Abstract
The aim of the study was to investigate maternal thyroid function in pregnancy by monitoring the circulating concentrations of thyroid stimulating hormone (TSH), free thyroxine (fT 4 ) and human chorionic gonadotrophin (hCG) in multifetal pregnancies before and after embryo reduction. We studied two groups of women: group 1 comprised singleton (n=12) and twin (n=12) pregnancies achieved after superovulation and in vitro fertilisation and embryo transfer (IVF-ET), and group 2 were multifetal pregnancies (n=39) undergoing selective fetal reduction to twin pregnancies. Blood samples were obtained initially at 10-12 weeks gestation (before fetal reduction) and then 4 and 8 weeks afterwards. Before fetal reduction, the circulating concentrations of fT 4 in multifetal pregnancies were significantly greater than those in singleton or twin pregnancies (singleton, mean 16·49 pmol/l (interquartile range14·09-18·13 pmol/l); twins, 15·84 (15·36-16·95 pmol/l); multifetal, 21·08 (16·64-26·29 pmol/l); P<0·005 for singleton and twins), and in a multiple regression analysis, fT 4 was significantly related to the number of fetuses (F=23·739, P=0·0001), but not to hCG. After fetal reduction to twins, the circulating concentrations of fT 4 in multifetal pregnancies decreased progressively towards those in control twin pregnancies, but remained significantly greater at both 4 (P=0·003) and 8 weeks (P=0·050). This pattern of change in the concentrations of fT 4 is similar to, but lags behind, that of hCG, which attains twin levels 4 weeks after fetal reduction. This may represent a delayed thyroid response to the decreasing concentrations of hCG, but the alternative is that the maternal thyroid function is controlled by a fetal factor in addition to hCG.
Introduction
Thyroid activity is increased during the first trimester of normal pregnancy, reflected by a decrease in the circulating concentrations of thyroid stimulating hormone (TSH) and an increase in the concentrations of free thyroxine (fT 4 ) and tri-iodothyronine (fT 3 ). The origin of the increased maternal thyroid function is almost certainly stimulation of the TSH receptor by human chorionic gonadotrophin (hCG) (Guillame et al. 1985 , Yoshikawa et al. 1989 , Glinoer et al. 1990 , Ballabio et al. 1991 . As the fetal thyroid is active only after 14 weeks gestation,it is suggested that the increased maternal thyroid activity ensures that normal neuronal development occurs in the fetus (Shepard 1975) . The circulating concentrations of TSH, fT 4 and fT 3 revert back to normal by the second trimester in most women, and remain within normal limits during the second and third trimesters (Harada et al. 1979) .
Introduction
The increase in thyroid activity in the first trimester is more profound in twin than in singleton pregnancy (Grun et al. 1997) , probably because peak concentrations of hCG are greater and remain increased for longer in twin pregnancies (Grun et al. 1997) . However, it has been suggested that factors other than hCG may be responsible for the pregnancy-associated increase in maternal thyroid function (Kennedy et al. 1992) . The aim of the present study was to test the hypothesis that a factor other than hCG is responsible for the changes in maternal thyroid function. To achieve this, we measured the circulating concentrations of TSH, fT 4 and hCG before and after selective fetal reduction.
Patients and Methods
Two groups of women were examined. Group 1 comprised singleton (n=12) and twin (n=12) pregnancies achieved after superovulation and in vitro fertilisation and embryo transfer (IVF-ET), recruited from the Assisted Conception Unit, King's College Hospital, and group 2 were multifetal pregnancies (three to eight fetuses, n=39: three fetuses n=22, four fetuses n=6, five fetuses n=7, six fetuses n=3 and eight fetuses n=1) undergoing selective fetal reduction to twin pregnancies, recruited from The Harris Birthright Research Centre for Fetal Medicine, King's College Hospital. The multifetal pregnancies occurred after a variety of assisted reproduction techniques that included ovulation induction, superovulation and intra-uterine insemination, and superovulation and IVF. They were referred from a variety of units. Fetal reduction to twins was carried out by ultrasound-guided transabdominal injection of potassium chloride into the fetal heart. Ultrasound demonstrated complete reabsorption of the feto-placental units 8-12 weeks after reduction. In group 1, blood samples were obtained initially at 10-12 weeks gestation and then 4 and 8 weeks afterwards. In group 2, blood was obtained before reduction at 10-12 weeks gestation and then at 4 and 8 weeks after reduction. The Ethics Committee of King's College Hospital approved the study and the women gave informed consent to participate.
