A study of teratogenicity of hydrosalpinx fluid using a whole rat embryo culture model

L.Y. Chan1,3, P.Y. Chiu1, L.P. Cheung1, C.J. Haines1, H.F. Tung2 and T.K. Lau1

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, People’s Republic of China

3 To whom correspondence should be addressed. e-mail: lyschan{at}cuhk.edu.hk


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Hydrosalpinx fluid may be toxic to sperm and early embryo growth. Information concerning the effect of hydrosalpinx fluid on embryo development during organogenesis is lacking. METHODS: Rat embryos at gestational day 9.5 were cultured for 48 h with 80% rat serum and 20% of either hydrosalpinx fluid (study group) or lactated Ringer’s solution (control group). Embryos were scored for growth and development at the end of the culture period. RESULTS: Hydrosalpinx fluid, collected from 10 patients, was tested for embryotoxicity individually. Median total morphological scores were significantly lower in embryos exposed to hydrosalpinx fluid from three of the 10 patients (43.0 versus 47.0, P = 0.01; 36.0 versus 45.0, P < 0.001; 36.0 versus 46.5, P = 0.003). This was accompanied by a significant reduction in median yolk sac diameter (4.0 versus 5.2 mm, P < 0.001 and 4.0 versus 5.0 mm, P < 0.001) and somite number (17.5 versus 22.5, P < 0.001 and 17.0 versus 21.5, P = 0.008) in the latter two patients. CONCLUSIONS: Hydrosalpinx fluid in some patients may contain toxin(s) that is potentially teratogenic.

Key words: hydrosalpinx/teratogenicity/whole rat embryo culture


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A hydrosalpinx is the accumulation of fluid in the Fallopian tube. As many as 30% of patients with tubal factor infertility have a hydrosalpinx (Aboulghar et al., 1998Go). Recent reports suggest that the presence of a hydrosalpinx significantly reduces the implantation and pregnancy rates after IVF by as much as 50% (Anderson et al., 1994Go; Strandell et al., 1994Go; Vandromme et al., 1995Go; Katz et al., 1996Go; Blazar et al., 1997Go). Early pregnancy losses after IVF have also been reported to be higher in patients with a hydrosalpinx than with those having tubal disease without a hydrosalpinx (Anderson et al., 1994Go). Several investigators reported improvement in IVF outcome in patients with a hydrosalpinx if the diseased tube is removed before IVF treatment (Strandell et al., 1999Go).

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., 1997Go) or an embryotoxic effect of hydrosalpinx fluid (Lass, 1999Go). Direct toxic effects of hydrosalpinx fluid on early non-human embryos at the 1–4-cell stage have been reported (Beyler et al., 1997Go). 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., 1997Go).

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hydrosalpinx fluid
Hydrosalpinx fluid was aspirated during laparoscopy or at the time of oocyte retrieval for IVF. After aspiration, the fluid was centrifuged at 4433 g for 15 min, decomplemented at 56°C for 30 min, sterile filtered, divided into aliquots and frozen at –70°C until used. The Clinical Research Ethics Committee of The Chinese University of Hong Kong approved the study.

Animals
Timed-gestation pregnant Sprague–Dawley 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, 1978Go). 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 crown–rump 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 Sprague–Dawley rat serum; (ii) 20% volume of hydrosalpinx fluid or lactated Ringer’s 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 patient’s hydrosalpinx fluid was used. We chose lactated Ringer’s solution as the diluting medium in the control group because its electrolyte content is similar to that of hydrosalpinx fluid (Amnon et al., 1969Go).

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., 1990Go), 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 Mann–Whitney 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.


    Results
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hydrosalpinx fluid was collected from ten patients. Table I shows the distribution of rat embryos in the study and control groups together with the total morphological scores for each group. The total morphological scores were significantly lower in embryos exposed to hydrosalpinx fluid from patients 7, 9 and 10 (Table I). There was also a significant reduction in yolk sac diameter among those embryos exposed to hydrosalpinx fluid from patients 8, 9 and 10 (Table II). Concerning the somite number, it was significantly fewer in embryos from patients 9 and 10 (Table III). There were no differences in CRL between experimental and control embryos except that in patient 2, embryonic CRL was significantly shorter for the control embryos (Table IV).


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Table I. Total morphological score of embryos in study and control groups
 

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Table II. Yolk sac diameter (mm) of embryos in study and control groups
 

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Table III. Somite number of embryos in study and control groups
 

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Table IV. Embryonic crown–rump length (mm) in study and control groups
 
Patient characteristics were reviewed. All hydrosalpinges had been evident sonographically. Patient 9 suffered from chronic pelvic inflammatory disease. She developed acute pelvic inflammatory disease and a pyosalpinx after her oocyte retrieval. Laparoscopy and drainage of the pyosalpinx was performed. Culture of the pyosalpinx fluid revealed E. coli.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous studies have shown that hydrosalpinx fluids are potentially toxic to gametes and embryos (Rawe et al., 1997Go). However, all studies so far have been limited to sperm (Ng et al., 2000Go) or to early embryos at either 1–4-cell stage or blastocyst development (Mukherjee et al., 1996Go; Rawe et al., 1997Go; Roberts et al., 1999Go). In the present study, we investigated the potential teratogenicity of hydrosalpinx fluid during the critical period of organogenesis using a whole rat embryo culture model. Our results showed that hydropsalpinx fluid from some patients had an adverse effect on organogenesis, while that from others had no effect. This variation in toxicity is in agreement with results of similar previous studies. Ng et al. (2000Go) showed that only three of their 15 fluid samples demonstrated toxicity towards sperm. Sachdev et al. (1997Go) showed that the toxic effect of hydrosalpinx fluid was observed at 0.3% concentration, while in Spandorfer’s study, hydrosalpinx fluid had no significant toxic effect until the concentration reached 50% (Spandorfer et al., 1999Go). The effect of hydrosalpinx fluid on blastocyst development is also variable. Some authors showed that 5–10% of hydrosalpinx fluid concentration is sufficient to cause impairment of blastocyst development (Mukherjee et al., 1996Go; Rawe et al., 1997Go), while other investigators showed that toxicity impairment is only evident at 100% concentration of hydrosalpinx fluid (Murray et al., 1997Go; Koong et al., 1998Go). Murray et al. (1997Go) suggested that this variation resulted from hydrosalpinx fluid being derived from bacterial infection of the Fallopian tubes and that different levels of infection-associated toxins may be produced depending on the nature of infection.

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., 1998Go), 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.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on September 19, 2002; resubmitted on November 27, 2002; accepted on January 10, 2003.