A study on placental transfer of diclofenac in first trimester of human pregnancy

S.S.N. Siu1,4, J.H.K. Yeung2 and T.K. Lau3

1 Department of Obstetrics and Gynaecology, Prince of Wales Hospital, 2 Department of Pharmacology and 3 Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Shatin, Hong Kong


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Diclofenac is a commonly used non-steroidal anti-inflammatory drug in women of reproductive age. It has teratogenic effects in animals. The aim of this study was to investigate the placental transfer of diclofenac in the first trimester of human pregnancy. Thirty patients undergoing surgical termination of pregnancy between 8 and 12 weeks gestation were given two doses of diclofenac before the procedure. Corresponding samples of maternal serum, amniotic fluid, coelomic fluid and fetal tissue were analysed by high-performance liquid chromatography. Diclofenac was detectable in all fetal tissue samples, with a concentration similar to that found in maternal venous samples. However, diclofenac was detectable in only 56.7 and 23.3% of the coelomic and amniotic fluid samples respectively, and the highest concentration attained was 80 and 5% of the maternal concentration respectively. In summary, we confirmed that diclofenac crosses the human placenta readily during the first trimester. Further studies are required to investigate the potential teratogenic effect of diclofenac in human embryos.

Key words: diclofenac/human/placental transfer/pregnancy/teratogenicity


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prostaglandins are important chemical mediators in the human body, being involved in both normal and abnormal function of virtually every organ and system. Non-steroidal anti-inflammatory drugs (NSAID) are prostaglandin synthetase inhibitors. They act on the cyclo-oxygenase, which is a crucial enzyme in the biosynthesis of prostaglandins from arachidonic acid (Van den Veyver and Moise, 1993Go). NSAID have been used extensively in the treatment of a variety of diseases and conditions, including arthritis, musculo-skeletal pain, dysmenorrhoea and menorrhagia (Dawood, 1993Go). This group of drugs is one of the commonest prescribed by general practitioners world-wide and it is not uncommon for newly pregnant women to report that they have been taking these drugs during the early conception period. This resulted in enormous anxiety and worry concerning the possible teratogenic effects on the fetus.

Aspirin and indomethacin are the two of the `oldest' NSAID, which have been used in the second and third trimester for the treatment and prevention of a wide range of obstetric complications (CLASP, 1994; Leitich et al., 1997Go; Reznikoff-Etievant et al., 1999Go). They were known to cross the placenta from second trimester onwards and to cause serious adverse fetal and neonatal effects (Ostensen, 1998Go).

Since disturbances in homeostasis of prostaglandins have been implicated in the teratogenesis of fetal malformations (Klein et al., 1984Go), NSAID are therefore potentially teratogenic. Teratogenic effects of the older generation of NSAID have been demonstrated in animal experiments (McGarrity et al., 1981Go), but conclusive evidence of teratogenicity in human embryos is lacking.

There is even less information concerning the newer generations of NSAID, of which diclofenac is the one of the most common. Diclofenac is widely prescribed to women of child-bearing age for the treatment of common gynaecological problems such as menorrhagia and dysmenorrhoea. Previous studies have confirmed the toxicity of diclofenac on rat embryos (Carp et al., 1988Go). Again, human data are lacking.

One of the possible reasons for the lack of reported teratogenic effect of NSAID in humans might be due to the absence of placental transfer in early pregnancy. The objective of this project was to investigate the placental transfer of diclofenac in the first trimester of human pregnancy by sampling the maternal serum, amniotic fluid, coelomic fluid and fetal tissue in women who are undergoing surgical termination of pregnancy. The result of this study will form the basis of further investigation on teratogenic effects of diclofenac.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Thirty pregnant women having legal termination of pregnancy under general anaesthetic between 8 and 11 weeks gestation were recruited. These patients received counselling on termination of pregnancy in the out-patient clinic. They were only approached to participate in this study after the decision to perform the termination of pregnancy had been made. The study was approved by the Clinical Research Ethics Committee of the local institution. Written informed consent for this study was obtained. In all cases, ultrasound examination had confirmed a singleton pregnancy and the gestational age.

