Abstracts of the 13th Annual Meeting of the ESHRE, Edinburgh 1997
damaged blastomeres is lower than for embryos with 100%
blastomere survival.
R-144. Sperm dysfunction, glucose in IVF culture
medium and embryo implanting ability
Ray B.D. I,2, Saad C.2, Williams J.A.C.2, Wilson p.2,
McDermott A. I and Hull M.G.R. 2
R-145. Comparative development of ICSI-derived human
embryos in glucose-free, phosphate-free simple media
solutions
1Regional Cytogenetics Centre, Southmead Hospital, Bristol
BSIO 5NB and 2University of Bristol IVF Unit, Redland Hill,
Bristol BS6 6UT, UK
High levels of glucose in IVF culture medium may be
deleterious, significantly reducing trophectoderm cell numbers,
but studies in this and other laboratories have failed to
show a convincing disadvantage in implanting ability for
normozoospermic patients. Here we report two studies of
sperm dysfunction defined by either reduced penetration of
cervical mucus or reduced recovery and survival in culture
medium.
Materials and methods: Study 1 compared Ham's F-lO
medium lacking glucose with Ham's F-lO medium containing
1 mM glucose. Study 2 compared Ham's F-lO medium lacking
glucose and other substances thought to be deleterious, with
standard Ham's F-lO medium containing 5.5 mM glucose.
Women aged <40 years were·· randomly allocated to the
different culture systems. Standard IVF techniques were used.
Cultures were supplemented with 10% human serum in all
cases, which added a low level of glucose to the cultures.
Pregnancy was defined by a gestational sac using ultrasound
scanning, and the implantation rate was calculated as the
number of sacs as a proportion of the number of embryos
transferred.
Results:
Effect of IVF culture medium on implantation
Study I
Ham's F-IO
- glucose
No. of oocytes retrieved
No. of mature oocytes
No. of normal embryos (%)
Median rate (95%
confidence interval)
No. of embryo transfers
No. of pregnancies
Rate per transfer (%)
No. of embryos (mean!
transfer)
No. of gestational sacs
Implantation rate (%)
14
131
38 (29)
26 (9-50)
12
3
25
30 (2.5)
4
13
Study 2
Ham's F-IO
+ I mM
glucose
Ham's F-IOglucose +
other"
20
23
189
186
66 (34)
66 (35)
42 (24-59)
3~ (18-61)
18
4
22
42 (2.3)
4
9
19
5
26
48 (2.5)
Ham's
F-IO
+ 5.5 mM
glucose
18
152
37 (24)
19 (7-50)
15
I
7
30 (2.0)
5
10
alron, copper, zinc and hypoxanthine.
There appeared to be a substantial reduction in implantation
and pregnancy rates associated with the relatively high concentration of glucose. The differences were not statistically significant, but the sample sizes were small and the studies could
not be concluded because of the introduction of ICSI treatment.
We would nevertheless speculate that a high glucose concentra298
tion may encourage excess production by already dysfunctional
spermatozoa of reactive oxygen species which harm the embryo
and reduce its ability to implant.
Schiewe M.C.2, Massacesi A.I, Santuari E.I, Amodei M.I,
Barbieri C. I, Ierardi K.I and Marrs R.p.I
lStudio Interdisciplinare Fecondazione e Transfer, Clinica
Villa Salaria, Roma 00139, Italy and 2Calijomia Fertility
Associates, Santa Monica, CA 90404, USA
Introduction: HTF is a simple medium commonly used in
human IVF. However, it is believed that the glucose and
phosphate components are unnecessary and potentially inhibitory to early embryo development. ICSI-derived human zygotes
(n = 337; 58 patients) were randomly assigned to one of three
simple media treatments including HTF (Irvine Scientific,
Santa Ana, CA, USA; n = 110; 21 patients), P-l (Irvine
Scientific; n = 102; 18 patients) and Q-XI (BioCare, San
Diego, CA, USA; n = 125; 19 patients). P-l and Q-XI are
new commercial formulations derived from HTF that are
devoid of glucose and phosphate, and use gentamycin (antibiotic source) and reduced amounts (3-fold) of Phenol Red.
