Human Reproduction vol.14 no.11 pp.2917-2919, 1999
Human live birth and sperm-sex ratios compared
J.Graffelman ' , E.F.Fugger , K.Keyvanfar , and
J.D.Schulman
1
3
2
2
2
University Pompeu Fabra, Department of Economics, Barcelona,
Spain and Genetics & IVF Institute, 3020 Javier Road, Fairfax,
VA 22031, USA
1
2
To whom correspondence should be addressed at: University
Pampeu Fabra, Ramon Trias Fargas 25-27, 08005 Barcelona, Spain
3
The human secondary sex ratio is compared with the
percentage of Y-chromosome bearing spermatozoa in
human semen. Live birth sex ratio is about 51.3%, whereas
the overall percentage of Y-chromosome bearing spermato¬
zoa in our study samples was 50.3%, i.e. 1% closer to
the proportion expected by Mendelian segregation. The
observed difference between live birth and sperm-sex ratios
was significant (P < 0.0001). A possible effect of male age
on the percentage Y-bearing spermatozoa was found to be
non-significant.
Key words: fluorescence in-situ hybridization/secondary sex
ratio/sperm-sex ratio
Introduction
The human secondary sex ratio (percentage of male live births)
is slightly male biased (Cavalli-Sforza and Bodmer, 1971).
This was probably first noted in the 17th century by Arbuthnott
(Kendall and Placket, 1977), whose data on London allow an
early estimate of 51.63% (SE 0.05%). Population statistics of
the 20th century give estimates for Western countries fluctuat¬
ing around 51.3%. There is little variation in the secondary
sex ratio, and there seems to be little or no genetic variation
for the sex ratio (Edwards, 1962; Maynard Smith, 1978).
Mendelian segregation theoretically implies a sex ratio of 50%.
What could be the cause of this small male bias of 1.3%?
With the recent advances in separation of X- and Y-bearing
spermatozoa by flow cytometry in mammals (Johnson, 1995)
and humans in particular (MicroSort®; Fugger et al., 1998),
more data on human semen are becoming available, allowing
us to compare the 'sperm-sex ratio' (percentage of Y-bearing
spermatozoa) with the live birth sex ratio, and to test the
hypothesis that a sperm-sex ratio bias underlies a live birth
sex ratio bias. Many factors have been postulated to affect the
human sex ratio (James, 1987), among which we find parental
age and child birth order. The sex ratio has been reported to
decrease slightly with increasing parental age and increasing
rank number of the child (Garfinkel and Selvin, 1976; Ruder,
1985). Multiparity has also been supposed to affect the sex
ratio (Juntunen et al., 1997), though another study found no
© European Society of Human Reproduction and Embryology
significant effects (Almagor et al., 1998). Paternal age, maternal
age and birth order are all associated, making it difficult to
assess their independent effects. Some large scale population
studies consider birth order and paternal age to be important
factors (Garfinkel and Selvin, 1976; Ruder, 1985). No associ¬
ation between sex ratio and time of insemination (Gray et al.,
1998) has been reported. If there is indeed such a paternal
effect, then the decrease in sex ratio could be explained by a
lower percentage of Y-bearing spermatozoa in older men. We
therefore evaluated the effect of male age on the sperm¬
sex ratio.
Materials and methods
Estimates of live birth sex ratios were obtained from data provided
by the national offices of population statistics from several European
countries. Live birth data are usually compiled annually, thus allowing
a very precise estimate of the secondary sex ratio. Percentages of
Y-bearing spermatozoa were determined by FISH (fluorescence in-situ
hybridization) studies of semen of men who accessed the Genetics
and IVF Institute for the purpose of gender selection (Fugger et al.,
1998). FISH analysis was performed as described earlier (Fugger
et al., 1998). Briefly, sperm cells were washed by centrifugation,
resuspended in a phosphate buffered saline and air dried on glass
slides. The spermatozoa were subsequently treated with 50 mmol/l
dithiothreitol and saline sodium citrate (SSC), and hybridized with X
and Y specific alpha-satellite DNA probes (Vysis Inc., Downers
Grove, IL, USA). Each sample was examined with a Nikon Optiphot2 fluorescence microscope (Nikon Inc., Melville, New York, USA)
equipped with an FITC/rhodamine filter. X-bearing spermatozoa were
identified by the presence of a red signal and Y-bearing spermatozoa
by the presence of a green signal.