Two oral doses of 50 mg diclofenac (Cataflam®; Ciba-Geigy Ltd., Basle, Switzerland) were given before surgery, the first at 2200 h the night before operation, the second at 2 h before the scheduled operation time. All operations were performed between 0830 and 1300 h. Surgical termination of pregnancy was performed under general anaesthesia. Maternal venous blood sample was taken just before induction of general anaesthesia. After induction of general anaesthesia, ultrasound scan examination was performed to confirm fetal viability and crown–rump length measurement. Coelocentesis and amniocentesis were performed by fine needle aspiration under ultrasound guidance transvaginally according to protocols previously reported (Lau et al., 1998Go). Surgical termination of pregnancy by suction curettage was then performed in the usual manner. Fetal parts were identified, washed with normal saline to clear maternal blood, and collected after the surgical procedure and were kept for further analysis.

Maternal serum was separated from maternal blood by centrifugation, using 1250 g for 10 min. All samples were stored at –70°C, pending further analyses. The fetus was weighed, homogenized in physiological saline and then centrifuged to clear samples before analysis. Diclofenac concentration in each of four specimens was measured by high-performance liquid chromatography with UV detection. The minimal detection limit of the assay was 0.5 ng/ml. The inter-batch assay of diclofenac was reproducible and precise with a coefficient of variation of 7.3%. Diclofenac metabolites in the samples were not analysed.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
The mean gestational age at termination was 10.2 weeks (range: 8.7–11.7). The mean duration between the first and second doses of diclofenac and termination of pregnancy was 13.5 and 2.5 h respectively.

Diclofenac was detected in all maternal serum and fetal tissue samples. Maternal serum diclofenac concentrations ranged from 24.6–842.4 ng/ml (mean 183.9 ± 209.0) and the fetal tissue drug concentration ranged from 17.4–665.7 ng/g (mean 279.2 ± 164.9).

Among 30 samples of amniotic fluid, diclofenac was detected in seven (23.3%), with the range of concentrations between 0.6–3.5 ng/ml. The highest ratio between amniotic fluid and maternal serum diclofenac concentrations was 0.047. In other words, the highest amniotic fluid drug concentration attained was <5% of the maternal concentration. Six of these positive samples were at gestational age >10 weeks, the remaining one was at 9 weeks and 5 days.

Diclofenac was detected in significantly more coelomic fluid samples (17 of 30, 56.7%) than amniotic fluid samples (23.3%) (P = 0.018). The drug concentration in coelomic fluid samples ranged from 1.1–33.4 ng/ml. The highest coelomic concentration measured was 80% of the maternal concentration.

There was an inverse relationship between fetal drug concentration and advancing gestational age (r = –0.40, P = 0.03) (Figure 1Go). There was no relationship between the drug concentration in coelomic fluid, amniotic fluid or maternal serum with gestational age.



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Figure 1. Significant inverse relationship between the concentration of diclofenac in fetal tissue and advancing gestational age.

 
Maternal/fetal diclofenac ratio was calculated by dividing the maternal serum concentration by the corresponding fetal tissue drug concentration. The mean ratio was 0.95 (range: 0.05–4.26). No association was demonstrated between this ratio and the gestational age (r = 0.25, P = 0.17) (Figure 2Go).



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Figure 2. Maternal/fetal ratio of diclofenac concentration plotted against gestational age: there was no significant relationship.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is well known that the use of NSAID in the second trimester onwards may lead to adverse fetal effects (Ostensen, 1998Go). Teratogenicity of aspirin and indomethacin has been demonstrated in animal experiments (Kimmel et al., 1971Go; Klein et al., 1981Go; Gupta and Goldman, 1986Go; Montenegro and Palomino, 1990Go) but observation studies in human pregnancies have produced conflicting results (Turner and Collins, 1975Go; Werler et al., 1989Go; Ostensen and Ostensen, 1996Go). Although NSAID are increasingly used for the prevention of obstetric complications such as pre-eclampsia or recurrent abortion, these drugs are either avoided or given in a lower dose in the first trimester of pregnancy (Reznikoff-Etievant et al., 1999Go). Therefore the absence of apparent teratogenic effects in this group of patients could not be generalized to those patients who conceive accidentally whilst taking NSAID. The apparent lack of teratogenic effect of NSAID in humans could be due to poor placental transfer in first trimester, and it was the objective of the current study to investigate this placental transfer.

