TITLE: The Effects of ISM1 Medium on Embryo Quality
and Outcomes of IVF/ICSI Cycles

  ABSTRACT.BACKGROUND:
The aim of this study is to investigate the effect of ISM1 culture medium
on embryo development, quality and outcomes of in vitro fertilization/intracytoplasmic
sperm injection (IVF/ICSI) cycles. This study compares culture medium commonly used
in the laboratory setting for oocyte recovery and embryo development with a medium
from MediCult. We have assessed the effects of these media on embryo development and
newborn characteristics.

ABSTRACT.MATERIALS AND METHODS:
In this prospective randomized study, fertilized oocytes
from patients were randomly assigned to culture in ISM1 (MediCult, cycles:
n=293) or routine lab culture medium (G-1TM v5; Vitrolife, cycles: n=290) according
to the daily media schedule for oocyte retrieval. IVF or ICSI and embryo
transfer were performed with either MediCult media or routine lab media. Embryo
quality on days 2/3, cleavage, pregnancy and implantation rates, baby take home
rate (BTHR), in addition to the weight and length of newborns were compared
between groups.

ABSTRACT.RESULTS:
There were similar cleavage rates for ISM1 (86%) vs. G-1TM v5 (88%). We
observed a significantly higher percentage of excellent embryos in ISM1 (42.7%) compared
to G-1TM v5 (39%, p<0.05). Babies born after culture in ISM1 had both higher
birth weight (3.03 kg) and length (48.8 cm) compared to G-1TM v5 babies that had a birth
weight of 2.66 kg and a length of 46.0 cm (p<0.001 for both).

ABSTRACT.CONCLUSION:
This study suggests that ISM1 is a more effective culture medium in
generating higher quality embryos, which may be reflected in the characteristics of
babies at birth.

BODY.INTRODUCTION:
The outcome of an assisted reproduction treatment
(ART) cycle is highly dependent on numerous
factors including the clinic location, equipment
and disposables used (1), patient age (2, 3), stimulation
regimens (4), sperm selection (5), culture
condition (pH) (6), O2 (7), and group culture (8).
It is recommended that the clinic select a suitable culture system that repeatedly produces high quality
embryos for embryo transfer and cryo-preservation
with low miscarriage rates that results in the
delivery of a healthy baby of normal gestational
age (9, 10).  In vitro culture and development of human embryos
for days 2 or 3 transfers is performed in a
variety of different culture media that range from
simple to complex (11-13). Increased knowledge
in the specific requirements for the development of
healthy human pre-implantation embryos has resulted
in the formulation of new culture media (2,
14, 15) that supports the development of embryos
from fertilization to the fully expanded blastocyst
stage (2, 16-24).  These products are commercially available, their
quality is meticulously tested, and they are readyto-
use. Therefore the use of these high quality
products should significantly improve embryo
quality, cost-efficiency of the treatment, and the
clinic's reputation (12).  The purpose of this study was to compare
the efficiency of two different commercially
available media products (ISM1, MediCult,
Denmark vs. G-1TM v5, Vitrolife, Sweden) in
supporting the development of embryos (days
2 and 3), their ability to implant and deliver a
healthy baby at term.

BODY.MATERIALS AND METHODS.PATIENT POPULATION:

This prospective randomized study was performed
during a five month period (February-
June, 2009). During this period all culture
conditions, routines and disposables were kept
constant in the laboratory. On the day of oocyte
pick up, there were 583 patients randomized
to both groups according to a randomization
list based on sequential numbers in sealed envelopes.
The ISM1 group (experimental) consisted
of 293 patients compared to 290 patients
in the routine lab procedure group (control).
Depending on the causes of infertility, patients
received either standard in vitro fertilization
(IVF) or intracytoplasmic sperm injection
(ICSI). Excluded from the study were patients
with previous fertilization failure [19 (experimental)
vs. 8 (control)]; those with no oocytes
retrieved [1 (experimental) vs. 1 (control)]; and
patients diagnosed with ovarian hyperstimulation
hormone syndrome [OHSS; 22 (experimental)
vs. 18 (control)].  Inclusion criteria were counseling patients undergoing
ICSI treatment, between 18 to 40 years
of age who had a minimum of 2 follicles (≥18 mm)
at the time of oocyte collection.  Patients were randomly assigned to have their
fertilized oocytes cultured in ISM1 (MediCult,
cycles: n=293, written informed consent was
taken from all the couples who received this culture
medium) or routine lab procedure (G-1TM v5;
vitrolife, cycles: n=290) according to the daily
medium schedule for oocyte retrieval. This study
was approved by Ethical Committee of Royan
Institute.

