influences on clinical outcomes and embryo development (Strandell et al., 1998). Because of the reported negative effects of hydrosalpinx on References IVF outcome, various treatment options including the use of Alvarez, J.G., Minaretzis, D., Barrett, C.B. et al. (1996) The sperm stress antibiotic (Sharara et al., 1996), aspiration of HF (Sowter test: a novel test that predicts pregnancy in assisted reproductive technologies. Fertil. Steril., 65, 400 405. et al., 1997; Van Voorhis et al., 1998), salpingostomy and Andersen, A.N., Zhou, Y., Fan, J.M. et al. (1994) Low implantation rate salpingectomy (Murray et al., 1998; Ejdrup Bredkjaer et al., after in-vitro fertilization in patients with hydrosalpinges diagnosed by 1999) have been proposed. A recent multi-centre trial in ultrasonography. Hum. Reprod., 9, 1935 1938. Scandinavia (Strandell et al., 1999) showed a clear benefit of Andersen, A.N., Lindhard, A., Loft, A. et al. (1996) The infertile patient with hydrosalpinges IVF with or without salpingectomy? Hum. Reprod., 10, salpingectomy only in patients with bilateral hydrosalpinges 2081 2084. and in patients with ultrasound-visible hydrosalpinges. How- Beyler, S.A., James, K.P., Fritz, M.A. et al. (1997) Hydrosalpingeal fluid ever, the preferred or best treatment for hydrosalpinges is not inhibits in-vitro embryonic development in a murine model. Hum. Reprod., yet settled and the debate still continues. 12, 2724 2728. Burkman, L.J. (1991) Discrimination between nonhyperactivated and classical Salpingectomy before IVF might jeopardize the blood supply hyperactivated motility patterns in human spermatozoa using computerized to the ovaries and reduce the ovarian reserve (Lass, 1999). analysis. Fertil. Steril., 55, 363 371. The results of our previous study (Ng et al., 1997) indicated Camus, E., Poncelet, C., Goffinet, F. et al. (1999) Pregnancy rates after invitro fertilization in cases of tubal infertility with and without hydrosalpinx: that patients with or without hydrosalpinx might have similar a meta-analysis of published comparative studies. Hum. Reprod., 14, implantation and pregnancy rates in their first treatment cycle. 1243 1249. The human sperm survival test using HF aspirated from Critchlow, J.D., Matson, P.L., Newman, M.C. et al. (1989) Quality control in patients with hydrosalpinx may be used as a bioassay to an in-vitro fertilization laboratory: use of human sperm survival studies. Hum. Reprod., 4, 545 549. evaluate the cytotoxic effects of HF. Those with survival Dickens, C.J., Maguiness, S.D., Comer, M.T. et al. (1995) Human tubal fluid: indices 85% might be advised to undergo salpingectomy in formation and composition during vascular perfusion of the Fallopian tube. order to optimize the chance of success. It is acknowledged Hum. Reprod., 10, 505 508. that the implantation problem in patients with hydrosalpinx Edwards, R.G. (1992) Why are agonadal and post-amenorrhoeic women so fertile after oocyte donation? Hum. Reprod., 7, 733 734. caused by the mechanical effect and abnormal endometrial Ejdrup Bredkjaer, H., Ziebe, S., Hamid, B. et al. (1999) Delivery rates after receptivity could not be addressed by this bioassay. in-vitro fertilization following bilateral salpingectomy due to hydrosalpinges: Tubal surgery with regard to salpingostomy in patients with a case control study. Hum. Reprod., 14, 101 105. hydrosalpinges carries poor results in terms of spontaneous Fleming, C. and Hull, M.G.R. (1996) Impaired implantation after in vitro fertilization treatment associated with hydrosalpinx. Br. J. Obstet. Gynaecol., conception and this is usually attributed to macroscopic and 103, 268 272. microscopic abnormalities of the diseased Fallopian tube Granot, I., Dekel, N., Segal, I. et al. (1998) Is hydrosalpinx fluid cytotoxic? (Vasquez et al., 1995). Close association occurs between Hum. Reprod., 13, 1620 1624. 776

Adverse effects of hydrosalpinx fluid Kassabji, M., Sims, J.A., Butler, L. et al. (1994) Reduced pregnancy outcome Williams, M., Hill, C.J., Scudamore, I. et al. (1993) Sperm numbers and in patients with unilateral or bilateral hydrosalpinx after in vitro fertilization. distribution within the human Fallopian tube around ovulation. Hum. Eur. J. Obstet. Gynecol., 56, 129 132. Reprod., 8, 2019 2026. Koong, M.K., Jun, J.H., Song, S.J. et al. (1998) A second look at the Wit, W., Gowrising, C.J., Kuik, D.J. et al. (1998) Only hydrosalpinges visible embryotoxicity of hydrosalpingeal fluid: an in-vitro assessment in a murine on ultrasound are associated with reduced implantation and pregnancy rates