Department of Obstetrics and Gynaecology, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, People's Republic of China
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: CASA/hydrosalpinx fluid/sperm motility and velocities
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the exact mechanisms are not fully known, there are several possibilities suggested for the implantation failure associated with the presence of hydrosalpinx. Fluid accumulation in the uterine cavity due to back passage of fluid can occasionally be observed during transvaginal ultrasound monitoring for IVF cycles in patients with hydrosalpinx (Mansour et al., 1991; Andersen et al., 1996
). Hydrosalpinx fluid (HF) within the uterine cavity may act as a mechanical barrier to implantation as the replaced embryos would be flushed out or the normal apposition of the endometrial cells is disrupted (Edwards, 1992
).
HF may contain micro-organisms, debris, toxins or cytokines or prostaglandins, which can exert a direct detrimental effect on the receptivity of the endometrium (Meyer et al., 1997) or on the developing embryos inside the uterus. The embryotoxic effects of HF were documented only in mouse embryos (Mukherjee et al., 1996
; Beyler et al., 1997
; Murray et al., 1997
; Rawe et al., 1997
; Sachdev et al., 1997
; Koong et al., 1998
) but not in human embryos (Granot et al., 1998
; Strandell et al., 1998
). Inter-species difference (mouse versus human) and small sample sizes of 210 HF samples in these studies may account for the contrasting results in relation to embryotoxicity.
The human sperm survival test (Critchlow et al., 1989) is a simple, reliable and objective quality control procedure to test the suitability of various materials and culture media for use in many human IVF laboratories. The objective of this study was to examine the effects of various concentrations of HF on sperm motility and survival after various periods of incubation.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 laparoscopy/hysteroscopy. The details of the ovarian stimulation regimen used at our centre had been previously published (Ng et al., 1997). In short, human menopausal gonadotrophin (Pergonal, Serono, Switzerland) injection was started after pituitary down-regulation with buserelin (Supracur, Hoechst, Frankfurt, Germany) nasal spray. The transvaginal ultrasound-guided oocyte retrieval (oocyte retrieval) was scheduled 3638 h after the human chorionic gonadotrophin (HCG) injection. Routine prophylactic antibiotics, 1 g ampicillin (Bristol-Meyers Squibb, NJ, USA) and 0.5 g metronidazole (McGaw Inc., Irvine, CA, USA), were given i.v. 30 min prior to oocyte retrieval.
Aspiration of HF after oocyte retrieval
Only those patients with hydrosalpinx visible on ultrasound scanning on the day of HCG were recruited. They underwent aspiration of HF after all follicles of >10 mm in diameter were aspirated and the needle was flushed several times with culture media. Both sides were aspirated in cases of bilateral hydrosalpinges and HF was pooled for analysis. The first part of HF was sent for culture of aerobic and anaerobic organisms. The fluid was then centrifuged at 500 g for 20 min and the supernatant was frozen at 20°C prior to further analysis and tests.
Hormonal and biochemical profile
After thawing at room temperature, HF samples were checked for oestradiol, progesterone, sodium, potassium, chloride, calcium, phosphate, bicarbonate, glucose, protein, pyruvate and lactate. Oestradiol was measured using a commercially available radioimmunoassay kit (Diagnostic Products Corporation, Los Angeles, CA, USA). The sensitivity of the assay was 8 pg/ml (conversion factor to SI unit, 3.67), and the inter-assay and intra-assay coefficients of variation were 4.2 and 4.0% respectively. Progesterone was measured using a commercially available radioimmunoassay kit (Chiron Diagnostics Corporation, MA, USA). The sensitivity of the assay was 0.11 ng/ml (conversion factor to SI unit, 3.18), and the inter-assay and intra-assay coefficients of variation were 9.4 and 8.4% respectively. The biochemical assays were performed in the general hospital laboratory.
Sperm motility analysis
HF samples were thawed at room temperature and diluted with Earle's balanced salt solution (EBSS: Flow Laboratories, Irvine, UK) supplemented with sodium bicarbonate, sodium pyruvate, penicillin-G, streptomycin sulphate and 0.3% bovine serum albumin (BSA), i.e. EBSS/BSA to 10 and 50% concentration. pH and osmolarity of EBSS/BSA and HF at different concentrations were measured.