BODY.MATERIALS AND METHODS.STIMULATION REGIMES AND OOCYTE RETRIEVAL TECHNIQUES:

Down regulation and ovarian stimulation were
performed in accordance with a previously reported
stimulation regimen (25). In brief, pituitary
gonadotropin secretion was suppressed with
the GnRH agonist buserelin acetate (Suprefact,
Hoechst AG, Germany) via subcutaneous (SC) injection
(500 mg/d) or by nasal spray (800 mg/d)
starting in the mid luteal phase of the preceding
ovarian cycle (day 21).  Once ovarian suppression was confirmed (serum
E2 ≤50 pg, FSH ≤12 IU and LH ≤5 IU), ovarian
stimulation was initiated with recombinant FSH
(Gonal F; SC injection, 150 IU/day, Serono, Switzerland).
The dose was adjusted according to the
response of the ovarian follicular development
and monitored by serial vaginal ultrasonography.
When at least three follicles reached 18 mm in
diameter, both GnRH agonist and hMG were discontinued
and a single dose of hCG (10000 IU;
Pregnyl; Organon, Netherlands) was administered.  Oocyte retrieval was performed via vaginal ultrasound-
guided follicle aspiration, 36-38 hours
after administration of hCG.

BODY.MATERIALS AND METHODS.INTRACYTOPLASMIC SPERM INJECTION (ICSI):
After egg collection, the cumulus-oocyte complexes were either placed in 50 μL droplets
of Universal IVF medium and covered with
liquid paraffin (MediCult, Denmark) as the
experimental group or in 50 μL droplets of
Ham's F-10, (Biochrom AG; Berlin, Germany),
which had been supplemented with 10%
recombinant human serum albumin (rHA, Vitrolife)
and covered with pre-incubated mineral
oil (Sigma, St. Louis, MO), as the control
group. Oocyte denudation was performed
with ICSI Cumulase (MediCult) in the experimental
group or with 80 IU of hyaluronidase,
(Sigma, USA) in the control group, two hours
after egg collection. Sperms were immobilized
in 10% PVP droplets (MediCult) in the experimental
group and in10% PVP droplets (Global)
in the control group by breaking the tail by
an injection pipette. Then, a single sperm with
head front was injected into each oocyte that
had a visible first polar body (MII) (26, 27).
All semen samples were prepared by swim-up
method.

BODY.MATERIALS AND METHODS.CONTROL OF FERTILIZATION AND EMBRYO CULTURE:

The fertilization rate was controlled 18-20
hours after sperm injection. Normally fertilized
oocytes that had evidence of two pronuclei
were washed and transferred to overnight
pre-incubated in 20 μl droplets of ISM1 or G-
1TM v5 supplemented with 10% recombinant
human serum albumin in the experimental and
control groups respectivly and covered with
either pre-incubated liquid paraffin (experimental
group) or mineral oil (control group).
All cultures incubated at 37 ̊C, in 5% O2 and
6% CO2.

