Volume 12 - IVFOnline.com

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VOLUME 12

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CONTENTS
Fertility Magazine
The First Magazine In Fertility TM
FEATURED ON THE COVER
Belgian legislation regarding IVF
by Guy De Groote, MD, Frank Vandekerckhove, MD . . . . . . . . . . . . .9
54
Successful vitrification in a closed carrier device of blastocysts
originating from infertile patients, egg donors, or in-vitro
maturation by J Lopez, NH Zech, P Frias, P Vanderzwalmen . . . . .40
Automated Robotic Human ICSI
by Navid Esfandiari, DVM, PhD, HCLD, Zhe Lu, PhD, Xuping
Zhang, PhD, Robert F. Casper, MD, Yu Sun, PhD . . . . . . . . . . . . . . .46
Improving air quality in ART laboratories by Giles Palmer . . . . . .54
Too Much of a Good Thing by Courtney Sirotin . . . . . . . . . . . . . . . .78
Improving air quality
in ART laboratories
FEATURES
12 Efficiency of human oocyte slow freezing: results from five assisted reproduction centres
by L Parmegiani, F Bertocci, C Garello, MC Salvarani, G Tambuscio, R Fabbri
22 Effect of growth hormone on oocyte competence in patients with multiple IVF failures
by A Hazout, AM Junca, Y Ménézo, J de Mouzon, P Cohen-Bacrie
32 S3 Vitrification System: a Novel Approach to Blastocyst Freezing by
James J. Stachecki, PhD, Jacques Cohen, PhD
44 Life-long impact of the first cell cycle by Dmitri Dozortsev, MD, PhD
49 An Alternate Method For Shipping Sperm: Aerogel Containers Fitted
With Data Loggers by Lakshmi Sharma, MSc, MPhil, ELD,
Susan Tarchala, MS, TS, Jon Nakagawa, CPA, John Perry, BS, MBA,
Richard Rawlins, PhD, HCLD
44
Life-long impact
of the first cell
4 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

52 Epigenetics and the ART Lab by Sangita Jindal, PhD, HCLD
58 The use of birefringence technology in assisted human reproduction
by Simon J Phillips, MSc, BSc
64 The identification of a toxic substance in the in vitro fertilization laboratory: the value of interlaboratory
communication by Tom Turner, MS ELD (ABB)
66 Clinical Implementation of the Halosperm ® Test Kit in Combination with SCA ®
by Kellie Williams, PhD and J. Kevin Thibodeaux, PhD, HCLD
69 A Prospective Randomized Comparison of global ® Medium with Sequential Media for
Culture of Human Embryos to the Blastocyst Stage by Don Rieger, PhD
72 “Morning Sickness” – An overview by Michele Brown, MD, FACOG
75 Vitamin D Deficiency and Pregnancy by Michele Brown, MD, FACOG
84 New Products
86 Books
87 Resources
92 Conferences
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WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
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INTERNATIONAL NEWS
Belgian legislation regarding IVF
by Guy De Groote, MD, Lab Director, CRI Laboratories, Gent, Belgium
Frank Vandekerckhove, MD, gynaecologist, Fertility Center, Ghent University, Gent, Belgium
In-vitro fertilization (IVF) units were first established in
Belgium in the 1980’s as private initiatives of interested
clinicians and laboratory directors, both in hospitals
and in private institutions. We all had the intention to help
couples with fertility problems using this exciting new
procedure. As the number of treatments grew and the
visibility of the new technique increased, IVF units also
became more professional, incorporating quality
standards. Almost as a side effect, they became subject to
new legislation.
The original legislation (1999) was not concerned with
medical practice or quality. Rather, it was intended to limit
the number of centers and the health care expenditure
related to the new treatment. The first quality standards
were suggested by the profession itself (e.g. the Flemish
Society of Clinical Embryologists, VVKE), anticipating
legislation.
As the possible applications of IVF related techniques
became clear, a public ethical and philosophical debate
arose. Health care workers in the field, together with local
ethical committees, had to cope with many difficult issues,
including age limits for patients and donors, a growing
number of spare embryos, the use of embryos for stem cell
research, sex selection, cloning, and remuneration of
donors. Of course, the decisions were often influenced by
pressure from the patient couple. The legislation on
ethical issues (2003 and 2007) provided answers to these
important questions, adding some strict rules, and partly
limiting the possibilities for health care and research.
Subsequent legislation also included quality
requirements. An important issue was single embryo
transfer (2003) in order to limit the health problems and
expenditure created by the increasing number of twins
and triplets.
The law of 2008 put IVF and intrauterine insemination
together with stem cell applications in a general legal
framework for human tissue banking. This law
introduced stringent quality requirements, established
control procedures by the national drug administration,
and incorporated the existing European directives.
In the following sections, I will try to summarize the
most important laws and royal decrees (RD) involved. For
full details the interested reader is referred to the original
publications in the Official Journal available through
http://www.just.fgov.be.
Guy De Groote, MD
Frank Vandekerckhove, MD
1. Creation of centers for reproductive medicine (RDs
Feb. 15, 1999)
• Incorporation of IVF units in centers for
reproductive medicine
• Limitation of the number of IVF centers
• Exclusion of extramural IVF centers
• Mandatory registration
This first legislation limited the (growing) number of
IVF centers and thus the health expenditure related to it.
This legislation incorporated the existing IVF units into
a limited number of newly-created hospital-based centers
for reproductive medicine. So-called type-B centers for
reproductive medicine were allowed to perform all IVF
related activities, whereas the activity in type-A centers was
mostly limited to procedures up to the stage of oocyte
pickup. For embryo culture, freezing, and transfer, type-A
centers must transfer the ova to a type-B center. Therefore,
every type-A center must have a formal agreement with a
type-B center.
The number of both types of IVF centers was limited;
two type-B IVF centers are allowed per province (1
university-based, 1 other), and one type-A center was
allowed per 700,000 inhabitants. Belgium consists of 10
provinces and has a population of approximately 10 million
(2009) inhabitants. Today, there are 18 type-B, and 15 type-A,
registered IVF centers. Their activity in the last published
CONTINUED ON PAGE 10
WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
9

INTERNATIONAL NEWS
CONTINUED FROM PAGE 9
report (data from 2007) totaled 26,836 cycles, of which 18,025
(67%) involved fresh transfers and 7,197 (27%) involved
transfer of frozen embryos.
This RD introduced obligatory multidisciplinary
cooperation, including psychological and social assistance,
surgery, andrology, and echography. The requirements for
Type-B type centers also included microsurgery and
reproductive endocrinology, and cooperation with a center
for medical genetics. Registration of data and participation in
a quality assessment program became obligatory. To this
end, participation in the Belgian Register for Assisted
Procreation (www.belrap.be) became mandatory for IVF
centers, ten years after this register had been started (1989).
2. Law on embryo research in vitro (Law May 11, 2003)
• Research requires agreement with university
based center for reproductive medicine and
specific approvals
• Embryo research allowed up to day 14 of
development
• Strictly forbids: sex selection, eugenics,
reproductive cloning, commercial use of embryos
According to this law embryo research is allowed on
embryos up to day 14 of development (disregarding a
possible freezing period) if needed for therapeutic
purposes or research contributing to better knowledge in
human (in)fertility, transplantation or diseases. This
research must be linked with one of the university-based
centers for reproductive medicine or medical genetic
departments, is subject to approval by an ethical
committee, and is controlled by a federal commission for
embryo research.
The defined penalties are severe, including large
monetary penalties and/or detention up to 5 years and/or
the prohibition from performing any medical activity or
research for 5 years.
This law clearly forbids reproductive human cloning,
implantation of human embryos into animals (some
limited exceptions), any commercial use or application for
eugenics or sex selection (exception for sex linked
diseases).
3. Reimbursement and single embryo transfer (RD
June 4, 2003)
• Reimbursement for IVF laboratory work
(maximum of. 6 cycles, limit of. 42 years of age)
• Single embryo transfer mandatory in first and (if
good quality) second cycles
This royal decree introduced reimbursement by the
national health insurance system (covering more than 95
% of the population) of laboratory procedures for IVF,
under specific conditions of patient age and the number of
embryos transferred.
For women up to 35 years of age: single embryo
transfer mandatory in the first treatment cycle. In the
second cycle one or two ( depending on quality) embryos
can be replaced. In the third and subsequent fresh cycles,
or in any frozen embryo transfer cycles, a maximum 2
embryos may be replaced. For older women up to 39 years
of age, a maximum 2 embryos can be transferred in the
first or second cycle and 3 in following cycles. For women
older than 39 years of age, no maximum number is
applied. The reimbursement is only applied for women
up to 42 years of age, and for a maximum of 6 IVF
treatment cycles.
This reimbursement (1182 EUR) covers all laboratory
expenses for regular IVF procedures (including germ cell
selection, insemination, embryo culture and evaluation,
and cryopreservation) and must be applied for all treated
patients covered by the Belgian national health insurance
system.
Later RD’s introduced a reimbursement for
gonadotrophin treatment (RD's of Sept 14 and 15, 2006,
RD of Oct 6, 2008, RD Dec 17, 2008), limited to 6 IVF cycles
and 6 IUI or other cycles each for (clomiphene resistant)
patients less than 43 years of age. A RD of July 2, 2008
included reimbursement for intrauterine or intracervical
insemination, including an obligatory registration.
4. Fate of supernumerary embryos and gametes (Law
of Jul 6, 2007)
• IVF, gamete and embryo freezing limited to
centers for reproductive medicine
• Maximum age limit to 45 years (pick-up) and to
47 years of age (transfer)
• Decision on the fate of supernumerary embryo’s:
personal use, donation or research
• Donation of gametes or embryos allowed, limited
to 6 successful recipients
• Preimplantation diagnostics on embryos allowed
• Strictly forbidden: eugenics or sex selection
(except for sex linked diseases)
• Embryo matching allowed
This law set a clear age limit of 45 years for oocyte
recovery and 47 years of age for embryo transfer. If a
woman has her own good-quality frozen embryo’s
10 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

INTERNATIONAL NEWS
available, then these must be used first before proceeding
to a next fresh cycle. Oocyte recovery from under-age girls
is permitted if required for medical reasons.
This law includes details on the information to be
provided to the patient, psychological support, and the
contract to be signed before proceeding. The last should
include the patient’s decision on the destination of
supernumerary embryos: personal use, destruction,
research, or donation. The contract also stipulates the fate
of frozen embryos in case of death or divorce. If the couple
decides for personal use by the surviving partner, then
these embryos can only be used for this purpose in the
period form 6 months to 2 years after the death of the first
partner. Gametes (oocytes, sperm or gonadal fragments)
may be collected for personal use, donation or research. A
similar contract is required.
The normal storage period for frozen embryos for
personal use is 5 years, for gametes 10 years (extension
possible). Embryo donation for eugenic purposes or sex
selection is not allowed. Embryos or gametes from one
donor may be used to produce a pregnancy in a maximum
of 6 recipients. Anonymity is mandatory and this law
absolutely excludes right of inheritance between donor
and receiving family or child. The commercial use of
gametes or embryos is not allowed.
Preimplantation diagnostics is allowed except for
eugenic purposes or sex selection (except in case of sex
linked hereditary diseases). The number of centers for
preimplantation diagnostics can be limited, but should not
be less than 8. Matching of embryos is allowed.
As for the law on embryo research severe penalties are
applicable. An official organization for control and
inspections is established (Law of Jul 24, 2008, RD Dec. 17,
2008 and Sept 20, 2009).
5. IVF and stem cell research included in a general law
on tissue banking for human application or
scientific research. (Law Dec 19, 2008 and Dec 23,
2009, several RD's Sep 28, 2009, MD Oct 14, 2009)
• Creation of tissue banks and related structures,
mostly limited to hospital environment
• Application: all aspects (extraction, storage,
processing, distribution and use) of stem cell
banking, reproductive applications (including
IVF, intra-uterine insemination, donations), any
human tissue materials for human application or
scientific research
• Detailed quality requirements, detailed
registration requirements, communication of
adverse events and non conformities to the
authorities
This extensive law and related decrees introduced the
structures for tissue banking in detail: obtaining,
extraction, processing, storage, distribution and use of any
human material or tissues for human therapeutic
application or scientific research. The application is very
large, including any kind of tissue and cells (i.e. any
collection of human cells that is not linked by fibrous
tissue), IVF, and every application of stem cells. Excluded
are organ transplantation and blood transfusion (both
subject of a separate legislation), immediate autologous
treatment (without processing) and direct diagnostic use
of the material for the person involved. Some tissues (hair,
nails, urine, feces, sweat, tears, and mother milk) are
excluded from this law.
This law established three different categories of
institutions: tissue banks for human body materials,
intermediary structures, and production units. The centers
for reproductive medicine are considered to be tissue
banks and remain the only structures allowed to deal with
gametes or embryos. Tissue banks must always be situated
in a hospital.
Part of the activity can be done in intermediary
structures (processing, storage, distribution), or in a
production unit. These should have a contractual link with
a tissue bank. If they are recognized as an intermediary
structure, then medical laboratories can perform
capacitation of sperm for partner donation.
Tissue banking without a preventive, therapeutic or
diagnostic purpose, or lacking a relevant scientific target
(as confirmed by the opinion of an ethics committee) is not
allowed.
Several royal decrees provided several additions to the
law: detailing quality requirements, infrastructure, cleanroom
specifications (including EU Grade A working
environment and Grade D background environment),
quality assurance, donor selection criteria, safety,
exclusion of risk of virus transmission, full traceability,
registration (data should be kept for 30 years), mandatory
communication of serious adverse effects, and nonconformities.
Approval of a tissue bank is for maximum 4 years with
a extensive inspection at least every 2 years. Control and
inspection organizations (mostly the federal drug
administration FAGG/AFMPS) are nominated.
You can contact Guy De Groote at: gdg@cri.be
WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
11

INTERNATIONAL NEWS
Efficiency of human oocyte slow freezing: results
from five assisted reproduction centres
L Parmegiani 1,7 , F Bertocci 2 , C Garello 3 , MC Salvarani 4 , G Tambuscio 5 , R Fabbri 6
1 Reproductive Medicine Unit, GynePro Medical Centres, Bologna, Italy; 2 Chianciano Salute, Centre for Reproductive Health,
Chianciano Terme (SI), Italy; 3 Livet Clinic, Turin, Italy; 4 Centre for Reproductive Medicine, Department of Obstetrics, Gynecology
and Neonatology, University of Parma, Parma, Italy; 5 Department of Gynecological Science and Reproductive Medicine, University
of Padua School of Medicine, Padua, Italy; 6 Human Reproductive Medicine Unit, University of Bologna, Bologna, Italy
7 Correspondence: e-mail: l.parmegiani@gynepro.it
c 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Dr. Lodovico Parmegiani, 2009 Efficiency of human oocyte slow freezing: results from five assisted reproduction centres. Reprod
BioMed Online, 18, 3, 352-359.
Lodovico Parmegiani obtained his degree in Biology in 1996 from the University of
Bologna and his specialization in Biochemistry and Clinical Chemistry in 2000 from
the University of Modena and Reggio Emilia. He trained as a clinical embryologist
at the Reproductive Endocrinology Centre, St Orsola Hospital, Bologna and
received a research scholarship (2001–2007) from the University of Bologna. Since
2002 he has been Laboratory Director, Reproductive Medicine Unit–GynePro
Medical Centres, Bologna. In 2008 he received certification as Senior Clinical
Embryologist from the European Society for Human Reproduction & Embryology.
His current research interests are cryobiology, gamete selection and
micromanipulation.
DR LODOVICO PARMEGIANI
Abstract
It has been demonstrated previously that freezing oocytes within 2 h of retrieval increases the efficiency of cryopreservation via
a slow-freezing/rapid-thawing protocol with 0.3 mol/l sucrose (SF/RT 0.3). The aim of this multicentre survey was to verify this
observation on a larger scale. This was a retrospective study on the clinical outcome of 510 SF/RT 0.3 cycles divided into two
groups: group A, freezing oocytes within 2 h of retrieval; group B, freezing oocytes more than 2 h after retrieval. The rate of bestquality
embryos was significantly higher (33.24%) in group A than in group B (16.20%, P < 0.001). Pregnancy and implantation
rates were 30.07% and 15.08% in group A versus 8.97% and 4.57% in group B (P < 0.001). Clinical pregnancy rates per thawed
and per injected oocyte in group A were 5.53% and 10.41%, versus 1.46% and 2.77% in group B (P < 0.001). The overall yield
from oocytes cryopreserved within 2 h of retrieval (group A) was 6.49 implantations per 100 oocytes thawed versus 1.74 for group
B (P < 0.001). Embryo quality, pregnancy and implantation rates, and clinical efficiency of thawing cycles were all significantly
improved when cryopreservation was carried out within 2 h of oocyte retrieval.
Keywords: human, oocyte cryopreservation, slow freezing, timing of ICSI, 0.3 mol/l sucrose concentration
Introduction
Oocyte cryopreservation can be used in conjunction
with conventional IVF, representing, as it does, an
alternative which circumvents many of the ethical issues
associated with embryo cryopreservation. Oocyte freezing
also allows extension or preservation of fertility in women
who intend to delay motherhood for family planning
reasons or in those at risk of losing their gonadal function.
While in Italy the adoption of oocyte cryopreservation as
a clinical tool represents the only alternative to embryo
cryopreservation, which is forbidden by the national IVF
12 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