We chose to study diclofenac in this experiment because its use is increasingly popular among women of child-bearing age and there is a lack of information on its placental transfer in early human pregnancy. We have shown that the drug crosses first-trimester human placenta readily. Its relatively low molecular weight of 318.15 Da probably explains why diclofenac crosses trophoblastic membranes easily. The mean maternal/fetal drug ratio was less than one, indicating the drug may accumulate in fetal tissue with time.

It should be noted that diclofenac concentrations in serum, amniotic and coelomic samples were expressed as amount of drug per unit volume (per ml), while that in fetal tissue was as drug per unit weight (per g). Therefore, the ratio involving fetal tissue drug concentration has to be interpreted appropriately. Since the density of fetal tissue should be higher than 1 g/ml, the numerical value of the drug concentration should be higher when expressed in ng/ml than the concentration as expressed in ng/g. Therefore, the true mean maternal/fetal ratio probably should be even lower than 0.95 when the same unit is used in both samples.

Our results showed that diclofenac was present in significantly more coelomic fluid samples than amniotic fluid samples, and the concentrations attained were also higher. This difference between compartments was probably a result of the different origins of these fluids. Coelomic fluid is produced by the chorionic plate (Jauniaux and Gulbis, 1998Go) and a high drug concentration indicates that diclofenac crosses the chorionic plate easily. On the other hand, amniotic fluid is essentially an exudate from fetal skin and amniotic membrane before 10 weeks gestation, while fetal urine becomes the major source as gestational age increases and when the fetal renal system starts to function (Gulbis et al., 1996Go). Our result indicates that fetal skin may not be permeable to diclofenac, therefore only a low concentration of drug was detectable in the amniotic fluid. It is possible that higher concentrations of diclofenac will be attained in pregnancy beyond the first trimester as the fetal renal system starts to function because the major source of metabolism of diclofenac is by renal excretion. Our results showing that drug concentration is higher in coelomic fluid confirmed the hypothesis reported previously (Jauniaux and Gulbis, 1998Go).

In clinical practice, diclofenac is usually given in divided doses in the range of 75–150 mg per day, resulting in a mean peak serum concentration of 1.5 µg/ml (unpublished results). The serum concentration in our study was lower, probably because only two doses of drug were given to our subjects. We would therefore expect that the corresponding mean fetal concentration would be higher in a real clinical situation, probably in the order of at least 1–10 µg/g.

In conclusion, we have shown that maternal administration of diclofenac results in a rapid accumulation of the drug in the fetus during the first trimester of pregnancy. Since an adverse embryonic effect of diclofenac has been demonstrated in animals (Carp et al., 1988Go), further investigations are required to elucidate whether the same effect occurs in humans. Before such a result becomes available, the use of NSAID should be considered potentially teratogenic. Patients at reproductive age who are taking NSAID regularly should be warned of the possible adverse fetal side-effects.


    Acknowledgments
 
The authors would like to thank Miss Penelope M.Y.Or for technical assistance. This work was supported by the Direct Grants for Research, The Chinese University of Hong Kong.