model. Hum. Reprod., 13, 2852 2856. after in-vitro fertilization. Hum. Reprod., 13, 1696 1701. Lass, A. (1999) What is the preferred treatment for hydrosalpinges? The World Health Organization (1992) WHO Laboratory Manual for the ovary s perspective. Hum. Reprod., 14, 1674 1677. Examination of Human Semen and Sperm Cervical Mucus Interaction, Leese, H.J. (1988) The formation and function of oviduct fluid. J. Reprod. third edn. Cambridge University Press, New York. Fertil., 82, 843 856. Yao, Y.Q., Yeung, W.S.B. and Ho, P.C. (1996) Human follicular fluid inhibits Lippes, J. and Wagh, P.V. (1989) Human oviductal fluid (hof) proteins. IV. the binding of human spermatozoa to zona pellucida in vitro. Hum. Reprod., Evidence for hof proteins binding to human sperm. Fertil. Steril., 51, 89 94. 11, 2674 2680. Mansour, R.T., Aboulghar, M.A., Serour, G.I. et al. (1991) Fluid accumulation Zeyneloglu, H.B., Arici, A. and Olive, D.L. (1998) Adverse effects of of the uterine cavity before embryo transfer: a possible hindrance for hydrosalpinx on pregnancy rates after in vitro fertilization embryo transfer. implantation. J. In Vitro Fertil. Embryo Transfer, 8, 157 159. Fertil. Steril., 70, 492 499. Meyer, W.R., Castelbaum, A.J., Somkuti, S. et al. (1997) Hydrosalpinges Received on August 26, 1999; accepted on December 15, 1999 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. et al. (1997) Inhibitory effect of human hydrosalpingeal fluid on mouse preimplantation embryonic development is significantly reduced by the addition of lactate. Hum. Reprod., 12, 2504 2507. Murray, D.L., Sagoskin, A.W., Widra, E.A. et al. (1998) The adverse effect of hydrosalpinges on in vitro fertilization pregnancy rates and the benefit of surgical correction. Fertil. Steril., 69, 41 45. Ng, E.H.Y., Yeung, W.S.B. and Ho, P.C. (1997) The presence of hydrosalpinx may not adversely affect the implantation and pregnancy rates in in vitro fertilization treatment. J. Assist. Reprod. Genet., 14, 508 512. Rawe, V.J., Liu, J., Shaffer, S. et al. (1997) Effect of human hydrosalpinx fluid on murine embryo development and implantation. Fertil. Steril., 68, 668 670. Sachdev, R., Kemmann, E., Bohrer, M.K. et al. (1997) Detrimental effect of hydrosalpinx fluid on the development and blastulation of mouse embryos in vitro. Fertil. Steril., 68, 531 533. Schats, R., Lens, J.W. and de Wit, W. (1997) Survival of spermatozoa in hydrosalpinx fluid is not impaired (Abstract). Presented at the 13 th ESHRE Annual Meeting, Edinburgh, June 1997. Hum. Reprod., 12, 111 112, O-226. Sharara, F.I., Scott, Jr R.T., Marut, E.L. et al. (1996) In-vitro fertilization outcome in women with hydrosalpinx. Hum. Reprod., 11, 526 530. Sowter, M., Akande, V.A., Williams, J.A.C. et al. (1997) Is the outcome of in-vitro fertilization and embryo transfer treatment improved by spontaneous or surgical drainage of a hydrosalpinx? Hum. Reprod., 12, 2147 2150. Steptoe, P.C. and Edwards, R.G. (1978) Birth after reimplantation of a human embryo. Lancet, 2, 366. Strandell, A., Waldenström, U., Nilsson, L. et al. (1994) Hydrosalpinx reduces in-vitro fertilization/embryo transfer pregnancy rates. Hum. Reprod., 9, 861 863. Strandell, A., Sjögren, A., Bentin-Ley, U. et al. (1998) Hydrosalpinx fluid does not adversely affect the normal development of human embryos and implantation in vitro. Hum. Reprod., 13, 2921 2925. Strandell, A., Lindhard, A., Waldenström, U. et al. (1999) Hydrosalpinx and IVF outcome: a prospective, randomized multicentre trial in Scandinavia on salpingectomy prior to IVF. Hum. Reprod., 14, 2762 2769. Tay, J.I., Rutherford, A.J., Killick, S.R. et al. (1997) Human tubal fluid: production, nutrient composition and response to adrenergic agents. Hum. Reprod., 12, 2451 2456. Vandromme, J., Chasse, E., Lejeune, B. et al. (1995) Hydrosalpinges in invitro fertilization: an unfavourable prognostic feature. Hum. Reprod., 10, 576 579. Van Voorhis, B.J., Sparks, A.E.T., Syrop, C.H. et al. (1998) Ultrasound-guided aspiration of hydrosalpinges is associated with improved pregnancy and implantation rates after in-vitro fertilization cycles. Hum. Reprod., 13, 736 739. Vasquez, G., Boeckx, W. and Brosens, I. (1995) Prospective study of tubal mucosal lesions and fertility in hydrosalpinges. Hum. Reprod., 10, 1075 1078. Wagh, P.V. and Lippes, J. (1989) Human oviductal fluid proteins. III. Identification and partial purification. Fertil. Steril., 51, 81 88. Wainer, R., Camus, E., Camier, B. et al. (1997) Does hydrosalpinx reduce the pregnancy rate after in vitro fertilization. Fertil. Steril., 68, 1022 1026. 777