INTERNATIONAL NEWS
law (Benagiano and Gianaroli, 2004), in other situations,
‘egg cryo-banking’ could represent a more efficient
approach in egg donor–recipient treatment. The early
successes using human cryopreserved oocytes were
reported 20 years ago (Chen, 1986; Al-Hasani et al., 1987)
but for a long period this technology was not investigated
further, being perceived as inefficient and unsafe. The
publication of later studies optimizing various
cryopreservation techniques (Gook et al., 1993, 1994,
1995a,b; Porcu et al., 1997; Kuleshova et al., 1999; Fabbri et
al., 2001; Kuwayama et al., 2005) opened new perspectives
on oocyte cryopreservation. Nowadays, oocyte
cryopreservation is seeing increasing clinical application
worldwide. Some authors maintain that oocyte
cryopreservation needs further studies on safety and
efficiency. Furthermore, the Practice Committee of
American Society for Reproductive Medicine (2006) has
stated that this technique should be considered as
experimental and that it should not be offered as a means
to defer reproductive ageing.
Most studies on human oocyte cryopreservation have
used the slow-cooling–computer-controlled protocol
(slow-freezing/rapid-thawing), which is probably the
main oocyte freezing method adopted in the majority of
assisted reproduction centres. The results of oocyte
cryopreservation using the slow freezing/rapid thawing
(SF/RT) protocol with 1,2-propanediol (1,2-PROH) and
high sucrose concentration (0.2 or 0.3 mol/l) as
cryoprotectants have shown a gradual improvement in
efficiency over time, with live birth rates per transfer
increasing during recent years (Jain and Paulson, 2006).
Furthermore, it has been demonstrated that the slowcooling
technique with high sucrose concentration allows
safe long-term oocyte cryopreservation (Yang et al., 2007;
Parmegiani et al., 2008b).
Oocyte freezing can be carried out up to several hours
after retrieval; following thawing, the oocytes are cultured
for a few hours before insemination to better evaluate the
survival after the freezing/thawing procedure (Gook et al.,
1994) and to allow the temperature-sensitive meiotic
spindle to fully restore (Rienzi et al., 2004; Bianchi et al.,
2005). The timing of intracytoplasmic sperm injection
(ICSI) is a critical factor determining embryo viability and
implantation as the developmental capacity of the oocyte
declines some hours after oocyte retrieval (Yanagida et al.,
1998). The optimal timing for insemination of fresh
oocytes seems to be 37–41 h after human chorionic
gonadotrophin (HCG) administration to trigger ovulation
(Dozortsev et al., 2004). Similarly, metabolic ageing at ICSI
of slow-cooled oocytes depends on the time of retrieval
after HCG administration and on pre-incubation, but also
on the post-thawing culture before insemination.
Furthermore, it seems possible that the freezing procedure
could influence oocyte ageing (Parmegiani et al., 2008a).
In a previous study, it was demonstrated that freezing
within 2 h from oocyte retrieval increases the efficiency of
oocyte cryopreservation when using a slow freezing/rapid
thawing protocol with 0.3 mol/l sucrose (SF/RT 0.3)
(Parmegiani et al., 2008a). The aim of the present multicentre
survey was to verify this result on a larger number
of oocyte thawing cycles and to definitively establish the
ideal time after oocyte retrieval for slow-cooling
cryopreservation.
Materials and methods
Study population
Five assisted reproduction centres in Italy were
involved in this retrospective survey: three private centres
(GynePro Medical Centres, Bologna; Livet Clinic, Turin;
and Chianciano Salute, Chianciano Terme) and two
University-based centres (Department of Gynecological
Science and Reproductive Medicine, University of Padua;
and Centre for Reproductive Medicine, University of
Parma). In Italy, the insemination of more than three
gametes at one time is prohibited, while cryopreservation
of surplus oocytes is allowed by the Italian law 40/2004
that regulates assisted reproductive technology
(Benagiano and Gianaroli, 2004). All the patients
undergoing an IVF treatment in these five assisted
reproductive centres were included in a salvage oocyte
cryopreservation programme by being offered the
opportunity to have their surplus oocytes cryopreserved.
The retrospective survey was carried out on 510 oocytethawing
cycles performed between March 2004 and
March 2008. The oocytes from 424 patients were
cryopreserved; the mean age ± SE of the patients at the
time of oocyte retrieval was 34.46 ± 0.17 years. All the
women included in this study were informed about the
procedure and written consent was obtained from each
one. The procedures were approved by the Institutional
Review Boards of each centre. Oocyte cryopreservation
was usually carried out from 1 h up to several hours after
oocyte retrieval. There was retrospective observation of
the influence of freezing within 2 h or more than 2 h from
oocyte retrieval on the outcome of the oocyte-thawing
cycles. To this aim, the thawing cycles were divided into
two groups: group A, oocytes were frozen within 2 h of
retrieval; group B, oocytes were frozen more than 2 h after
retrieval.
CONTINUED ON PAGE 14
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13

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CONTINUED FROM PAGE 13
Ovarian stimulation, oocyte retrieval and selection
Ovarian stimulation was achieved using
gonadotrophin-releasing hormone analogues in
combination with a graded gonadotrophin administration.
Transvaginal ultrasound-guided oocyte retrieval was
performed 35.82 ± 0.02 h (mean ± SE; range 35–36 h) after
ovulation induction with either 5000 or 10,000 IU of HCG,
depending on the procedures of each assisted
reproduction. After retrieval, oocytes were cultured for
between 1 and 7 h at 37°C in an atmosphere of either 5% or
6% CO2 before the complete removal of cumulus mass and
corona cells by enzymatic digestion of hyaluronidase, and
by gentle mechanic aspiration with plastic denuding
pipettes. The denuded oocytes were then evaluated to
assess their nuclear maturation stage. The oocytes that had
released the first polar body [metaphase II (MII)]
underwent a strict selection by morphological features
(zona pellucida thickness, perivitelline space size, oocyte
shape, cytoplasm colour and granularity, presence of
vacuoles and first polar body morphology) under an
inverted microscope with Hoffman modulation contrast.
The oocytes classified as ‘high quality’ were those which
were colourless and of regular shape, with regular zona
pellucida and small perivitelline space without debris,
homogeneous cytoplasm and no vacuoles or granulations
(De Sutter et al., 1996; Xia, 1997; Ebner et al., 2003).
Amongst the ‘high quality’ oocytes, the presence of an
intact, round or ovoid polar body with smooth surface was
considered as a selection criterion (Ebner et al., 2000).
Immediately after decumulation and quality evaluation,
the three best available MII oocytes were inseminated by
ICSI, according to the Italian law regulating assisted
reproductive technology. Only the supernumerary MII
oocytes reaching the ‘high quality’ standards were
cryopreserved.
Cryopreservation protocol
The cryopreservation protocol consisted of a slowfreezing–rapid-thawing
method. Oocyte freezing and
thawing solutions (OocyteFreeze–OocyteThaw;
MediCult, Jyllinge, Denmark) contained Dulbecco’s
phosphate-buffered saline (PBS) supplemented with
human serum albumin, α- and β-globulins, and 1,2-PROH
and sucrose as cryoprotectants.
Freezing procedure
After washing in a PBS solution (vial 1,
OocyteFreeze; MediCult), the oocytes were equilibrated
for 10 min at room temperature in 1.5 mol/l 1,2-PROH
(vial 2, OocyteFreeze; MediCult) and then transferred
into the loading solution of 1.5 mol/l 1,2-PROH and 0.3
mol/l sucrose (vial 3, OocyteFreeze; MediCult). Between
one and three oocytes were loaded in plastic straws
(Paillette Cristal 133 mm; Cryo Bio System, Paris, France)
and transferred into an automated biological vertical
freezer (Kryo 360-1.7; Planer, Sunbury, UK). The cooling
process was initiated reducing chamber temperature from
20°C to –7°C at a rate of 2°C/min. Ice nucleation was
induced manually at –7°C. After a hold time of 10 min at
–7°C, the straws were cooled slowly to –30°C at a rate of
0.3°C/min and then rapidly to –150°C at a rate of
50°C/min. After 10–12 min at stabilization temperature,
the straws were transferred into liquid nitrogen and
stored for later use.
Thawing procedure
The straws were air-warmed for 30 s and then
immersed in a 30°C water bath for 40 s. The cryoprotectant
was removed at room temperature by stepwise dilution of
1,2-PROH in the thawing solutions: the contents of the
straws were expelled in 1.0 mol/l 1,2-PROH and 0.3 mol/l
sucrose solution (vial 1, OocyteThaw; MediCult) and
the oocytes were equilibrated for 5 min. The oocytes were
then transferred into 0.5 mol/l 1,2-PROH and 0.3 mol/l
sucrose solution (vial 2, OocyteThaw; MediCult) for 5
min and then into 0.3 mol/l sucrose solution (vial 3,
OocyteThaw; MediCult) for 10 min before final dilution
in PBS solution (vial 4, OocyteThawTM; MediCult) for
20 min (10 min at room temperature and 10 min at 37°C).
The oocytes were finally cultured at 37°C in an
atmosphere of 5% or 6% CO2 in air for 2.79 ± 0.05 h (range
2–5 h) before ICSI.
Survival evaluation, ICSI and embryo culture
After the post-thaw culture the three best surviving
oocytes, according to the previously described
parameters, were inseminated by ICSI, as allowed under
the Italian IVF act. The evaluation of survival was carried
out by inverted microscope with Hoffman modulation
contrast, and thawed oocytes were considered to have
survived in the absence of negative characteristics: dark or
contracted ooplasm, vacuolization, cytoplasmic leakage,
abnormal perivitelline space, cracked zona pellucida. The
surviving oocytes were selected prior to ICSI following
the centre’s own ‘high quality’ standards. Amongst the
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INTERNATIONAL NEWS
‘high quality’ thawed oocytes, the presence of an intact
polar body was considered as a selection criterion; as far as
possible, injecting thawed oocytes presenting an atretic
polar body or a so-called ‘ghost polar body’, which is an
empty membrane without cytoplasm, was avoided (La
Sala et al., 2006). Fertilization and embryo development
were examined by inverted microscope. Embryos were
graded 1–5 (1 best, 5 worst), with grade 1 assigned to the
best quality embryos containing equally sized
symmetrical blastomeres with no fragmentation,
according to the criteria previously described by Veeck
(1999). The embryo development rating (EDR) as
described by Cummins et al. (1986) was calculated to
define the growth rate of transferred embryos obtained by
thawed oocytes. The formula for calculating the EDR was
as follows: EDR = (TE/TO) × 100 (TE = time expected, TO =
time observed). The ideal EDR is 100: this value is obtained
when a hypothetical ‘normally’ growing embryo is at the
2-cell stage at 33.6 h, at the 4-cell stage at 45.5 h, and at 8-
cell stage at 56.4 h.
Endometrial preparation and embryo transfer
Preparation of the endometrium for the embryo
transfer involved the natural ovulatory cycle or a hormone
replacement cycle. An endometrial thickness .8 mm was
considered to be optimal for performing an embryo
transfer. Embryo transfer was carried out after 2 (day 2) or
3 days (day 3) from oocyte thawing and ICSI. In one case
(group B), the embryo transfer was carried out after 6 days
(day 6 blastocyst stage) from thawing and ICSI; testing for
HCG was performed 14 days after embryo transfer.
Clinical pregnancy was defined as the presence of a
gestational sac with or without fetal heart beat at
ultrasound examination 2 weeks after positive HCG
testing.
Statistical analysis
Continuous variables are presented as means ± SE.
Categorical variables are presented as percentages.
Normality of distribution of continuous variables was
assessed with a Kolmogorov–Smirnov test (with Lillefor
correction). Between-group differences of normally
distributed continuous variables were assessed with
parametric statistic (Student’s t-test), whereas nonparametric
statistics (Mann.Whitney Rank Sum Test) were
employed when the normality test was not passed.
Between-group differences in non-continuous variables
were assessed using the chi-squared method with Yates
correction if needed, or with Fisher’s exact test. A
difference was considered significant when a P-value was

INTERNATIONAL NEWS
CONTINUED FROM PAGE 15
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INTERNATIONAL NEWS
methods). A total of 1139 oocytes fertilized after ICSI
(fertilization rate: 82.60%) and 1037 cleaved (cleavage rate:
91.04%). Oocyte survival, fertilization and cleavage were
comparable in all the groups (Table 3). Embryo
development rating (EDR) was significantly higher (85.54
± 1.00) in group A than in group B (83.66 ± 0.85, P = 0.047).
The best quality embryo rate (grade 1) in group A was
significantly higher (33.24%) than in group B (16.20%, P <
0.001) and in total (14.1%, P = 0.002).
Discussion
Initial studies on human oocyte cryopreservation
(Chen, 1986; Al-Hasani et al., 1987) were mainly conducted
using a slow-freezing/rapid-thawing method (SF/RT)
based on criteria optimized for embryo freezing (Trounson
and Mohr, 1983; Lassalle et al., 1985). The original SF/RT
methodology underwent various modifications (Gook et
al., 1993, 1994, 1995a,b), such as the introduction of
elevated dehydrating sucrose concentrations (Yang et al.,
1998; Fabbri et al., 2001; Bianchi et al., 2007), designed to
improve oocyte survival and clinical results (Winslow et
al., 2001; Yang et al., 2002; Fosas et al., 2003; Chen et al.,
2005; Li et al., 2005; Borini et al., 2006b). The optimal time
for ICSI of a SF/RT oocyte is strictly dependent on the
complete restoration of cellular function and in particular
the organization of the meiotic spindle; a post-thaw
incubation of approximately 3 h is recommended to allow
spindle reappearance in slow frozen oocytes (Rienzi et al.,
2004; Bianchi et al., 2005). The timing of ICSI can affect
embryo implantation: even though it is possible to achieve
implantation of embryos derived from aged oocytes (Chen
and Kattera, 2003), it has been shown that the
developmental capacity of the oocyte declines 10 h after
retrieval (Yanagida et al., 1998). Insemination of fresh
oocytes between 37 and 41 h after HCG administration to
trigger ovulation determines the highest embryo
implantation rate (Dozortsev et al., 2004). Therefore, the
metabolic ageing at ICSI of slow-cooled oocytes depends
on: (i) time of retrieval after HCG administration; (ii) preincubation
before cryopreservation; and (iii) post-thawing
culture before insemination (Parmegiani et al., 2008a). In
this multicentre study, oocyte retrieval was performed
35.82 ± 0.02 h (range 35.36 h) after HCG administration
and thawed oocytes were injected after 2.79 ± 0.05 h (range
2–5 h) of post-thaw culture. Thus, the total timing of
insemination (i + ii + iii) was (35.82 h + ≤2 h + 2.79 h) for
oocytes in group A, and (35.82 h+ >2 h + 2.79 h) for those
in group B. When using a SF/RT protocol, the time of
incubation between oocyte retrieval and cryopreservation
is critical in order to avoid injecting ‘aged’ oocytes. In this
survey, ICSI was performed with ideal timing (

INTERNATIONAL NEWS
CONTINUED FROM PAGE 17
Regarding the preservation of the cellular function
(fertilization, cleavage and implantation) the results of this
study were at least comparable with the other studies
using SF/RT with high sucrose concentration (Gook and
Edgar, 2007).Fertilization and cleavage rates did not
significantly vary among the study groups, even though
the embryo quality was influenced by the time of freezing:
a significant increase of top quality embryo rate by
freezing the oocytes within 2 h from oocyte retrieval rather
than freezing them after 2 h was observed (Table 3). It was
observed that the growth rate of embryos (EDR) obtained
from thawed oocytes (84.31 ± 0.65) was lower than the
hypothetical ‘normally’ growing embryo rate (100)
theorized by Cummins et al. (1986). This retarded
development of embryos obtained fro m oocytes
cryopreserved in 0.3 mol/l sucrose has already been
reported by Parmegiani et al. (2008a) in the previous study
on SF/RT 0.3. Nevertheless, in the present multicentre
survey, a significantly higher mean EDR was seen in group
A than in group B confirming the positive effect of timely
cryopreservation (Table 3).
Efficiency of oocyte freezing can be measured as
implantation and pregnancy rates obtained per oocytes
thawed or per embryos transferred (Oktay et al., 2006). The
number of oocytes frozen and the number of thawing
cycles performed vary widely among published reports on
oocyte cryopreservation. Highest implantations per oocyte
thawed were reported in thawing cycles of SF/RT high
sucrose cryopreserved donor oocytes: 21/158 (13.3%) by
Yang et al. (2002), and 10/81 (12.3%) by Li et al. (2005). The
best clinical results in larger studies on SF/RT (Porcu et al.,
2000, Borini et al. 2006a,b; La Sala et al., 2006; Levi Setti et
al., 2006; De Santis et al., 2007) were reported by Bianchi et
al. (2007), with implantation and pregnancy rates per
transfer of 13% and 21% respectively, resulting in 6.0%
(24/403) of implantation rate per thawed oocyte. In the
present multi-centre study, the overall pregnancy rate per
transfer reported was 16.25%: 72 clinical pregnancies were
obtained after 443 embryo transfers, with 85 implantations
from 1037 transferred embryos (8.20% of implantation
rate) (Table 1) and from 2608 thawed oocytes (3.26%)
(Table 2). The data are comparable with the results
obtained in the wider studies on SF/RT. A high miscarriage
rate (32%) was observed in this study, especially when
compared with the miscarriages observed in fresh cycles
(about 22%) in the five centres involved in this multicentre
survey in the same period as the study (March 2004 to
March 2008). Nevertheless, the miscarriage rate observed
in this study is lower than that reported by La Sala et al.
(2006) in a wide study on SF/RT 0.3 (performed under the
same legal restrictions and using the same SF/RT 0.3
protocol) involving 518 cryopreservation treatments. In
fact, the authors of that study reported a miscarriage rate
of 47% and a take-home baby rate/embryo transfer of 1.5%
(7/456) resulting in a probability of one live birth in 65
embryo transfers. The results on salvaging oocyte
cryopreservation observed in the present multicentre
survey can be considered more encouraging: there was a
take-home baby rate/embryo transfer of 9.7% (43/443)
with a probability of one live birth in 10 embryo transfers.
Furthermore, the lowest miscarriage rate observed in this
study for group A (28%) suggests a better trend with
oocytes frozen within 2 h.
In this multicentre study, observing the results in
group A, in which thawing cycles were performed with
oocytes previously frozen within 2 h from oocyte retrieval,
pregnancy and implantation rates were 30.07% and
15.08%, with 6.49 implantations per 100 oocytes thawed.
The pregnancy and implantation rates per transfer and
per thawed–injected oocytes observed in group A were all
significantly higher than in group B, in which oocytes
were frozen after more than 2 h of pre-incubation. The
better efficiency rates observed when oocytes were frozen
within 2 h from oocyte retrieval confirmed, on a larger
number of thawing cycles, the preliminary observation of
Parmegiani et al. (2008a), which reported the highest
implantation rate per oocytes thawed (8.1) in studies
regarding homologous SF/RT.
A very promising alternative to the slow-cooling
protocol is vitrification, in which the gametes, immersed
in a viscous solution with a high concentration of
cryoprotectants, are cooled at an extremely rapid rate. In
the wider studies with vitrification protocols, an
implantation rate per thawed oocyte of 11.2% (12/107) was
reported by Kuwayama et al. (2005), and 11.8% (39/330) by
Antinori et al. (2007). It was encouraging that the results of
this multicentre survey suggest that freezing oocytes via
SF/RT 0.3 within 2 h allows optimal clinical results to be
achieved that are almost comparable with those obtained
with vitrification.
In conclusion, this multicentre study confirmed on a
large scale the fact that the efficiency of oocyte SF/RT 0.3 is
improved if the freezing procedure is carried out within 2
h of oocyte retrieval. In fact, embryo quality and EDR,
clinical pregnancies and implantations per transfer and
per oocytes thawed/injected, were all significantly
increased when oocytes were frozen within 2 h of
retrieval. Thus, it is concluded that freezing within 2 h of
retrieval optimizes oocyte cryopreservation when using
SF/RT 0.3.
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Acknowledgements
The authors wish to thank Ms Maggie Baigent for
revising the manuscript. The authors also wish to thank
Mr Giovanni Ermini (MediCult, Italy), Ms Susanne
Hauschildt Bendz (MediCult, Denmark) and Ms Francesca
Granella (Livet Clinic, Turin, Italy) for their valuable
collaboration in the collection of the data.
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20 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