Patients and Methods
TSH and fT 4 were measured using the Elecsys electrochemiluminescence immunoassay analyser (Roche Diagnostic Ltd, Lewes, UK). Inter-assay precision (coefficient of variation) was as follows: fT 4 , 4·7% (8·8 pmol/l) and 5·2% (34·5 pmol/l); TSH, 6·9% (0·32 mU/l), 5·3% (7·8 mU/l) and 5·1% (26·0 mU/l). hCG was measured by non-competitive fluoroimmunoassay (Pharmacia Wallac, Milton Keynes, Bucks, UK.). The variation (intra-and inter-assay) for these assays over the period of the study was less than 10%.
Patients and Methods
The data were not normally distributed, and nonparametric tests (Mann-Whitney U) were used to analyse the data. Simple and multiple regression analysis were used to investigate the associations between analytes and fetal number.
Results
There was no significant difference in the circulating concentrations of fT 4 between singleton and twin pregnancies (P=0·95 at 10-12 weeks, P=0·40 at 14-16 weeks, and P=0·21 at 18-20 weeks). However, before fetal reduction, the fT 4 concentrations were greater in multifetal pregnancies when compared with singleton or twin pregnancies (P<0·005) ( Table 1) . After fetal reduction to twins, the circulating concentrations of fT 4 in multifetal pregnancies decreased progressively, but remained significantly different from those in twin 
Results
*P<0·05, **P<0·01 compared with multifetal values.
Results
O OGUEH and others · Maternal thyroid function in multifetal pregnancypregnancies at 8 weeks (P=0·003 and P=0·0502 at 4 and 8 weeks after reduction respectively) ( Table 1) . There was no significant difference in the circulating concentrations of TSH between singleton and twin pregnancies (P=0·20 at 10-12 weeks, P=0·41 at 14-16 weeks, and P=0·14 at 18-20 weeks). However, before fetal reduction, the TSH concentrations were significantly lower in multifetal pregnancies compared with singleton, but not twin, pregnancies (P=0·005 and P=0·23 respectively) (Table 1) . After fetal reduction to twins, the circulating concentrations of TSH in multifetal pregnancies increased to twin pregnancy values by 8 weeks (P=0·025 and P=0·19 at 4 and 8 weeks after reduction) ( Table 1) .
Results
Before fetal reduction, there was a positive association between the circulating concentrations of fT 4 and both hCG (r=0·52, P=0006; Fig. 1 ) and fetal number (r=0·63 and P<0·0001 ; Fig. 2) ; and a negative association with TSH concentrations (r= 0·52, P=0·0001; Fig. 3) . Thus, for a given hCG concentration, fT 4 concentration is proportionately greater when there are multiple fetuses. However, using multiple regression analysis, including all the above variables, fT 4 was significantly related to the number of fetuses (a positive relationship) and TSH (a negative relationship, Overall F=23·739; for number of fetuses Partial F=18·199 and P=0·0001; for TSH Partial F=10·629 and P=0·0019), but not to hCG.