BODY.MATERIALS AND METHODS.EMBRYO SCORING AND TRANSFER TECHNIQUES:

On the day of embryo transfer (44-72 hours after
injection), we scored embryo morphology according
to the following quality criteria:  ExcellentDay 2: 2-4 even size blastomeres with ≤10% fragmentationDay 3: 6-8 even size blastomeres with ≤10% fragmentationGoodDay 2: 2-4 even or uneven size blastomeres with
10%-20% fragmentationDay 3: 6-8 even or uneven size blastomeres with
10%-20% fragmentationPoorUneven and few blastomeres with >20% fragmentation  Based upon the embryo quality, female age and
number of previous cycles, we selected a maximum
of four high quality embryos and incubated
them in UTM (MediCult) for the experimental
group or Embryo Glue (Vitrolife) for the control
group, for a period of 20-120 minutes before embryo
transfer.  All embryo transfers were performed with a
Labotect catheter (Labotect, Germany) against a
filled urine bladder with the expertise of gynecologists
and embryologists.

BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS:

We used the Statistical Package for the Social
Sciences (SPSS 11.5; Chicago, IL, USA, http://
www.spss.com) software to analyze differences
amongst the variables of both groups by Chisquare
and Fisher's exact tests for categorical
variables or the student's t test for continuous
variables, as appropriate. A p value <0.05 was
considered to be statistically significant.

BODY.RESULTS:
Patient characteristics of the two groups are
summarized in table 1. The number of cycles,
mean female age, indications for infertility, number
of previous treatment cycles, retrieved oocytes
and percentage of cleaved embryos were similar in
both groups.  There were no significant differences between
the groups in terms of cleavage rates, number
of embryos transferred, clinical pregnancy and
implantation rates and multiple pregnancies.
There were a significantly higher percentage
of excellent embryos in the experimental group
(42.7%; ISM1) compared to the control group
(39%, p<0.05). There was also a trend towards
higher clinical and multiple pregnancy rates as
well as implantation rates (Table 2) for the experimental
group.  In terms of abortion, the experimental group (20.5%) was not significantly different from the
control group (21.1%). There was no significant
difference in baby take home rates (BTHR) between
both groups, however both the length [48.8
cm (experimental) vs. 46.0 cm (control)] and weight
[3.03 kg (experimental) vs. 2.66 kg (control)] of babies
born were significantly higher in the expreimental
group (p<0.001 for both parameters).   Table 1 Patients’ ART cycle characteristics in the MediCult (experimental) and lab routine media (control) Parameters Experimental group Control group P value No. of cycles 293 290 0.37 Female age (Y) (mean ± SD) 32.4 ± 5.7 31.8 ± 5.6 0.18 Male factor infertility 170/293 (58%) 170/290 (58.6%) 0.88 Female factor infertility 66/293 (22.5%) 62/290 (21.4%)  0.73  Mixed infertility 44/293 (15%) 46/290 (16%) 0.77 Unexplained infertility 13/293 (4.4%)  12/290 (4.1%)  0.83 Previous ART cycles (mean ± SD) 1.7 ± 1.2 1.8 ± 1.0 0.21  No. of retrieved oocytes (mean ± SD) 9.1 ± 5.6 8.8 ± 5.1 0.52   Table 2 Fertilization, cleaved embryo, embryo quality and clinical pregnancy in the MediCult (experimental) and lab routine
media (control) groups Parameters Experimental group Control group *P value Cleavage rate 86% (1471/1706) 88% (1455/1678) 0.68 Embryo quality Excellent 43% (628) 39% (567) 0.04a Good 39% (580) 41% (599) 0.33 Poor 18% (261) 20% (288) 0.15 No. of transferred embryos (mean ± SD) 2.4 ± 0.8 2.4 ± 0.8 0.83 Clinical pregnancy rate/embryo transfer 32.1% (94/293) 27.6% (80/290) 0.23 Implantation rate 15% (102/697) 12% (84/693) 0.16 Multiple pregnancy rate 8.5% (8/94) 3.8% (3/80) 0.19 Single 91.5% (86/94) 96.2% (77/80) 0.19 Twin 8.5% (8/94) 2.5% (2/80) 0.09 Triplet 0% (0/94) 1.3% (1/80) 0.46 a; Statistically significant differences between the two groups and *; Fisher’s exact test.   Table 3 Clinical outcome, abortion and take-home baby rate in the MediCult (experimental) and lab routine media (control) groups Parameters Experimental group Control group *P value Abortion rate 20.5% (18/88) 21.1% (16/76) 0.9 First trimester  77.8% (14/18) 87.5% (14/16) 0.58 Second trimester   16.7% (3/18)  12.5% (2/16) 0.48 Third trimester  5.5% (1/18) 0% (0/16) 0.35 Baby take home rate (BTHR)  84.3% (86/102)  80.4% (78/97) 0.57 Multiple pregnancy rate 8.5% (8/94) 3.8% (3/80)  0.19 Single  78.9% (56/71)   66.7% (40/60) 0.1 Twin  21.1% (15/71)  31.7% (19/60) 0.1 Triplet 0% (0/71) 1.7% (1/60)  0.1 Sex Male 44.1% (38/86) 51.3% (40/78) 0.3 Female 55.8% (48/86) 48.7% (38/78) 0.3 Length (cm, mean ± SD) 48.8 ± 0.52 46.0 ± 0.66 0.001 a Weight (kg, mean ± D) 3.03 ± 0.07 2.66 ± 0.08 0.001 a a; Statistically significant differences between the two groups and *; t test. Of all pregnant patients, 10 (n=6 for expreimental group and n=4 for control group) patients were not available and were
excluded from table 3. 