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ARTICLES
Effect of growth hormone on oocyte competence in
patients with multiple IVF failures
A Hazout 1,3 , AM Junca1, Y Ménézo 1 , J de Mouzon 2 , P Cohen-Bacrie 1
1 ART Unit, EYLAU, 55 Rue Saint Didier, 75016 Paris, France; 2 Unité, INSERM, U822 Kremlin-Bicêtre, France
3 Correspondence: e-mail: ahazout@hotmail.com
c 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Dr. André Hazout, 2009 Effect of growth hormone on oocyte competence in patients with multiple IVF failures. Reprod BioMed Online,
18, 5, 664-670.
André Hazout was co-leader of the assisted reproduction program of Dr Frydman’s
team in Clamart from 1983 to 2003 and leader of the private assisted reproduction
unit ‘Eylau la Muette’ until 2008. From 2003 to 2008 he also led the assisted
reproduction program of the University Paris VII. His recent research interests
include male infertility, sclerotherapy of endometriosis cysts and embryo
implantation markers in endothelium explants. Throughout his career he has been
responsible for many initiatives and has also published extensively in both national
and international journals. He is a past President of the French Society of
Reproductive Medicine.
DR ANDRÉ HAZOUT
Abstract
In a preliminary, unpublished randomized study conducted in 2000 on 39 patients, including a placebo group, it was observed that
the addition of growth hormone (GH) during ovarian stimulation in patients with poor-quality oocytes increased the pregnancy
rate. However, the results were not statistically significant due to the small number of patients in each group. A protocol with 8 IU
GH was tested in 291 patients with three or more previous failures of embryo transfer for no clearly identifiable reasons. The
analysis was restricted to patients receiving either recombinant FSH or human menopausal gonadotrophin (HMG) (n = 245). They
were compared retrospectively to all patients with three or more failures during the same period of time but stimulated only with
recombinant FSH or HMG, without GH, in an observational study design. Co-stimulation with GH gave better results in terms of
number of oocytes collected and embryos obtained. Pregnancy rate per retrieval was higher than in the control group (25.7%
versus 18.2%, P < 0.01) and reached a level similar to the one observed in the study centre for the whole population. Ovarian
stimulation associated with GH can be proposed for patients with a history of repeated assisted reproduction failures. An
improvement of cytoplasmic competence is proposed as an explanation.
Keywords: growth hormone, ICSI, IVF, ovarian stimulation
Introduction
Recurrent IVF failure is always a source of distress in
patients, especially in the group of so-called normoresponders
where ovarian stimulation is expected to give
acceptable results. The oocytes of this group of patients
are often classified as dysmorphic (Van Blerkom and
Henry, 1992); this includes abnormal aspects of cytoplasm,
perivitelline space and zona pellucida. Early embryo
preimplantation development is under maternal control
until maternal to zygotic transition. The first cleavages are
under control of mRNA and proteins stored during
maturation; the quality of these stores is directly related to
the cytoplasmic maturation, i.e. competence in order to
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allow early preimplantation development in a correct
timing. Even if the pivotal role of gonadotrophins cannot
be underestimated, other co-effectors such as insulin-like
growth factor-I (IGF-I), leukaemia inhibitory factor (LIF)
and growth hormone (GH) cannot be neglected especially
for their role in enhancing cytoplasmic maturation (Ptak et
al., 2006; De Matos et al. 2008). In cultured cow (Langhout
et al., 1991) and human (Mason et al., 1990), GH stimulates
steroidogenesis either directly or through enhancement of
the effect of FSH. The positive impact of GH, i.e. the
improvement of nuclear and cytoplasmic maturation,
acting independently of IGF-I, is well documented in
mouse, cow and monkey (Izadyar et al., 1996, 1998, 2000;
Pantaleon et al., 1997; Modina et al., 2007, de Prada and
VandeVoort, 2008). GH allows a full maturation of naked
oocytes in humans (Hassan et al., 2001; Ménézo et al.,
2006). The GH receptor is present in cumulus cells and in
the oocyte for all these animal species as well as in humans
(Ménézo et al., 2003). GH signalling is two-fold: it uses the
signal transducer and activator of transcription (STAT) or
the cAMP response element-binding (CREB), mitogenactivated
protein (MAP) kinase pathways (Izadyar et al.,
1999). Studies evaluating the benefit of co-stimulation
treatment with GH have given discordant results
(Schoolcraft et al., 1997; Howles et al., 1999) for a variety of
aetiologies such as aged patients (Tesarik et al., 2004), poor
responders and patients having GH deficiency (Rajesh et
al., 2007).
The most recently updated Cochrane database
(Harper et al., 2003) showed a positive effect of GH in
poor-responder patients (odds ratio [OR] 4.37, 95%
confidence interval [CI] 1.06–18.01). The reviewer’s
conclusion, based on the large CI and high upper value of
18.01 and the fact that the database was from three rather
small studies, was that ‘before recommending GH in IVF,
further research is necessary to fully define its role.
Meanwhile GH should only be considered in the context
of a clinical trial’. The aim of the current study was to
investigate the effect of GH co-stimulation in ovarian
stimulation in normo-responder patients, who had
previously had at least three failed IVF/intracytoplasmic
sperm injection (ICSI) cycles. In a preliminary,
unpublished randomized study involving 39 patients,
including a placebo group, conducted in 2000, it was
observed that the addition of GH during ovarian
stimulation in patients with poor-quality oocytes
increased the pregnancy rate (50% in the arm with 4 IU
GH daily [n = 13], 55% in the arm with 8 IU GH daily [n =
13] and 18% in the placebo group [n = 13]). The dose of GH
used in the current study was based on this previous
investigation.
Materials and methods
An open, non-comparative and non-randomized
study on the effect of the addition of recombinant GH (r-
GH, Saizen ® ; Serono, Lyon, France) to gonadotrophins on
assisted reproduction treatment outcome was performed
in voluntary patients enrolled in the study centre, Clinique
de la Muette (Paris, France) collaborating with the IVF
laboratory (Laboratoire d’Eylau, Paris, France), between
January 2002 and September 2007. The study was given
approval by the ethical committee of the Assisted
Reproduction Unit, Eylau Laboratory and by the official
ethical committee of Comité Consultatif de Protection des
Personnes en Recherche Biomédicale of Saint Germain en
Laye Hospital. The patients received information on the
protocol and they signed an informed consent form.
Growth hormone group
Inclusion criteria
Patients were eligible if they met the following criteria:
(i) at least three previous assisted cycle failures; (ii) regular
spontaneous menstrual cycles of 25–30 days; (iii) FSH, LH,
oestradiol, inhibin B and anti-Müllerian hormone
concentrations in the normal range during the early
follicular phase; (iv) unexplained infertility with normal
spermatozoa before IVF, or subnormal spermatozoa
justifying ICSI; (v) less than 50% of dysmorphic oocytes in
their previous assisted cycles; (vi) no treatment with
gonadotrophins within 1 month of the treated cycle for the
study; (vii) normal uterine cavity; (viii) negative
pregnancy test; (ix) willingness to participate and to
comply with the protocol.
Exclusion criteria
Exclusion criteria were the following: abnormal
gynaecological bleeding of undetermined origin,
gonadotrophin allergy, known evolutive tumour, any
unstabilized chronic pathology, medical treatments with
corticoids, known human immunodeficiency virus and
hepatitis B and C positive serology (according to the
French legislation) or inability to comply with the
protocol.
Stimulation protocol
Starting on day 20 of the previous cycle, patients
received daily injections of gonadotrophin-releasing
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hormone agonist (GnRHa, Decapeptyl ® ; 0.1 mg, Ipsen
Laboratory France). The daily dose was decreased to 0.05
mg after the confirmation of down-regulation and this
reduced dose was maintained until the day of ovulation
induction with 10,000 IU urinary human chorionic
gonadotrophin (HCG; Gonadotrophine chorionique
endo ® , Organon, France). Pituitary down-regulation was
confirmed by an ultrasound scan showing no evidence of
ovarian activity and/or serum concentrations of oestradiol
16 mm in diameter and
an oestradiol concentration of 140 pg/ml per follicle) were
reached. An injection of HCG was given within 24 h of the
last gonadotrophin administration. Oocytes were
retrieved 36 h after HCG administration. Assessments of
oocytes, 2 pronucleate zygotes and embryos were made on
days 1, 2 and 3 after retrieval. Embryos were replaced on
day 3. The luteal phase was supported with natural
progesterone (Utrogestan ® ; Besins, France) administered
via the vaginal route (300 mg/day) combined or not with
HCG (1500 IU the day after transfer and 3 days later)
depending on the oestradiol concentration the day of HCG
administration. In total, 291 patients were treated with GH
during the period, of whom 245 were considered for the
comparison study. The other 46 patients were excluded
because the stimulation protocol involved either urinary
FSH (n = 15) or clomiphene citrate (n = 2), associations of
rFSH and HMG (n = 21), no gonadotrophin (n = 2), or was
not clearly specified (n = 6).
Comparison group
The patients treated with GH were compared with all
the patients with three or more previous assisted cycle
failures, treated during the same period, without GH, with
a stimulation protocol involving either rFSH only or HMG
only (n = 2780).
The efficacy of GH with rFSH and HMG was analysed.
The total dose of rFSH (IU) or HMG, plasma oestradiol on
the day of HCG, total number and quality of retrieved
cumulus–oocyte–complexes, oocyte quality, particularly
before ICSI (if any) (dark cytoplasm and large blurred
perivitelline space and/or thick zona pellucida), number of
embryos obtained and their quality, number of embryos
replaced, clinical pregnancy rate, number of multiple
pregnancies, miscarriages and live birth rates were
recorded.
Statistical analysis
The analysis was performed in two steps. In the first
step, the two groups were compared with
monovariate analysis, using chi-squared analysis or
variance analysis according to the nature of the variable. In
the second step, a multivariate logistic model was applied
to analyse the effect of the main prognostic factors on the
pregnancy chance per oocyte retrieval and per transfer. In
this case, OR and 95% CI were calculated. Analysis was
carried out using SAS software, release 9.
Results
Patient characteristics
The patients in the GH group were slightly younger
than in the control group and although the difference was
not large it was statistically significant (35.5 years ± 4.0
versus 36.6 ± 4.1, P < 0.001). They also had a significantly
lower FSH dosage on day 3 (6.7 ± 2.0 IU/l, versus 7.1 ± 2.0
IU/l, P = 0.04). The other parameters evaluating the ovarian
reserve were not statistically different in the two groups
(Table 1). The study covers a 5-year period from 2002
(when only a few cycles were included) to 2007. The
percentages of cycles with GH were 5.5, 14.7, 6.7, 9.6, 6.5,
for 2003, 2004, 2005, 2006, and 2007, respectively, without
any significant tendency.
Ovarian response
The average total dose of gonadotrophins (IU) used to
perform stimulation was lower in the GH group (2586 ±
1237 versus 2886 ± 1300 in the control group), and the
oestradiol concentration on the day of HCG was higher
(2215 ± 1286 versus 1945 ± 1132, P < 0.01), whereas the
duration of ovarian stimulation was almost the same
(Table 1).
Efficacy results
The numbers of oocytes retrieved and inseminated
were higher in the GH group (respectively, 10.6 ± 5.9
versus 9.0 ± 5.8, and 8.8 ± 5.5 versus 7.6 ± 5.1, respectively;
P < 0.001 for both), as was the number of embryos cleaved
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at day 2 (5.4 ± 4.0 versus 4.7 ± 3.7, P < 0.001). The cleavage
rates were equivalent, as were the mean numbers of
transferred embryos, while clinical pregnancy rate was
significantly higher in the GH group (25.7% versus 18.7%,
P < 0.01). The effects of GH on enhancing the number of
oocytes retrieved and pregnancy rate were significant
when adjusting for the women’s age (P = 0.01 and P = 0.03,
respectively; Table 2), even if there were few significant
differences in each strata, mainly because of low numbers.
Pregnancy evolution was comparable in the two groups,
with 70.4% delivery per pregnancy in the GH group,
compared with 73.2% in the control group. Abortion and
ectopic pregnancy rates were 23.7% versus 25.4% and 1.8%
versus 2.5%, respectively.
Oocyte characteristics
The percentage of atretic oocytes (small, brownish
without visible organelles) was the same in the two groups
(7.5 ± 10.2% in the GH group versus 8.9 ± 14.3% in the
control group), as was the percentage of metaphase II
(MII) oocytes among the retrieved oocytes (79.9 ± 18.4%
versus 81.0 ± 19.6%, respectively). The number of
dysmorphic oocytes was higher in the GH group (3.7 ± 4.6
versus 3.0 ± 3.7, P < 0.01), as was the total number of
oocytes, but the proportion among the MII oocytes was
similar (46.0 ± 42.3% in the GH group versus 46.0 ± 44.5%).
The impact of the gonadotrophin choice (rFSH or
HMG) in association with GH is shown in Table 3. The
numbers of oocytes retrieved and inseminated were
higher in the group GH + rFSH (11.4 ± 6.3 versus 8.8 ± 4.3,
P < 0.01 and 9.5 ± 5.9 versus 7.0 ± 3.7, P < 0.001,
respectively) while the pregnancy rate was the same
(25.4% versus 26.4%) The two groups did not differ
significantly in age or in the ovarian reserve tests.
The multivariate model, applied to take into account
the effect of women’s age and the gonadotrophin nature
on the chances of pregnancy (Table 4) revealed a very
significant effect of age, with a decreased OR of 0.67 (95%
CI 0.53–0.85) for women aged 38–39 years compared with
age 37 years and less, and OR of 0.50 (95% CI 0.40–0.52) for
those aged 40 years or more. The positive effect of GH was
also significant (OR = 1.48, 95% CI 1.12–1.96), whereas the
gonadotrophin choice had no significant effect on the
pregnancy chance. A second model including the year of
aspiration did not find any significant effect for year of
aspiration (OR 0.97, 95% CI 0.91–1.03), the odds ratios for
GH, women’s age and gonadotrophins remained at the
same level.
Discussion
This study shows results in favour of using GH in
addition to gonadotrophins in recurrent assisted cycle
failures. In the GH group, the number of oocytes retrieved
and the pregnancy rate were close to those usually
observed in a first cycle and were much higher than in the
comparative group with three previous failures, having no
GH co-treatment. It has to be recognized here that this
study has some methodological limitations. The most
important one lies in the protocol itself, since it was not a
double-blinded randomized study. The comparative
group was not chosen at random, because of the results of
the preliminary study conducted in the study centre
during the early 2000s, demonstrating that GH addition
enhanced embryonic development in patients with
dysmorphic oocytes and also at least three IVF failures
(Hazout et al., 2003). The patients, knowing those results
before filling and signing the consent form, did not want
to face the probability of being in the placebo group. Thus,
it was decided to use the patients who were not offered
GH as a comparison group, which is susceptible to some
bias since the reason for having or not GH may be related
to underlying prognostic factors. The multivariate
analysis, taking into account the main factors, including
women’s age, made it possible to partly bypass this aspect.
Moreover, only FSH was significantly higher in the
comparison group among the ovarian reserve testing,
together with age, and the introduction of age in the model
probably also controlled for FSH, since the strong
relationship between age and FSH is well known (Toner et
al., 1993). Thus, the difference in age cannot explain the
difference found for GH. A second bias possibility could
be due to the study length. Recruiting a sufficient number
of cycles needed 5 years because the percentage of cycles
with three previous failures or more is relatively low, and
also because only a few physicians of the centre
participated and the patients had to agree to a new
treatment. However, the percentage of cycles with GH
among the study period did not vary significantly, and the
multilogistic model including the year of aspiration did
not find any significant effect for year of aspiration, and
the OR for GH, women’s age and gonadotrophins
remained at the same level. Finally, a prescription bias
cannot be totally excluded, but the positive results of this
study encourage the design of a new, randomized study,
double-blinded if possible, in order to get the scientific
proof of GH action.
Gonadotrophins are the most important hormones
that regulate major ovarian functions including
folliculogenesis, steroidogenesis and oocyte maturation. In
addition, locally produced growth factors and metabolic
hormones like GH and insulin growth factors play a role
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either alone or in combination with gonadotrophins
(Moreira et al., 2002).
The results of this study confirm in humans the
observations obtained by Izadyar on in-vitro maturation
(IVM) of bovine oocytes. These workers found that the
addition of GH in the culture medium accelerates nuclear
maturation and promotes subsequent embryonic
development. GH receptors have been described in mouse
and bovine oocytes and early preimplantation embryos
(Izadyar et al., 1996, 1998, 1999, 2000; Pantaleon et al., 1997).
GH has a positive effect, in animal models, on maturation
process through a favourable increase in cytoplasmic
competence leading to better embryo quality obtained
either in vitro or in vivo (Folch et al., 2001; Moreira et al.,
2002; de Prada and VandeVoort, 2008). The increase of MII
bovine oocytes obtained after 16 h of culture pointed to an
effect of GH on the kinetics of meiosis rather than an
enhancement of the proportion of oocytes that reached to
the MII stage after 24 h of maturation. The acceleration of
meiosis by GH affected the first cleavage divisions rather
than the process of fertilization. mRNA for GH receptor
(GHR) in humans is present in the oocyte and cumulus
cells during the final stage of oocyte maturation (Ménézo
et al., 2003). This argues in favour of a role for GH during
oocyte maturation, especially in terms of cytoplasmic
competence. GHR mRNA is also present in human
embryos during preimplantation development. In
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humans, the results are more controversial: its positive
action seems to be limited to a special population of age
and/or poor-responder patients (Harper et al., 2003;
Tesarik et al., 2005). More recently, it was proposed that
GH supplementation might improve embryo quality in
patients with GH deficiency (Rajesh et al., 2007), fitting
with the previous observations of Mendoza et al. (2002). In
any case, GH increases the maturation rates of human
naked germinal vesicle (GV) oocytes (Hassan et al., 2001;
Ménézo et al., 2003, 2006). It resulted in live births after
maturation of naked GV oocytes (Ménézo et al., 2006) and
it increases the rate of maturation in an IVM programme
(Ali et al., 2006). Mendoza et al. (2002) noted that GH
concentration in follicular fluid is associated with assisted
reproduction treatment failures: it is obvious that, in some
patients, the GH concentration can be below a critical
threshold impairing ovarian function (Spiliotis, 2003),
especially during the late phases of oocyte maturation.
Exogenous GH tends to correct this negative effect (Rajesh
et al., 2007) and the present study suggested that GH may
be effective in assisted reproduction treatment in a specific
group of young women who failed to conceive after at
least three IVF/ICSI cycles without a rationale explanation
and no specific aetiology.
For all patients in the GH group, more than three
transfers of three or more embryos were unsuccessful.
Ovarian response to the combined treatment (GnRHa +
rFSH +rHGH) showed that follicular maturation occurred
at the same time as in cycles without rHGH treatment.
This is not in agreement with previous studies that found
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that HGH enhances FSH-induced oestradiol production
by isolated human ovary in vivo (Lanzone et al., 1996),
FSH-independent oestradiol production by isolated
human granulosa cells (Mason et al., 1990) and FSHinduced
formation of LH receptor in rat granulosa cells
(Jia et al., 1986). The total number of good-quality oocytes
and embryos obtained was significantly improved without
a real change in the classical scheme of maturation
(stimulation length, hormone dose, oestradiol
concentration). GH seems to act per se. In this way, this
study seems to be partly in agreement both with the
Cochrane database (Kotarba et al., 2000) rather than the
European–Australian multicentre study on the absence of
effect on stimulation parameters. The increase in the
number of good-quality oocytes collected in the GH group
was not found in the Cochrane analysis. The costimulation
treatment rFSH was as efficient as HMG. The
impact also seems to be related to oocyte quality leading to
better embryo competence in terms of cleavage and
further development to term. GH supplementation in this
group of patients resulted in a pregnancy rate similar to
the overall patient population.
Growth hormone signalling is multiple: it uses the
STAT (Ras/Raf/MEK/extracellular signal-regulated) kinase
pathway, the MAP kinase/CREB pathways. It is interesting
to note that the RAS/RAF/MEK extracellular signalregulated
kinase pathway interacts with the LIF Janus
kinase/STAT pathway: LIF receptor is present in the oocyte
and the preimplantation embryo. GH and LIF increase
gene regulation in cumulus cells and oocytes. A possible
target for this signalling could be the oocyte DNA repair
capacity, which is of major importance, especially for ICSI
for poor sperm quality (Ménézo et al., 2007). Liver and
embryo have many common metabolic similarities and
GH increases liver DNA repair capacity (Thompson et al.,
2000; Herubel et al., 2002). However a direct effect on the
endometrium positively affecting implantation cannot be
totally excluded. This leads to the idea that HGH plays a
direct role in the oocyte at the final stage of maturation,
particularly in terms of cytoplasmic maturation. In a
recent case report, Ménézo et al. (2006) demonstrated that
pregnancy and delivery could be obtained after IVM of
naked GV human oocytes with HGH and transfer of a
frozen–thawed blastocyst.
In conclusion, although this study was not a
randomized controlled trial, the data obtained here are
indicate a positive effect of GH on IVF outcome in a special
population of patients with no clear explanation for their
multiple failure of embryo transfer. These results need to
be confirmed by a large-scale randomized controlled trial.
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Schoolcraft W, Schlenker T, Gee M 1997 improved controlled
ovarian hyperstimulation in poor responders IVF patients
with a microdose follicle stimulating hormone flare, growth
hormone protocol. Fertility and Sterility 67, 93–97.
Spiliotis BE 2003 Growth hormone insufficiency and its impact
on ovarian function. Annals of the New York Academy of
Sciences 997, 77–84.
Tesarik J, Hazout A, Mendoza C 2005 Improvement of delivery
and live birth rates after ICSI in women aged >40 years by
ovarian co-stimulation with growth hormone. Human
Reproduction 20, 2536–2541.
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INTRODUCING THE
global ® DMSO Blastocyst Vitrification System
global ® DMSO Blastocyst Vitrification Kit
• 2 X 1 ml vial of Equilibration Solution containing DMSO, Ethylene Glycol and 10 mg/ml HSA in global ® w/HEPES
• 2 X 1 ml vial of Vitrification Solution containing DMSO, Ethylene Glycol, Sucrose, and 10 mg/ml HSA in global ® w/HEPES
• 4 X SunIVF embyo GPS ® dishes, sterile, endotoxin and MEA tested
global ® DMSO Blastocyst Warming Kit
• 1 X 1 ml vial of Warm 1 Solution containing Sucrose and 10 mg/ml HSA in global ® w/HEPES
• 1 X 1 ml vial of Warm 2 Solution containing Sucrose and 10 mg/ml HSA in global ® w/HEPES
• 2 X 1 ml vial of Warm 3 Solution containing 10 mg/ml HSA in global ® w/HEPES
• 4 X SunIVF embryo GPS ® dishes, sterile, endotoxin and MEA tested
Reference
Stehlik E, Stehlik J, Katayama KP, Kuwayama M, Jambor V, Brohammer R, Kato O (2005) Vitrification demonstrates significant
improvement versus slow freezing of human blastocysts. Reprod Biomed Online 11, 53-7.
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INTRODUCING THE
global ® Blastocyst Vitrification System – Based on S 3
global ® Blastocyst Vitrification Kit – Based on S 3
• 2 X 1 ml vial of Vitrification Solution 1 • 5 X 1.2 ml vial of Vitrification Solution 3
• 2 X 1 ml vial of Vitrification Solution 2 • 4 X SunIVF Universal GPS ® dishes
global ® Blastocyst Vitrification Thawing Kit – Based on S 3
• 1 X 1 ml vial of Thawing Solution 1 • 1 X 1 ml vial of Thawing Solution 4
• 1 X 1 ml vial of Thawing Solution 2 • 1 X 1 ml vial of Thawing Solution 5
• 1 X 1 ml vial of Thawing Solution 3 • 4 X SunIVF Universal GPS ® dishes
References
Stachecki JJ, Cohen J (2008) S 3 Vitrifcation System: A novel approach to blastocyst freezing. J. Clin. Embryol. 11, 5-14.
Stachecki JJ, Garrisi J, Sabino S et al. 2008 A new safe, simple and successful vitrification method for bovine and human
blastocysts. Reprod Biomed Online 17, 360-367.
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ARTICLES
S 3 Vitrification System: a Novel Approach to
Blastocyst Freezing
James J. Stachecki, Ph.D., Jacques Cohen, Ph.D.
Tyho-Galileo Research Laboratories, 3 Regent Street, Suite 301, Livingston, NJ 07039
Email: james @galileoivf.com
Stachecki JJ, Cohen J (2008) S 3 vitrification system: A novel approach to blastocyst freezing. J. Clin. Embryol, 11, 5-14
This article was first published in The Journal of Clinical Embryology, 11, 5-14 (2008). It is reproduced here with permission from The
Journal of Clinical Embryology.
Reducing multiple pregnancies is a concern of IVF
clinics everywhere. Blastocyst transfer is the
method of choice for the replacement of just a
single embryo. As more clinics become proficient at
culturing embryos to the blastocyst stage there is an
increasing need to store extra blastocysts. Slow-cooling
regimes have been around for over 30 years, and
although thousands of babies have been produced
throughout the world from this technique, there is room
for improvement, especially when it comes to storing
blastocysts. Modern, faster methods of cryopreservation,
namely vitrification, are enticing because of their
apparent ease, reduced procedure time, and published
success rates. Rapid freezing has been the focus of
research in recent years and, in several laboratories, is
now the preferred method for storing human embryos.
The concept of vitrification, or achieving a glasslike
state, is not new and was first described in 1860. Rall and
Fahy showed that vitrification is a potential alternative to
slow-cooling of embryos over 100 years later (Rall and
Fahy, 1985). Since then it has been the topic of numerous
publications in the IVF field (Ali, 2001; Antinori et al.,
2007; Chen et al., 2000; Chung et al., 2000; Cremades et al.,
2004; Hiraoka et al., 2004; Kasai and Mukaida, 2004;
Kuleshova and Lopata, 2002; Liebermann and Tucker,
2002; Liebermann et al., 2003; Vanderzwalmen et al., 2003;
Vanderzwalmen et al., 2002; Wininger and Kort, 2002; Wu
et al., 2001; Yoon et al., 2003). Vitrification by rapid
cooling proved the only effective method to store the
cold-sensitive oocytes and embryos of pigs, cows, and
sheep (Beeb et al., 2002; Fahning and Garcia, 1992; Szell et
al., 1990; Vajta et al., 1998). Soon after these reports were
published, the method of vitrification by rapid cooling
was applied to human embryo storage. Many of the
recent manuscripts clearly show improved results in
terms of survival and clinical pregnancy rates, when
using vitrification. However, current vitrification
methods have potential problems.
In order to understand rapid-cooling or vitrification
techniques, let us compare them to the slow-cooling
method. During slow-cooling using a programmable
freezer, embryos are exposed to relatively low
concentrations of permeable and non-permeable
cryoprotectants (such as 1.5M Propanediol (PrOH) in
conjunction with 0.2M sucrose), equilibrated for 10-25 min
at room temperature, loaded into a straw or vial, sealed
and placed into a controlled-rate freezer. Ice formation is
initially induced extracellularly by seeding and, as a result
of the solute gradient created, freezable water flows out of
the cells minimizing the chance of intracellular ice
formation during cooling. As the temperature is gradually
lowered, the concentration of cryoprotectant in the liquid
phase (which includes intracellular fluid) increases
correspondingly until a level is reached at which
additional formation and growth of ice crystals, although
possible, are unlikely even if the temperature drops
further (Luyet, 1970). Rather, this remaining liquid phase
turns immediately into a glassy substance upon plunging
into liquid nitrogen and solidifies without further crystal
formation. The unfrozen liquid phase remaining within
the cells should now ideally consist of this glassy
substance with all original cell solutes remaining in
solution (Luyet, 1970). This suggests that when we slowcool
cells using a penetrating cryoprotectant such as
PrOH, and standard slow-cooling protocols, we are
actually vitrifying the cells. Indeed, when we slow-cool
human embryos, typical survival rates range between 80%
and 100%, for many IVF centers. These survival rates
would not be possible, at least according to Mazur
(Mazur, 1963), if intracellular ice formation were
occurring. This correlates well with the theory that
slowcooling is vitrification. Now let’s examine modern
day rapid-cooling (vitrification). All of the rapid-cooling
forms of vitrification procedures for human embryos
described in the recent literature are, in principle, the
same. They all involve exposure of oocytes or embryos to
high concentrations of cryoprotectant(s) for brief periods
of time at or near room temperature followed by loading
onto or into a tiny container (cryo-loop, cryo-top, cryoleaf,
cryo-tip, etc.) that may or may not be sealed, then
submerged directly into liquid nitrogen and stored. The
high osmolarity of the vitrification solution rapidly
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dehydrates the cell, some of the cryoprotectant enters the
cell and binds the remaining water, and submersion into
liquid nitrogen quickly solidifies or vitrifies the cell so that
any remaining intracellular water not bound by
cryoprotectants, does not have time to form a lethal
amount of ice crystals. The embryo is effectively vitrified
without intracellular ice, similar to slow-cooling. From
these descriptions, both techniques, although seemingly
very different, have the same outcome of vitrifying the cell.
Therefore, the term vitrification can be used to describe
both slow-cooling or rapid-cooling, as long as the outcome
is the formation of an amorphous glass-like solid.
Compared with slow-cooling, rapid cooling
vitrification has allowed for improved survival and
pregnancy rates. Reasons for this are numerous, despite
the fact that both procedures vitrify the cell. The methods
are different enough that, despite posing a greater risk
from the potential toxicity of the highly concentrated
cryoprotectants used and the relatively high exposure
temperature, rapid-cooling has met with greater success in
most instances (Hotamisligil et al., 1996; Mukaida et al.,
1998). To combat the toxic effects of elevated
cryoprotectant levels, exposure to the final vitrification
solution is usually limited to around 45-90 seconds or less
before plunging in liquid nitrogen (Chung et al., 2000;
Hong et al., 1999; Hunter et al., 1995; Shaw et al., 1992; Wu
et al., 2001; Yoon et al., 2003). Also, to promote faster
solidification, minute amounts of vitrification media are
used, usually under 2ul. For example, when using the
cryo-top device, the cell(s) are placed on the tip and excess
media is removed leaving the cell(s) covered in a very thin
film of media before plunging directly into liquid nitrogen
This allows for an extremely rapid cooling rate of over
20,000°C/min as shown in Table 1.
Similar vitrification devices to those in Table 1 allow
cooling rates of >15,000°C/min and have resulted in high
survival rates (Antinori et al., 2007; Cremades et al., 2004;
Hiraoka et al., 2004; Hong et al., 1999; Huang et al., 2005;
Isachenko et al., 2005; Kuwayama et al., 2005a; Lane and
Gardner, 2001; Liebermann et al., 2003; Martino et al., 1996;
Mukaida et al., 2003; Son et al., 2003; Wu et al., 2001). In
fact, the combination of cryoprotectants used in
conjunction with very rapid cooling rates has allowed for
these results (Table 2), whereas slower cooling rates have
yielded poor survival rates (Escriba et al., 2006;
Vanderzwalmen et al., 2002).
Despite the increase in survival and pregnancy rates,
and relative abundance of recent reports on rapid-cooling
vitrification (Table 2), there are numerous potential
shortcomings associated with these protocols that have
prevented its widespread application and acceptance
(Kuleshova and Lopata, 2002). Viral contamination from
direct contact to liquid nitrogen is a concern despite
Table 1. Cooling rates for modern vitrification devices.
Device Media (ul) Freezing Rate
0.25cc straw 25ul 4460°C/min
Open-pulled straw 1.5ul 16,340°C/min
Cryo-Top 0.1ul 22,800°C/min
Cryo-Tip

