Human Reproduction Update 1996, Vol. 2, No. 4 pp. 355–363
European Society for Human Reproduction and Embryology
The predetermination of embryonic sex using
flow cytometrically separated X and Y
spermatozoa
D.G.Cran1,3 and L.A.Johnson2
1Mastercalf Limited,
Scottish Agricultural College, Parkhead, Bucksburn, Aberdeen AB21 9TN, UK and 2Germplasm and
Gamete Physiology Laboratory, Agricultural Research Service, US Department of Agriculture, Beltsville, MD 20705, USA
TABLE OF CONTENTS
Introduction
Basis for successful and repeatable separation
of X and Y spermatozoa
Flow cytometric sperm sorting
Validation of sperm sorting
Sex bias of offspring
Factors affecting sperm sorting
Effect of sorting on fertility and embryonic
development
Implications for the human
References
Key words: DNA contents/flow cytometry/human/
sexing/spermatozoa
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A review is given of the predetermination of sex in
various domestic animals and in the human using
sperm samples enriched for X- or Y-chromosome
bearing spermatozoa obtained by flow cytometry and
cell sorting. A comparison of other putative methods
of sperm separation is made. In separating human X
and Y spermatozoa, measurements of the DNA content in each individual gamete using the Hoechst fluorochrome 33342 remains the only validated method.
The difference in DNA content between human X and
Y spermatozoa is ∼2.8%, and cell sorters have been
adapted to take account of this and the asymmetrical
nature of the sperm head. DNA analyses and PCR
have been used to validate the method for animal spermatozoa. In the human, fluorescence in-situ hybridization (FISH) has confirmed sorting accuracy. Many
correctly-diagnosed normal offspring have been born
in various animal species and any potential mutagenic
or cytotoxic effects are being closely monitored as are
the cost and efficiency of the technology.
3To
whom correspondence should be addressed
Introduction
The potential of producing offspring of a predetermined
sex has been a goal which has exercised the imagination of
mankind over many generations. Numerous suggestions,
some more fanciful than others, have been proposed, particularly for the human, and a variety of popular articles
continue to be written purporting to allow gender selection.
One of the more recent (Ridley, 1994) provides tantalizing
evidence that, under certain circumstances, such as following
major wars, the sex of children may be non-random, but
how this is effected is still far from clear. In the human, a
desire to predetermine sex is generally based on social
values or a wish to avoid the potential conception of a child
with an X-linked recessive disorder, of which more than
370 have been listed (McKusick, 1992). In farm animals
the driving force has been economic advantage through
genetic gain and optimization of farm management. For
example, dairy farmers, in general, would prefer to have a
preponderance of female offspring for herd replacement
with rather few males. Farmers who gain their livelihood
from beef production would clearly prefer the converse
since growth rate is sex linked.
With the advent of polymerase chain reaction (PCR)
analysis and the derivation of DNA probes specific for the
sex chromosomes it has become a relatively simple matter
to remove a few cells from an embryo for sex determination for a variety of species (e.g. Itagaki et al., 1993; Kudo
et al., 1993). In addition, progress is being made on noninvasive approaches of embryo sexing (Leese, 1987).
