Molecular Human Reproduction Vol. 1, pp. 59-62, 1995
DNA-based X-enriched sperm separation as an adjunct to
preimplantation genetic testing for the prevention of X-linked
disease
Gene Levinson1-24, Keyvan Keyvanfar1, Joy C.Wu1, Edward F.Fugger1-3, Rita A.Fields1,
Gary L.Harton1, Frances T.Palmer1, Michael E.Sisson1, Karin M.Starr1, Lisa Dennison-Lagos1,
Lucrecia Calvo1, Richard J.Sherins1, David Bick1-2, Joseph D.Schulman1-2 and
Susan H.Black13
4
To whom correspondence should be addressed
We report the world's first clinical pregnancy resulting from DNA-based enrichment for X-bearing human
spermatozoa, for prevention of X-linked hydrocephalus. Sperm separation was followed by embryo biopsy
and nested multiplex polymerase chain reaction (PCR) for gender determination. Enriched populations of Xbearing spermatozoa ranging from 80 to 89% pure as determined by fluorescence in-srtu hybridization (FISH)
resulted in in-vitro fertilization (IVF) rates indistinguishable from normal IVF procedures (65%). In two separate
biopsy procedures, 7/9 and 15/16 of the resulting embryos were determined to be female by multiplex PCR.
Embryo transfer resulted in a karyotypically normal female fetus. This technique should be widely applicable
to gender selection for the prevention of genetic disorders.
Key words: flow cytometry/fluorescence in-situ hybridization/gender selection/hereditary diseases/spermatozoa
Introduction
Amniocentesis and chorionic villus sampling represent safe
and accurate methods for the prenatal diagnosis of many
disorders (D'Alton and DeChemey, 1993). However, when an
abnormality is found, couples face the difficult choice of either
bearing an affected child or terminating the pregnancy. Invitro fertilization (IVF) followed by preimplantation genetic
testing provides an opportunity for couples to reduce the risk of
conceiving an affected individual. Successful preimplantation
genetic testing has been reported for a number of genetic
disorders (Harper and Handyside, 1994).
For prevention of X-linked diseases, determination of
embryo sex, followed by transfer of putative female embryos,
greatly reduces the risk of bearing an affected son. However,
the chance for a successful pregnancy is diminished because
on average only half of the embryos will be female. In addition,
the disposition of the remaining male embryos, of which half
should be genetically unaffected, remains a dilemma for some
couples. Increasing the proportion of female FVF embryos
would improve both of these situations.
Flow cytometric separation of viable X and Y chromosomebearing spermatozoa (Johnson and Pinkel, 1986) was first
reported in animals by Johnson et al. (1989). His team and
our own have recently adapted the sperm sexing technology
for separation of human spermatozoa (Johnson et al., 1993).
This separation is based on the mean 2.8% difference in DNA
content of human X- and Y-bearing spermatozoa. In the present
study, we describe the combined use of sperm separation with
© Oxford University Press
preimplantation genetic testing for the prevention of X-linked
hydrocephalus, an approach that significantly improved the
yield of female embryos available for transfer.
Materials and methods
Patient history
The patient was 34 years old, gravida 4 parity 2. Three of her
brothers had died with congenital hydrocephalus. The patient's
first two pregnancies resulted in a healthy girl and a healthy
boy. In the third pregnancy, amniocentesis revealed a 46,XY
karyotype. At 18 weeks of gestation, ultrasound examination
demonstrated hydrocephalus, and the patient elected to terminate the pregnancy. DNA analysis of the family using probe
SH4.1 was consistent with linkage to the X-linked hydrocephalus locus at Xq28 (Lyonnet et al., 1992). In the fourth
pregnancy, chorionic villus sampling was performed at 11
weeks gestation. The karyotype was 46,XY and DNA analysis
showed that the fetus had inherited the same allele as the
previously affected fetus. This pregnancy was also terminated.
