Human Reproduction vol.8 no.10 pp.1733-1739, 1993
Gender preselection in humans? Flow cytometric
separation of X and Y spermatozoa for the prevention
of X-linked diseases
Lawrence A.Johnson1"3, Glenn R.Welch1,
Keyvan Keyvanfar2, Andrew Dorfmann2,
Edward F.Fugger2 and Joseph D.Schulman2
3
To whom correspondence should be addressed
Human X- and Y-chromosome-bearing spermatozoa were
separated based on their DNA content, using modified flow
cytometric cell sorting technology. The resulting separation
purity of the X-bearing from Y-bearing spermatozoa was
evaluated using in-situ hybridization with alpha satellite DNA
probes for the X- and Y-chromosomes. In the putative
X-enriched-sorted populations, an average of 82% of the
spermatozoa showed a hybridization signal with the X probe.
Similarly, in the Y-sorted population 75% gave a signal
with the Y probe. Sorted X- and Y-bearing spermatozoa
were found to maintain their viability for several hours
after sorting. These results demonstrate that the human
sperm sex ratio can be significantly shifted to favour the
selection of female-producing (X) spermatozoa or maleproducing (Y) spermatozoa when spermatozoa are flow cytometrically sorted on the basis of DNA content. We propose
that flow cytometrically sorted human spermatozoa, used in
conjunction with in-vitro fertilization or intra-oviductal
insemination, could be used by families who are at risk for
X-linked diseases to preferentially produce female offspring.
Sorted spermatozoa could also be used to pre-select for male
offspring if that were medically indicated.
Key words: DNA/flow cytometry/spermatozoa/X and Y
chromosomes
Introduction
Controlling the gender of offspring has been of interest to
scientists and laymen alike for generations. Such control has
significant economic value in agriculture where different livestock
production systems favour progeny of one sex or the other.
Pre-selecting sex in the human population is also of interest for
the prevention of X-linked diseases in at-risk families. There are
~ 6000 heritable defects in the human. About 370 of these defects
are known to be X-linked (McKusick, 1992). Generally the
X-linked diseases are expressed by sons of carrier mothers who
inherit the X chromosome with the defective gene. The ability to
© Oxford University Press
1733
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'US Department of Agriculture, Agricultural Research Service,
Germplasm and Gamete Physiology Laboratory, Beltsville,
MD 20705 and 2Genetics & IVF Institute, Fairfax, VA 22031,
USA
selectively separate X- from Y-chromosome-bearing spermatozoa
and use the X spermatozoa to preferentially produce female
offspring reduces, or may even eliminate, the probability of
conceiving affected males. This represents a powerful approach
to disease prevention. Attempts to sex semen by physical or
immunological means are well documented (Gledhill, 1988). A
common characteristic of most of the reported attempts to sex
spermatozoa has been lack of repeatability. One method that has
been used clinically in the human attempts to take advantage of
proposed differences in swimming ability between X- and
Y-bearing spermatozoa (Ericsson etal., 1973). The method
utilizes serum albumin as a column medium and has been claimed
to produce male offspring >70% of the time in clinical use
(Beernink et al., 1993). However, a study designed to validate
the albumin column procedure by chromosome analysis of
penetrated hamster eggs was not able to confirm the claims made
for the clinical data (Brandriff et al, 1986).