However, the most effective means of achieving sex
preselection involves the separation of the individual X and
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D.G.Cran and L.A.Johnson
Y chromosome-bearing sperm populations prior to their
use for artificial insemination or in-vitro fertilization (IVF)
followed by embryo transfer. In the case of cattle, the clearly
preferred route is to supplant current artificial insemination, which uses frozen semen from selected bulls, by the
use of semen that has been pre-selected for sex. Numerous
attempts have been made to bulk-separate semen from both
cattle and humans and apply it on a wider scale. To date
none has proven effective. Most of these procedures have
tried to utilize the real or apparent physical differences
between X and Y chromosome-bearing spermatozoa. Most
of these protocols have attempted to take advantage of
differences in mass, variations in surface charge, antigenic
differences and differences in buoyant density as the basis
for separation. Several reviews have been written on this
subject (e.g. Kiddy and Hafs, 1971; Amann and Seidel,
1982; Gledhill, 1988; Cran, 1992; Johnson, 1992, 1994;
Windsor et al., 1993). These approaches will not be dealt
with in detail here. A major problem in the past has been the
lack of a reliable method to determine the potential efficacy
of separation methods. Flow cytometric separation and the
advent of fluorescence in-situ hybridization (FISH) to
detect the presence of X and Y chromosomes in spermatozoa have allowed a re-evaluation of some of the earlier
procedures. Numerous ‘physical separation’ methods have
been evaluated using flow cytometry to measure DNA
difference. No separation method tested has provided irrefutable evidence of any enrichment of X or Y spermatozoa
(Pinkel et al., 1982; Johnson, 1988, D.G.Cran, unpublished; L.A.Johnson, unpublished), although Cartwright et
al. (1993), using DNA hybridization as a marker, have
claimed limited separation using thin-layer counter current
distribution. The H-Y antigen has long been thought to
differ in its presence on the X and Y spermatozoa, but
Hendriksen et al. (1993) using flow cytometrically sorted
X and Y populations of spermatozoa found no difference in
H-Y antigen binding on the X and Y sorted populations.
Such a marker would, of course, lend itself to the development of a bulk separation method for X and Y spermatozoa.
The procedure that has been most widely used and
claimed to allow separation of X- and Y-bearing spermatozoa in the human is that described by Ericsson et al. (1973)
employing albumin gradients. The basis of this is the
suggestion that Y-bearing spermatozoa have a greater ability
than X-bearing spermatozoa to penetrate interfaces
between fluids and between discontinuous gradients of an
albumin-based isolation medium. Beernink et al. (1993)
have reported that, of 1034 births following insemination
with purportedly separated Y spermatozoa, 72% were
males. In addition, using a different protocol in association
with the administration of clomiphene citrate to patients, it
is claimed that separation of X-bearing spermatozoa is
possible. Using this approach, Beernink et al. (1993)
observed that 69% of those born were female (133/193). In
a similar study, based on eight pregnancies, Dmowski et al.
(1979) reported seven births and one spontaneous abortion,
with six males (75%) and two females.
It is perhaps not surprising that the basis for this skewness
in the sex ratio at birth has been a matter of some controversy
(Carson, 1988; Zarutskie et al., 1989; Ericsson, 1994;
Martin, 1994; Pyrzak, 1994). A key feature of the procedure
appears to be the recovery of a very motile fraction of spermatozoa in the most concentrated region of the gradient. It
has been indicated that the highly motile Y-bearing fraction
represents ∼5–9% of the total motile spermatozoa population
(Beernink et al., 1993; Ericsson, 1994) and that the contrary
results of Vidal et al. (1993) and Wang et al. (1994) were due
to not following the precise protocol and, in particular,
obtaining a ‘purified’ fraction that contained too high a percentage of motile spermatozoa that possibly had relatively
poor motility. It is clear that independent repeatability of the
albumin separation method presents a major problem. Perhaps more crucially, it seems likely that general acceptability,
in the UK at least, of manipulation of the human sex ratio is
only likely to be forthcoming in cases in which there is an
inherited X-linked disorder since, as Beernink et al. (1993)
state, the accuracy of their method is not sufficient to guarantee 100% assurance of success and the likely route for sperm
separation may be through the use of flow cytometry and cell
sorting.
Basis for successful and repeatable separation
of X and Y spermatozoa
The DNA content is still the only scientifically validated
and consistently measurable difference between X and Y
spermatozoa. The autosomes carried by X or Y spermatozoa have identical DNA content and the difference in DNA
mass of the X and Y chromosomes has formed the basis
upon which flow cytometric analysis of DNA in X and Y
spermatozoa has been carried out. One of the first reports
describing the analysis of sperm DNA using flow cytometry was made by Gledhill et al. (1976). That report was
followed by the ability to distinguish mouse X and Y spermatozoa using flow cytometry (Pinkel et al., 1982). The
use of flow cytometric cell sorting for viable spermatozoa
was first reported in 1989 by Johnson et al. This work led to
the birth of normal offspring in a variety of species following
either tubal insemination or IVF (Johnson et al., 1989;
Johnson, 1991; Cran et al., 1993, 1995; Rath et al., 1996).