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CONTINUED FROM PAGE 33
Table 2. Published survival and pregnancy rates of vitrified human cells.
Study Cell Type n Survival Rate (%) Pregnancy Rate (%)
(Lane et al., 1999) Blastocyst 18 83% N/A
(Choi et al., 2000) Blastocyst 93 51.6% 25%
(Cho et al., 2002) Blastocyst 120 84.2% 34.1%
(Reed et al., 2002) Blastocyst 15 100% 25%
(Vanderzwalmen et al., 2002) Blastocyst 75 70.6% 22.9%
(Vanderzwalmen et al., 2003) Blastocyst 186 78.5% 32.4%
(Mukaida et al., 2003) Blastocyst 725 80.4% 37%
(Son et al., 2003) Blastocyst 90 90 48%
(Cremades et al., 2004) Blastocyst 33 82% N/A
Teramoto et al., 2004 (abs) Blastocyst 197 100 57.7%
(Hiraoka et al., 2004) Blastocyst 49 98% 50%
(Huang et al., 2005) Blastocyst 96 77.1% 53.8%
(Takahashi et al., 2005) Blastocyst 1129 85.7% 44.1%
(Stehlik et al., 2005) Blastocyst 41 100 50%
(Kuwayama et al., 2005a) Blastocyst 6328 90 53%
(Liebermann and Tucker, 2006) Blastocyst 547 96.5% 46.1%
(Stachecki et al., 2008) Blastocyst 93 89 65%
(Isachenko et al., 2003) Embryo 59 71 N/A
(Kuwayama et al., 2005a) Embryo 5881 100% 44%
(Kuwayama et al., 2005a) Embryo 897 98 32
(Sher et al., 2008) Embryo 78 96% 63%
(Balaban et al., 2008) Embryo 234 94.8% 49%
(Katayama et al., 2003) Oocyte 46 94% 33.3%
(Kuwayama et al., 2005b) Oocyte 64 91% 45.4%
(Selman et al., 2006) Oocyte 24 75% 33%
(Lucena et al., 2006) Oocyte 159 96.7% 56.5%
(Antinori et al., 2007) Oocyte 330 99% 32.5%
(Cobo et al., 2008) Oocyte 27 86.9% N/A
Cao et al., 2008 Oocyte 292 91.8% N/A
pipeting the blastocyst in and out of a fine bore pipette or
rupturing it using an ICSI needle or similar device
(Hiraoka et al., 2004; Son et al., 2003; Vanderzwalmen et
al., 2002). Although survival can be increased using these
methods, the obvious drawback is that there is an
additional step involved that is potentially damaging to
the embryo.
In this study we describe a new method to vitrify
human blastocysts that is safe, successful, and relatively
easy to learn and use. This method and media can be used
to vitrify blastocysts of all stages (from cavitating to fully
hatched) without reduction of the blastocoel or using
DMSO in the media. Our technique uses a standard 0.25cc
sterile straw, up to 3 times longer cryoprotectant exposure,
ample loading time, and heat-sealing protection. These
factors should permit adequate recovery time in cases of
operator-error. This alternative method to slow-cooling,
entitled S 3 -vitrification, has been described in a recent
study of ours (Stachecki et al., 2008) and we report here
updated clinical outcomes from several clinics using this
technique.
Materials and Methods
Collection of Blastocysts
Luteal phase GnRH agonist regimes, also called luteal
phase lupron protocols, were used for all patient hormonal
stimulations and oocytes were collected by standard
means with fertilization occurring using ICSI or IVF. Only
high quality blastocysts that had a well formed blastocoel,
trophectoderm with many cells, and a well-formed visible
ICM were chosen for clinical vitrification. The Gardner
scale was used to grade blastocysts, where the ICM or
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ARTICLES
trophectoderm was either an A or B (Gardner et al., 2000).
None of the patients had HCV.
Training
Several embryologists at 5 different IVF clinics in
North and South America were trained in the S3
Vitrification procedure over a period of 1 to 2 days. After
training, the embryologists practiced vitrifying spare
blastocysts consented for research. Upon reaching a level
of comfort with the technique and their results, which
varied in time from 1 week to 2 months (depending on
how much material was available to practice with) they
began clinical use.
Vitrification
A series of 3 solutions (V1, V2, V3) were used to vitrify
blastocysts according to (Stachecki et al., 2008). Blastocysts
were exposed to V1 for 5 min at room temperature (RT),
transferred to V2 for 5 min at RT, and then to V3. Once in
V3, the cells were immediately loaded into a standard,
sterile 0.25cc cryopreservation straw Figure 1. The straws
were frozen per straw.
Thawing and Embryo Replacement
Straws were thawed by holding them in room
temperature air before immersion in a water bath
(Stachecki et al., 2008). After thawing, the cryoprotectants
were removed by dilution at room temperature through a
series of five media (T1-T5) at 5 min per step. The
blastocysts were warmed on a heated surface (37°C)
before being placed in culture at 37°C and then analyzed
before being transferred. Embryos deemed to have
survived thawing were selected for replacement on an
individual patient basis, per the clinic’s guidelines.
Results
Table 3 shows each clinic’s initial results with S3
vitrification along with the totals for all five clinics
combined. Note that these results are from the first set of
blastocysts that the clinics had vitrified and extensive
training and practice was not necessary. Additionally,
there were multiple embryologists performing the
Figure 1. Straw Loading Diagram
were then heat-sealed at both ends. A 0.5cc straw with
patient information was heat-sealed to one end of the
0.25cc straw. The total time it took to load a straw and seal
it was under 120 seconds. Straws were then vitrified by
pre-cooling in liquid nitrogen vapors (–95°C to –105°C)
before being stored in liquid nitrogen. This method of
loading and cooling was simple and easily accomplished,
within the given time frame, and in most cases there was
time to spare prior to cooling. Either 1 or 2 blastocysts
vitrification and thaw procedures at each clinic. A total of
884 blastocysts were thawed. Overall survival rates
ranged between 83% and 100% with the average being
88.7%. Fetal heart beat (FHB) rates ranged between 32.8%
and 58.7%. Pregnancy rates per transfer ranged between
CONTINUED ON PAGE 36
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CONTINUED FROM PAGE 35
Table 3. Clinical results of S3 Vitrification on blastocysts.
Clinic Thawed Intact Transfers Replaced FHB Preg/Transfer
A 104 86 (83%) 45 80 47 (58.7%) 32/45 (71.1%)
B 160 141 (88.1%) 77 131 43 (32.8%) 37/77 (48.0%)
C 41 35 (85.4%) 19 35 16 (45.7%) 12/19 (63.1%)
D 566 509 (89.9%) 209 509 N/A 116/209 (55.5%)
E 13 13 (100%) 8 13 5 (38.5%) 5/8 (62.5%)
Total 884 784 (88.7%) 358 768 111 (42.8%) 202/358 (56.4%)
48-71% with the average being 56.4%. All babies born to
date were born healthy with no reported abnormalities.
Discussion
Previous reports show that fast-rate vitrification of
blastocysts offers a feasible and often better approach to
storage than slow-cooling techniques (Table 2).
Refinement of earlier methods has led to the use of tiny
containers and rapid cooling rates that coincide with a
marked increase in blastocyst survival. However, these
procedures are not as easy to use for some individuals and
have other potential problems as described above.
Concerns regarding sterility and ease of use will continue
to grow as more and more regulations are placed upon
IVF clinics by outside inspecting bodies such as CAP and
the FDA. An alternative methodology that avoids these
problems, conforms to potential future regulations, and
provides for high survival and pregnancy rates, would be
very useful. The new technique, S 3 -vitrification, described
here and elsewhere (Stachecki et al., 2008), has been used
effectively and reproducibly, at least based upon the few
clinics that have used this method. The relatively high
survival and pregnancy rates obtained from five clinics in
North and South America were collected from their initial
attempts at using the procedure and, with continued use,
could improve further.
The results varied between clinics, but overall, the
rates were similar to other reports in the scientific
literature. Although the slow-cool vitrification procedure
was similar between the clinics, differences in patient
selection, stimulation, patient age, methods of grading
embryo survival, etc. could all be expected to differ among
embryologists and clinics. For example, Clinic A had the
lowest survival rate with 83%, yet had the highest clinical
pregnancy rate with 71.1%, over a reasonable amount of
transfers (n=45). They appear to have a much more
stringent grading system, and even though survival
seemed to be lower, the blastocysts that they deemed to
have survived led to the highest pregnancy rate among the
clinics. We cannot, and did not want to control for
individual differences within and between clinics. We feel
that this strategy will lead to a more accurate picture of
overall success. Likewise, not every clinic will have the
same success rate for embryo culture, despite the use of
the same culture medium. The data from Table 2 also show
a high variability of survival (51.6% to 100%) and
pregnancy rates (22.9% to 55.6%) for blastocyst storage,
despite the fact that the techniques are essentially the
same. A storage technique that is highly effective and
robust should produce results that, despite the myriad
differences within and between labs, will ultimately
produce reasonably high pregnancy rates. Although there
are higher survival rates reported (see Table 2) collectively,
our results are similar to those reported. Rather than
spending time making extensive comparisons to other
studies, the technique of
S 3 vitrification simply represents an alternative method of
storing embryos (and oocytes, data not shown), that can
potentially improve outcomes and has collectively yielded
over 200 pregnancies to date.
What makes S 3 vitrification unique is that it uses a
relatively large 0.25cc straw, that can be loaded and sealed
easily in a timely manner, and that uses a significantly
slower cooling rate of