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 used in this study. One semen sample was used for each HF sample. After complete liquefaction at room temperature, semen samples were processed through a two-gradient Percoll preparation, i.e. 45 and 90% Percoll (Pharmacia, Uppsala, Sweden). Sperm suspensions were adjusted with EBSS/BSA to a concentration of 20x106/ml and spermatozoa from each sample were divided into four equal portions. The adjusted sperm suspension was centrifuged at 500 g for 5 min and the supernatant was discarded as much as possible and the sperm pellet was overlaid with 0.5 ml of 10, 50 and 100% of HF (test) and with 0.5 ml of EBSS/BSA (control).
Sperm motility and velocities were analysed by computer-aided sperm analysis (CASA) at 30 min, 3 and 5 h after incubation at 37°C under 5% CO2 using the Hobson Sperm Tracker System (HST; Hobson Tracking Systems Ltd, Sheffield, UK) as previously described (Yao et al., 1996). A 6 µl aliquot of the sperm suspension was transferred to a pre-warmed Cell-VU disposable semen analysis chamber (Fertility Technologies, Inc., MA, USA) with a chamber depth of 20 µm placed on a warmed microscope stage at 37°C. The fields were selected randomly during evaluation, and 500 spermatozoa were analysed in each specimen.
The following parameters were determined: percentage of motile spermatozoa (MOT), mean curvilinear velocity (VCL, µm/s), mean straight line velocity (VSL, µm/s), average path velocity (VAP, µm/s), mean linearity (LIN: VSL/VCL), amplitude of lateral head displacement (ALH, µm), head beat cross frequency (BCF, Hz), and percentage of spermatozoa exhibiting hyperactivation (HA). Spermatozoa satisfying the criteria LIN <65, VCL >100 µm/s, ALH >7 µm (Burkman, 1991) were classified as hyperactivated spermatozoa.
Human sperm survival test
One semen sample was again used for each HF sample. Semen samples were processed by a swim-up method after complete liquefaction and the sperm concentration was adjusted to 5x106/ml. Spermatozoa from each sample were divided into four equal portions. A 0.5 ml aliquot of the adjusted sperm suspension was centrifuged at 500 g 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 0.5 ml of 10%, 50% and 100% of HF (test) and 0.5 ml of EBSS/BSA (control).
The test and the control tubes were placed inside a modular chamber gassed with 5% CO2 and left at room temperature for 4 days. The sperm suspensions were agitated every 24 h and a drop was removed from each tube for examination of sperm motility. For the assay to be regarded as valid, >70% of spermatozoa in the control tube had to show progressive motility after 4 days of culture. The progressive motility of the test sample on day 4 was then used to calculate a survival index:
![]() |
A survival index of <0.85 was considered to be potentially cytotoxic (Critchlow et al., 1989).
Statistical analysis
Data were expressed as median (range). Correlation was assessed by the Pearson method and comparison was carried out using the KruskalWallis test. Difference between groups, if present, was compared by the MannWhitney test. Paired data were analysed with the Friedman test. A P-value (two-tailed) of <0.05 was considered to be significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Despite the use of routine prophylactic antibiotics and antiseptic solution to clean the vagina prior to oocyte retrieval, positive cultures were present in nearly 75% of HF samples. The majority of organisms found were commensal flora present in the lower genital tract and of scanty amount only. The high incidence of positive growth was probably due to contamination of the needles after aspirating follicles over both sides through the transvaginal route. None of the patients reported febrile episodes within a week after aspiration of HF.