Predetermination of embryonic sex
357
Figure 1. (A) Diagram of an orthogonal sperm sorting system. A modified flow cytometer/cell sorter is used to analyse and separate X from Y
chromosome-bearing spermatozoa. The standard sample injection needle is bevelled at the exit orifice to properly orientate (as measured by the
90° detector) a higher percentage of spermatozoa for X or Y chromosome determination by the additional 0° fluorescence detector. Individual
spermatozoa (within a droplet) are then sorted by deflecting their respective carrier droplet either to the left (Y-bearing) or to the right (X-bearing). Spermatozoa are routinely sorted and collected in each direction at ∼100/s. (B) Histogram of signals received by the 90° detector. The peak
at the right represents sperm edge on to the 90° detector and therefore parallel to the 0° detector. The region gated for examination by the 0°
detector is within the horizontal lines. (C) The orientated population as seen by the 0° detector. Peaks representing X (right) and Y spermatozoa
are clearly visible. The positions of X and Y sorting regions are indicated by the vertical lines.
Flow cytometric sperm sorting
The difference in DNA content of X and Y spermatozoa
varies, with a 2.8% difference in the human to ∼4% in
sheep and horses (Johnson, 1992; Johnson et al., 1993).
This is a much smaller difference between the two populations when compared to most other cell types which have
been separated by flow cytometry/cell sorting. In addition,
the DNA of spermatozoa is exceedingly compact and the
head is, in the case of farm animals, paddle shaped. This,
together with the fact that they have a long tail and are
motile, make spermatozoa one of the more difficult cell
types to deal with successfully. It is possible to distinguish
the small DNA difference between X and Y spermatozoa
only when the laser beam passes through the flat face of the
head (Johnson and Pinkel, 1986). Clearly, in a random
population only a small fraction of spermatozoa will be in
this orientated position. However, the compactness of the
cell and the difference in refractive index between the spermatozoa and the surrounding medium can be used to detect
this population since there is a preferential emission of light
from the edge of the cell. Thus, the modified flow cytometer/
cell sorter (Johnson and Pinkel, 1986) has photodetectors
situated at right angles to one other (Figure 1), one of which
can be used to detect maximum emission from the edge of
the sperm heads and the other to measure the DNA in these
cells since only they are at right angles to the laser beam.
With a standard blunt sample needle only ∼10% of the cells
can be analysed in this way. If, however, the end of the
injection needle is bevelled, ∼25% of the live spermatozoa
can be distinguished by their DNA differences (Johnson et
al., 1989). At present, only this arrangement of a flow
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D.G.Cran and L.A.Johnson
Figure 2. DNA reanalysis of separated X and Y sheep spermatozoa. (A and B) Two-dimensional plots of fluorescence detected by the 90° (horizontal axis) and 0° (vertical axis) photodetectors for X and Y separated spermatozoa. The population to the right of the vertical line represents
that part of the population which is parallel to the 0° detector and the horizontal line represents the point of overlap of the X and Y fractions. (C)
Histogram of separated X and Y sperm populations. The X and Y spermatozoa are clearly distinguishable from each other.
cytometer/cell sorter has been successfully used to sort live
sperm cells. This modified system is also essential for the
reanalysis of the DNA of the separated X and Y sperm
populations.
Validation of sperm sorting
Following sorting, it is clearly desirable to be able to obtain
information regarding the purity of the populations. This
can be obtained in a number of ways.
The first approach which provides immediate information is that in which the sperm populations are reanalysed
on the basis of their DNA content as described by Johnson
et al. (1987, 1989). Following sorting, the putative X and Y
populations are re-stained and passed back through the cell
sorter so that the purity of the populations, which is a
reflection of the overlap of one population on the other, can
be readily ascertained (Figure 2). The validity of this
approach has recently been demonstrated by Welch et al.