BODY.DISCUSSION:
There are a variety of different in vitro culture
systems available for laboratory procedures applied
to gamete maintenance and embryonic
growth. These media products range from simple
solutions to more complex culture media systems,
which have been adapted to the changes that the
developing embryos face during their passage
through the female reproductive tract (11-13).
Many recent discoveries have been fundamental
in describing different biochemical, physiologic,
genetic, and epigenetic characteristics of the embryo,
resulting in recent innovations in this field.
Culture media formulations are presumed to be of
essential importance for normal development of
human embryos in the controlled environment of
an incubatorin addition to their effects on developing
embryos (22, 23, 28).  A general agreement does not exist regarding
the efficacy of different commercially available
in vitro culture media, since some studies have
reported no differences in embryo quality, pregnancy
and implantation rates (18, 21, 29), whereas
others have observed subtle differences in day 3
embryo quality [G1.2 vs. Sydney IVF sequential
media (16), G1.2 vs. HTF (20) and GIII series versus
ISM1(30)].  Several studies have investigated the effect of
ISM1 and G-1TM v5 media on embryo quality and
implantation rate. Embryo morphology on days 2
and 3 significantly enhanced when the embryos
were cultured in GIII series versus ISM1 (18).
Another study showed that "in a day 3 embryo
transfer program, G1.2/G2.2 media were superior
to Sydney IVF media, whereas both media yielded
similar outcomes in blastocyst transfer program"
(20).  A prospective study on sibling oocytes showed
that embryo quality in the day 2 stage improved
under ISM1 culture rather than FertiCult culture
medium, even if data regarding pregnancy and
implantation rate were not reported (31). Another
study demonstrated that ISM1 culture medium
seemed to improve the performance of embryonic growth and development, as well as increased the
percentage of pregnancies (32).  For this reason, we conducted a prospective randomized
trial at Royan Institute to compare embryo
development parameters, IVF outcome and
BTHR at the end of the gestational period between
ISM1 and G-1TM v5 media, which is used routinely
in our laboratory.  The result of the study suggests that both ISM1
and G-1TM v5 support embryo development in vitro,
whereas culture in ISM1 gives a higher proportion
of excellent quality embryos.  Good embryo quality, cleavage rate, and single,
triplet pregnancy rate in the control group were
higher than ISM1 medium, but there were no significant
differences. In ISM1, the implantation,
clinical pregnancy and twin pregnancy rates were
higher than the G-1TM v5 medium. However, this
difference was not significant. The percentage of
excellent embryos was significantly higher than
the control group which might be reflected in the
characteristics of babies born. Babies born after
culture in ISM1 had both significantly higher birth
weight and were also longer compared to the control
group, both of which were significant.  In vivo as well as in vitro fundamental factors for
proper embryonic development could be summarized
as oxygen consumption, the balance between
reactive oxygen species and antioxidant defense,
energetic metabolites (Na+, K+, adenosine triphosphatase),
and amino acid turnover. The oxygen
requirement appears constant over the course of
the early embryonic development phases up to the
blastocyst stage, and later increases (33).  Although in vivo physiologic reactive oxygen
species production is controlled continuously by
a strong cellular defense, the lack of this system
during the in vitro culture could cause oxidation
of proteins, lipids, DNA, and the misregulation
of metabolic pathways that may lead to cell death
(34-36). On the basis of these observations, it can
be speculated that the antioxidant elements present
in ISM1 culture medium sustain the cultured