The electrolyte concentrations of HF were similar to that found in the serum (Table II) as human tubal fluid is mainly derived from transudation of the plasma. Glucose, pyruvate and lactate concentrations in human tubal fluid as assessed by fluorescence micro-analysis were 0.531.11, 0.140.17 and 5.48.58 mmol/l respectively (Dickens et al., 1995
; Tay et al., 1997
). In this study, glucose and pyruvate concentrations of HF were comparable but lactate concentrations were below the normal range. The pH of HF in this study ranged 7.58.1 and this was similar to that found in human tubal fluid (Leese, 1988
). A more alkaline condition (pH 8.458.70) of HF was reported by others (Mukherjee et al., 1996
; Granot et al., 1998
). Although the median osmolarity of HF was 271 mmol/kg, the osmolarity of one sample was only 180 mmol/kg, whereas that of another sample reached 344 mmol/kg. A patient with HF of very low osmolarity was also found by other workers (Strandell et al., 1998
). Our findings differed from those of another study (Granot et al., 1998
). In that study, all four HF samples (268280 mmol/kg) were within the physiological range.
All the sperm motility and velocities were unchanged after a relatively short period (5 h) of incubation with various concentrations of HF, when compared to the control media. It appears that even incubation with 100% HF does not lead to acute cytotoxic effects on human spermatozoa. The HF sample with osmolarity of 180 mmol/kg was an exception: there was a remarkable reduction in sperm motility and velocities after 30 min incubation and actually all spermatozoa became immotile after 3 h of incubation.
Prolonged incubation (14 days) of HF was clearly associated with a reduction of the percentage of progressive motile spermatozoa. It appears that higher concentrations of HF would be associated with a more rapid decline in the sperm motility as shown by significant differences in sperm motility between control and 100% HF and between 10 and 100% HF from day 1 onwards. The differences between control and 50% HF and between control and 10% HF reached significance from day 2 onwards and on day 4 respectively. The survival indices of 50 and 100% HF were 79.8 and 58.8% respectively.
The rate of loss of sperm motility in vitro is related in part to the rate of endogenous lipid peroxidation. This results in extensive damage to the plasma and acrosomal membranes, 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 hypo-osmotic swelling test. Lactate acts as an energy substrate for spermatozoa and was found to be low in HF. The median total protein concentration in HF was 2.15 g/l, but the concentrations ranged from 0.2 to 59.0 g/l. The detailed composition of the proteins present in HF was not examined in this study and they may adversely affect the sperm motility. A protein of 54 kDa molecular weight with an isoelectric point of 4.5 and containing carbohydrate was identified in human oviductal fluid and the protein could bind to the surface of the entire spermatozoon (Lippes and Wagh, 1989
; Wagh and Lippes, 1989
).
Positive growth of aerobic and/or anaerobic organisms in HF was unlikely to be the cause of toxicity because the proportion of positive cultures was similar in the toxic (6/8) and non-toxic (2/3) HF samples (Table I). The electrolytes, glucose and pyruvate concentrations in HF were all within the physiological range found in normal human tubal fluid. It is difficult to assess the effects of hormonal concentrations on sperm motility because of the small number of HF samples available.
The range of survival indices of 100% HF was large and three (20%) HF samples had survival indices >85%, i.e. non-toxic. 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 to provide a possible explanation for the difference in clinical results among studies. There might be individual variation in 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 IVF outcome, various treatment options including the use of antibiotic (Sharara et al., 1996), aspiration of HF (Sowter et al., 1997
; Van Voorhis et al., 1998
), salpingostomy and salpingectomy (Murray et al., 1998
; Ejdrup Bredkjaer et al., 1999
) have been proposed. A recent multi-centre trial in Scandinavia (Strandell et al., 1999
) showed a clear benefit of salpingectomy only in patients with bilateral hydrosalpinges and in patients with ultrasound-visible hydrosalpinges. However, the preferred or best treatment for hydrosalpinges is not yet settled and the debate still continues.
Salpingectomy before IVF might jeopardize the blood supply to the ovaries and reduce the ovarian reserve (Lass, 1999). The results of our previous study (Ng et al., 1997
) indicated that patients with or without hydrosalpinx might have similar implantation and pregnancy rates in their first treatment cycle. The human sperm survival test using HF aspirated from patients with hydrosalpinx may be used as a bioassay to evaluate the cytotoxic effects of HF. Those with survival indices <85% might be advised to undergo salpingectomy in order to optimize the chance of success. It is acknowledged that the implantation problem in patients with hydrosalpinx caused by the mechanical effect and abnormal endometrial receptivity could not be addressed by this bioassay.