(1996), who sorted single X or Y spermatozoa into the
wells of microtitre plates and performed PCR analyses for
both X and Y chromosomes. Results from 100 amplifications of each X and Y sort demonstrated a purity of 94% for
the X sort and 90% for the Y. These values were not different
from those which had been ascertained by reanalysis of the
sorted spermatozoa for DNA content.
Predetermination of embryonic sex
359
Table I. Sex bias of offspring resulting from fertilization with X or Y spermatozoa
Treatment
No. of females
inseminated or
receiving ET
No. kindled/
farrowed/
calved
No. of
offspring
born
% Male
% Female
Rabbit (Johnson et al., 1989)
Sorted Y
16
5
21
81
19
Sorted X
14
3
16
6
94
Sorted X+Ya
17
5
14
43
57
Pig (Johnson, 1991)
Sorted Y
8
4
37
68
32
Sorted X
10
5
34
26
74
Controlb
18
5
86
43
57
4
2
3
100
0
Cattle (Cran et al., 1993, 1995)
Sorted Y
Sorted X
4
2
3
0
100
Sorted Y
106
34
41
90
10
aSorted
for X and Y and recombined.
stained or unstained.
ET = embryo transfer.
bUnsorted,
Another validation method involves microscopic examination of the separated spermatozoa with FISH. Using
primers for specific base sequences on the X and Y
chromosomes, the proportions of X and Y spermatozoa can
be readily quantified. This has been particularly useful in
the analysis of separated human X and Y spermatozoa
which, due to the small DNA difference (2.8%), gives
variable results when reanalysis is done using flow cytometry (Johnson et al, 1993).
In cattle, IVF has been used as the favoured procedure to
generate embryos. PCR has been frequently used as a
method for determining embryonic sex (e.g. Herr et al.,
1990). Embryos produced by fertilization from sorted spermatozoa have been analysed by PCR (Cran et al., 1993)
and the results gave a close correlation to those obtained by
flow cytometric DNA analysis.
Sex bias of offspring
The first offspring whose sex was determined by separation of X and Y spermatozoa were derived from rabbits
(Johnson et al., 1989) which underwent intrauterine insemination. The sex ratios of those first litters were 94% female
(n = 16) for those inseminated with X spermatozoa and
81% male (n = 21) for those inseminated with Y spermatozoa. Pigs treated similarly gave birth to 74% females (n
=34) and 68% males (n = 37) (Johnson, 1991) (More recent
results with pigs have shown 85% females born following
insemination with X spermatozoa; L.A.Johnson, unpublished data.) In a recent study, Rath et al. (1996) reported
the birth of 10 piglets derived from embryos fertilized in
vitro with X spermatozoa. All were female. For economic
and practical reasons the use of tubal insemination in cattle
has not been attempted. Over the past decade there have
been significant advances in the efficiency of IVF in this
species (Lu et al., 1987) and separated X and Y spermatozoa have been introduced successfully into this system. To
date, most calves have been produced using spermatozoa
separated for the Y population (Table I; Cran et al., 1995).
Eggs fertilized with these Y-sorted spermatozoa have given
rise to 42 live calves of which 38 (90%) were male. Only
three calves resulting from fertilization using spermatozoa
which were specifically sorted for the X population have
been born. All were female.