All men were healthy and fertile, aged between 23 and 56 years
(one outlying exception). The analysis was based on 176 males of
Caucasian background only, as there were too few observations
among other races. Semen samples were collected over the years
1994-1998, and observations were more or less uniformly spread
over the 12 months in the year. About 200 spermatozoa from each
man were analysed by FISH for their sex-chromosomal content (X
or Y).
Results
Live birth sex ratios calculated from data aggregated over time
give estimates of about 51.3% (e.g. Austria 51.39%, Belgium
51.27%, Denmark 51.38%, England and Wales 51.26%, France
51.18%, Germany 51.47%, Italy 51.35%, Netherlands 51.38%,
Spain 51.44%, USA 51.38%; SE ^0.02%). The combination
of these data sets gives us a secondary sex ratio of 51.33%,
which we use as a point estimate. Estimates of the sperm-sex
ratio of 176 Caucasian men (repeated samples of the same
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J.Graffelman et al.
Figure 1. Sperm sex ratio of Caucasian males. Percentages of
Y-bearing spermatozoa (open circles) are plotted for 176 men. Dots
indicate 9 5 % confidence limits. Mendelian (solid line) and live
birth (dashed line) sex ratios are indicated for reference.
man being aggregated), are shown in Figure 1, where dots
indicate the 95% confidence limits. The confidence intervals
(CI) always contain the Mendelian proportion and practically
always (two exceptions) the live birth sex ratio, 51.3%. For
an individual man, the sample size is too small to allow an
appreciation of small bias from Mendel or the live birth sex
ratio. However, the pooled data of all men provides us with a
more precise estimate: 50.31% (95% CI: 49.86-50.76) which
differs significantly from the live birth sex ratio ( P = 5.5X
10 ), but not from the Mendelian ratio. Computer-intensive
methods confirm this result: bootstrap resampling (Noreen,
1989) with 2000 samples also gives a CI for the overall sperm¬
sex ratio excluding the live birth sex ratio: 50.31% (95% CI:
50.00-50.61), where the 2.5 and 97.5 percentiles of the
bootstrap distribution are used to construct this interval.
In order to assess a possible effect of male age on sperm¬
sex ratio, we regressed sperm-sex ratio on age, and found the
slope to be insignificant [a = 0.498 (t = 52.66, P = 0.00),
b = 0.0001 (t = 0.51, P = 0.61)]. We performed weighted
regression, weights being total sperm counts. The regression
line is shown in Figure 2. We omitted one outlier exerting
high leverage on the slope of the regression line [estimates
including the outlier: (a = 0.499, P = 0.00, b = 0.00008,
P = 0.72)].
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Figure 2. Sperm sex ratio versus age. Weighted regression of
sperm-sex ratio on age. Dot size roughly proportional to total
sperm count. Slope is insignificant (b = 0.0001, SE 0.0002,
P = 0.611, R = 0.00015).
2
ratio than live births, e.g. Austria 54.27% (SE 0.23),
Netherlands 53.87% (SE 0.14) and Spain 57.80% (SE 0.24).
The high reported rates of spontaneous abortion (Boklage,
1990; Forbes, 1997) also show that prenatal mortality is
important from a quantitative point of view, and suggest that
changes in mortality in the womb might affect the sex ratio
observed at birth. Our finding is also corroborated by a FISH
study (Chevret et al., 1995) where few men were sampled,
but a large number of cells scored. The pooled X and Y counts
from their study give a sperm-sex ratio of 49.67%, and also
differs significantly from the live birth sex ratio at the 5% level.
As for the effect of age, our data failed to reveal a
relationship between male age and semen sex ratio. Observed
effects of paternal age on live birth sex ratio are based on
population studies with enormous sample size, and effects on
sex ratio were of a tiny order of magnitude (Garfinkel and
Selvin, 1976; Ruder, 1985). Our own sample size is probably
too small to detect such tiny effects, if present, in the sperm¬
sex ratio.
Acknowledgements
This work was partially supported by the Spanish DGICYT grant
PB96-0300.
Discussion
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Our data suggest that the male bias observed in live births
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biased sex ratios arise in utero: still births have a higher sex
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Human live birth and sperm—sex ratios compared
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Received on May 10, 1999; accepted on August 24, 1999
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