Chromosomal constitution is the only established and
scientifically validated difference on which to base X- or
Y-bearing spermatozoa separation. The DNA content of human
X- and Y-chromosomes has been reported to differ by 3.0%
(Sumner and Robinson, 1976). We have shown (Johnson, 1992)
that several other mammals have differences in DNA, from 3.0
to 12.5%. We have applied flow cytometry/cell sorting to the
analysis and sorting of individual animal spermatozoa based on
this DNA content difference. To our knowledge the flow
cytometric method is the only method fully validated by
laboratory and birth data (Johnson et al., 1989; Johnson, 1991;
Cran et al., 1993). We have demonstrated in numerous animal
studies the usefulness of this technology to (i) verify me
proportions of X and Y spermatozoa in a sample of semen, (ii)
predict the proportions of males and females to be born based
on DNA reanalysis of sorted spermatozoa, and (iii) determine
the efficacy of the method by evaluating the phenotypic sex of
the resulting offspring. Spermatozoa from cattle, rabbits and
swine have been flow cytometrically separated into X and Y
sperm populations. The sorted populations have been inseminated
into females and the sex of the resulting offspring verified against
the predicted sex based on DNA reanalysis of an aliquot of the
sorted spermatozoa. Proportions of females ranged from 75 %
in swine to 94% in rabbits and males from 70% in swine to 86%
in rabbits (Johnson etal., 1989; Johnson, 1991).
In this communication, we report the first successful separation
by flow cytometric cell sorting of human X- and Y-chromosomebearing spermatozoa into populations of more than 80% purity
for X spermatozoa and 75% purity for Y spermatozoa. We
directly verified the proportions of X and Y spermatozoa using
L.A.Johnson et al.
alpha satellite DNA probes for the X and for the Y chromosome
and fluorescence in-situ hybridization (FISH).
Materials and methods
Preparation of intact viable sperm
Fresh, viable spermatozoa from mature healthy donors were
obtained from the Fairfax Cryobank, under conditions described
above, suspended at 10 x 106 sperm/ml, in modified Tyrode's
medium (117.5 mM NaCl, 0.3 mM NaH2PO4, 8.6 mM KC1,
2.5 mM CaCl2, 0.4 mM MgCl 2 -6H 2 O, 2.0 mM glucose,
25 mM HEPES, 19 mM Na lactate, 0.25 mM Na pyruvate,
100 IU/ml penicillin) and immediately transported to Beltsville,
MD, USA.
Fluorescent staining of spermatozoa
All preparations (nuclei or intact) were stained by adding a vital
fluorochrome, bisbenzimide (Hoechst 33342, Calbiochem-Behring
Corp., La Jolla, CA, USA) to a final concentration of 9 jtM
(Johnson et al., 1987). Samples were then incubated for 1 h at
35°C (Johnson et al., 1989). All samples were flow-analysed
or flow-sorted at room temperature.
Flow cytometric analysis and sorting
Spermatozoa were sorted using a flow cytometer/cell sorter (Epics
753 or Epics V, Coulter Corporation, Hialeah, FL, USA)
modified specifically for the analysis of spermatozoa for DNA
content and for their flow sorting. Modifications have been
previously described in detail (Johnson and Pinkel, 1986). Briefly,
the forward angle light scatter detector was replaced with a
fluorescence detector and the standard cylindrical sample injection
tip was bevelled to produce a ribbon-shaped sample core stream.
These modifications are essential if one is to control the orientation
of the spermatozoa to the laser beam (bevelled needle) and
measure thefluorescencefrom the properly orientated spermatozoa
(forward fluorescence detector) and thus distinguish the small
differences in DNA content between individual X- or Y-bearing
spermatozoa. The stained spermatozoa were excited with the
1734
Collection of sorted spermatozoa
Sperm nuclei, intact non-motile or intact motile spermatozoa were
sorted directly onto standard microscope slides coded so they
could be evaluated blind. These spermatozoa were then probed
for the presence of the X or Y chromosome. Approximately
40 000 spermatozoa were sorted onto a 1 cm2 area of the slide
using a Lief bucket (Lief et al., 1971; Coulter Corporation) as
the slide holder and sort collection well. The sorted cells could
then be treated with liquids and centrifuged as required. After
sufficient spermatozoa were sorted onto the slide the bucket was
centrifuged for 10 min at 300 g, the supernatant was drawn off,
and 1 ml of fixative (75% methyl alcohol, 25% acetic acid) was
added dropwise; the bucket was again centrifuged and the fixative
was removed. The bucket was disassembled and slides removed.
