The Sex Differences in Cord-Blood Cholesterol and Fatty-Acid Levels Among Japanese Fetuses

We examined serum cholesterol and fatty-acid levels of cord blood and maternal blood samples collected from 193 Japanese fetuses and their mothers. Our study, which is the largest study of this kind ever conducted in Japan, is the first Japanese study reporting that total, high density lipoprotein (HDL) and non-HDL cholesterol levels in females were statistically significantly higher than those in males; the sex differences of total, HDL and non-HDL cholesterol levels were 8.5 mg/dl (P= 0.002), 4.5 mg/dl (P= 0.004) and 4.1 mg/dl (P= 0.045), respectively. The sex difference of total cholesterol was attributable to both HDL and non-HDL cholesterol. The sex of fetuses didn't show evident differences in cholesterol levels in maternal sera. Fatty-acid levels in cord blood were also higher in female fetuses than in male fetuses. However, none of the differences except for monoene fatty acids were statistically significant. Further investigations seem warranted to elucidate the mechanisms involved in our results. J Epidemiol, 1997; 7 : 226-231.

In the last decade, the number of children with elevated blood cholesterol levels has been rapidly increasing in many countries and therefore ever more attention has been focused on the determinants of cholesterol and other lipid levels in the early life periods.Barker and his colleagues recently reported that lower birth weights and fetal undernutrition were related to high cardiovascular disease rates in adulthood 1-3).Although their studies indicate the possibility that fetal and neonatal life may affect the health in adulthood, little is known about the determinants of cholesterol and other lipid levels in fetal and neonatal periods.
In epidemiology, gender is one of the major factors that should always be given careful consideration.To date, as many as 20 studies have examined the sex differences of cholesterol concentrations [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] in cord blood.With a few exceptions, total cholesterol levels in cord blood were higher in female neonates than in male neonates.HDL cholesterol was also measured by six studies and all except one study reported the sex difference in the same direction as total cholesterol.In Japan, there are only two studies reporting cholesterol levels of male and female cord blood, separately.
The number of neonates examined in Japanese studies are too small to draw any conclusion.
In this study we determined the levels of total and HDL cholesterol and fatty acids in blood samples obtained from Japanese fetuses and their mothers and examined the sex differences of cord-blood cholesterol and fatty-acid levels.

SUBJECTS AND METHODS
We decided to exclude neonates with gestational periods shorter than 28 weeks, with birth weights less than 2000 g, and with congenital anomalies.Mothers with severe pregnancy induced hypertension and mothers with a history of using hormonal drugs or diuretics regularly were also excluded because their lipid levels might have been affected by medication.There were 232 pairs of eligible newborn infants and their mothers at the Kagoshima Prefectural Ohshima Hospital in Amami-Ohshima from June 14 to October 13, 1991.Thirtynine pairs (23 male and 16 female neonates) could not be used in our analysis because a sufficient volume of blood or essential information about the pregnancy and other relevant factors was not collected.Medical records were reviewed to retrieve information on the pregnancy and delivery.There were 33 caesarean sections.Blood samples were collected, obtaining informed consent.

Lipid Analysis
Cord-blood samples were collected from the placental end of the umbilical vein.Maternal blood samples were taken from the basilic vein on the fifth day after delivery.Blood samples were stored at 4°C for less than 3 days, and their sera were then separated by centrifugation.
The specimens were stored at -20°C before cholesterol and fatty-acid analysis.
All lipid analysis was conducted by an author (T.A.), who did not know the sex of neonates, age of mothers or other relevant information.

Statistical Analysis
We conducted linear regression analysis, using the SAS GLM procedure.The dependent variables were cord blood cholesterol or fatty acids levels and the independent variables were sex of neonates and other relevant factors.All P-values presented were two-sided.

RESULTS
We analyzed the data for 101 male and 92 female neonates and their mothers.Table 1 shows the distributions of matemofetal factors, which are quite similar between the two sexes.Table 2 shows that total, HDL and non-HDL cholesterol levels of cord-blood samples were higher in female fetuses than in male fetuses whereas cholesterol levels in maternal sera didn't show evident sex differences.The sex differences of total, HDL and non-HDL cholesterol in cord-blood sera were estimated to be 8.5 (SE = 2.7), 4.5 (SE = 1.5) and 4.1 (SE = 2.0) mg/dl, respectively (Table 2).We also examined sex differences of fatty-acid levels.Fatty-acid levels in cord blood were also higher in female fetuses than in male fetuses (Table 3).However, none of them except for the sex difference of monoene fatty acids were statistically significant at 5% level.

