Download Pregnancy and birth resulting from transfer of a blastocyst observed

Human Reproduction vol.14 no.7 pp.1869–1871, 1999
CASE REPORT
Pregnancy and birth resulting from transfer of a
blastocyst observed to have one pronucleus at the time
of examination for fertilization
Lyn Gras1,3 and Alan O.Trounson2
1Monash
IVF, Clayton, Victoria, Australia, 3168 and 2Monash
Institute of Reproduction and Development, Monash University,
Clayton, Victoria, Australia, 3168
3To
whom correspondence should be addressed
This case report describes a successful full-term pregnancy
and birth after the transfer of a zona-free blastocyst derived
from an oocyte observed at fertilization check as having
only one distinct pronucleus (PN). The patient had previously undergone four in-vitro fertilization (IVF) cycles and
three frozen embryo transfer cycles, with one pregnancy
resulting. In this IVF cycle, 7/19 oocytes were fertilized
exhibiting two distinct PN; however, all these oocyctes
failed to develop in culture and had arrested or totally
fragmented by day 6 after insemination. One oocyte (1/19)
displayed only one PN 18 h after insemination, but culture
of this oocyte led to development of an early cavitating
blastocyst by day 6. Since no other embryos were available
for transfer to the patient, this embryo was transferred,
resulting in a full-term pregnancy with delivery of a normal
healthy boy. Observation of a single PN at the normal time
of fertilization assessment would not appear to be an
absolute indicator of developmental incompetence. In-vitro
culture to 6 days post-insemination provides the opportunity to assess embryological development after activation of
the embryonic genome. Formation of a morphologically
normal blastocyst may be an indicator of a fertilized
embryo with normal developmental capacity.
Key words: blastocyst transfer/fertilization/IVF/parthenogenetic activation/single pronucleus
Introduction
Normal fertilization is determined in in-vitro fertilization (IVF)
by the presence of two distinct pronuclei (PN) at 16–20 h
post-insemination. Absence of PN indicates fertilization failure.
Occasionally only one PN is visible in the oocyte at fertilization
check, and the absence of a second PN may be explained as
oocyte parthenogenetic activation, irregular pronuclear formation resulting from asynchrony of pronuclear appearance, or
possibly male and female pronuclear fusion. In-vitro culture
of these 1PN oocytes shows that although the majority are
incapable of development past early cell divisions, a small
proportion exhibit further cleavage, are indistinguishable from
© European Society of Human Reproduction and Embryology
2PN embryos and are capable of forming morphologically
normal blastocysts (Plachot and Crozet, 1992).
The experimentally induced parthenogenetic nucleus is
incapable of supporting normal early development. Likewise,
embryonic development of human parthenogenetic oocytes
would be expected to become impaired after activation of the
embryonic genome, at ~2 days post-insemination, thus leading
to early embryo arrest. Standard IVF culture to day 2 or 3
does not provide an opportunity to observe embryo development once the maternal message has ceased. Oocytes that
appeared initially as 1PN but are actually fertilized, cannot be
distinguished from parthenogenetic embryos at this stage.
By extending in-vitro culture to day 6, further embryonic
development and formation of a morphologically normal
blastocyst may possibly be used as an indicator of fertilization
and normal developmental capacity.
This case report involves a full-term pregnancy and birth
resulting from transfer of a blastocyst observed to have only
one PN at 18 h post-insemination, but which developed to
form a morphologically normal blastocyst after 6 days of invitro culture.
Case report
The patient presented with subfertility in 1993 with a tortuous
right tube blocked by a hydrosalphinx, and proceeded through
two gamete intra-Fallopian transfers (GIFT), two IVF and three
frozen–thawed embryo cycles with one resulting pregnancy and
birth. In 1998 the patient, then aged 31 years, had ovulation
stimulation for her fifth cycle of artificial reproductive treatment. Oocytes were retrieved transvaginally 36 h after human
chorionic gonadotrophin (HCG) administration, inseminated
with 150 000 motile spermatozoa/ml and cultured in IVF-50
medium (Scandinavian Sciences), with 10% human serum
albumin (HSA) in 5% CO2, 5%O2 and 90% N2. Oocytes were
mechanically denuded of corona cells 18 h post-insemination
and carefully examined with an inverted microscope. Fertilization was determined by the presence of two distinct PN and
two polar bodies. Oocytes with only one PN were considered
potentially competent for development. These oocytes were
identified and cultured in vitro, as were 2PN derived oocytes,
in 20 µl microdrops of IVF-50 medium with HSA under oil
to day 3. Embryos were then morphologically assessed and
changed to 50 µl drops of G2 medium (Barnes et al., 1995).