The slides were washed three times in sodium chloride sodium
citrate (2 x SSC) and allowed to air dry. Additionally 105
spermatozoa were sorted into 0.5 ml microfuge tubes that had
been pre-treated with 1 % BSA and contained 50 /d of Test-Yolk
(Johnson et al., 1989) to determine sperm motility after sorting.
In all cases, X- and Y-enriched populations were sorted from
the same stained sample prepared for that day, one population
in each direction. The flow rate was 1000 cells/s, resulting in
sort rates of 30—40 cells/s for each population. Sorted populations from initially viable samples were examined under phase
and fluorescent microscopy for estimation of percentage and
quality of motility.
Molecular probing of sorted spermatozoa
Fifty-three individual sorts were performed. Each half of the slide
contained a sample of ~40 000 spermatozoa either sorted X,
sorted Y, sorted control (oriented sperm as seen by the 90°
detector both populations inclusive) or control samples created
by pipetting onto slides rather than by sorting (pipetted control
spermatozoa were an equivalent number of spermatozoa removed
from the unsorted sample by pipette and then placed on the
slide in the Lief bucket). Each sample could be probed once
with either the X or Y DNA probe. Sorted and pipetted samples
were generated in a random fashion so that no pattern was
discernible to the microscopist who was quantifying the number
of hybridization signals.
Information from several reports (Garner et al., 1984; Pinkel
et al., 1986; Pieters et al., 1990) was used to develop the method
for FISH of sorted spermatozoa. Spermatozoa were sorted onto
slides, fixed as previously described, treated with 0.05 M
dithioerythritol (DTE) in 0.1 M Tris-HCl (pH 8.0 at room
temperature) for 15 min, washed three times with 2 x SSC, then
air dried at room temperature. Treated sperm preparations and
lymphocyte preparations (obtained using standard cytogenetic
techniques) were then denatured in 70% formamide and 2 x SSC,
and then dehydrated in a series of ethanol washes at 70, 80, 95
and 100%, and air-dried. The X- or Y-centromere-specific
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Preparation of sperm nuclei
Human sperm were obtained from healthy donors by masturbation
and frozen by established cryotechnique for human spermatozoa
(Mahadevan and Trounson, 1984) by Fairfax Cryobank, a
division of the Genetics & IVF Institute, Fairfax, VA. The five
donors used had participated in the donor programme for a
minimum of 2 years, during which they were tested every 30
days for human immunodeficiency virus (HIV) antibody. Frozen
sperm specimens were routinely quarantined for 6 months prior
to use. The spermatozoa were thawed at room temperature,
washed two times by centrifugation (500 g) in 10 mM phosphatebuffered saline (PBS) and resuspended at 10 X 106 sperm/ml.
Samples of sperm heads (nuclei) were prepared by sonication
of the suspension for 10 s to break the tail from the head (Branson
Sonifier Cell Disrupter 200 with tapered microtip; Danbury, CT;
Johnson et al., 1987). Nuclei were then centrifuged for 20 s at
12 800 g (Eppendorf 5415, Brinkmann Instruments, New York,
USA), decanted and resuspended again in 1 ml PBS.
ultraviolet (351, 364 nm) lines of a 5 W 90-5 Innova argon-ion
laser (Coherent, Inc., Palo Alto, CA, USA) operating at 175 mW
and fluorescence detected through 418 nm long pass filters. A
76-^m jet-in-air flow tip was used. Data were collected as 256
channel histograms. Sheath fluid was 10 mM PBS.
Separation of human X- and Y-bearing spermatozoa
biotinylated alpha satellite DNA probes (Oncor, Inc. Gaithersburg,
MD, USA) were denatured in a solution of 70% formamide,
2 x SSC, 10% dextran sulphate and 500 ng herring sperm DNA
at 70 °C for 5 min. The sperm DNA was denatured and
hybridized for 15 h in a humidified chamber at 37°C. The
biotinylated probes were detected with fluorescein isothiocyanate
(FITC)-avidin as described by Pinkel et al. (1986). Cells were
counterstained with propidium iodide (PI). The FTTC and PI were
excited at 450—490 run and observed using a Leitz Orthoplan
microscope equipped with an epifluorescence plume illuminator.