ARTICLES
removed and enough cryoprotectant is around to prevent
damage to the trophectoderm and ICM cells based on the
survival and pregnancy rates obtained. After thawing and
removal of cryoprotectants, blastocysts could easily be
graded, and were replaced anytime from 30 minutes to 2
hours after thawing.
The results from the various clinics presented here
demonstrate that blastocysts can be vitrified using a
simple easy-to-use protocol, in a relatively large, sterile,
sealable container without the need for DMSO, and in a
manner that allows time for equilibration and loading. So
far, S 3 vitrification seems to be easy to learn, effective, and
reproducible, yielding high survival and pregnancy rates
in a number of reproductive clinics using the procedure
for the first time.
Acknowledgements
We thank all of the embryologists at the Institute for
Reproductive Medicine and Science, Highland Park IVF
Center, Northwest Center for Reproductive Sciences,
ProCriar, Advanced Reproductive Science, and Bryn
Mawr Center for Reproductive Medicine (Main Line
Health) for their assistance. We also thank all those who
have supported our endeavors and are currently
investigating the use of S 3 vitrification in their clinics.
Many thanks to everyone at Tyho-Galileo Research
Laboratories for their continued support and hard work.
Also, to those women who consented to have their
blastocysts frozen with our technique, we wish you the
best and a healthy and happy pregnancy.
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Lucena E, Bernal DP, Lucena C et al. 2006 Successful ongoing
pregnancies after vitrification of oocytes. Fertil Steril 85, 108-
111.
Luyet B. (1970) Physical changes occurring in frozen solutions
during rewarming and melting. In Wolstenholme, G. and M,
O.C. (eds.), The Frozen Cell. J & A Churchill, London, pp. 27-
50.
Martino A, Songsasen N, Leibo SP 1996 Development into
blastocysts of bovine oocytes cryopreserved by ultra-rapid
cooling. Biol Reprod 54, 1059-1069.
Mazur P 1963 Kinetics of water loss from cells at subzero
temperatures and the likelihood of intracellular freezing.
Journal of general physiology 47, 347-369.
Mukaida T, Nakamura S, Tomiyama T et al. 2003 Vitrification of
human blastocysts using cryoloops: clinical outcome of 223
cycles. Hum Reprod 18, 384-391.
Mukaida T, Wada S, Takahashi K et al. 1998 Vitrification of
human embryos based on the assessment of suitable
conditions for 8-cell mouse embryos. Hum Reprod 13, 2874-
2879.
Rall WF, Fahy GM 1985 Ice-free cryopreservation of mouse
embryos at -196 degrees C by vitrification. Nature 313, 573-
575.
Reed ML, Lane M, Gardner DK et al. 2002 Vitrification of human
blastocysts using the cryoloop method: successful clinical
application and birth of offspring. J Assist Reprod Genet 19,
304-306.
Selman H, Angelini A, Barnocchi N et al. 2006 Ongoing
pregnancies after vitrification of human oocytes using a
combined solution of ethylene glycol and dimethyl
sulfoxide. Fertil Steril 86, 997-1000. Epub 2006 Sep 1011.
Shaw PW, Bernard AG, Fuller BJ et al. 1992 Vitrification of mouse
oocytes using short cryoprotectant exposure: effects of
varying exposure times on survival. Molecular Reproduction
& Development 33, 210-214.
Sher G, Keskintepe L, Mukaida T et al. 2008 Selective vitrification
of euploid oocytes markedly improves survival, fertilization
and pregnancygenerating potential. Reprod Biomed Online
17, 524-529.
Son WY, Yoon SH, Yoon HJ et al. 2003 Pregnancy outcome
following transfer of human blastocysts vitrified on electron
microscopy grids after induced collapse of the blastocoele.
Hum Reprod 18, 137-139.
Stachecki JJ, Garrisi J, Sabino S et al. 2008 A new safe, simple and
successful vitrification method for bovine and human
blastocysts. Reprod Biomed Online 17, 360-367.
Stehlik E, Stehlik J, Katayama KP et al. 2005 Vitrification
demonstrates significant improvement versus slow freezing
of human blastocysts. Reprod Biomed Online 11, 53-57.
Szell A, Zhang J, Hudson R 1990 Rapid cryopreservation of
sheep embryos by direct transfer into liquid nitrogen
vapour at -180 degrees C. Reproduction, Fertility, &
Development 2, 613-618.
Takahashi K, Mukaida T, Goto T et al. 2005 Perinatal outcome of
blastocyst transfer with vitrification using cryoloop: a 4-year
follow-up study. Fertil Steril 84, 88-92.
Teramoto S, Uchiyama K, Aono F, et al. 2004 The efficacy of
selected single embryo transfer (SET) with vitrification.
Fertil Steril 82, S207.
Vajta G, Holm P, Kuwayama M et al. 1998 Open pulled straw
(OPS) vitrification: a new way to reduce cryoinjuries of
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Vanderzwalmen P, Bertin G, Debauche C et al. 2003 Vitrification
of human blastocysts with the Hemi-Straw carrier:
application of assisted hatching after thawing. Hum Reprod
18, 1504-1511.
Vanderzwalmen P, Bertin G, Debauche C et al. 2002 Births after
vitrification at morula and blastocyst stages: effect of
artificial reduction of the blastocoelic cavity before
vitrification. Hum Reprod 17, 744-751.
Wininger JD, Kort HI 2002 Cryopreservation of immature and
mature human oocytes. Semin Reprod Med 20, 45-49.
Wu J, Zhang L, Wang X 2001 In vitro maturation, fertilization
and embryo development after ultrarapid freezing of
immature human oocytes. Reproduction 121, 389-393.
Yoon TK, Kim TJ, Park SE et al. 2003 Live births after vitrification
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transfer program. Fertil Steril 79, 1323-1326.
38 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

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ARTICLES
Successful vitrification in a closed carrier device of blastocysts
originating from infertile patients, egg donors, or in-vitro maturation
by Lopez J 1 , Zech NH 2 , Frias P 1 , Vanderzwalmen P 2,3
1 IVF Center, Cochabamba, Bolivia
2 IVF Centers Prof. Zech, Bregenz, Austria
3 Centre Hospitalier Inter Regional Cavell (CHIREC), Braine l’Alleud - Bruxelles, Belgium
Vitrification in reduced cooling conditions
Vitrification is a cryopreservation procedure by which
solutions are converted into a glass-like amorphous solid
free of any crystalline structures. In order to reduce the
likelihood of lethal ice crystal formation during transit
through the crystalline phase, open embryo carrier
devices allowing direct contact of the biological sample
with liquid nitrogen were designed (Vajta and Nagy,
2006). This allows extremely high cooling rates (more
than 20.000 C°/min), instantaneously bringing the
embryos below the glass transition temperature where
intra and extracellular parts are captured in an amorphous
state. The advantage of ultra-rapid cooling is that a
vitrified state is obtained even if embryos are exposed to
high concentrations of cryoprotectant solutions for only a
very short period of time, long enough to permit the
protection of the biological material. Similarly, extremely
high warming rates (> 20,000 C°/min) can be achieved,
preventing recrystallization.
However, a major drawback of such ultra-rapid
cooling procedure is the possible risk of bacterial or viral
contamination of the biological sample during cooling and
during long-term storage (Bielanski, 2005). Even though
the question of contamination during storage in liquid
nitrogen (LN2) remains debatable (Kyuwa et al., 2003), it
was important to modify the strategy and develop a
vitrification technique ensuring total protection and
isolation of the sample from LN2 during cooling
procedure and long-term storage. Stachecki et al. (2008)
developed a vitrification technique of blastocysts in closed
0.25 ml straws. After warming, a survival rate of 89% was
obtained and, out of 43 transfers, clinical pregnancy and
implantation rates of 60% and 45%, respectively, were
obtained.
Consequently, a hermetically closed carrier device
(VitriSafe, VitriMed, Austria) that is easy to handle and
guarantees the medical safety of vitrified human embryos
and oocytes was developed. This closed system is a
modification of the original open hemi-straw plug carrier
device (Vanderzwalmen et al., 2003). The VitriSafe
consists of a large gutter in which a small quantity of
cryoprotectant containing the blastocysts can be deposited
(Figure 1) Before plunging the biological material into
LN2, the VitriSafe is completely inserted into a high
security 0.3 ml straw (CBS,
Cryo Bio System, France).
Both ends of the outer
protective are heat-sealed
before being plunged into
LN2, ensuring hermetic
isolation of the sample. The
VitriSafe guarantees high
warming rates of >20,000 PIERRE VANDERZWALMEN, PHD
C°/min,
without
compromising aseptic conditions during extraction from
the outer protective straw.
The protective straw, which is required to achieve
aseptic vitrification conditions, inevitably leads to an
important loss in the cooling rate (~1300 C°/min),
increasing the possibility of ice crystal formation if the
intra-cellular concentration of cryoprotectant is not well
adapted to the need of the cells. Because the probability of
fixing the intracellular parts into a glass-like state depends
on the rate of cooling and re-warming, and the
concentration of cryoprotectant solutions (Yavin et al.,
2007), it is obvious that the risk of ice crystal formation will
increase if the cells have not been exposed long enough to
the cryoprotectant solutions. Consequently, the step of
exposure of the embryo to the cryoprotectant solutions
before cooling at a lower rate, has to be considered as a
crucial one. In fact, the decrease in the rate of cooling as
observed with the VitriSafe device has to be compensated
for by longer exposure to the cryoprotectant solutions in
order to increase the intra-cellular concentration.
In order to determine the optimal concentration of the
cryoprotectant solutions and the duration of contact to
these solutions, a microscopic cinematographic evaluation
was undertaken. This study showed how embryos react
during the dehydration and entrance steps with different
cryoprotectant concentrations and times of exposure to the
solutions. The morphometric analysis permitted us to
determine the shrinkage/swelling response of embryos to
increased concentrations of cryoprotectant and thereby
stay within the limits of volume variation that are
compatible with good survival rate. When a longer
exposure to the cryoprotectant solutions is required, a
gradual exposure in three steps induces less shrinkage –
swelling stress to the cell than does a two-step addition.
40 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

ARTICLES
Figure 1: The use of the VitriSafe system for the vitrification of human embryos. a) the VitriSafe embryo carrier (G = gutter),
b) embryos in the gutter of the VitriSafe embryo carrier (CPS = cryoprotectant solution), c) a 0.3 ml CBS straw, d) the VitriSafe embryo
carrier inserted in a 0.3 ml hermetically closed CBS straw before plunging into LN2.
Clinical application
The protocol that we have devised consists of a
gradual exposure of the embryos to 5%-5%, 10%-10% and
20%-20% DMSO-ethylene-glycol (EG) before aseptic
vitrification using the VitriSafe carrier (Table 1).
As shown in Table 2, this approach resulted in
satisfactory clinical outcomes of 233 aseptic blastocyst
vitrification/warming cycles despite reduced cooling rates
to

ARTICLES
CONTINUED FROM PAGE 41
Table 1. The procedure for vitrification and warming of
blastocysts with the VitriSafe device.
Vitrification
Step 1 5% DMSO/5% EG 5 to 10 min
Step 2 10% DMSO/10% EG 4 min
Step 3 20% DMSO/20% EG 40 to 60 sec
Warming
Step 1 Plunge the tip of the Vitrisafe in 1 ml of 1.0 M
sucrose for 1 min 30 sec.
Step 2 0.75 M Sucrose 1 min 30 sec
Step 3 0.50M Sucrose 2 min
Step 4 0.25M Sucrose 2 - 3 min
Step 5 0.125M Sucrose 2 - 3 min
Culture
Embryo culture in Global medium (LifeGlobal, Ontario, Canada)
It is well accepted that lower implantation rates are
seen after a fresh embryo transfers with IVM embryos as
compared to standard IVF/ISCI. One of the reasons might
be the sub-optimal quality of the endometrium. The
challenge with IVM is to retrieve immature oocytes from
small antral follicles – usually in mid to late follicular
phase, and to avoid the occurrence of dominant follicles.
The endometrium is exposed to relatively low levels of
estradiol when immature oocytes are retrieved from the
small antral follicles. This explains the reduced thickness
of the endometrium. Additionally, embryos are placed
back into the uterus much earlier than after normal
IVF/ICSI with respect to the number of days that the
endometrium encounters increasing estradiol levels.
Taken together, with IVM and fresh ET, a synchrony
between the endometrium and embryos has to take place
in a greatly accelerated time schedule compared to other
types of ART. For these reasons, one alternative to fresh
embryo transfer could be the cryopreservation of embryos,
with subsequent transfers in an estradiol/progesterone
supplemented cycle.
Ongoing twin pregnancy after vitrification of
blastocysts produced after IVM from a woman with PCOS
was reported by Son et al. (2002). To the best of our
knowledge, this is the first report of pregnancies from a
large series of vitrified blastocysts produced from IVM
oocytes retrieved from PCOS women.
Compared to the transfers of IVM-derived embryos in
fresh cycles, vitrification in the VitriSafe system resulted in
a significant increase in the implantation rate. A study was
undertaken to compare the outcome of IVM cycles after
embryo transfers in fresh cycles or after vitrification and
Table 2. Clinical outcomes of aseptic vitrification of blastocysts generated from different origins.
Origin of blastocysts Male and/or Egg donation IVM Total
female factor
infertility
Number of patients 103 91 22 216
Patient age (years, mean + SD) 34.5 + 3.7 28.8 +3.1 32.3 + 4.9
Number of vitrification – warming cycles 120 91 22 233
Number of vitrified blastocysts 348 427 64 839
Number of warmed blastocysts 348 218 64 630
Survival after warming 285 (82%) 203 (93%) 53 (83%) 541 (86%)
Survival before embryo transfer 254 (73%) 191 (88%) 44 (69%) 489 (78%)
Number of embryo transfers 111 91 21 223
Number of blastocysts transferred (mean) 231 (2.1) 186 (2.0) 39 (1.9) 456 (2.0)
Pregnancies (per vitrification – warming cycle) 66 (55%) 54 (59%) 15 (68%) 135 (58%)
Miscarriages 15 (23%) 7 (13.0%) 4 (27%) 26 (19%)
Ongoing pregnancies (per vitrification – warming cycle) 51 (43%) 48 (53%) 11 (50%) 110 (47%)
Number of fetal heart beats 60 64 15 139
Implantation rate 26% 34% 38% 30%
Deliveries 14 35 5
42 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

ARTICLES
replacement in an estradiol/progesterone
supplementation transfer cycle. A total of 34 fresh transfers
and 49 vitrified transfers were performed. The ongoing
pregnancy rates in the fresh and vitrified group were 21%
and 44%, respectively, with implantation rates of 10 and
30%.
In spite of reduced cooling rates due to aseptic
vitrification conditions, acceptable results are obtainable if
the intracellular concentrations of cryoprotectants are well
adapted to the needs of the cells. If such results are
confirmed on a large scale, aseptic vitrification has the
potential to become the standard cryopreservation
technique, not only for human blastocysts but also for
early embryo developmental stages.
References
Bielanski A 2005 Non-transmission of bacterial and viral
microbes to embryos and semen stored in the vapour phase
of liquid nitrogen in dry shippers. Cryobiology 50, 206-210.
Kyuwa S, Nishikawa T, Kaneko et al. 2003 Experimental
evaluation of cross-contamination between cryotubes
containing mouse 2-cell embryos and murine pathogens in
liquid nitrogen tanks. Experimental Animal 52, 67-70.
Son WY, Yoon SH, Park SJ, et al., 2002 Ongoing twin pregnancy
after vitrification of blastocysts produced by in-vitro
matured oocytes retrieved from a woman with polycystic
ovary syndrome: case report. Human Reproduction; 17:2963-
2966.
Stachecki J, Garrisi J, Sabino S et al., (2008) A new safe, simple
and successful vitrification method for bovine and human
blastocysts. Reproductive BioMedicine Online 17, 360-367.
Vanderzwalmen P, Bertin G, Debauche CH et al. 2003
Vitrification of human blastocysts with the Hemi-Straw
carrier: application of assisted hatching after thawing.
Human Reproduction 18, 1504-1511.
Vanderzwalmen P, Ectors F, Grobet L, et al., 2009, Development
of an aseptic vitrification technique: application to
blastocysts originating from infertile patients, egg donors
and after in vitro maturation. Reproductive BioMedicine
Online. (in press).
Vajta G, Nagy Z 2006 Are programmable freezers still needed in
the embryo laboratory Review on vitrification. Reprod
BioMed Online 7, 623-633.
Yavin S, Arav A 2007 Measurement of essential physical
properties of vitrification solutions. Theriogenology 67, 81-89.
You can contact Pierre Vanderzwalmen, PhD at: pierrevdz@hotmail.com.
A unique PGD Biopsy Medium formulation
based on global ® medium
PGD Biopsy Medium is ready-to-use and designed to maintain embryos during
the biopsy procedure, allowing ease in removal of blastomeres.
PGD Biopsy Medium was designed along with the staff of Reprogenetics, led by
Santiago Munné, in conjunction with our ongoing development of global ® based
on Simplex Optimization and the entire line of LifeGlobal ® media.
embryo GPS ®
– The best dish for PGD cases
PGD Biopsy Medium Advantages
• PGD Biopsy Medium is calcium- and magnesium-free to break the
cadherin bonds between the blastomeres and thereby allow for the
removal of one or two blastomeres for PGD.
• PGD Biopsy Medium contains sucrose to cause a mild shrinkage of the
blastomeres and thereby facilitate removal of the blastomere(s) for PGD.
• PGD Biopsy Medium contains all the other components of global ®
embryo culture medium including energy substrates and amino acids.
This reduces the stress on the embryo and promotes better development
of the embryo after it is returned to culture.
• PGD Biopsy Medium is HEPES-buffered for use outside of a CO 2
incubator, and contains gentamicin and HSA so that it is ready to use.
• PGD Biopsy Medium was designed in consultation with Santiago
Munné and his scientific staff at Reprogenetics, one of the foremost PGD
laboratories in the world.
www.LifeGlobal.com
WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
43