Factors affecting sperm sorting
Two makes of flow cytometer/cell sorter can be adapted to
sort X and Y spermatozoa, and to date it is known that 11
instruments manufactured by either Becton Dickinson or
Coulter have been modified for this purpose (L.A.Johnson,
unpublished data). The preparative steps for sperm sorting
are straightforward and include the staining of the spermatozoa with the vital fluorochrome Hoechst 33342 at
35°C. This ensures uniform quantitative binding to the
DNA such that, on illumination by a UV laser at ∼125–200
MW, resolution of the two populations is possible (Figure
3). The maximum rate at which spermatozoa may pass
through the flow cytometer is ∼2500/s. Increasing this rate
reduces the resolution of the populations and also decreases
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D.G.Cran and L.A.Johnson
Figure 3. (A) Two-dimensional plot of stained sheep nuclei. The X and Y spermatozoa in the orientated fraction can be detected. The rectangles
labelled R1 and R2 represent sorting frames which would allow separation of X and Y spermatozoa with a purity of at least 90%. (B) Histogram
of fluorescence (horizontal axis) against cell number of the orientated population shown in A. The peaks for X and Y spermatozoa are clearly
resolved.
the size of the population (orientated population) passing
with their flat surfaces parallel to the 0° detector. In practice, the rate at which X and Y spermatozoa may be sorted
depends on the degree of purity required. To obtain 90%
purity only ∼10% of the entire population may be sorted
into X and Y fractions. Thus, ∼100 cells/s of each may be
sorted. This relatively slow rate of sorting limits the delivery system in which the spermatozoa may be used. In the
case of cattle, artificial insemination requires ∼20 × 106
spermatozoa for a single insemination. Recent evidence
suggests that deep uterine insemination may be useful for
delivering small numbers of spermatozoa to the site of
fertilization in the cow (Seidel et al., 1995, 1996). Sufficient spermatozoa can easily be sorted for use in intracytoplasmic sperm injection (ICSI), intratubal insemination,
intrauterine insemination and IVF and it is these techniques
in which sorted spermatozoa will be most used, until
advances in instrumentation allow for a higher rate of
sorting.
Not only do the above considerations apply to the rate at
which spermatozoa may be sorted but the relative difference in values of DNA content of X and Y spermatozoa
between species also has a significant impact. The large
farm animals (i.e. cow, sheep, pig and horse) have X and Y
DNA differences of between 3.5 and 4.2% and, in general,
the greater the difference the easier it is to sort the spermatozoa and the higher the rate possible for a given purity.
Thus, in the case of Chinchilla laniger (Johnson et al.,
1987), which has a DNA difference of 7.5%, very clear
resolution of the X and Y populations can be achieved
(Figure 4), and sorting rates of ≥200 cells/s can be achieved
in this species without marked loss of purity. In the case of
the human, the difference is ∼2.8% (Johnson et al., 1993),
and therefore on that basis alone a relatively slow sort rate
would be expected. In addition, human spermatozoa do not
have the flat paddle shape of the domestic animal species
but are rather more angular. They do, however, have an
‘edge’ and a ‘flat’ side which allows them to be treated in
the same way as other spermatozoa, although the conditions required to obtain pure populations may be rather
more stringent. Because of the small DNA difference and
the cell shape, reanalysis of sorted human sperm cell
populations is inconsistent. However, as described above,
FISH, using probes against both X and Y chromosomes, is
an equally satisfactory alternative. With this approach,
Johnson et al. (1993) were able to demonstrate that human
spermatozoa could be sorted to a purity of 80–85% for X
spermatozoa and ∼75% for Y spermatozoa.
Predetermination of embryonic sex
361
matozoa stained with Hoechst 33342 have been born, with
no evidence of morphological abnormality. In addition, all
three species have been bred to at least the second generation without any evidence of an effect of the dye on normal reproductive performance or morphology.
Implications for the human
Figure 4. Plot of fluorescence emission from stained chinchilla spermatozoa. Due to the large DNA difference, the X and Y populations
are readily distinguished.
Effect of sorting on fertility and embryonic
development
Since the DNA stain Hoechst 33342 can be mutagenic and
may have cytotoxic effects, particularly at high dosage
(Durand and Olive, 1982; Wiczoreck, 1984), attention has
been drawn to possible harmful effects of the dye on fertility,
embryonic development and the normality of offspring.