Positive signals from interphase nuclei of the blood lymphocyte
served as controls and verifiers of optimum probe response. A
total of 200 spermatozoa were counted for each population and
the number of cells containing hybridization signals was recorded.
The mean percentage of positive signals were tested statistically
according to Moore and Gledhill (1988).
SHEATH
. ' FLUID
90°
DETECTOR
SAMPLE
. • STREAM
EDGE
.-SPERM
0"
DETECT.
(I
B
LASER BEAM I
9 0 ° DETECTOR
Human spermatozoa are not easily sorted or analysed for DNA
content for three reasons. First, the head morphology of the
human spermatozoon is more angular than the paddle shape of
spermatozoa of domestic animals. The sorting process relies on
orienting the flattest side of the spermatozoon to the laser beam
so that variation in fluorescent signal can be minimized and
separation achieved (Johnson and Pinkel, 1986). Fluorescence
emitted from the edge of the spermatozoon is much brighter than
the fluorescence emitted from its larger flat surface. This
fluorescence heterogeneity (which is due to the compactness
of the chromatin and high index of refraction between the
spermatozoon and surrounding media) is maximal when the
fluorescence is detected at a right angle (90°) to the incident
excitation source. Secondly, human spermatozoa are more
polymorphic and have greater heterogeneity of chromatin
composition than animal spermatozoa. Thirdly, such a small
difference (2.8-3.0%) in DNA content requires that the
resolving capability (near 1.0 coefficient of variation) of the
instrumentation be pushed to its inherent limits. We found that
flow cytometric reanalysis of sorted human spermatozoa for DNA
gives indicative but inconclusive results (data not presented).
Consequently, we chose to use DNA probes in combination with
FISH to validate the proportions of X and Y human spermatozoa.
The fluorescence heterogeneity can be greatly diminished
by not only detecting the fluorescence at a 0° angle, but by
detecting the 0° fluorescence from only those spermatozoa
(properly oriented) whose edge is nearly perfectly aligned with
the 90° detector.
The sorting system configuration (Figure 1A) allows the
generation of a fluorescent signal from spermatozoa stained with
Hoechst 33342 (Figure 2A). The signal is proportional to sperm
orientation in the laser beam or DNA content depending on the
angle of fluorescence detection. Those spermatozoa positioned
with their brightest edge (Figures 1A and 2A) toward the 90°
detector are represented in the right-most peak (shaded area,
Figure IB).The fluorescent signals of the oriented spermatozoa
falling within the shaded area (gate) are simultaneously seen by
the 0° detector (Figure 1C). The fluorescence signal collected
o
a
UJ
DC
0
DETECTOR
XY=51
X-Y=2.8S
FLUORESCENCE INTENSITY
Fig. 1. (A) Schematic drawing of an orthogonal flow cytometer
modified for resolution of X- from Y-chromosome-bearing
spermatozoa based on their difference in DNA content. This plan
view shows the sperm head properly orientated. Proper orientation
occurs when the opposing flatter surfaces of the sperm head
simultaneously face the laser and 0° forward detector. The
excitation source was an argon laser that emitted 175 mW of
ultraviolet (351, 364 nm) power. Fluorescence emission was
collected at 0° and 90° to the excitation source through 418 nm
long pass filters. The fluorescence distribution from the 90"
detector (B) shows electronic gating of the properly orientated
spermatozoon (shaded region). The simultaneously collected 0°
fluorescence distribution of the properly orientated spermatozoon
(from the shaded region in B) shows a partially resolved bimodal
distribution (C). Spermatozoa containing the smaller Y chromosome
and thus less total DNA comprise the dimmer (left) population, and
similarly the larger X-chromosome-bearing spermatozoa comprise
the brighter population. Even when a bimodal distribution has no
apparent split between the two populations, the same principle of
separation holds true for the left and right portions of the
populations within the distribution. The X and Y sperm populations
are selected by the electronic sort windows (shaded areas, C) and
electrostatically separated by the flow cytometer/cell sorter.
by the 0° detector is highly proportional to sperm DNA content.