Sperm separation
Methods for separating human X- and Y-bearing spermatozoa
have been described previously (Johnson et al, 1993). Briefly,
using a modified flow cytometer, spermatozoa are separated
based on DNA content by Hoechst 33342 forward fluorescence,
using 90° scatter for purposes of orientation. Current purifica59
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Genetics & IVF Institute, 3020 Javier Road, Fairfax, VA 22031, department of Human Genetics and department of
Obstetrics and Gynecology, Medical College of Virginia, Richmond, VA, USA
G.levimson et al.
tion yields range from 0.2 to 0.4% of input spermatozoa. On
the day of oocyte retrieval, spermatozoa (40X10 6 ) obtained
from the husband yielded a total of 77 000 X-enriched
spermatozoa in the first IVF cycle. The second IVF cycle
yielded 193 000 X-enriched spermatozoa from an original
sample of 50X10 6 . Spermatozoa were collected in 200 |il of
HEPES-buffered Tyrode's modified medium supplemented
with 2% bovine serum albumin at room temperature. Approximately 20 000 sperm cells were removed for fluorescence
in-situ hybridization (FISH) analysis to provide an adequate
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Figure 1. Flow cytometric sperm separation combined with preimplantation genetic testing for the prevention of X-linked hydrocephalus.
(A) Human spermatozoa stained with Hoechst 33342 prior to flow cytometric separation. Photographed with combined bright-field and
fluorescence illumination. (B) Histogram output from modified (see Johnson et al, 1993) Coulter Epics 752 flow cytometer: (a) 90°
fluorescence profile for sperm orientation used for primary gating (red rectangle; see Johnson et al, 1993); (b) forward fluorescence profile
indicating DNA content of X- and Y-bearing spermatozoa; the rectangle encloses the gated, chosen X-bearing population. (C) Representative
example of an X chromosome-enriched population of human spermatozoa subjected to fluorescence in-situ hybridization with X (orange)
and Y (green) directly labelled probes. (D) Polyacrylamide gel electrophoresis of Rsal digests of amplified X- and Y-linked human
amelogenin gene segments (X, Y) and Y-linked DYZ1 satellite DNA (D) (see Levinson et al, 1992); 1-8 = single blastomeres from each
of the eight biopsied embryos; M = molecular weight markers; N = no DNA negative control; W = cell-free fluid wash negative control.
60
Sperm separation for prevention of X-linked disease
IVF and embryo biopsy
Gonadotrophin-releasing hormone analogue (Lupron; Tap
Pharmaceutical, North Chicago, IL, USA) and follicle stimulating hormone (Metrodin; Serono, Randolph, MA, USA) were
used to achieve pituitary suppression and ovarian stimulation,
using standard protocols. After human chorionic gonadotrophin
administration, oocytes were recovered by transvaginal ultrasound-guided follicle aspiration. For IVF cycle no.l, 22 eggs
were retrieved, of which 15 were viable. In the second IVF
cycle, 27 eggs were retrieved, of which 20 were viable. Each
viable egg was inseminated in a microdroplet under oil with
2500 spermatozoa from the separated X chromosome-enriched
fraction, 6 h after sorting and egg retrieval. Embryo biopsies
were performed as previously described (Fields et al, 1991;
Levinson et al, 1992).
Nested polymerase chain reaction
The sex of the blastomeres obtained at biopsy was determined
by nested polymerase chain reaction (PCR) as previously
described (Levinson et al, 1992), with the following modification: second-round amplification of Y-specific repetitive probe
DYZ1 was limited to 11 cycles total.
Results
first IVF cycle
Purity of the X chromosome-enriched sperm fraction was
determined to be 86% X-bearing by FISH (179 X, 22 Y; n =
201). Of the 15 eggs, 10 (67%) were successfully fertilized
in vitro. Nine of these embryos were two pronuclear (2PN;
presumptive diploids). These nine embryos were biopsied at
the 3- to 6-cell stage, 72-77 h post-retrieval. Eight of the nine
embryos survived the biopsy procedure and were subjected to
gender determination. For five of the eight surviving embryos,
it was possible to assay two blastomeres independently, for
added reliability.
As shown in Figure 1 and Table I, seven of the eight survivors
(87%) were sexed as female, while one was determined to
be male. This ratio was concordant with the 86% purity
ascertained by FISH. Six embryos were viable 24 h post-
biopsy, three of which had cleaved. Five of the viable embryos
were female. Three female embryos were transferred, but a
pregnancy did not result.
The two remaining female embryos were cryopreserved.