On day 6, all embryos were assessed for blastocyst formation.
The zonae were enzymatically removed from blastocysts
selected for transfer using 0.2% pronase for ~1 min, then
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L.Gras and A.O.Trounson
washed three times in G2 medium and incubated at 37°C
until transfer.
A total of 19 oocytes was retrieved, of which seven (37%)
were fertilized as shown by the presence of two PN. One
oocyte was observed to have only one PN with fragmented
polar bodies. Culture of the seven fertilized oocytes produced
embryos ranging from two to eight cells on day 3; however,
all these embryos were either arrested or totally fragmented
by day 6 post-insemination and therefore not suitable for
transfer. Culture of the single 1PN oocyte produced an eightcell embryo on day 3 and a morphologically normal early
nascent blastocyst on day 6. Since no other embryos were
suitable for transfer to the patient and this embryo exhibited
apparently normal developmental capacity in vitro, this embryo
was transferred to the patient. On day 16 after oocyte collection,
pregnancy was confirmed with a positive blood pregnancy test
and 6 weeks later ultrasound showed a viable singleton
intrauterine gestational sac and embryo dimensions consistent
with transfer dates. The pregnancy proceeded uneventfully and
an ultrasound scan at 23 weeks revealed a normal baby with
no congenital abnormalities. An uneventful delivery at 38
weeks resulted in birth of a 3860 g normal healthy boy.
Discussion
Observation of 1PN after IVF is generally considered to be
the result of parthenogenetic activation of the oocyte and these
oocytes are usually discarded. A proportion of these oocytes,
however, may actually be fertilized, resulting from irregular
PN formation such as asynchrony of male and female PN, or
pronuclear fusion (Levron et al., 1995). A single observation
of one PN, therefore, may not distinguish between oocyte
activation or fertilization. Reassessment of single PN oocytes
4–6 h after initial fertilization check can reveal the presence
of the second PN; however by this time evidence of either PN
may also have completely disappeared, reducing the reliability
of this second assessment as a further fertilization check. For
example, reassessment (Staessen et al., 1993) revealed 62.2%
of oocytes still with one PN 4–6 h after initial check, whereas
25% of oocytes showed a second PN but 12.8% showed
complete disappearance of the original PN. From time-lapse
recording of events involved in PN formation (Payne et al.,
1997), only 63% of oocytes actually showed simultaneous
appearance of male and female PN. Asynchrony of PN
formation in normally fertilized oocytes may be explained by
faster or earlier formation of either male or female PN. This
asynchrony has been observed to occur in similar frequencies
irrespective of the source of spermatozoa for ICSI (ejaculated
or testicular), but more frequently in IVF than ICSI (Nagy
et al., 1998).
Pronuclear fusion is exhibited in species where fertilization
occurs after completion of meiosis. In humans, however, this
event has never been observed and PN have only been identified
as separate bodies prior to nuclear membrane breakdown. It
has been suggested (Levron et al., 1995) that a normal variant
of fertilization may involve formation of a common pronuclear
membrane during syngamy, leading to a diploid zygote
appearing with only one PN. The single 1PN oocyte that
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resulted in the livebirth in this case report was assessed only
once for fertilization 18 h after insemination and no second
PN was observed. Therefore no conclusion can be drawn as
to either pronuclear asynchrony or fusion being the mechanism
leading to normal development in this case.
