scientific program • symposia - American Society for Reproductive ...

seconds and time-lapse digital recordings are analyzed
for phenotypic abnormalities. Representative findings from
seventy tri-pronuclear embryos are displayed in this video,
along with an explanation for observed events.
MATERIALS AND METHODS: TLM is a powerful tool to analyze
early embryo development. Analysis of TLM images
recorded from tri-pronuclear embryos provides important
insight into the mechanisms of early mitotic and other
developmental events.
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V-4 11:52 AM
RESCUE OF AGED OOCYTES USING TRANSFER METAPHASE -II
NUCLEAR TRANSFER.
A. Tanaka1, M. Nagayoshi1, I. Tanaka1, H. Kusunoki2, S.
Watanabe3. 1Saint Mother Hospital, Kitakyushu, Fukuoka,
Japan; 2Faunal Diversity Sciences Graduate School
of Agriculture, Kobe University, Kobe, Hyogo, Japan;
3Department of Anatomical Science, Hirosaki University
Graduate School of Medicine, Hirosaki, Aomori, Japan.
OBJECTIVE: Nuclear transfer into the metaphase-II(M-II)
oocytes shows promise as a means of repairing female
infertility due to ooplasmic deficiency and abnormalities.
We therefore conducted nuclear transfer of in vitro matured
metaphase-II oocytes (recipient oocytes) into enucleated
freshly ovulated metaphase-II oocytes (donor oocytes).
DESIGN: Recipient and donor oocytes were placed in
a microdrop containing 5 μ/ml of cytochalasinB (CCB).
The aspirated M-II karyoplast of the recipient oocyte was
transferred into the perivitelline space of an enucleated
donor oocyte. The grafted oocyte was transferred in a 295
μm mannitol solution containing 0.05 μm MgCl2. Membrane
fusion was facilitated by electrical stimulation (5V for 1
second AC + 15V for 33 microseconds DC) with an electro
cell fusion generator (LF 201). After fusion, the constructed
oocytes were cultured in HTF medium for 2 hours and ICSI
was performed.
The percentage of identification of M-II chromosome was
90.1 % (82 out of 91) in freshly ovulated oocytes and 96.0%
(48 out of 50) in vitro-matured oocytes.
The M-II karyoplast was removed successfully in 72 of 82
(87.8%) of the donor oocytes and 81 of 95 (85.2%) of the
recipient oocytes. All of 81 karyoplasts of recipient oocytes
were replaced in the periviteleine space of enucleated
donor oocytes and 64 of these 79.0% were fused to form a
reconstituted oocyte.The fertilization rate, cleavage rate
and blastocyst formation rate following ICSI for constructed
oocytes and recipients oocytes were(68.8%(44/64),
64.1%(41/64), 25.0%(16/64)),(59.0%(58/98), 26.1%(25/98),
3.4%(3/98))respectively. Chromosomal analysis of 6 embryos
following nuclear transfer indicated they were all diploid sets
of 46 chromosomes.
MATERIALS AND METHODS: These results demonstrate that
this technique can be applied to the treatment of female
infertility due to ooplasmic deficiency and abnormalities in
aged oocytes.
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V-5 12:00 PM
SUCCESSFUL HUMAN SPERMATID INJECTION INTO OOCYTES.
A. Tanaka, M. Nagayoshi, I. Tanaka. Saint Mother Hospital,
Kitakyushu, Fukuoka, Japan.
OBJECTIVE: In cases where the patient’s most developed
spermatogenic cell is a spermatid, the spermatid injection
into the oocyte is the sole treatment available to conceive.
However the success rate is very low. From this low success
VIDEO PROGRAM
96
rate we can infer that the oocyte activating substance in
spermatids might be less sufficient than that of in matured
spermatozoa. Electrical stimulation of oocytes revealed the
beneficial effects on oocyte activation and subsequent
embryonic development. We here explain the procedure of
spermatid injections into electrically activated oocytes.
DESIGN: This study deals with non-obstructive azoospermic
men whose spermatogenesis was arrested at the level of
early spermatid (Sa,Sb) or late spermatid (Sc, Sd) (Clermont
1963) or whose testicular spermatozoa were completely
abnormal in shape. Biopsied testicular tissues were in D-PBS
containing 0.125% collagenase and 0.01% DNase. Oocytes
were transferred to 295mM solution of mannitol with 0.1mM
CaCl2 and 0.05mM Mgcl2 and exposed to an alternating
current of 8V / cm 1000KHz for 8s, followed by a single
1200V / cm pulse of direct current for 99μs. Each spermatid,
transferred into PVP-HTF, was drawn into an injection pipette
(7 μm I.D. for Sa or Sb, 5 μm I.D. for Sc, 3μm I.D. for Sd) and
the separated nucleus and cytoplasm were injected into
the ooplasm. Injected oocytes were cultured in sequential
media and their embryonic development was observed
until the blastocyst stage.
MATERIALS AND METHODS: Fertilization rate, cleavage rate
and blastocyst rate following spermatid injection with
or without electrical stimulation were [ 63.8%(162/254),
72.2%(117/162), 16.7%(27/162)] and [30.0%(93/310),
70.8%(34/48), 8.3%(4/48)] respectively. Pregnancy rate and
miscarriage rate between two different stage of spermatids
(early : Stage a-b, late: stage c-d) were [14.0%(310/2214),
46.6%(48/103)], [20.0%(414/2073), 29.0%(120/414)]. The
spermatid injection into the electrically activated oocytes
was revealed to be useful in clinical application.
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V-6 12:07 PM
CRYOPRESERVATION OF OVARIAN TISSUE BY VITRIFICATION.
S. J. Silber1, N. Kagawa2, K. Lenahan1, J. Hicks1, M.
DeRosa1, M. Kuwayama3. 1Infertility Center of St. Louis, St.
Luke’s Hospital, St. Louis, MO; 2Kato Ladies Clinic, Tokyo,
Shinjuku, Japan; 3Repro Support Medical Research Centre,
Tokyo, Shinjuku, Japan.
OBJECTIVE: To virtually eliminate oocyte loss from the
freezing technique using a vitrification protocol instead of
classic slow freeze methods.
DESIGN: Virtually all of the primordial follicles of the ovary are
in the thin fibrous cortex within one millimeter of the surface.
This has allowed the freezing, thawing, and transplantation
of ovarian tissue slices resulting in live healthy offspring in
both humans and experimental animals. However, the
procedure resulted in substantial egg loss from both the
transplantation technique and from the freezing process
itself. Ischemic loss of eggs from the transplant technique
can be reduced greatly by using very thin slices and by
avoiding micro-hematoma formation underneath the
graft, exactly like with skin grafts in plastic surgery. In this
video, we interview a typical such cancer patient and
then demonstrate the technique for both vitrification and
thawing of ovarian tissue. Then with a different patient we
demonstrate the surgical technique which minimizes oocyte
loss from ischemia as well.
MATERIALS AND METHODS: Histology and vital staining fail
to reveal any difference between fresh ovarian tissue and
tissue cryopreserved by vitrification.
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