Tubal surgery with regard to salpingostomy in patients with hydrosalpinges carries poor results in terms of spontaneous conception and this is usually attributed to macroscopic and microscopic abnormalities of the diseased Fallopian tube (Vasquez et al., 1995). Close association occurs between human spermatozoa and tubal epithelium (Williams et al., 1993
). Based on our results, it is tempting to speculate that some of the failure after tubal surgery might be due to the cytotoxic effects of HF on human spermatozoa because after the tubal surgery, the diseased tube will continue to produce tubal fluid, which might have a similar content to that of hydrosalpinx. Human sperm survival test of HF from hydrosalpinx may be incorporated as part of the evaluation in the fertility outcome after surgery. Such information would be useful in counselling patients prior to surgery.
In summary, sperm motility and velocities remained unchanged after a relatively short period (5 h) of incubation with various concentrations of HF. The percentage of motile spermatozoa was significantly reduced after 24 h of HF incubation and the detrimental effect seemed to be dependent on the concentrations of HF used. Both 50 and 100% HF were potentially cytotoxic as the survival indices were <85%. Low osmolarity, low lactate concentrations and protein content may be responsible for the loss of sperm motility. A human sperm survival test using HF may be useful in selecting appropriate treatment options for patients with hydrosalpinx undergoing IVF treatment or tubal surgery.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andersen, A.N., Zhou, Y., Fan, J.M. et al. (1994) Low implantation rate after in-vitro fertilization in patients with hydrosalpinges diagnosed by ultrasonography. Hum. Reprod., 9, 1935 1938.[Abstract]
Andersen, A.N., Lindhard, A., Loft, A. et al. (1996) The infertile patient with hydrosalpingesIVF with or without salpingectomy? Hum. Reprod., 10, 20812084.
Beyler, S.A., James, K.P., Fritz, M.A. et al. (1997) Hydrosalpingeal fluid inhibits in-vitro embryonic development in a murine model. Hum. Reprod., 12, 27242728.[Abstract]
Burkman, L.J. (1991) Discrimination between nonhyperactivated and classical hyperactivated motility patterns in human spermatozoa using computerized analysis. Fertil. Steril., 55, 363371.[ISI][Medline]
Camus, E., Poncelet, C., Goffinet, F. et al. (1999) Pregnancy rates after in-vitro fertilization in cases of tubal infertility with and without hydrosalpinx: a meta-analysis of published comparative studies. Hum. Reprod., 14, 12431249.
Critchlow, J.D., Matson, P.L., Newman, M.C. et al. (1989) Quality control in an in-vitro fertilization laboratory: use of human sperm survival studies. Hum. Reprod., 4, 545549.[Abstract]
Dickens, C.J., Maguiness, S.D., Comer, M.T. et al. (1995) Human tubal fluid: formation and composition during vascular perfusion of the Fallopian tube. Hum. Reprod., 10, 505508.[Abstract]
Edwards, R.G. (1992) Why are agonadal and post-amenorrhoeic women so fertile after oocyte donation? Hum. Reprod., 7, 733734.[ISI][Medline]
Ejdrup Bredkjaer, H., Ziebe, S., Hamid, B. et al. (1999) Delivery rates after in-vitro fertilization following bilateral salpingectomy due to hydrosalpinges: a case control study. Hum. Reprod., 14, 101105.
Fleming, C. and Hull, M.G.R. (1996) Impaired implantation after in vitro fertilization treatment associated with hydrosalpinx. Br. J. Obstet. Gynaecol., 103, 268272.[ISI][Medline]
Granot, I., Dekel, N., Segal, I. et al. (1998) Is hydrosalpinx fluid cytotoxic? Hum. Reprod., 13, 16201624.[Abstract]
Kassabji, M., Sims, J.A., Butler, L. et al. (1994) Reduced pregnancy outcome in patients with unilateral or bilateral hydrosalpinx after in vitro fertilization. Eur. J. Obstet. Gynecol., 56, 129132.[ISI][Medline]
Koong, M.K., Jun, J.H., Song, S.J. et al. (1998) A second look at the embryotoxicity of hydrosalpingeal fluid: an in-vitro assessment in a murine model. Hum. Reprod., 13, 28522856.