In contrast to HTF, P-l possesses taurine and citrate, while QXI has EDTA and glutamine additives.
Materials and methods: In two ICSI series (October 1996
and January 1997),688 oocytes were aspirated from 58 women
(mean age 33 years) undergoing ovarian stimulation with a
GnRH antagonist (Decapeptile) and purified FSH (MetrodinHP). In all, 590 mature oocytes were injected with a single
immobilized spermatozoon (Schiewe, 1996) and placed in
HTF, P-l or Q-XI medium containing 10% SSS (Irvine
Scientific) under oil in organ culture dishes. 2PN zygotes were
transferred individually into 100 11 microdroplets under oil and
maintained in 5% CO 2 in a humidified air atmosphere at 37°C.
Embryos were evaluated at 48 and 72 h for cell number,
fragmentation and blastomere shape/size, and then assigned a
subjective quality grade (4 = excellent, 1 = poor). Embryos
were transferred on day 3, with or without assisted hatching.
Data were evaluated using the X2 analysis and ANOVA, with
differences determined by Fisher's LSD.
Results: A total of 430 2PN zygotes were produced (73.3%).
Of the 337 zygotes cultured, 329 (98.5%) cleaved. There were
no differences in the cleavage rate or cell number at 48 h
between treatments. However, the percentage fragmentation
was reduced (P ~ 0.05) at 48 and 72 h for embryos cultured
in P-l medium (7.6 ± 0.8 and 11.2 ± 1.2%) compared with
Q-XI (12.2 ± 1.5 and 16.5 ± 1.8%), but was not different
from HTF (9.0 ± 1.1 and 12.5 ± 1.7%). In tum, quality
grades were higher (P = 0.02) at 48 h for P-l- and HTFtreated embryos (3.2 ± 0.6) compared with Q-XI treatment
(3.0 ± 0.8). Cell numbers tended (P = 0.07) to be greater in
P-l cultured embryos at 72 h (6.4 ± 0.2) compared with HTF
and Q-XI (5.8 ± 0.2). The latter observation was significant
Abstracts of the 13th Annual Meeting of the ESHRE, Edinburgh 1997
(P = 0.03) in 74 IVF control embryos, with more cells
produced in P-l (7.1 :::': 0.5) than Q-XI (5.4 :::': 0.4) and an
intermediate response in HTF (6.1 :::': 0.5). Blastocyst formation
was observed on day 4 in P-l and Q-XI medium compared
with day 5 or 6 in HTF. However, in series I, these invitro evaluated developmental differences did not result in
differences in the pregnancy rate. A high clinical pregnancy
rate was achieved in each media treatment group: HTF 75%
(6/8), P-l 50% (4/8) and Q-XI 66% (4/6). Series II results are
pending, as are additional results from Series I-derived frozen
embryos.
Conclusions: Additional trials are necessary to fully determine
significant differences between media treatment groups. However, it is evident that glucose and phosphate are unnecessary
additives for culturing human embryos up to day 3. In fact,
excellent blastocyst development (50%) through to day 6
occurred in P-l and Q-XI media.
Reference:
Schiewe (1996) 1. Assist. Reprod. Genet.
R-146. Effect of embryonic stage on the success of
cryopreservation
Nematollahi N.!, Rezazadeh M.2 and Hosseini A.3
Royan Institute, Tehran, 1Department of Anatomy, Medical
School of Kerman University, Kerman, 2Department of
Anatomy, Tarbiat Modarres, School of Medicine, Tehran and
3Department of Anatomy, Shahid Beheshti School of
Medicine, Tehran, Iran
The aim of this study was to investigate the sensitivity of
different developmental stages of mouse embryos to cryopreservation. Some 1-, 2-, 4- and 8-cell Swiss white mouse
embryos were cryopreserved using a slow freezing (0.3°CI
min) and rapid thawing (440°C/min) protocol with 1.5 M 1,2propanediol and 0.1 M sucrose as cryoprotectant. Data show
that the cryopreservation protocol did not affect the survival
rate of different embryonic stages [102/112 (91 %), 1431
167 (86%), 104/127 (82%) and 164/197 (83%) respectively].
