1 Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong and 2 Department of Obstetrics and Gynaecology, Princess Margaret Hospital, Kowloon, Hong Kong, Peoples Republic of China
3 To whom correspondence should be addressed. e-mail: lyschan{at}cuhk.edu.hk
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Abstract |
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Key words: hydrosalpinx/teratogenicity/whole rat embryo culture
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Introduction |
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The exact mechanisms by which a hydrosalpinx could adversely affect IVF outcome remain unclear. Possible mechanisms include mechanical factors, reduced endometrial receptivity (Meyer et al., 1997) or an embryotoxic effect of hydrosalpinx fluid (Lass, 1999
). Direct toxic effects of hydrosalpinx fluid on early non-human embryos at the 14-cell stage have been reported (Beyler et al., 1997
). It was demonstrated that the deleterious effect of hydrosalpinx fluid on mouse embryos was preventable if cultures were performed under an oil overlay. They postulated that lipophilic embryotoxic factor(s) might be responsible for the embryotoxicity (Beyler et al., 1997
).
Since hydrosalpinx fluid may be toxic to sperm and early embryo growth, it might also affect early embryo development during organogenesis. The aim of the current study was to evaluate the potential embryotoxicity and teratogenic effect(s) of hydrosalpinx fluid during the critical period of organogenesis using a whole rat embryo culture model.
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Materials and methods |
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Animals
Timed-gestation pregnant SpragueDawley rats were supplied by the Laboratory Animals Services Centre of the University. The day on which sperm were found in the vaginal smear was defined as day 0 of pregnancy. The Animal Research Ethics Committee of The Chinese University of Hong Kong approved this study.
Whole embryo culture
The whole embryo culture system was based on the model previously described (New, 1978). Animals were killed by diethyl ether overdose (Merck, Germany) at gestational day 9.5 between 09:00 and 10:00 and embryos were explanted. To minimize variation, only embryos with crownrump length (CRL) of 1.5 ± 0.3 mm were used for the experiment. Embryos were explanted from a maximum of four pregnant rats at one time. They were then mixed together and three to five embryos were assigned to a culture bottle, which belonged either to the experimental or control group. The investigator who assigned the embryos was unaware of the group the embryos had been assigned to. Embryos were then cultured for 48 h using a rotating-bottle culture unit (BTC Engineering, UK), rotating at a constant rate of 60 rpm. During the period of culture, the system was continuously aerated initially with a gas mixture of 5% CO2, 5% O2 and 90% N2 (24 h), followed by 5% CO2, 20% O2 in 75% N2 for 8 h and then 5% CO2, 40% O2 in 55% N2 for the remaining 16 h. The switching of aerating gas was performed automatically by a timer-controlled system. Different types of gas mixtures were premixed and prepared commercially.
Embryos were randomly assigned to the study or control group. Each culture bottle contained 1 ml of culture medium per embryo. Each millilitre of culture medium contained (i) 80% volume of SpragueDawley rat serum; (ii) 20% volume of hydrosalpinx fluid or lactated Ringers solution (Thai Otsuka, Thailand) in the study and control groups respectively; and (iii) penicillin G (Sigma, UK) and streptomycin sulphate (Sigma) with a final concentration of 60 and 100 µg/ml respectively. During each experiment, only one patients hydrosalpinx fluid was used. We chose lactated Ringers solution as the diluting medium in the control group because its electrolyte content is similar to that of hydrosalpinx fluid (Amnon et al., 1969).
Morphological assessment
Embryos were examined after 48 h of culture at the equivalent of 11.5 days of gestation by a researcher who was not aware of the study group assignment. Mean yolk sac diameter and CRL were measured. Embryonic morphologies were studied according to a standard morphological scoring system (Van Maele-Fabry et al., 1990), which gives a numerical score [0 (worst) to 5 (best)] to 17 morphological features depending on their stage of development. Embryos with a total morphological score of <2 were most likely damaged due to explantation and were excluded from the analysis.
Statistical evaluation
Between-group differences were analysed by MannWhitney U-test. Analyses were performed by the Statistical Package for Social Sciences for Windows version 10.0 (SPSS Inc., USA). P < 0.05 was considered statistically significant.
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Results |
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Discussion |
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In this study, we have shown that hydosalpinx fluid from some patients was embryotoxic. In patient 7, the significant reduction in morphological score was not accompanied by reduction in yolk sac diameter and somite number. Because we had made four statistical comparisons in each of the 10 patients, it is possible that this may represent a type I error. On the contrary, hydrosalpinx fluids from patients 9 and 10 were clearly embryotoxic with a large reduction of morphological score, yolk sac diameter, and somite number. While the toxicity of hydrosalpinx fluid from patient 9 might have been due to the presence of bacteria in the fluid introduced during transvaginal oocyte retrieval, no specific risk factors could be identified in patient 10. The toxicity of hydrosalpinx fluid was unlikely due to the lack of growth factors because embryos in the control group were exposed to the same conditions. These adverse effects were therefore most probably due to the presence of toxic substances in the hydrosalpinx fluid. Further studies are required to elucidate the exact nature of the toxic substances.