ARTICLES
Life-long impact of the first cell cycle
by Dmitri Dozortsev, MD, PhD
Advanced Fertility Center of Texas, Houston, TX, USA, American College of Embryology
Whether you believe that life begins at conception
or not, the life of each and every one of us began
at the pronuclear stage, when two mortal cells
came together and transcended into the continuum of
immortality of humans as a species. There are many
critical stages in the development of a human being but
the first cell cycle stands out in its significance, ever more
so as we advance our knowledge about it. In this brief
article I will discuss the three most crucial events of the
first cell cycle, and how they may be affected by
embryology practitioner now, and into the future.
First, during natural fertilization, the development is
set into motion by calcium ion waves elicited by a factor in
the sperm (1), most likely phospholipase C-zeta (PLCzeta)
(2), located initially in the head of a spermatozoon
and subsequently migrating into the pronucleus.
Experimental evidence suggests that full term
development is also possible following parthenogenetic
activation (3). Those studies, and Ozil’s work (4) in
particular, clearly demonstrate that the duration and
amplitude of calcium oscillations affect embryonic
developments after implantation. On the other hand, from
clinical experience with sharing of donor eggs, we have
learned that embryonic development is affected by a
fertilizing spermatozoon. Therefore, it is plausible that
even though the activation itself is all or nothing
phenomenon, the amount of PLC-zeta carried by a given
sperm cell could affect the duration and amplitude of
oscillations and thus produce an effect similar to that
observed in Ozil’s experiments. In the same way that
intracytoplasmic sperm injection turned out to be more
effective than natural fertilization, we may one day find
that artificial activation is more efficient and allows more
control over the activation process than natural activation.
The second crucial event involves the telomeres. There
is accumulating evidence of the importance of the length
of the telomeres for the life span, probability of cancer, and
the length of reproductive life. Because it has been well
established that the length of the telomeres shortens with
every cell cycle due to inability of telomerase to replicate
in 5’-3’ direction (5), it is obvious that at some point during
reproduction length of the telomeres must be restored.
However, until recently, the stage and mechanism of this
restoration remained a complete mystery. It was not until
2007 that this puzzle was solved. It turned out that
telomere length abruptly increases during pronuclei stage
by a telomerase independent mechanism through sister
chromatid exchange (6).
This is the only
opportunity to reset the
length of the telomeres in
an individual’s lifetime. It
is possible that a better
understanding of how
telomere length is restored
may allow us to extend and
improve life span. Only the
embryology practitioner
will be in a position to
DMITRI DOZORTSEV, MD, PHD
accomplish that.
Furthermore, we must
realize that we may already be unknowingly affecting this
process, and will have to wait for a few more decades to
see if our in-vitro interventions are affecting life span.
The repair of DNA damage is the third critical event
happening during the first cell cycle. By the time of
fertilization, both gametes, particularly the oocyte, have
accumulated some damage. Of particular concern is DNA
damage by by-products of oxidative phosphorylation,
which may result in as many as 1 million individual
molecular lesions per day. Data suggest that, in the oocyte,
the telomeric region of the chromosomes is particularly
vulnerable, leading to chromosomal non-disjunctions, the
majority of which take place during meiosis I (7).
In the sperm, no regional preference for chromosomal
damage has been clearly demonstrated, while there is
overwhelming evidence that sperm cells accumulate a
very significant damage to their DNA, particularly in cases
with abnormal sperm parameters. Unlike the oocyte, the
spermatozoon has virtually no mechanism to repair DNA
damage. Thus, one of the critical missions of the first cell
cycle is to repair the DNA from both gametes. The
accuracy requirements for this repair are much higher
than for any other cell, as it will serve as the template for
all other cells. Reparative DNA synthesis must precede
DNA replication. If the damage was extensive, it may
increase the length of the first cell cycle. In any other cell,
this increase would not matter much, because of the
checkpoint that prevents premature activation of p34
kinase, which does not begin phosphorylation of histones
before the DNA is replicated. However, during the first
cell cycle cell, division is not as tightly coupled with
chromosomal replication as it is in any other cell. In fact,
experiments show that the enucleated zygote will undergo
44 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

ARTICLES
cell division, albeit disorganized, around the time when it
would normally take place. Furthermore, an experiment
with okadaic acid (OA) clearly demonstrated uncoupling
between completion of DNA replication and nuclear
envelope break down (8). The exact mechanism for that is
unknown. One hypothesis states that OA specifically
activates H1 kinase, although it is more plausible that it
simply gives advantage to H1 kinase, removing a
counterbalancing influence of a phosphatase. However,
whatever the mechanism, remarkably it only forces the
zygote into premature chromosome condensation, but
does not affect 2-cell or any later-stage embryos. These
and other observations suggest that the normal progress
of the first cell cycle relies, at least
in part, on a general balance of phosporylation/
dephosphorylation, and that shifting it one way or the
other will delay or accelerate its pace. One of the factors
that has a powerful impact on this balance is the pH of the
culture medium, which is in turn affected by CO2
concentration, specific for each culture medium.
Therefore, ‘playing’ with pH gives us a unique
opportunity to control the first cell cycle in a multitude of
ways. Beyond the cell cycle, it is likely to also impact DNA
methylation, responsible for gene imprinting, which was
shown recently, may differ drastically between in-vitro
and in-vivo generated embryos (9). The significance of this
is to be understood in the future.
In summary, from the moment of fertilization, our
future existence is molded by epigenetic pressures which
come in the variety of forms. Therefore, embryology
practitioners are to a large extent, responsible for
establishing a strong foundation for the future of the
human being that may be born as a result of their efforts.
synthesis of polynucleotides and biological significance of
the phenomenon. J. Theor. Biol. 41: 181-90
6. Liu, Lin; Bailey, Susan M; Okuka, Maja; Munoz, Purificacion;
Li, Chao; Zhou, Lingjun; Wu, Chao; Czerwiec, Eva; Sandler,
Laurel; Seyfang, Andreas; Blasco, Maria A; Keefe, David L.
(2007) Telomere lengthening early in development. Nature
Cell Biol.; 9: 1436-41
7. Keefe, D L; Liu, L; Marquard, K. (2007) Telomeres and agingrelated
meiotic dysfunction in women. Cell. Mol.Life Sci: 64:
139-43
8. Dyban AP, De Sutter P, Verlinsky Y. (1993) Okadaic acid
induces premature chromosome condensation reflecting the
cell cycle progression in one-cell stage mouse embryos. Mol
Reprod Dev 34:402-15.
9. Gomes MV, Huber J, Ferriani RA, Amaral Neto AM, Ramos
ES. (2009) Abnormal methylation at the KvDMR1
imprinting control region in clinically normal children
conceived by assisted reproductive technologies. Mol Hum
Reprod 15:471-7.
You can contact Dmitri Dozortsev at: dmitrid385@hotmail.com
References
1. Dozortsev D, Rybouchkin A, De Sutter P, Qian C, Dhont M.
(1995) Human oocyte activation following intracytoplasmic
injection: the role of the sperm cell. Hum Reprod. 10:403-7.
2. Parrington J, Swann K, Shevchenko VI, Sesay AK, Lai FA.
(1996) Calcium oscillations in mammalian eggs triggered by
a soluble sperm protein. Nature. 379(6563):364-8.
3. Rybouchkin AV, Van der Straeten F, Quatacker J, De Sutter P,
Dhont M. (1997) Fertilization and pregnancy after assisted
oocyte activation and intracytoplasmic sperm injection in a
case of round-headed sperm associated with deficient
oocyte activation capacity. Fertil Steril. 68:1144-7.
4. Ozil JP (1990) The parthenogenetic development of rabbit
oocytes after repetitive pulsatile electrical stimulation..
Development 109:117-27.
5. Olovnikov AM. (1973) A theory of marginotomy. The
incomplete copying of template margin in enzymic
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Automated Robotic Human ICSI
Navid Esfandiari, DVM, PhD, HCLD 1 ; Zhe Lu, PhD2; Xuping Zhang, PhD 2 ; Robert F. Casper, MD 1 ; Yu Sun, PhD 2
1 Toronto Centre for Advanced Reproductive Technology (TCART), Department of Obstetrics and Gynecology, and
2 Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, Canada.
Before the introduction of micromanipulation, the
majority of cases of severe male infertility were
generally not treatable. In-vitro fertilization (IVF)
cycles were cancelled in about one-third of these cases,
due to failed fertilization. The introduction of
micromanipulation techniques such as zona drilling,
partial zona dissection, and subzonal insemination
reduced the incidence of failed fertilization and salvaged
many IVF cycles. However, there was little improvement
on fertilization and pregnancy outcome because these
procedures required a relatively high number of
progressive motile sperm which in turn frequently
resulted in a high incidence of polyspermy in inseminated
eggs. To overcome these limitations, microinjection of a
single sperm into the ooplasm (intracytoplasmic sperm
injection, ICSI) was developed. ICSI resulted in a dramatic
improvement in fertilization rates and revolutionized the
treatment of infertile couples with severe male factor.
ICSI, however, is a labor-intensive laboratory
procedure and is technically challenging. Manual
microinjection and micromanipulation of human oocytes
requires a long period of training, and is further limited
by low speed, human errors, and inter-operator
variability. Although ICSI is now considered routine, it
remains a very difficult technique to master, due partly to
its inherent technical difficulty and partly to the
heterogeneity of the oocytes. It is generally agreed that the
ICSI procedure is subject to a learning curve and that not
depositing the spermatozoon within the oocyte cytoplasm
is a common technical failure.
The degeneration of oocytes after ICSI is often a result
of a fault in the ICSI technique, for example an injection
pipette that is either too large or not sharp enough. Proper
orientation of the polar body and needle position are also
important, because improper positioning can damage or
disrupt the metaphase plate during needle entry.
Moreover, disturbances in the nuclear spindle may
dispose oocytes to aneuploidy or maturation arrest. Thus,
perturbation of the oocyte cytoskeleton may critically
influence the fate of the embryo. During ICSI, the location
of the first polar body is commonly used as an indication
of the spindle position, with the assumption that they are
located in close proximity. To avoid damage to the spindle,
oocytes are injected at the 3 o’clock position with the first
polar body at the 6 or 12 o’clock position.
NAVID ESFANDIARI, DVM, PHD, HCLD
The skill of the embryologist conducting the ICSI
procedure is considered a significant predictor of
fertilization, whereas laboratory conditions (i.e.
incubators, culture of oocytes individually versus
grouped, etc.) do not affect the rates as much. The lack of
automated systems capable of high-throughput human
oocyte injection hinders both clinical practice and
research. The goal of our present research is to develop a
robotic system that can quickly and reproducibly inject a
single sperm into the cytoplasm of a human oocyte, or
manipulate the oocyte/zygote.
At present, the automated ICSI system comprises the
following elements:
• a standard inverted microscope (Bright field
imaging, 20X objective, Nikon Ti-S)
• a CMOS camera (601f, Basler)
• an in-house developed vacuum-based cell holding
device for immobilizing multiple oocytes
• an in-house developed precision vacuum pump
• an in-house developed motorized rotational stage
placed on a motorized X-Y translational stage
(ProScan, Prior Scientific Inc.) for oocyte
positioning and orientation control
• a straight ICSI micropipette (MIC-50-0,
Humagen) connected to a 25 microliter glass
syringe (Hamilton) filled with mineral oil and
mounted on a linear stage (eTrack, Newmark
System Inc.) for computer-controlled sperm
aspiration and deposition
• a three-dimensional motorized micromanipulator
(MP285, Sutter Inc.) for positioning the ICSI
micropipette (45° tilting angle) to diagonally
penetrate the oocyte
• a heated stage (THN-60-10, LINKAM) to maintain
oocytes and sperm at 37°
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• a host computer for controlling multiple motion
control devices and processing images in real time
• a vibration isolation table (9100 series, KSI)
• a dissecting microscope (SZX 12, Olympus) for
loading oocytes and sperm onto the cell holding
device.
The procedure begins with loading sperm and oocytes
onto the cell holding device under the dissecting
microscope and the culture medium is covered with
mineral oil. A low vacuum holds an oocyte on top of each
through-hole in the cell holding device. The cell holding
device is then transferred onto the rotational stage on the
inverted microscope. The first oocyte is positioned at
center of the microscope field by moving the X-Y
translational stage. A straight ICSI micropipette is lowered
until the tip of the micropipette roughly appears in the
image. The system integrates a vision-based contact
detection algorithm to automatically determine the vertical
position of the micropipette tip and device surface in the
oocyte area. The micropipette is then automatically moved
to the sperm area to perform contact detection in order to
determine the vertical position of the micropipette tip and
device surface in the sperm area. The position of the sperm
area is also recorded by the system. To inject a sperm into
an oocyte, the user first selects a sperm by mouse clicking
the sperm head on the monitor of the host computer
(Figure 1).
Figure 1: The monitor of the host computer
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The system automatically tracks the motion of the
sperm and taps its tail to immobilize it. The user then
aspirates the sperm into the micropipette through the
control program interface. When a sperm is positioned in
the proximity of micropipette opening, an oocyte is
automatically brought into the field of view. The user
identifies the position of the polar body on the monitor,
and, if needed, the system automatically rotates the polar
body away from the penetration site. The system performs
penetration, sperm deposition, and micropipette
retraction all through computer control. For the next
injection, the system moves the sperm area into the field of
view for the user to select the next sperm for injection.
This process is repeated until all oocytes have been
injected. At the end of ICSI, the cell holding device is taken
off the rotational stage and put under the dissecting
microscope. A low positive pressure is applied to release
the oocytes for collection and transfer to proper culture
dishes for further culture.
In our preliminary study we aimed to quantify the
efficiency of the robotic microinjection system using
mouse zygotes. The robotic ICSI system was first tested to
inject one-cell mouse embryos with phosphate buffered
saline. The embryos were cultured in the same condition
(KSOM, 37°C, 5% CO 2 ) as the control group. The system
demonstrated an injection speed of 12 mouse zygotes per
minute; a lysis rate of 1.1%; and a high blastocyst
formation rate of 89.8% which was similar to the control
group.
The system is currently being tested by injecting
human sperm into hamster oocytes before moving on to
clinical ICSI evaluation. Figure 2 shows a sperm being
injected into a hamster oocyte by the robotic system. We
expect the automated system to produce a high success
rate and consistency in human ICSI, which, together with
other features such as skill independence, and short
learning curve, will make the system a useful tool for
clinical ICSI practice.
References:
1. Esfandiari N, Javed M, Gotlieb L, Casper RF. (2005)
Complete failed fertilization after intracytoplasmic sperm
injection - analysis of 10 years data. Int J Fertil Women’s Med
50:187-92.
2. X.Y. Liu and Y. Sun (2009) Automated mouse embryo
injection moves toward practical use. IEEE International
Conf. on Robotics and Automation (ICRA2009), Kobe, Japan,
May 12-17.
3. Javed M, Esfandiari N, Casper RF. (2010) Failed fertilization
after intracytoplasmic sperm injection. Reprod. Biomed. Online
20: 56-67.
You can contact Navid Esfandiari at: navid.esfandiari@utoronto.ca
Figure 2: Hamster ICSI: (A) held hamster oocyte, (B, C) a human sperm is being injected into a hamster oocyte
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AN ALTERNATE METHOD FOR SHIPPING SPERM: AEROGEL
CONTAINERS FITTED WITH DATA LOGGERS
By Lakshmi Sharma M.Sc, M.Phil, ELD ., Susan Tarchala M.S, T.S ., Jon Nakagawa* CPA
John Perry * B.S, MBA ., Richard Rawlins PhD, HCLD.
Rush Center for Advanced Reproductive Care, Chicago, Illinois
*Marathon Products, San Leandro, CA
Temperature maintenance and documentation play a
vital role in the IVF laboratory. Media and sperm
samples used in the laboratory must be stored and
shipped within specific temperature ranges in order to
maintain the integrity of the products and to produce
optimal end results. Dry shippers charged with liquid
nitrogen are the current method of choice for shipping
sperm, and they are safe and ensure temperature
maintenance at -196 C for up to 5 days. However, dry
shippers are bulky and typically weigh around 20 lbs
when fully charged. Freight costs for these vessels average
between $200 and $250 for 2-day delivery within the
continental United States, and this cost is passed down to
the patient. We propose an alternative method for
shipping sperm to the laboratory using carbon based
aerogel containers that are filled with dry ice.
Aerogels are produced by extracting the liquid
component of a gel through supercritical drying and
replacing it with a gas. This allows the liquid to be slowly
drawn off without causing the solid matrix in the gel to
collapse from capillary action, as would happen with
conventional evaporation (Pierre and Pajonk, 2002).
Aerogels are good thermal insulators because they almost
nullify three methods of heat transfer (convection,
conduction and radiation) (Fricke and Emmerling, 1992).
For example, NASA uses aerogel for thermal insulation of
the Mars Rover and space suits.
(http://stardust.jpl.nasa.gov/tech/aerogel.html).
A modification of this material to produce boxes that
are then filled with dry ice and can maintain temperature
of -70 to -80 C for extended time periods have been used
in this study. Documentation of maintenance of cold chain
is ensured by a data logging device, the microDL
(Marathon Products, San Leandro CA). Figure 2.
containing 1.5 cc of
cryopreserved sperm
samples frozen in EYB
(Irvine Scientific) by
conventional methods were
taken out of liquid nitrogen
storage and placed onto the
dry ice. The flat ribbon-like
probe of the microDL was
LAKSHMI SHARMA, MSC, MPHIL, ELD
placed next to the vials and
secured in place by taping it to the walls of the box. The
cryovials were then covered with another 0.5 lbs of dry ice
so the sperm samples lay sandwiched between the 2 layers
of dry ice. The entire box was placed into a standard
corrugated shipping box lined with Styrofoam. One box
was labeled as day 3 and the other day 5. The final weight
of the boxes ranged from 2.0 to 2.5 lbs. The microDL was
programmed to measure temperature inside the aerogel
containers every 4 minutes. The temperature data from the
microDL units were downloaded on to a microcomputer.
We used both internal and external controls for this
study. Internal controls were cryopreserved sperm in 3.0
ml Nunc vials prepared for shipping in a standard fully
charged MVE dry shipper, and external controls were
sperm received from a commercial cryobank.
Post thaw motility and forward progression on sperm
samples was measured on day 3 and day 5. The box
labeled day 3 was opened on the third day and post thaw
motility and forward progression noted and compared to
controls in the dry shipper. Similarly, the box labeled as
day 5 was opened the fifth day and post thaw motility and
forward progression noted and compared to controls.
Materials and Methods
Two aerogel boxes (Figure 1b) were prepared for
shipping in the following manner. The boxes were placed
overnight in a freezer and pre-equilibrated to -10 C prior
to being filled with 0.5 lbs of dry ice. Nunc cryovials
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Figure 1a: Carbon based aerogel boxes.
Figure 1b: Carbon based aerogel boxes prepared for shipping
with dry ice.
Figure 2: Micro dl data loggers
Results
The results of temperature measurements from the
microDL showed that the temperature inside the boxes
was maintained between -70 and -80 C, as long as the box
was unopened for a maximum period of 5 days (Figure 3).
The box was opened on day 3 and the samples evaluated
for post thaw sperm motility and forward progression
which were comparable to the control. The box opening
resulted in rapid rise of the internal temperature to reach
room temperature within 24 hrs. A similar pattern of in the
rising of temperature after the box was opened on day 5
was seen. Post thaw motility and forward progression of
the samples day 3 and day 5 were comparable controls
(Table 1). Poor post-thaw viability was noted for frozen
thawed 1-cell mouse embryos on both day 3 and day 5,
using this method.
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Table 1: Sperm motility and forward progression on Day 3 and Day 5. 1cell mouse embryo viabilty post thaw.
Internal Control External Control
Dry Shipper Cryobank Sample 1 Sample 2
Day 3 percent motility 62% 64% 60% _
Day 3 forward progression 61% 60% 54% _
D5 percent motility 63% _ _ 61%
Day 5 forward progression 61% _ _ 56%
1 cell mouse embryos – 19/21 6/21 6/20
Viability at thaw (90.4%) (28.5%) (30%)
Figure 3: Temperature data on day 3 and day 5.
Discussion and Conclusions
In conclusion the carbon based aerogel boxes provide
a viable alternative to liquid nitrogen dry shippers
without compromising the post thaw quality of semen
samples over a short time period of 3 to 5 days but are not
optimal for mouse embryo shipping. It has previously
been shown that sperm can be stored in dry ice for short
period of time with relatively modest loss of motility (CRC
handbook of laboratory diagnosis and treatment of
fertility, Brooks A. Keel and Bobby W. Webster page 235)
The microDL unites provide an added measure of quality
assurance documenting the maintenance of the cold chain.
This method is also cost effective; the container weighs 2-
2.5 lbs, and freight costs are one-tenth of that for a dry
shipper, and their lighter weight makes them easier to
handle.
References
Pierre A. C. and Pajonk G. M. (2002). Chemistry of aerogels and
their applications. Chemical Reviews 102: 4243-4266.) .
Fricke, J and Emmerling, A (1992). Aerogel – preparation,
properties, applications. Structure & Bonding 77:37-87.
You can contact Lakshmi Sharma at: lakshmisharma63@aol.com
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Epigenetics and the ART Lab
by Sangita Jindal, PhD, HCLD
Montefiore’s Institute for Reproductive Medicine and Health, Albert Einstein College of Medicine Hartsdale, NY, USA
The concept of epigenetics has been discussed with
greater frequency in genetics literature in recent
years. This brief article summarizes the most up-todate
understanding of the connection between epigenetics
and assisted reproductive technologies (ART).
Genomic imprinting is a modification of the genome
in which genes from only one, rather than two, parental
alleles are expressed. Epigenetics is the mechanism by
which genomic imprinting occurs. A sex-specific mark or
imprint on certain chromosomal regions has been
identified for approximately 200 genes in the human
genome (Luedi et al., 2007). In these regions, the paternal
and maternal genomes are functionally non-equivalent,
differing in DNA methylation or histone modification of
the genome, rather than in alterations of DNA sequences.
These stable but reversible epigenetic modifications
determine whether genes in those regions of the genome
are expressed.
During development, only specific sets of genes are
active. Accessibility of transcription factor binding sites on
DNA is determined by specific patterns of chromatin
modification and DNA methylation. Chromatin exists in a
transcriptionally competent or transcriptionally silent
state. For example histone acetylation is typically
associated with rendering transcription binding sites
competent while histone methylation renders them silent.
We know that sweeping changes in DNA methylation
and chromatin remodeling take place in developing germ
cells and in the pre-implantation embryo, rendering them
particularly vulnerable to environmentally induced
epigenetic modifications. Epigenetics in the germ line has
been most extensively studied in the mouse, in which the
primary imprints are established during gametogenesis.
This is followed by a second wave of epigenetic erasure
after fertilization with asymmetric demethylation of the
male (active) and female (passive) pronuclei. Finally, a
third wave of de-novo methylation of the embryonic
genome occurs at the blastocyst stage (Grace and Sinclair,
2009; Laprise, 2009).
Of the 200 imprinted genes in the human genome,
approximately 50 have been associated with a phenotype
or clinical syndrome (Amor and Halliday, 2008). Of those,
a link has most commonly been suggested between ART
procedures and Beckwith-Wiedemann Syndrome (BWS)
and Angelman Syndrome (AS). BWS is characterized by
prenatal overgrowth, umbilical hernia and increased risk
of developing tumors, and AS is characterized by severe
developmental delay,
absent speech and
hypotonia. These
syndromes both involve
an epigenetic defect of
hypomethylation on the
maternal allele of the
relevant differentially
methylated regions. The
incidence of BWS in the
general population is 0.8% SANGITA JINDAL, PHD, HCLD
but BWS occurred in 4.6%
of the ART babies examined in the study by (DeBaun et al.,
2003). Studies have yielded inconsistent results but
together they suggest that epigenetic errors may originate
from specific aspects of ART such as the reason for the
subfertility itself, ovulation induction, use of IVF or ICSI,
and finally in-vitro culture and embryo transfer.
One theory proposes that there exist built-in buffers
that prevent every mutation from being expressed as a
phenotype; if these buffers are removed, hidden genetic
variation within a population can be phenotypically
expressed (Horsthemke and Ludwig, 2005). ARTconceived
babies usually develop normally, although ART
bypasses many biological filters such as selective gamete
resorption, selective sperm uptake and sperm competition.
ART also introduces many environmental stresses to the
gametes and embryos such as exogenous hormones
during ovulation induction, chemical and pH fluctuations
in culture media, lab fluctuations in temperature,
humidity, light, and air quality. Evidence suggests that
maturational or environmental changes caused by ART,
both in the ovary and in the lab, have greater effects on the
oocyte than on the sperm (Niemitz and Feinberg, 2004).
Khosla et al. (2001) showed that serum addition to culture
media changed the methylation pattern of imprinted
genes in mouse embryos, and was related to a reduction in
fetal birth weight. It has been postulated that the absence
of the amino acid methionine in media may be involved in
the hypo-methylation of genes during the preimplantation
phase of mouse embryo culture (Niemitz and Feinberg,
2004), and Menezo (2006) stresses the importance of
including methionine in embryo culture medium at all
stages of development.
Follow up studies of ART-conceived babies are
reassuring, but many studies are difficult to interpret due
to small sample size, absence of correction for etiology of
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infertility or maternal age, and the reliance on selfreporting
surveys (Laprise, 2009). As a result, there
remains a lack of reliable data on relative and overall risk
of rare imprinting syndromes in ART-conceived children.
Further study is needed to detect subtle phenotypes
affecting health, growth, behavior, predisposition to
disease, all of which may be partially due to aberrant
imprinting. Imprinting may have a wider impact on
neurological development and behavior. Some reports
suggest parent-specific imprinting defects are involved in
childhood expression of autism, bipolar disorder,
schizophrenia, alcohol abuse and audiogenic seizures
(Paoloni-Giacobino and Chaillet, 2004).
In conclusion, it is clear that the extracellular
environment during critical periods of development is a
key mechanism underlying epigenetic modifications
(Thompson et al., 2002). In-vitro culture and manipulation
in the laboratory can alter oocyte and embryo cell
physiology by stress-induced cellular responses such as
production of reactive oxygen species and cytokines, and
can therefore lead to altered early gene expression
through epigenetic mechanisms. Given the strong but
circumstantial evidence linking an increased risk of
epigenetic syndromes to ART, long term follow up in welldefined
studies on children conceived from ART are
recommended.
References
Amor, D.J., Halliday, J. (2008) A review of known imprinting
syndromes and their association with assisted reproduction
technologies. Hum Reprod 23, 2826-2834.
DeBaun, M.R., Niemitz, E.L., Feinberg, A.P. (2003) Association of
in vitro fertilization with Beckwith-Wiedemann syndrome
and epigenetic alterations of LIT1 and H19. Am J Hum Genet
72, 156-160.
Grace, K.S., Sinclair, K.D. (2009) Assisted reproductive
technology, epigenetics, and long-term health: a
developmental time bomb still ticking. Semin Reprod Med 27,
409-416.
Horsthemke, B., Ludwig, M. (2005) Assisted reproduction: the
epigenetic perspective. Hum Reprod Update 11, 473-482.
Laprise, S.L., 2009, Implications of epigenetics and genomic
imprinting in assisted reproductive technologies. Mol Reprod
Dev 76, 1006-1018.
Luedi, P.P., Dietrich, F.S., Weidman, J.R., Bosko, J.M., Jirtle, R.L.,
Hartemink, A.J. (2007) Computational and experimental
identification of novel human imprinted genes. Genome Res
17, 1723-1730.
Menezo, Y. (2006) Paternal and maternal factors in
preimplantation embryogenesis: interaction with the
biochemical environment. Reprod BioMed Online 12, 616-621.
Niemitz, E.L., Feinberg, A.P. (2004) Epigenetics and assisted
reproductive technology: a call for investigation. Am J Hum
Genet 74, 599-609.
Paoloni-Giacobino, A., Chaillet, J.R. (2004) Genomic imprinting
and assisted reproduction. Reprod Health 1, 6.
Thompson, J.G., Kind, K.L., Roberts, C.T., Robertson, S.A.,
Robinson, J.S. (2002) Epigenetic risks related to assisted
reproductive technologies: short- and long-term
consequences for the health of children conceived through
assisted reproduction technology: more reason for caution
Hum Reprod 17, 2783-2786.
You can contact Sangita Jindal at: sangita.k.jindal@gmail.com
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Improving air quality in ART laboratories
by Giles Palmer
ESHRE Senior Clinical Embryologist, Director of Assisted Conception Unit, Mitera Hospital, Athens, Greece
Air in urban areas can contain high levels of
pollutants such as carbon monoxide, nitrous
oxide, sulphur dioxide, and heavy metals.
Indoors, construction materials, MDF, PVC flooring,
paints and adhesives are major sources of volatile organic
compounds (VOCs) leading to the “sick building
syndrome”. Other sources of indoor chemical hazards are
cleaning fluids, floor waxes, cosmetics and cigarette
smoke.
National health and safety authorities set safe limits of
VOC exposure for humans and give guidelines for
building ventilation-but there is little evidence in the IVF
literature of the toxicological effect of these on embryos
in vitro.
Pollutants can settle on work surfaces and dissolve in
aqueous solutions of embryo culture medium. Embryos,
lacking an immune system, cannot protect themselves
against these environmental contaminants.
Cohen et al. (1997) illustrated that high levels of
aldehydes and other noxious compounds are present not
only in the IVF laboratory (higher than outside air and the
average house), but also in the incubators. Controlling air
quality in an ART laboratory has shown beneficial effects
on fertilization and embryo development (Boone et al.,
1999).
In Europe the EU directive 2004/23/EC, which
stipulates quality requirements when Human tissue and
cell are handled (a critical point being clean air), has led to
most centers making, at the very least, slight structural
changes to their IVF units. The implementation of the
directive has created problems in the IVF industry, some
measures being contradictory to temperature control
associated with ART procedures, but do not mention
reduction of damaging compounds in the air such as
VOCs.
Laboratory design
If one is fortunate and can design an IVF facility from
the beginning, then the most important consideration
should be its location. The challenges of reducing air
pollution should be factored into your initial design
phase.
Control of the air quality may be hindered in an urban
area, or in units close to busy roads and car parks. Within
a hospital, difficulties may be encountered with working
in an area adjacent to the laundry, sterilizing or histology
departments.
If embryo development
and implantation depends
so highly on its culture
environment, the IVF
laboratory should do well
to follow the stringent
regulatory standards of the
food and pharmaceutical
industry.
Clean room technology
GILES PALMER
creates a carefully
controlled sealed environment in which the number of
particles and contamination is significantly minimized.
This is achieved using highly filtered air under positive
pressure being flushed through High Efficiency Particle
Air (HEPA) filters. All furniture, cleaning methods and
clothing must be appropriate for use in a clean room,
consistent with the level of air quality required.
The laboratory should have smooth, non-porous
walls, impervious unbroken surfaces with no difficult-toclean
corners and ledges, no sliding doors, sinks and
drains. The lighting should be within sealed units.
Partition wall systems specially designed for clean rooms
or made from a non-porous material such as Corian® are
expensive but provide an inert, hypoallergenic, easy-toclean
wall surface. Ducts and pipes can be hidden between
wall panels or covered within an alcove to prevent dust
accumulation.
Traditionally copper pipes have been used to transport
the gas from cylinder to incubator. Copper, however, is
prone to oxidation and therefore inert stainless steel
tubing, certified for use with medical gasses, is now
recommended.
Egg collection and embryo transfer involve
cooperation with operating theatre, and this can creates a
challenge for maintaining clean air conditions.
Hermetically-sealed doors and pass- through windows
may help these critical steps.
Changing rooms should be as clean as possible,
flushed with filtered air, and adjacent to the laboratory.
Positive pressure in the laboratory will be easier to
maintain and not require such a high velocity output if the
adjoining operating theater also has sealed doors and
ceiling.
Provision should be given to how bulky ART
equipment will access the unit after the construction
phase. A removable sealed section of wall allows not only
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ventilation in the final stages of building but an easy way
to bring large equipment such as incubators and laminar
flow cabinets into the completed laboratory space.
New equipment
Whenever possible, new incubators should be “run
in” in a room other than the laboratory for a certain time
period to dissipate latent VOCs produced in their
manufacture.
This may make the difference between a poor initial
pregnancy rate and a triumphant start to a new IVF unit!
How can an existing laboratory improve its air quality
Other than install a central air handling system that
includes activated carbon pre-filters and HEPA filters,
several commercial mobile filter units exists which clean
air quite efficiently- purifying the air of damaging volatile
compounds, bacteria and moulds (Forman et al., 2004). Air
is forced through the device and either passed through
carbon filters and/or ultra violet light for photo catalytic
purification.
Compressed gas can contain harmful compounds
such as benzene, isopropanol and pentane (Cohen et al.,
1997). Medical grade O 2 and CO 2 can be filtered before
entering the incubators by gas line filters. Because high
levels of VOCs have been shown to be present in
incubators, internal air filtering systems may be added to
decrease VOC levels inside incubators themselves. (Hall et
al., 1998., Mayer et al., 1999).
Anesthetic gasses used in oocytes retrieval may linger
in the air, and therefore a suitable extraction system may
be installed in the operation theatre.
Renovation and building work
Renovating an existing laboratory can introduce a
variety of compounds and emission of harmful
compounds used in finishing can have an adverse effect
on embryo development and pregnancy rate either
temporarily or long term! Paints, adhesives and sealants
can contain alkanes, aromatics, alcohols, aldehydes and
ketones, among others. Epoxy paints emit VOCs and take
several weeks to cure, and thus their use should be
avoided.
When renovating an older laboratory, there are
materials that can be used with less harmful chemicals:
Solvent-free adhesives used for floor covering are
available with low VOC emissions, as well as “ecological”
water based paints that contain non volatile chemicals.
On site supervision is essential to assure no materials
or methods are used that could jeopardize later IVF
success.
Removal of wooden furniture
To reduce VOC emissions from laboratory shelves,
cupboards and tables can be replaced with stainless
furniture and workbenches, as used in the pharmaceutical,
electronic, and food industries.
Access
Entry of unauthorized personnel, packaging, and
other materials into the laboratory should be restricted.
An air lock chamber before entering the laboratory offers
both a physical barrier and acts as an "air shower" if
flushed with filtered air.
Laboratory dress code
Staff can be one of the biggest contaminants in a clean
environment. Clean room clothing should provide
maximum control against microbial and particulate
contamination. Cotton hospital “greens” should be
replaced by pocket-less trouser suits, or two piece suits
with covered wrists and neck. Made specifically for clean
rooms, these polyester garments act as particle barriers
and shed no fibers into the environment.
Purging
Enough time to purge the room of harmful chemicals
should be allocated before laboratory start up. For at least
for one week, room-temperature and positive air pressure
should be increased to hasten the removal of VOCs
introduced during construction.
We renovated our laboratory in Athens in August
2009, following the above measures. The laboratory
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55