There is little direct effect of the stain on the capacity to
fertilize bovine eggs. Cran et al. (1994) found cleavage
rates of 79% for X spermatozoa (723/921 eggs) and 80%
using Y spermatozoa (752/938 eggs). This is very similar
to values obtained with untreated spermatozoa. There is,
however, a clear impact of the dye and the sorting procedure on embryonic development. Reduced litter sizes
were found in both rabbits and pigs inseminated intratubally
(Johnson et al., 1989; Johnson, 1991), and, in a study on the
development of sex-predetermined rabbit embryos,
McNutt and Johnson (1996) found a delay in development
compared with embryos resulting from fertilization by
untreated spermatozoa. In cattle, the developmental rate of
in-vitro cultured embryos is delayed by ∼12 h compared
with controls. In addition, the blastocyst development rate
was ∼17% (Cran et al., 1994), compared with 35% for
controls. It would clearly be preferable to use a different
dye which would fluoresce in a visible wavelength. However, there is currently no alternative available which
allows for normal spermatozoal action and resolution of
the two populations. To date, several hundred offspring
from rabbit, pig and cattle embryos fertilized with sper-
In the human, following separation of X spermatozoa into a
highly enriched population, the spermatozoa may be used
in a standard IVF system, intrauterine insemination or with
ICSI. Clinical trials are in progress at the Genetics and IVF
Institute, Fairfax, VA, USA. One birth has occurred and
other pregnancies are in progress (Levinson et al., 1995).
These trials are taking place within clearly laid down
regulations to ensure that defined criteria are satisfied. If
this method is to have any widespread application in the
human, the nature of the supervisory authorities and public
opinion will be important. The high set-up cost, the low
numbers of spermatozoa available and the demands in
terms of technical expertise would indicate that, for the
foreseeable future the predetermination of sex in the
human is likely to have a limited role, and thus the dangers
discussed by ethicists are probably not warranted.
Questions are frequently raised regarding the potential
for misuse of the technology as well as the safety of its use
(Ashwood-Smith, 1994; Johnson and Schulman, 1994;
Munné, 1994). Once a technology has passed from the
developmental stage to be applied in practice there exists
the potential for misuse or for the use of the application by
those with questionable motives. Sex selection in humans
has always been the subject of controversy, with forceful
arguments being proffered both for and against its use. In
general, secular society has always allowed freedom in
matters of family planning. The advent of the capacity to
preselect the sex of one’s child is a matter for society to
decide and, in the UK at least, organizations have been
specifically established to examine and come to an
informed opinion on these matters. It is the authors’ view
that the use of this sex preselection technology for medical
indications is a useful and important undertaking. The use
of sexing technology for social reasons, on the other hand,
is quite another matter and has been the subject of much
debate. At present, at the site of application, the decision as
to the use of the technology rests largely on the family units
concerned. Whether restrictions will be placed on this use
remains to be determined and is likely to vary from country
to country. It must be emphasized, however, that there are
severe limitations to the technology in terms of cost and
throughput which will limit its application on a world-wide
basis.
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With regard to the issue of safety raised by AshwoodSmith (1994), the theoretical possibility of chromosomal
damage cannot be completely ruled out (Johnson and
Schulman, 1994). However, every animal born to date has
shown normal morphology. As described above, a lower
rate of fertilization occurs with sorted spermatozoa (Johnson
et al., 1989; McNutt and Johnson, 1995) and embryo
development is slower. Efforts are continuing to find a
fluorochrome which does not require ultraviolet excitation,
since its use would help alleviate such concerns. However,
once pregnancy is established, the majority go to term and
the offspring breed successfully. Those embryos which are
transferred and do not establish a full-term pregnancy die at
an early stage (McNutt and Johnson, 1995; D.G.Cran,
unpublished results). Consequently, there have been normal births throughout all trials conducted. At this time this
is the best evidence available that the process is safe.
In summary, the use of this technology in the human
opens the potential for improving the quality of life for
many. How widespread it will become depends on many
factors, including cost, sophistication of the technology,
delivery of small numbers of spermatozoa to the site of
fertilization and general acceptability.
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Received on February 16, 1996; accepted on June 26, 1996