Because hydrosalpinx fluid may reflux into the endometrial cavity and have direct contact with the developing embryo (Aboulghar et al., 1998), it potentially may affect early embryonic development. Although the number of patients in the present study was small, based on our results, it appears that hydrosalpinx fluid in some patients may contain toxin(s) that not only affects early embryo development, but may also be teratogenic. Further studies involving more patients will provide additional information concerning the teratogenicity of hydrosalpinx fluids. Future research is also required to identify those patients whose hydrosalpinx fluids are toxic. Although results from animal teratogenicity studies may not reflect the circumstances in humans, our findings suggest that further investigation and monitoring of teratogenic effects of hydrosalpinx fluid is warranted.
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References |
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Amnon, D., Celso-Ramon, G. and Bernard, C. (1969) Human hydrosalpinx. Histologic study and chemical composition of fluid. Am. J. Obstet. Gynecol., 105, 400411.[ISI][Medline]
Anderson, A.N., Yue, Z., Meng, F.J. and Petersen, K. (1994) Low implantation rate after in-vitro fertilization in patients with hydrosalpinges diagnosed by ultrasonography. Hum. Reprod., 9, 19351938.[Abstract]
Beyler, S.A., James, K.P., Fritz, M.A. and Meyer, W.R. (1997) Hydrosalpingeal fluid inhibits in-vitro embryonic development in a murine model. Hum. Reprod., 12, 27242728.[Abstract]
Blazar, A.S., Hogan, J.W., Seifer, D.B., Frishman, G.N., Wheeler, C.A. and Haning, R.V. (1997) The impact of hydrosalpinx on successful pregnancy in tubal factor infertility treated by in vitro fertilization. Fertil. Steril., 67, 517520.[ISI][Medline]
Katz, E., Akman, M.A., Damewood, M.D. and Garcia, J.E. (1996) Deleterious effect of the presence of hydrosalpinx on implantation and pregnancy rates with in vitro fertilization. Fertil. Steril., 66, 122125.[ISI][Medline]
Koong, M.K., Jun, J.H., Song, S.J., Lee, H.J., Song, I.O. and Kang, I.S. (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 ovarys perspective. Hum. Reprod., 14, 16741677.
Meyer, W.R., Castelbaum, A.J., Somkuti, S., Sagoskin, A.W., Doyle, M., Harris, J.E. et al. (1997) Hydrosalpinges adversely affect markers of endometrial receptivity. Hum. Reprod., 12, 13931398.[Abstract]
Mukherjee, T., Copperman, A.B., McCaffrey, C., Cook, C.A., Bustillo, M. and Obasaju, M.F. (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. and Tan, S.L. (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]
New, D.A.T. (1978) Whole embryo culture and study of the mammalian embryo during organogenesis. Biol. Rev. Camb. Phil. Soc., 53, 81112.[ISI][Medline]
Ng, E.H., Ajonuma, L.C., Lau, E.Y., Yeung, W.S. and Ho, P.C. (2000) Adverse effects of hydrosalpinx fluid on sperm motility and survival. Hum. Reprod., 15, 772777.
Rawe, V.J., Liu, J., Shaffer, S., Compton, M.G., Garcia, J.E. and Katz, E. (1997) Effect of human hydrosalpinx fluid on murine embryo development and implantation. Fertil. Steril., 68, 668670. [CrossRef][ISI][Medline]
Roberts, J.E., Clarke, H.J., Tulandi, T. and Tan, S.L. (1999) Effects of hydrosalpingeal fluid on murine embryo development and implantation. J. Assist. Reprod. Genet., 16, 421424.[CrossRef][ISI][Medline]
Sachdev, R., Kemmann, E., Bohrer, M.K. and el-Danasouri, I. (1997) Detrimental effect of hydrosalpinx fluid on the development and blastulation of mouse embryos in vitro. Fertil. Steril., 68, 531533.[CrossRef][ISI][Medline]
Spandorfer, S.D., Liu, H.C., Neuer, A., Barmat, L.I., Davis, O. and Rosenwaks, Z. (1999) The embryo toxicity of hydrosalpinx fluid is only apparent at high concentrations: an in vitro model that stimulates in vivo events. Fertil. Steril., 71, 619626.[CrossRef][ISI][Medline]
Strandell, A., Waldenstrom, Nilsson, L. and Hamberger, L. (1994) Hydrosalpinx reduces in-vitro fertilization/embryo transfer pregnancy rates. Hum. Reprod., 9, 861863.[Abstract]
Strandell, A., Lindhard, A., Waldenstrom, U., Thorburn, J., Janson, P.O. and Hamberger, L. (1999) Hydrosalpinx and IVF outcome: a prospective, randomized multicentre trial in Scandinavia on salpingectomy prior to IVF. Hum. Reprod., 14, 27622769.
Vandromme, J., Chasse, E., Lejeune, B., Rysselberge, M.V., Delvigne, A. and Leroy, F. (1995) Hydrosalpinges in in-vitro fertilization: an unfavourable prognostic feature. Hum. Reprod., 10, 576579.[Abstract]
Van Maele-Fabry, G., Delhaise, F. and Picard, J.J. (1990) Morphogenesis and quantification of the development of postimplantation mouse embryos. Toxicol. In Vitro, 4, 149156.[CrossRef][ISI]
Submitted on September 19, 2002; resubmitted on November 27, 2002; accepted on January 10, 2003.