ARTICLES
CONTINUED FROM PAGE 55
premises were already fitted with activated-carbon pre
filters, HEPA filters, and in-line filters, but an upgrade to
a clean room GMP Class C (ISO class 7) was sanctified.
Completion took one month and VOC measurements
of the environment using ACS badges were measured
during each critical phase of reconstruction and the
following two weeks. The highest VOC levels were
observed in the adhesion of Corian© wall panels where
acetone reached a level of 15.5 ppm. Vinyl glue used in the
final stages of floor surfacing also indicated emissions of
hazardous VOCs.
One week of purging was performed after
completion. Two weeks after completion, no detectable
chemicals were present. Figure 1 illustrates the VOC
emissions in various phases before and after renovation.
Figure 1: Chemicals detected during renovation of the ART
laboratory.
better environment for the culture of oocytes and
embryos. It is up to every IVF facility to access the clean air
requirements and determine if changes need to be made.
Maintaining good air quality
To maintain the clean air environment, good
laboratory practice must be continued. Frequent
measurements of air flow, and servicing of equipment
safeguards against a drop in air quality due to exhausted
HEPA filters.
Positive pressure between the laboratory and adjacent
rooms can be monitored by the pressure differential
recorded daily to indicate filter efficiency.
Validation of air quality can be measured periodically
by particle counters, and VOC levels should be monitored
either electronically, or using inexpensive organic
chemical sensors that analyze absorbed ion levels on
chromatography paper which can measure workplace
exposure of toxic vapors.
Agar contact plates or commercially available kits are
used to count microbial load on surfaces. Establishing
bench marks for conventional in-vitro fertilization rate,
embryo quality, and pregnancy rates adds to your total
quality management.
References
Although not statistically significant, an improvement
of IVF outcome was observed after renovation of our
clinic in Athens (Table 1). This reassures us that the
reconstruction of the laboratory did not have a
detrimental effect on our assisted conception program,
and an adequate air filtration system was possibly already
in place. We are pleased, however, with our new working
environment and are satisfied that we have created a
W.R. Boone, J.E. Johnson, A. Locke, M.M. Crane, T.M. Price
(1997). Control of air quality in an assisted reproductive
technology laboratory. Fertil. Steril. 71, 150-154.
J. Cohen, A. Gilligan, W. Esposito, T. Schimmel, B. Dale (1997).
Ambient air and its potential effects on conception in vitro.
Hum. Reprod. 12, 1742-1749.
M. Forman, V. Polanski, A. Gilligan, D. Rieger. (2004) Reduction
in volatile organic compounds, adehydes, and particulate
air contaminants in an IVF laboratory by centralized and
stand alone air filtration systems. Fertil Steril. 82, Suppl.2. P-
535 (Abstract).
J. Hall, A. Gilligan, T. Scchimmel, M. Cecchi, J. Cohen (1998). The
origin, effects and control of air pollution in laboratories for
human embryo culture. Hum. Reprod. 13, 146-155.
J.F. Mayer, F. Nehchiri, V.M Weedon et al. (1999) Prospective
randomized crossover analysis of the impact of an IVF
incubator air filtration system (Coda. Gen X) on clinical
pregnancy rates. Fertil Steril. 72, Suppl. 1. S42.
You can contact Giles Palmer at: gpalmer@mitera.gr
Table 1: Pregnancy and implantation rates before (May to July) and after (September to November) renovation of the ART laboratory.
May June July September October November
No. Transfers 97 76 74 54 96 98
No. Positive β-HCG 37 (38%) 32 (42%) 24 (32%) 22 (41%) 55 (57%) 53 (54%)
Clinical Pregnancy rate 25 (26%) 18 (24%) 14 (19%) 15 (27%) 27 (28%) 37 (38%)
Implantation rate 39 (16%) 30 (17%) 19 (11%) 25 (18%) 53 (22%) 64 (24%)
56 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

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and to HELP our environment, giving you the new Coda Tower ®
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The ECO TM has a smaller footprint, using less laboratory space, while
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Our Coda ® Canister filters remove and reduce the levels of VOCs, (volatile
organic contaminants) and CACs (chemical air contaminants) while our 99.997%
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ARTICLES
The use of birefringence technology in
assisted human reproduction
by Simon J Phillips, MSc, BSc
OVO Fertility, 8000 Boulevard Decarie #100, Montréal, Québec H4P 2S4, Canada
Introduction
Polarization technology is capable of visualizing
highly ordered structures, since these structures split the
polarized light beam, change the plane of vibration, and
retard the polarised light. This effect is called
birefringence. Such highly ordered structures found in
gametes include the meiotic spindle, the inner and outer
layers of the zona pellucida, and sperm heads. The use of
birefringence technology in ART was first suggested by
Wang et al (2000), although the filamentous nature of the
spindle observed by birefringence was first noted by
Inoué (1953). By using alternating perpendicularorientated
polarising and analyser lenses, nonbirefringent
structures will remain dark. A birefringent
structure will become visible if the compensator optics are
rotated so that the polarised light that has been changed in
its plane of vibration by the object can pass through. The
limiting factor had been that conventional polarized light
microscopy can assess only relatively large differences in
changes of retardance and biological structures have
relatively little birefringence. Orientation-independent
polarizing microscopy (PolScope) uses circularly
polarized light and an electronically controlled liquid
crystal analyser to assess objects. In combination with a
CCD camera and specific software, this permits the user to
analyse small changes in the region of a few nanometres
associated with biological samples.
Using a PolScope, Wang et al (2000) assessed the
meiotic spindle of oocytes to ensure maturation prior to
ICSI being used. Since then, studies have been published
proposing the use of the PolScope to the morphology in
terms of the oocyte spindle and the zona pellucida.
Applications including the selection of embryos for
transfer, assuring the integrity of cryopreserved/thawed
oocytes for ICSI and assessing the location of the meiotic
spindle for ICSI have been suggested. (Cohen et al., 2004;
De Santis et al., 2005; Kilani et al., 2006). Retardance
measurements in addition to meiotic spindle length and
azimuth can be recorded using the PolScope, as well as
spindle location and physical evidence of a poorly formed
spindle. (Figure 1)
The following data are from two studies; prospective
data collection using the PolScope in stimulated IVF
cycles, and using the PolScope in controlled, natural cycle
IVF.
SIMON J PHILLIPS, MSC, BSC
Materials and Methods
Subjects
All patients presenting during the test period for
either stimulated or controlled natural cycle IVF and
having need of ICSI were included in the study.
Clinical Protocol
Our clinical protocol for controlled natural cycle IVF
has been described previously. (Phillips et al., 2007;
Kadoch et al., 2007). Ovarian stimulation for stimulated
IVF cycles was performed using standard GnRH agonist
long protocols, GnRH antagonist protocols, or GnRH
agonist short microdose protocols, depending on the
patient profile and the physician choice.
Laboratory Protocol
At the time of ICSI, all oocytes were assessed using the
Oosight (CRI, USA) to evaluate the position of the meiotic
spindle in the oocyte. ICSI was performed at 38-40 hours
post-hCG. In addition an image was taken using the
software associated with the Oosight which allows for
analysis of various measurement parameters of the
meiotic spindle once the oocyte has been returned to the
incubator.
Results
A total of 1112 metaphase II oocytes from stimulated
IVF cycles were assessed using the PolScope. In 81.6% of
oocytes, it was possible to visualize the spindle. The
pregnancy rate and clinical pregnancy rate were 51% and
40% respectively. (Table 1)
When looking at data from stimulated IVF cycles and
comparing those cycles resulting in a pregnancy with
cycles not resulting in a pregnancy, there were 68 pregnant
cycles and 66 non pregnant cycles. There were no
significant differences in the number of assessed oocytes
58 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