However, development was affected by freezing. The I-cell
embryos showed a good survival rate but, because of the strain
of mice, these embryos arrested at the 2-cell stage, although
82 out of 102 (82%) reached the 2-cell stage. Out of 104 4cell embryos, 94 (90%) developed to expanded blastocyst
stage while only 59 out of 143 (41 %) in the 2-cell group and
130 out of 164 (79%) in the 8-cell group developed to this
stage. The rate of hatching was greater in 4-cell embryos [61
69 (10%), 39/94 (41%) and 44/130 (34%) for 2-,4- and 8cell embryos respectively]. Data show that the freeze-thaw
procedure used in this study did not affect the survival rate
at different embryonic stages but the long-term effects of
cryopreservation are stage dependent.
R-147. Effects of granulocyte-macrophage colonystimulating factor on incisional wound healing of diabetic
rats
Vural B., Canttirk N.Z., Esen N., Ozbay O. and Yticesoy I.
Kocaeli University, School of Medicine, Kocaeli, Turkey
Introduction: Wound healing is an important problem in
diabetics in whom granulocyte function is impaired. Granulocyte-macrophage colony-stimulating factor (GM-CSF) specifically stimulates granulocyte, macrophage and eosinophil
colonies. These cells have been implicated in the process of
wound healing.
Materials and methods: A total of 45 rats were divided into
three groups: controls (group I; n = 15) and two diabetic
groups (groups II and III; n = 15 for each group) induced by
streptozotocin. Each rat in the three groups had 4 cm paravertebral incisions made. Group III rats were given a s.c. injection
of GM-CSF (1000 ng) in PBS solution every 12 h for 2 days
before wound healing; injections were continued for 5 days
after wounding. Neutrophil count, neutrophil phagocytosis and
adherence index were determined in each group 1 day after
wounding. Animals were killed on day 14. Fresh breaking
strengths were recorded with a tensinometer. Hydroxyproline
concentrations in the wound tissue were measured. A histological analysis was performed by light and electron
microscopy.
Results: White blood cell counts, neutrophil counts and
neutrophil function decreased significantly in group II (P <
0.05). Systematic GM-CSF injections improved these parameters significantly in group III rats (P < 0.05). Histological
studies revealed an increase in wound cellulity at day 14 in
GM-CSF-treated wounds.
Conclusions: These results suggest that GM-CSF affects both
blood and tissue granulocyte count and granulocyte function.
We could not determine any positive effect of GM-CSF on
wound healing in diabetic rats.
General programme
R-148. Magnetic resonance diagnosis of an ectopic ovary
in an infertility clinic
Ombelet W.!, Grieten M. 2, Eerdekens C.! and Huysmans v.I
IGenk Institute for Fertility Technology and 2Department of
Radiology, ZOL Campus St.Jan, Schiepse Bos 6, 3600 Genk,
Belgium
A 34 year old male and a 35 year old female were referred to
our infertility centre for donor insemination. The couple had
been primary infertile for 3 years. A previous investigation of
the man had revealed a diagnosis of azoospermia without signs
of genital tract obstruction, with high serum FSH and small
bilateral testicular size. A genetic examination turned out to
be normal (46,XY). An infertility work-up of the female,
including an endocrine evaluation and a local and systemic
immunological evaluation, was normal. A hysterosalpingography showed a herni-uterus with only one patent tube. Subsequently, a laparoscopy and hysteroscopy were carried out,
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