Subsequent transfer failed to result in a pregnancy.
Second IVF cycle
A control sample of unenriched spermatozoa was assessed by
FISH and determined to be 49.5% (99/200) X and 50.5%
(101/200) Y. Purity of the X-enriched sperm fraction was
determined to be 80% by FISH (320 X, 82 Y; n = 402). Of
the 20 eggs, 13 (65%) were successfully fertilized in vitro to
form 2PN embryos. These 13 embryos were biopsied at the
2- to 8-cell stage, 72-77 h post-retrieval. In addition, three
embryos that fertilized but were not 2PN were also biopsied.
These 16 embryos were subjected to gender determination.
For 13 of the 16 embryos, it was possible to assay two
blastomeres independently.
As shown in Table I, 15 of these embryos (94%) were sexed
as female, and one embryo produced ambiguous results. All
13 embryos which were viable 24 h post-biopsy were female.
Three female embryos were transferred, resulting in an ongoing
pregnancy that is in its 16th week at the time of submission.
Chorionic villus testing confirmed the female gender of the
fetus. The 10 remaining female embryos were cryopreserved.
Discussion
This case represents the first reported clinical application of
fluorescence^activated sperm separation to human embryos,
as well as the first separation procedure combined with
preimplantation genetic testing. A recent review reported that
sex determination for prevention of X-linked disease is the
most common application of preimplantation genetic testing
worldwide (Harper and Handyside, 1994); patients have undergone 80 stimulation cycles for X-linked disease diagnosis,
compared to 63 cycles for all other diagnoses combined. With
more than 300 described X-linked disorders (McKusick, 1994),
this trend is likely to continue.
The use of flow cytometry for separation of X and Y sperm
populations has been demonstrated in several animal species
(Morrell and Dresser, 1989; Johnson, 1991, 1992; Cran et al.,
1993; Johnson et al, 1994) as well as in man (Johnson et al,
1993). Pregnancy outcomes in a variety of mammalian species
have confirmed significant alteration of the sex ratio among
resulting offspring (Johnson, 1991; Cran et al, 1993; Johnson
et al, 1994). Our results show that separated human spermatozoa will fertilize human eggs. The 65—67% fertilization rate
Table I. Results from clinical sperm separation followed by embryo biopsy
for gender determination
Biopsy
X purity by
FISH (%)
No.
sexed
Female
(n)
85.9
79.6
16
7
15
Male
(")
Outcome
(i)
no pregnancy
ongoing pregnancy
FISH = fluorescence in-situ hybridization.
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concentration of cells on the slide. Remaining spermatozoa
were incubated at 35°C for 20 min; this was followed by
sperm count and motility, progression and viability measurements. The pelleted sample was resuspended in Menezo B2
medium (Fertility Technologies, Inc. Natick, MA, USA) at
3000 motile spermatozoa/u.1 for IVF. For FISH analysis,
enriched sperm aliquots were pelleted and fixed with 3:1
methanol:acetic acid on a microscope slide followed by incubation with 50 mM dithiothreitol in 0.1 M Tris-HCl for
8 min. Hybridization and detection procedures were performed
according to the manufacturer's protocol, using direct-labelled
dual colour X and Y probes (Spectrum CEP direct chromosome
enumeration system; Vysis, Framingham, MA, USA). For each
purity assessment, random fields were counted until 200
spermatozoa were scored for X or Y hybridization signals.
Typical hybridization efficiencies for X and Y probes with
this protocol exceed 95%.
G.Levinson et al.
for spermatozoa subjected to the sorting procedure compares
favourably to our fertilization rate of 65% in routine IVF (when
there is no male infertility or proven failure of fertilization in
prior IVF cycles; unpublished observations).
These results provide compelling evidence that the number of
female IVF embryos available for transfer can be substantially
altered by flow cytometric sperm separation. Clinical application of this technique should therefore provide larger numbers
of female embryos for preimplantation genetic testing programmes, and reduce the numbers of embryos unsuitable for
transfer. We are currently investigating the possible application
of sperm separation to a number of other research and clinical
situations.
We thank Larry Johnson and Glen Welch for helpful technical advice
and comments on the manuscript.
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Received on February 2,1995; accepted on February 17, 1995
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Acknowledgements