Since misoriented spermatozoa have been removed from the
analysis, it is possible to resolve the X and Y spermatozoa
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Results
L.A Johnson et al.
Downloaded from http://humrep.oxfordjournals.org/ at Pennsylvania State University on May 11, 2016
1736
Separation of human X- and Y-bearing spermatozoa
Table I. Percentages of sperm binding the X- or Y-chromosome-specific
probe after flow' cytometric sorting for the enrichment of X or Y sperm
population
Sorted for
Probed for
n
Mean % positive
signal ± SEM
X
X
Y
Y
Control
Control
X
Y
X
Y
X
Y
10
g
9
10
7
9
82.2
19.0
20.8
75.5
52.6
44.1
±
±
±
±
±
±
1.7
1.9
2.3
1.8
2.2
3.3
1991) where motility of sorted spermatozoa ranged from 60 to
80%. Indications are that human spermatozoa show no significant
deleterious effect due to staining and cell sorting. They are likely
to fertilize ova under either in-vivo or in-vitro conditions.
Although no human fertilization experiments were conducted,
it is important to note that out of > 150 animal offspring born
to date, all offspring have proved morphologically and functionally
normal (Johnson etal., 1989; Johnson, 1991). Other workers
have used the Hoechst 33342 stain on bull and rabbit spermatozoa
along with cell sorting and have reported similar morphological
data on offspring (Morrell and Dresser, 1989).
Discussion
Mammalian spermatozoa, with the exception of the Microtus
oregoni (Ohno, 1963; Johnson and Clarke, 1990), all carry an
X or a Y chromosome. To our knowledge, the X-chromosome
is always larger than the Y-chromosome due to a greater amount
of heterologous chromatin. The DNA content of spermatozoa
is directly related to the chromosome complement, thus DNA
can serve as a marker to differentiate X- and Y-chromosomebearing spermatozoa. Species vary significantly in the amount
of DNA carried by the X versus the Y chromosome (Moruzzi,
1979; Johnson, 1992). The relative fluorescence of X and Y
spermatozoa is dependent on the amount of DNA determined
by flow cytometric analysis (Johnson, 1992). Agriculturally
important mammals, cattle, swine, sheep, differ in X and Y
chromosome DNA content by 3.9, 3.6 and 4.2%, respectively
(Johnson et al., 1987). DNA content of the human spermatozoa
in this study was shown to differ by only 2.8%. This flow
cytometric determination of human spermatozoa DNA content
difference agrees with the value reported by Sumner and
Robinson (1976), which was determined on the basis of dry mass.
The morphological shape of the human sperm head is smaller
and more angular than the sperm head of many domestic animals.
Since the original modifications to the flow cytometer cell sorter
were designed to take advantage of the flatness and paddle shape
of animal spermatozoa, the consistency of human sperm orientation
was a problem. However, it was found that the angular head of
the human spermatozoa also gives off preferential emission of
fluorescence. This fact and extraordinary attention to machine
settings and parameters led us to successfully orientate the human
spermatozoon, select the oriented spermatozoa through electronic
gating and measure the differential DNA with the forward
fluorescence detector. The spermatozoa could then be sorted in
a manner similar to what we have done with other species.