Discussion
It has been reported that hCG has a thyroid-stimulating effect in vitro (Davies & Platzer 1986 ). The thyrotrophic activity of hCG is believed to be due to the homology between the TSH and hCG molecular structures, and receptors with only a 15% sequence difference between them (Nisula et al. 1974 , Yoshikawa et al. 1989 . However, thyroid stimulation by hCG is reported to be weak and to be effective only at high concentrations of hCG (Guillaume et al. 1985) . These data would explain why overt hyperthyroidism is seen only in some women and then only in the first trimester, when hCG concentrations are greatest, and the occurrence of clinical and biochemical hyperthyroidism in women with hCG-producing tumours such as hydatidiform mole and choriocarcinoma (Miyai et al. 1976) . Indeed, the spectrum of increasing thyroid activity correlates with the severity of disease and the concentration of hCG, suggesting a causal relationship (Kenimer et al. 1975) . At the cellular level, hCG is believed to increase intracellular release of cAMP in the thyroid, which leads to a degranulation response, and the secretion of thyroxine. In vitro data have not fully supported this view, as only a minimal increase in secretion occurs in response to hCG (Kraiem et al. 1994) . However, the conflict may well be due to the isoform of hCG used, as its effect at the TSH receptor is determined by its glycosylation and can range from stimulation to inhibition (Hoermann et al. 1994 , Poertl et al. 1995 . Such data suggest that hCG is probably the only factor involved in the alteration of maternal thyroid function in early pregnancy, but the possibility remains that other factors are also involved.
Discussion
After fetal reduction, the concentrations of hCG decrease to those of control twin pregnancies by 4 weeks, suggesting that fetal reduction causes placental death and therefore production of hCG by the remaining pregnancies at a rate similar to that in control twins . In contrast, after fetal reduction, the maternal concentrations of alpha fetoprotein ( -FP) increase, and remain increased for at least 8 weeks ). This pattern is probably due to the gradual breakdown of fetal tissue, the release of -FP into amniotic fluid, and its subsequent absorption into the maternal circulation . We found that multifetal pregnancy reduction was associated with a decrease in the circulating concentrations of fT 4 to those of the control group by 8 weeks, but at 4 weeks the concentrations of fT 4 were still significantly greater. The relative delay in the decrease in fT 4 compared with hCG may reflect a delay in the thyroid response to decreasing hCG concentrations. However, as fT 4 was related to the number of fetuses but not to hCG concentrations in the multiple regression analysis, it is possible that a fetal factor is also involved in the alteration of maternal thyroid function in pregnancy. If this were so, it would explain the similarity in the pattern of fT 4 decline to that of -FP described above. A further possibility is that the form of hCG, which has thyroid stimulating-properties, may be cleared at a slower rate and so continues to stimulate thyroid activity for longer than anticipated by the total circulating concentrations of hCG. However, to account for the relationship between fT 4 concentrations and fetal number, concentrations of this form of hCG would have to be more closely related to fetal number than are total hCG concentrations.
Discussion
We have shown that, in multiple pregnancy, thyroid activity increases with fetal number. This is consistent with the greater circulating concentrations of fT 4 and lesser circulating concentrations of TSH in twin, compared with singleton, pregnancies (the measurements between 8-15 weeks were pooled; Grun et al. 1997) . However, the difference in TSH was not statistically significant and that of the fT 4 was significant at the P=0·046 level (Grun et al. 1997) . With twin pregnancies, we have demonstrated a similar pattern for TSH, but not fT 4 . The lack of difference between singleton and twin pregnancies in our study may be because our samples were collected at 10-12 weeks gestation (rather than at 8 weeks onwards), when the altered concentrations of TSH and fT 4 may have reverted to normal (Harada et al. 1979) . In contrast, the alteration in thyroid physiology is more profound in multifetal pregnancies and still apparent at 10-12 weeks.
Discussion
As we do not have pre-pregnancy thyroid function tests, it is possible that the women with multifetal pregnancies had greater concentrations of fT 4 , that this promoted implantation of multiple fetuses, and that this is the origin of the observed differences in maternal thyroid function. However, we would not have expected to see a decline in maternal fT 4 concentrations after reduction, had this been the case. Similarly, it seems unlikely that maternal thyroid function is materially altered by the treatment used to achieve pregnancy, but this cannot be excluded.
Discussion
In conclusion, there is profound alteration in thyroid physiology in multifetal pregnancies, which may be due to a combination of hCG and a fetal factor. Further studies are needed to elucidate whether such a fetal factor exists and, if so, what it is.

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