Lass, A. (1999) What is the preferred treatment for hydrosalpinges? The ovary's perspective. Hum. Reprod., 14, 16741677.
Leese, H.J. (1988) The formation and function of oviduct fluid. J. Reprod. Fertil., 82, 843856.[Medline]
Lippes, J. and Wagh, P.V. (1989) Human oviductal fluid (hOF) proteins. IV. Evidence for hOF proteins binding to human sperm. Fertil. Steril., 51, 8994.[ISI][Medline]
Mansour, R.T., Aboulghar, M.A., Serour, G.I. et al. (1991) Fluid accumulation of the uterine cavity before embryo transfer: a possible hindrance for implantation. J. In Vitro Fertil. Embryo Transfer, 8, 157159.[ISI][Medline]
Meyer, W.R., Castelbaum, A.J., Somkuti, S. et al. (1997) Hydrosalpinges adversely affect markers of endometrial receptivity. Hum. Reprod., 12, 13931398.[Abstract]
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, 851853.[ISI][Medline]
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, 25042507.[Abstract]
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, 4145.[ISI][Medline]
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, 508512.[ISI][Medline]
Rawe, V.J., Liu, J., Shaffer, S. et al. (1997) Effect of human hydrosalpinx fluid on murine embryo development and implantation. Fertil. Steril., 68, 668670.[ISI][Medline]
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, 531533.[ISI][Medline]
Schats, R., Lens, J.W. and de Wit, W. (1997) Survival of spermatozoa in hydrosalpinx fluid is not impaired (Abstract). Presented at the 13th ESHRE Annual Meeting, Edinburgh, June 1997. Hum. Reprod., 12, 111112, 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, 526530.[Abstract]
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, 21472150.[Abstract]
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, 861863.[Abstract]
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, 29212925.
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, 27622769.
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, 24512456.[Abstract]
Vandromme, J., Chasse, E., Lejeune, B. et al. (1995) Hydrosalpinges in in-vitro fertilization: an unfavourable prognostic feature. Hum. Reprod., 10, 576579.[Abstract]
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, 736739.[Abstract]
Vasquez, G., Boeckx, W. and Brosens, I. (1995) Prospective study of tubal mucosal lesions and fertility in hydrosalpinges. Hum. Reprod., 10, 10751078.[Abstract]
Wagh, P.V. and Lippes, J. (1989) Human oviductal fluid proteins. III. Identification and partial purification. Fertil. Steril., 51, 8188.[ISI][Medline]
Wainer, R., Camus, E., Camier, B. et al. (1997) Does hydrosalpinx reduce the pregnancy rate after in vitro fertilization. Fertil. Steril., 68, 10221026.[ISI][Medline]
Williams, M., Hill, C.J., Scudamore, I. et al. (1993) Sperm numbers and distribution within the human Fallopian tube around ovulation. Hum. Reprod., 8, 20192026.[Abstract]
Wit, W., Gowrising, C.J., Kuik, D.J. et al. (1998) Only hydrosalpinges visible on ultrasound are associated with reduced implantation and pregnancy rates after in-vitro fertilization. Hum. Reprod., 13, 16961701.[Abstract]
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction, third edn. Cambridge University Press, New York.
Yao, Y.Q., Yeung, W.S.B. and Ho, P.C. (1996) Human follicular fluid inhibits the binding of human spermatozoa to zona pellucida in vitro. Hum. Reprod., 11, 26742680.[Abstract]
Zeyneloglu, H.B., Arici, A. and Olive, D.L. (1998) Adverse effects of hydrosalpinx on pregnancy rates after in vitro fertilizationembryo transfer. Fertil. Steril., 70, 492499.[ISI][Medline]
Submitted on August 26, 1999; accepted on December 15, 1999.