Fig. 2. (A) A fluorescent micrograph of intact human spermatozoa stained with the DNA binding dye Hoechst 33342. The sperm tails do
not show fluorescence. Note the greaterfluorescentintensity of the spermatozoon marked by the arrow. This spermatozoon is exhibiting
edge fluorescence which is brighter thanfluorescencefromthe flatter surface of the human sperm head as exhibited by the other two
spermatozoa in the micrograph. When the brighterfluorescingsperm edge (arrow) is towards the 90° detector the sperm fall within the
electronic gate (Fig. IB). (B and C) Fluorescent micrographs of spermatozoa that were sorted for the Y chromosome (B) and for the X
chromosome (C). The spermatozoa in both micrographs illustrate the results of hybridization with the X-chromosome-specific alpha satellite
DNA probe. THe background stain (reddish colour) is the DNA stain propidium iodide. The yellow/greenfluorescence,present on only two
spermatozoa in (B) and on numerous spermatozoa in (C), represents the presence of the X chromosome.
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(Figure 1C). The two peaks are —2.8% apart in terms of
DNA content, resulting in an overlap of the two populations.
The shaded areas of Figure 1C are sort windows. Those windows
represent six to eight channels out of a total of 256 channels on
the intensity scale. The spermatozoa falling within the windows
are deflected to the left and right, respectively, and collected in
tubes or onto slides. These sort windows were chosen to select
the region of the peaks that contained the greater proportion of
the X or Y spermatozoa.
The results of X- or Y-chromosome-specific DNA probe
hybridization to human spermatozoa that had been sorted for
bearing the X or Y chromosomes are shown in Table I.
Illustrations of hybridization response are shown in Figure 2B
and C. Each half of the slide contained a sample of —40 000
spermatozoa either sorted X, sorted Y, sorted control [orientated
spermatozoa as seen by the 90° detector (arrow, Figure 2A),
both populations inclusive] or pipetted control spermatozoa. The
values refer to the percentage of positive hybridization signals
recorded for the various X or Y sperm sorts. Statistical comparisons indicate a significant difference (P < 0.02-0.04) from
the theoretical human sex ratio of 107/100. Neither control
differed from the theoretical (P > 0.05; Table I).
The independent verification of X and Y sperm enrichment
on 53 sorts shows that the proportions of X and Y spermatozoa
can be altered from a 50/50 ratio by cell sorting based on DNA.
Although it was not essential to use both X and Y probes, using
both probes served as additional validation to the efficacy of
the separation technique.
The motility of neat semen, stained with Hoechst 33342 sorted
and collected into Test-Yolk extender, was determined by
microscopical examination. Sperm motility of sorted spermatozoa
after 1 h ranged from 65 to 75%. This was similar to earlier
results with the domestic animals (Johnson et ah, 1989; Johnson,
L.A.Johnson et al.
With the ever-increasing improvement of in-vitro fertilization
(IVF) protocols, fewer and fewer spermatozoa are being required
to fertilize a single ovum. On that basis, the likelihood of
achieving the sex of choice could well be increased since if
only a few spermatozoa are required, the sorting technique could
then become more stringent. Another application for X- and
Y-sorted human spermatozoa would involve micro-injection of
the whole spermatozoa or sperm heads from the sorted population
into eggs. The successful use of sorted sperm micro-injection
in several species has been demonstrated (Johnson and Clarke,
1988; Clarke etal., 1989). Others (Goto et al., 1990) have
produced calves from micro-injected spermatozoa and ova.
Recendy Katayose et al. (1992) demonstrated that freeze-drying
and long-term storage of human sperm nuclei at 5°C did not
eliminate fertilizing capacity.
The data described illustrate the usefulness of DNA as a marker
by which viable human X-bearing spermatozoa can be separated
from viable Y-bearing spermatozoa. Although die fertilization
capacity of stained and sorted spermatozoa has not been
determined, one would expect sorted spermatozoa to be capable
of fertilization based on ongoing animal studies. Accurate sex
pre-selection would improve the dierapeutic alternatives in cases
of X-linked genetic diseases. Use of die sexing technology
described here could in time reduce or eliminate the use of
selective abortion as a means of decreasing the incidence of
X-linked genetic disorders.
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The ability to monitor, by laboratory assessment, the purity
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Received on April 23, 1993; accepted on June 8, 1993
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