embryos, thereby avoiding developmental failure
and also achieving the best quality embryos
for implantation (32). The result of this study has
demonstrated that ISM1 medium improved embryo
development. The percentage of excellent
embryos was significantly higher in ISM1 medium
compared with the control group (G-1TM v5). This
result was possibly due to the antioxidant elements
present in ISM1 culture medium.  Singleton children born after IVF have a significantly
lower birth weight compared with their
spontaneously conceived peers (37-39), even after
adjustments for maternal age and parity. This
phenomenon has produced concern, because low
birth weight is associated with morbidity over both
the short and long term (40). Speculations about
possible causes, including epigenetic alterations
by ovarian stimulation, have been published (41).
Theoretically the lower birth weight in IVF singletons
could be attributed to i. maternal and/or
paternal characteristics associated with infertility;
ii. the effects of ovarian stimulation on oocytes, endometrium
or the endocrinology of the luteal phase
or early pregnancy; or iii. IVF laboratory procedures
such as ICSI or embryo culture conditions (42, 43).
The implementation of suitable quality control has
focused on the effectiveness of culture media in improving
embryo morphology and it is fundamental to
the success of an IVF laboratory (44).  "The possible interference of assisted reproduction
techniques (ART) with epigenetic reprogramming
during early embryo development has recently
sparked renewed interest about the reported
lower birth weight among infants born as a consequence
of infertility treatments" (45).  Both the shorter duration of pregnancy and the
reduced neonatal birth weight could be caused by
inactivation of paternal alleles during the imprinting
process. If the shorter duration of pregnancy
and the lower birth weight after IVF and ICSI can
be attributed to a distinct defect of epigenetic phenomena
at work during parental imprinting, then
this defect may have later effects on the individual
(46, 47) and has the potential to be transferred to
other generations (48).  ISM1 culture medium was formulated to promote
correct imprinting for ensuring correct development
and implantation (32). As demonstrated
by Cassuto et al. (49), different timing and culture
conditions can have different effects on embryo
development and implantation rate. Extended culture
time and particular culture conditions result in
an increase in monozygotic twinning. In this study
the use of only ISM1/ISM2 culture conditions resulted
in lower monozygotic twin pregnancies, independent of the technology used (IVF/ICSI)
(49). The result of our study demonstrated that
babies born after culture in ISM1 had both higher
birth weight and were also longer than those in
control group. This result might be due to correct
imprinting that ensured correct development and
implantation.

BODY.CONCLUSION:
This study suggests that on the one hand ISM1
contains antioxidant elements and on the other
hand it is formulated to promote correct imprinting
to ensure correct development and implantation.
ISM1 is presumed to be a more effective culture
medium that generates higher quality embryos,
which is reflected in the characteristics of babies
born. Since numerous factors that include mother's
diabetes and multiple pregnancies, among others
may also impact the height and the weight of the
newborn, we cannot directly relate the change in
these two characteristics solely to the culture media.
For reaching a definitive conclusion, additional
studies that consider these factors are warranted.

Question: Compared to routine lab culture medium (G-1TM v5; Vitrolife, what was the result of culture in ISM1 (MediCult) on percentage of excellent embryos?
Evidence: There were a significantly higher percentage
of excellent embryos in the experimental group
(42.7%; ISM1) compared to the control group
(39%, p<0.05). 
Answer: significantly increased