    Notes
 
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Prince of Wales Hospital, Shatin, Hong Kong. E-mail: nelsonsiu{at}cuhk.edu.hk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Carp, H.J.A., Fein, A. and Nebel L. (1988) Effect of diclofenac on implantation and embryonic development in the rat. Eur. J. Obstet. Gynecol. Reprod. Biol., 28, 273–277.[ISI][Medline]

CLASP Collaborative Group (1994) CLASP: a randomised trial of low-dose aspirin for the prevention and treatment of pre-eclampsia among 9364 pregnant women. Lancet, 343, 619–629.[ISI][Medline]

Dawood, M.Y. (1993) Nonsteroidal antiinflammatory drugs and reproduction. Am. J. Obstet. Gynecol., 169, 1255–1265.[ISI][Medline]

Gulbis, B., Jauniaux, E., Jurkovic, D. et al. (1996) Biochemical investigation of fetal renal maturation in early pregnancy. Pediatr. Res., 39, 731–735.[Abstract]

Gupta, C. and Goldman, A. (1986) The arachidonic acid cascade is involved in the masculinizing action of testosterone on embryonic external genitalia in mice. Proc. Natl Acad. Sci. USA, 83, 4346–4349.[Abstract]

Jauniaux, E. and Gulbis, B. (1998) In vivo study of placental drug transfer during the first trimester of human pregnancy. Trophoblast Res., 12, 257–264.

Kimmel, C.A., Wilson, J.G. and Schumacher, H.J. (1971) Studies on the metabolism and identification of the causative agent in aspirin teratogenesis in rats. Teratology, 4, 15–24.[ISI][Medline]

Klein, K.L., Scott, W.J. and Wilson, J.G. (1981) Aspirin-induced teratogenesis: a unique pattern of cell death and subsequent polydactyly in the rat. J. Exp. Zool., 216, 107–112.[ISI][Medline]

Klein, K.L., Clark, K.E. and Scott, W.J. (1984) Prostaglandin synthesis in rat embryo tissue: the effect of non-steroidal anti-inflammatory drugs in vivo and ex vivo. Prostaglandins, 27, 659–672.

Lau, T.K., Fung, T.Y., Wong, Y.F. et al. (1998) A study of fetal sex determination in coelomic fluid. Gynecol. Obstet. Invest., 45, 16–18.[ISI][Medline]

Leitich, H., Egarter, C., Husslein, P. et al. (1997) A meta-analysis of low dose aspirin for the prevention of intrauterine growth retardation. Br. J. Obstet. Gynaecol., 104, 450–459.[ISI][Medline]

McGarrity, C., Samani, N., Beck, F. et al. (1981) The effect of sodium salicylate on the rat embryo in culture: an in vitro model for the morphological assessment of teratogenicity. J. Anat., 133, 257–269.[ISI][Medline]

MIMS (1994) MIMS Annual, MIMS Australia, pp. 5–310.

Montenegro, M.A. and Palomino, H. (1990) Induction of cleft palate in mice by inhibitors of prostaglandin synthesis. J. Craniofac. Genet. Dev. Biol., 10, 83–94.[ISI][Medline]

Ostensen, M. (1998) Nonsteroidal anti-inflammatory drugs during pregnancy. Scand. J. Rheumatol., 27 (Suppl. 107), 128–132.

Ostensen, M., and Ostensen, H. (1996) Safety of non-steroidal antiinflammatory drugs in pregnant patients with rheumatic disease. J. Rheumatol., 23, 1045–1049.[ISI][Medline]

Reznikoff-Etievant, M.F., Cayol, V., Zou, G.M. et al. (1999) Habitual abortions in 678 healthy patients: investigation and prevention. Hum. Reprod., 14, 2106–2109.[Abstract/Free Full Text]

Turner, G. and Collins, E. (1975) Fetal effects of regular salicylate ingestion in pregnancy. Lancet, 2, 338–339.[ISI][Medline]

Van den Veyver, I.B. and Moise, K.J. (1993) Prostaglandin synthetase inhibitors in pregnancy. Obstet. Gynecol. Surv., 48, 493–502.[Medline]

Werler, M.M., Mitchell, A.A. and Shapiro, S. (1989) The relation of aspirin use during the first trimester of pregnancy to congenital cardiac defects. N. Engl. J. Med., 321, 1639–1642.[Abstract]

Submitted on February 14, 2000; accepted on July 14, 2000.