DISCUSSION
Our study showed that total cholesterol levels in females were higher than those in males and that the sex difference of total cholesterol was attributable to both HDL and non-HDL cholesterol.There are a number of studies reporting the sex difference of cord-blood cholesterol levels.Among the studies listed in Table 4, majority of the studies reported cord-blood cholesterol levels to be higher in females than in males.There are three studies reporting no sex difference or the sex difference in direction opposite to that observed in our study.Some of those studies don't look reliable, though.Among them is the study by Kleeberg et al e, which lacked the information on sex for 22 cases with relatively low cholesterol levels.Another study is the one by Glueck et al15), where 58 white and 59 black neonates were examined.In their study, cord-blood cholesterol levels were higher in white females than in white males while the sex difference observed in black neonates was in the direction opposite to that in white neonates.It should be noted here, however, that the number of black male neonates is more than twice the number of black females, suggesting a possibility that a sizable number of female neonates were excluded from the blackneonate study.Two other studies, which are not listed in Table 4, should also be mentioned here.One is the report by Kaplan and Lee22), which examined cord-blood samples from 56 neonates.In their report they stated ; "There was no sex difference in lipid distribution." The other is the study by Glueck et al who examined 1800 live births23).They reported the absence of sex difference in the neonates with elevated cord blood cholesterol levels but they didn't mention the sex differences of lipid levels for the entire subjects.There are two Japanese studies  ; poly unsaturated fatty acids that reported cord-blood cholesterol levels separately for male and female neonates.Sato et al examined 49 cord-blood samples and reported total cholesterol levels to be 66 and 78 mg/dl for males and females, respectively").
On the other hand, Gotoh et al measured total and HDL cholesterol levels in 34 male and 4lfemale blood samples and reported that total and HDL cholesterol levels showed a little sex difference, if any).
According to the literature, HDL cholesterol in cord blood Table 4. Sex difference of total cholesterol in cord blood * Sex difference = male -female Table 5. Sex defference of HDL cholesterol in cord blood Table 6.Sex defference of triglyceride in cord blood *Sex difference = male -female *Sex difference = male -female also showed the sex difference (Table 5) in the same direction as observed in this study.An exception is the study of black neonates by Glueck et al15).The drawback of this study was already mentioned.Although the sex difference of triglyceride levels in cord blood was also noted by several studies (Table 6), the results were not entirely consistent.The difference was not evident in the study by Carlson et al13), which had the largest number of study subjects.In our study we didn't measure triglyceride levels.
To our knowledge, no study reported the presence of sex dif-ferences in fatty-acid levels in cord blood.Fatty-acid levels in cord blood were higher in female than in male fetuses in our study.However, none of the differences except for monoene fatty acids were statistically significant.Further investigations are necessary to confirm the observation.As discussed above, the sex difference of cord blood cholesterol levels was observed in a number of studies including our own.The mechanisms that created the sex difference are, however, yet to be elucidated.The major proportion of fetal cholesterol is produced by fetal liver, adrenal and testicular tissue25) The Sex Differences in Cord-Blood Cholesterol while direct transfer of maternal lipids through the placenta accounts for no more than 20% of the cholesterol in a human fetus, and lipogenesis in the placenta is considered to be negligible').It is well known that human chorionic gonadotropin (hCG) levels in maternal and cord-blood levels in late pregnancy are higher in the pregnancy with female fetuses than that with male fetuse27).The mechanisms involved in hCG sex difference is yet unclear.However, this fact indicates that the sex difference in cord blood is not limited to cholesterol.
Cholesterol levels of fertile females are lower than those of males of similar ages due to the effect(s) of estrogen.After menopause, cholesterol levels in females become higher than levels in males.Higher total and HDL cholesterol levels in females than in males are also observed in blood samples obtained from preadolescent children28,29).Our results showed the sex differences of blood cholesterol levels can be detected as early as in the fetal period.Whether a common factor is involved in the sex difference observed in cord blood, preado-

Table 1 .
Distribution of materno-fetal factors * For two subjects , the information is missing.** The cases of cesarean section were excluded .

Table 2 .
Sex difference of serum cholesterol levels * Sex difference = male -female

Table 3 .
Serum levels of fatty acids in cord blood lescent children and old people is unclear.Further investigations seem warranted to elucidate the mechanisms involved in our results., estradiol, cortisol, and testosterone in cord sera and their correlations.Am J Obstet Gynecol, 1982; 143: 918-921.28.The lipid research clinics program epidemiology committee.Plasma lipid distributions in selected North American populations: The lipid research clinics program prevalence study.Circulation, 1979; 60: 427-439.29.Yamamoto A. Blood lipid in Japanese.Sougou Rinshou, 1994; 43: 861-863(in Japanese). mone