Analysis of the chromosome constitution of 1PN oocytes
after standard IVF and intracytoplasmic sperm injection (ICSI)
further supports the presence of fertilization in some 1PN
oocytes. Y-specific hybridization signals have been revealed
in 35.6% of embryos (Staessen et al., 1997), therefore indicating that in 70–75% of such cases a spermatozoon had penetrated
the oocyte and that only 25–30% of the 1PN oocytes were
actually parthenogenetic. Since 48.7% of IVF and 27.9% of
ICSI-derived 1PN oocytes were found to be diploid, with
equal ratios of XX and XY embryos, formation of a single
PN in a diploid embryo was interpreted as indicative of normal
fertilization followed by asynchronous formation of PN.
In-vitro culture of oocytes observed as 1PN reveals that
although the majority arrest in early cleavage stage, a small
proportion are capable of further development and are observed
to form morphologically normal blastocysts indistinguishable
from other 2PN-derived blastocysts. It has been observed
(Plachot et al., 1992) that 77% of 1PN oocytes proceeding to
the two to four-cell stage by day 2, whereas 14.3% can reach
early blastocyst by day 5 in culture. Those 1PN-derived
embryos capable of blastulation with adequately developed
inner cell mass may in fact be derived from fertilized oocytes
with a diploid complement. Differences in cleavage performance of diploid and haploid 1PN embryos have been observed
(Levron et al., 1995) with a higher incidence of haploidy noted
in zygote stage and the rarity of haploid 1PN embryos after 3
days of culture. This provides evidence of selection against
haploid eggs during culture.
Diploid 1PN-derived embryos may indicate fertilization,
or result from non-extrusion of the second polar body or
spontaneous diploidization. Observations of these diploid parthenogenomes are rarely reported; however, the existence has
been reported (Staessen et al., 1997) of a possible diploid,
androgenetic one PN-derived eight-cell embryo with two Y and
two 18-specific signals. It is possible that diploid androgenetic
oocytes do occur but this is most likely to be very rare, since
the fertilizing spermatozoa would need to be diploid and the
female chromosomes lost (no female PN formed). It would
seem more likely that these oocytes would contain two (diploid
sperm and haploid oocyte chromatin) or even three (two haploid
sperm and haploid oocyte chromatin) PN. Consequently, it
is probably less likely that diploid androgenomes occur when
only one PN is observed, than when two or more PN are
observed. The developmental competence of diploid androgenomes to the blastocyst stage is unknown but could be
expected to be irregular because of possible multiple sperm
centrioles (Sathananthan et al., 1991).
Culture of normal fertilized oocytes to the blastocyst stage
has been shown to allow a more objective selection of viable
embryos for transfer (Jones et al., 1998). This extended culture
beyond 2–3 days in vitro allows elimination of embryos
incapable of development once maternal message ceases and
activation of the embryonic genome is initiated (Braude et al.,
Blastocyst transfer
1988). Extension of in-vitro culture to day 6 provides the
opportunity to observe further embryonic development of these
embryos and to identify those capable of blastocyst formation
and therefore most likely to be normally fertilized.
In this report, transfer of a blastocyst derived from a 1PN
oocyte resulted in a full-term pregnancy and birth of a normal,
healthy boy at 38 weeks. It appears that the initial fertilization
check observation of one PN did not reflect the developmental
capacity of the embryo. This is not the first report of birth
following transfer of 1PN embryos. The occurrence has been
reported (Staessen et al., 1997) of 38 single embryo transfers
where the only embryo available for transfer originated from
a 1PN oocyte, resulting in three pregnancies with two livebirths
and one preclinical abortion. Pregnancy and livebirth following
transfer of 1PN embryos following round spermatid injection
at ICSI have also been reported (Barak et al., 1998). However,
all these embryos were transferred after culture only to early
cleavage stage, allowing no further assessment. By extending
the culture period to allow for blastocyst formation, an opportunity is provided to select further those 1PN-derived embryos
with the greatest developmental potential. Further culture to
establish blastulation may provide a better opportunity to
determine fertilization. These blastocysts may then provide an
additional source of embryos for the patient, particularly if no
other embryos are available.
References
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after transfer of embryos that developed from single-nucleated zygotes
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Received on December 4, 1998; accepted on April 18, 1999
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