ARTICLES
Figure 1: Meiotic spindle (top left) at ICSI assessed using the Cri Oosight system. It is clear that the microfilaments of the spindle have
not formed correctly, increasing the chance of aneuploidy in the resulting embryo. Indeed this oocyte resulted in an embryo with 2
pronuclei but with a z-score of Z4. (Central image) Previous work (Scott et al 2000) associated an increased risk of aneuploidy with
Z4 pronuclei.
Table 1: Information for all cycles in stimulated cycles
sIVF
Number of cycles 134
Av. Patient age 34.4
Number of oocytes 1112
Proportion of oocytes with visualised spindle 81.6%
Pregnancy rate / cycle 51%
Clinical pregnancy rate / cycle 40%
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CONTINUED FROM PAGE 59
or age of patients. (Table 2) There was no difference
between the proportions of visualized spindles in the two
groups. (83.1% versus 79.7%)
Spindle measurements were compared between the
two groups and there was no significant difference
between the average retardance of spindles from
pregnant cycles versus non pregnant cycles for both all
oocytes and also when only the oocytes that resulted in
transferred embryos were compared. However, a trend
towards a difference was noted. (p=0.07, p=0.08
respectively). There was, however, a statistically
significant difference between the two groups for spindle
length. (p=0.02) (Table 3)
In addition the data from controlled natural IVF
cycles was collected; the retardance and spindle length
were compared with those from stimulated IVF cycles.
Forty-eight nIVF cycles were included from the same
study time period. There were no differences seen
between the nIVF and sIVF groups in either spindle
retardance or spindle length. (Table 4)
Discussion
We assessed a large number of oocytes and, in
addition, compared oocytes derived from controlled
natural cycle IVF as well as traditional stimulated IVF
cycles from which previous studies have performed their
evaluations. This allowed us to compare conception cycles
with non-conception cycles and verify whether meiotic
spindle measurements were predictive of outcome.
Our data suggest that the spindle length may be
indicative of oocyte competency because there was a
significant difference in spindle length between
conception cycles and non-conception cycles. This
confirms the findings of Ramu Raju et al (2007) whose
looked at embryo development relative to various spindle
parameters and found a correlation between spindle
length and blastocyst rate. However, Kilani et al (2009)
showed shorter spindle length in conception cycles than
in non-conception cycles. This apparent contradiction
may come down to a question of numbers, since their
study, although very well designed, only assessed 22
versus 30 oocytes, whereas we were able to look at over
1000 oocytes.
We saw no significant correlation between spindle
retardance was demonstrated between conception and
Table 2: Standard information of cycles with positive and those with negative pregnancy tests
sIVF Conception cycles Non-conception cycles
Cycles (n) 68 66
Oocytes (n) 620 492
Av. Age 33.5 35.3
Av. no. Eggs 9.1 7.5
Table 3: Comparison of spindle measurements between pregnant and non-pregnancy cycles
sIVF Conception cycles Non-conception cycles P
Av. Retardance -all eggs (sd) 2.11 (0.55) 2.03 (0.61) 0.066
Av. Retardance - transferred eggs (sd) 2.10 (0.57) 1.95 (0.59) 0.08
Av. Spindle Length (sd) 13.2 (1.93) 12.48 (2.06) 0.015
Table 4: Comparison of spindle measurements between stimulated and controlled natural IVF cycles
nIVF sIVF P
Av. Retardance - transferred eggs (sd) 1.8 (0.64) 2.0 (0.58) NS
Av. Spindle length (sd) 12.7 (1.95) 12.6 (2.16) NS
60 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

ARTICLES
non-conception cycles, again in contradiction of Kilani et
al. (2009). However, we did see a trend towards increased
spindle retardance in the conception cycles. Furthermore,
the range of retardance values seen for spindles is small
and there is some overlap between the groups, so that it
may be difficult to clearly define limits for ‘good’ and
‘poor’ spindle retardance. In our data, a value of
approximately 2.0 seemed to represent a shift between
these two groups, however other published data have
shown higher values attributable to ‘good’ spindle
retardance. This may be related to system set-up and
therefore may mean that intra-laboratory retardance
values may be of limited value.
No differences were seen between parameters
evaluated in controlled natural cycle IVF derived oocytes
and traditional IVF cycles with ovarian stimulation
derived oocytes. Insufficient numbers in the nIVF group
prevented us from analyzing conception versus nonconception
cycles in this particularly interesting group of
oocytes. These data are currently being prospectively
collected for future analysis.
To date there is increasing evidence of the interest and
possible application of birefringence technology with ART.
However, further research is still required to quantify
those applications.
As research develops with birefringence technology it
may become a more widely used indicator of oocyte
quality and embryo selection in ART cycles, especially
since it is, importantly, a non-invasive method of
assessment. A recent review of this technology proposed
that it may be a useful tool for aneuploidy research in
oocytes. (Shen et al., 2008) The report of Gianaroli et al
(2008) has suggested that birefringence technology may be
useful in sperm selection for ICSI, and further research is
required to confirm potential benefits to this approach as
well as to explain the reason behind any benefit.
Suggestions that sperm head birefringence could be
related to sperm DNA damage certainly warrant further
investigation. (Damasceno et al., 2008)
References
Cohen Y, Malcov T, Schwartz N et al. (2004) Spindle imaging: a
new marker for optimal timing of ICSI Human Reproduction
19, 649-654.
De Santis L, Cino I, Rabellotti E, et al. (2005) Polar body
morphology and spindle imaging as predictors of oocyte
quality. Reproductive Biomedicine Online. 11, 36-42.
Damasceno-Vieira A, Silva C, Ying Y et al. (2008) A non-invasive
method to assess DNA damage in individual sperm. Fertility
and Sterility 90, Suppl., S11 (abstract O-30).
Gianaroli L, Magli MC, Collodel et al. (2008) Sperm head’s
birefringence: a new criterion for sperm selection. Fertility
and Sterility. 90, 104-112
Gianaroli L, Magli MC Ferraretti AP et al. (2008) Birefringence
characteristics in sperm heads allow for the selection of
reacted spermatozoa for intracytoplasmic sperm injection.
Fertility and Sterility. 2008. Epub ahead of print.
Inoué S. 1953. Polarisation optical studies of the mitotic spindle.
1. The demonstration of spindle fibers in living cells.
Chromosoma 5, 487-500.
Kadoch IJ, Al-Khaduri M, Phillips SJ, Lapensee L, Couturier B,
Hemmings R, Bissonnette F. (2007) Spontaneous Ovulation
Rate before Oocyte Retrieval in Controlled Natural Cycle In
Vitro Fertilisation (nIVF) with and without Indomethacin.
Reproductive Biomedicine Online. 16, 245-249.
Kettel LM, Roseff SJ, Chiu TC et al (1991) Follicular arrest during
the midfollicular phase of the menstrual cycle; a
gonadotropin-releasing hormone antagonist imposed
follicular-follicular transition. The Journal of clinical
endocrinology & metabolism. 73, 644-649.
Kilani SS, Cooke S, Kan AK, Chapman MG. (2006) Do age and
extended culture affect the architecture of the zona pellucida
of human oocytes and embryos Zygote. 14, 39-44.
Kilani S, Cooke S, Kan A, Chapman M. (2009) Are there noninvasive
markers in human oocytes that can predict
pregnancy outcome Reproductive Biomedicine Online. 18,
674-680.
Phillips SJ, Kadoch IJ, Lapensée L, et al (2007) Controlled natural
cycle IVF: our experience in a world of stimulation.
Reproductive Biomedicine Online. 14, 356-359.
Rama Raju GA. (2007) Meiotic spindle and zona pellucida
characteristics as predictors of embryonic development: a
preliminary study using PolScope imaging. Reproductive
Biomedicine Online. 149,166-174.
Rongières-Bertrand C, Olivennes F, Righini C et al (1999) Revival
of the natural cycles in in-vitro fertilization with the use of a
new gonadotrophin-releasing hormone antagonist
(Cetrorelix): a pilot study with minimal stimulation. Human
Reproduction. 14, 683-688.
Shen L, Betzendahl L, Tinneberg HR, Eichenlaub-Ritter U. (2008)
Enhanced polarizing microscopy as a new tool in
aneuploidy research in oocytes. Genetic Toxicology and
Environmental Mutagenesis. 651, 131-140.
Wang WH, Meng L, Hackett RJ et al. (2001) The spindle
observation and its relationship with fertilisation after
intracytoplasmic sperm injection in living human oocytes.
Fertility and Sterility. 75, 348-353.
You can contact Simon J Phillips at: s.phillips@cliniqueovo.com
WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
61

• Proven Results
• Over 20 independent studies with published results
• Stringent Quality Control
Figure 1. Development of human embryos cultured in global ® from Day 1 to Day
3 and Day 3 to Day 5, or in G1 from Day 1 to Day 3 and then in G2 from Day 3 to Day
5. The proportion of embryos reaching the blastocyst stage by Day 5 was significantly
great in global ® . The pregnancy and implantation rates were significantly greater
for embroys cultures in global ® than for those cultured in G/G2. (Zech
et al., Human Reprod. 21, Suppl. 1, i162, 2006)
Figure 2. Development of human embryos cultured in global ® from Day 1 to Day 3
and Day 3 to Day 5, or in BAS1 from Day 1 to Day 3 and then in BAS2 from Day 3 to
Day 5. The proportion of embryos having 6 or more cells on Day 3 was significantly
greater in global ® . The blastocyst rate on Day 5 and the pregnancy rate were not
different between media treatments (Matsubara et al., Proc. 24th Ann. Meet. Japan
Soc. Fert. Implant. 206, 2006)
Figure 3. Development of human embryos cultured in global® from Day 1 to Day 3
and Day 3 to Day 5, or in G1 from Day 1 to Day 3 and then in G2 from Day 3 to
Day 5. The proportion of embryos reaching the blastocyst state by Day 5 was
significantly great in global®. The pregnancy rates were not different between the
culture media treatments. (Angus et al., Fert. Steril. 86 Suppl 2, S229, 2006)
Figure 4. Development of human embryos cultured in global® from Day 1 to Day 3
and Day 3 to Day 5, or in ECM from Day 1 to Day 3 and then in Multiblast from
Day 3 to Day 5. The proportion of embryos ³ 6 cells on Day 3, and at the
blastocyst by Day 5, were significantly great in global®. The clinical pregnancy
rate was not different between the culture groups. (Sepulveda et al., 2008)
Figure 5. Development of human embryos cultured in global® from Day 1 to Day 3
and Day 3 to Day 5, or in Cleavage from Day 1 to Day 3 and then in Blastocyst
from Day 3 to Day 5. The proportion of embryos > 6 cells on Day 3, and at the
blastocyst by Day 5-6, were significantly greater in global®. The clinical pregnancy
rate was not different between the culture groups. (Carrilo & Yalcinkaya, 2008)
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Protect Embryos
…Embryos do not have an “immune system”
• VOCs can have significant detrimental effects on embryos.
• VOCs are too small to be trapped by HEPA filters.
• Coda ® filters are common-sense safety devices that serve to protect embryos from
unforeseen Volatile Organic Contaminants (VOCs) and other contaminants.
Common VOCs found inside incubators, such as, Benzene,
Acetone, Ethanol, and Formaldehyde are 100 to 1000 times
smaller than the pore size of the HEPA filter.
Air contaminants present in research and clinical
laboratories interact with specimens, samples,
tissues, media and oils. Studies show that Chemical
Air Contaminants (CACs) and Volatile Organic
Contaminants (VOCs) introduced from many
sources may seriously distort your results. The
Coda ® Air and Gas Filtration System can maximize
the air quality of your incubators and laboratories by
removing up to 99.97% of these contaminants. The
following table shows the filtration power of the
Coda ® System compared to existing HVAC systems
and HEPA filters used with your equipment. HEPA
filters are designed to stop materials down to 0.3
microns.
μg/m 3
25
20
15
10
5
0
Outside air
Return air
Hallways
Procedure
Room
Laminar
flow units
Incubators
Benzene
Toluene
Xylenes
Styrene
Volatile Organic Contaminants in Air Outside and Inside an ART Laboratory
(Cohen et al., Human Reprod. 12, 1742-1749, 1997)
HEPA filters are designed to stop materials down to
0.3 microns. Unfortunately many of the gases found in
IVF labs; benzene, acetone, ethanol, formaldehyde, etc;
are 100 to 1000 times smaller than the pore size of the
HEPA filter. If improving results is your goal, then Coda ®
is the solution. For more information call IVFonline or
visit our website at www.IVFonline.com.
FDA 510(k) Cleared ISO13485:2003 ISO9001:2000
Laboratories
Gas Lines
Incubators

ARTICLES
The identification of a toxic substance in
the in vitro fertilization laboratory: the
value of inter-laboratory communication
by Tom Turner, MS ELD (ABB), Director of Embryology
Austin IVF / Texas Fertility Center, Austin, TX, USA
The quick identification and elimination of toxic
substances within the IVF laboratory is critical for the
growth of quality embryos. In spite of having a wellconstructed
lab and using tested products, unknown
substances can enter the culture system from a variety of
sources and are difficult to pinpoint and eliminate. These
toxins can have a negative impact on embryo
development and lower patients' chances for pregnancy.
We have a well-established, successful IVF clinic that
has been in operation since 1984. In January 2007, we
moved our IVF laboratory into a new facility that was
constructed with all of the current knowledge about A/C,
air filtration, construction materials, and laboratory
techniques.
The culture system used in our IVF laboratory
consists of triple gas incubators that are 1-4 years old.
These are maintained properly and checked daily for
temperature, CO2, and O2. The pH of the equilibrated
culture media is checked weekly. Medical grade gases are
used, and the media are of a sequential system from a
well-known, respected company offering many IVF
products. Oil is purchased from the same company that
provides the culture media.
Notably, after fertilization, the embryos are cultured
individually in 25 microliter drops over-layered with
11 ml of oil, within 60 mm Petri dishes.
All of the plastic products used in the laboratory are
mouse embryo tested before they are used in the IVF lab.
They are allowed to aerate while out of their package
before they are used.
The first observations of deteriorating quality of
embryo development occurred in sporadic cases in
November. We observed that day-3 embryos appeared
healthy, while most day 5 blastocysts were still
developing, but not sufficiently developed to freeze. Some
of the embryos that appeared healthy, but not sufficiently
developed to freeze on day 5, had degenerated by day 6.
To clarify, degeneration was the death of all or most of the
cells in the blastocyst. Our concern with these
observations led to a review of our techniques of
blastocyst handling, the embryologists involved,
incubators used, and the
aeration time of our
polystyrene dishes. No
cause was identified.
Some improvement in
blast development was seen
in early December, with no
obvious reason. But by mid
December, we began seeing
more degeneration of
blastocysts on day 6. There
was also a decrease in the
TOM
percent of patients who had
TURNER, MS ELS (ABB)
blastocysts left after transfer that were suitable to freeze.
Since we had a laboratory shutdown scheduled for
late December, we examined all of the options for change
and improvement that we could accomplish during the
month-long break.
During the scheduled shutdown, all of the potassium
permanganate, carbon, and HEPA filters in the laboratory
A/C system were changed. A thorough cleaning,
sterilization and calibration of each piece of equipment
that heated or provided an atmosphere was undertaken.
We ran mouse embryo assays (MEA) on each incubator
and on all of the plastics used in the andrology lab and in
the IVF lab. All of the equipment and the supplies passed
the mouse embryo assay. The lab staff reviewed all
protocols and discussed possible causes of the
deterioration in embryo development.
Patient retrievals began in January with high hopes
that the problem had been eliminated. Immediately, there
appeared the same sporadic degeneration of embryos
from day 5 to day 6.
The manufacturer of our culture medium was
contacted to inform them of the problems that we were
seeing. Because we were told that no other lab had
reported problems to them, we assumed the problem was
internal. The company had supplied our lab with quality
culture medium for many years and we receive a
certificate of analysis with each product, so it was easy to
look internally for our problems.
64 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

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At this point, we talked to embryologists in other parts
of the country who were using the same products. We
found that other programs were having similar problems
that they had yet to identify. Some suspected the medium
or protein. These conversations led to a change of our
blastocyst medium to one that was compatible with the
other products we were using. When there was no
improvement, we replaced all of our sequential media and
protein with products from the second company.
Because numerous emails and phone conversations
with the initial company failed to offer solutions, we again
began networking with other embryologists. At the same
time, the use of EmbryoMail or ivf.net was not used
because we did not want the company to be criticized by
individuals who had an agenda for another company.
Continuation of degenerate blastocysts coupled with
poor re-expansion or degeneration of thawed blastocysts,
led us to suspect that our oil might be the problem.
Immediately, we replaced the oil from the original
company and began using oil from the new company for
all of our human embryo culture.
We also ran two MEA’s with medium from the new
company and each type of oil, deciding to grow the mouse
embryos to day 6. This was because the growth from day
5 to day 6 is where we saw the greatest degeneration.
The results were definitive. All two-celled mouse
embryos cultured in the new company’s medium overlayered
with the new company’s oil made hatched
blastocysts by day 6. All mouse embryos cultured with the
new company’s medium over-layered with the original
company's oil died by day 6.
Meanwhile, no human blastocysts showed signs of
degeneration while grown in the new medium and the
new oil. The original company was notified of our
findings.
The identification of the toxic oil that was present in
our lab took much time and effort by the embryologists
and at the expense of human blastocysts. The method to
identify it was painfully slow.
What we learned was;
1. Other labs using the same oil had a similar experience.
2. Labs using the same oil that did not have the same
experience were washing their oil or were growing
embryos in 4-wells with larger volumes of medium
and smaller volumes of oil overlay.
3. Finally, we learned the importance of communicating
with other IVF labs and the need for an electronic
forum for discussion of similar problems without
worrying about the expression of commercial
agendas.
You can contact Tom Turner at: tom@austinivf.com
WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
65

ARTICLES
Clinical Implementation of the Halosperm ® Test Kit in
Combination with SCA ®
by Kellie Williams, PhD and J. Kevin Thibodeaux, PhD, HCLD
Tulsa Fertility Center, 115 East 15th St., Tulsa, OK 74119
Introduction
Efforts to further evaluate the sub-fertile male beyond
less objective parameters such as sperm count, motility
and morphology, have focused on the estimation of sperm
DNA damage. Chromatin within the spermatozoa consists
of DNA, RNA and nuclear proteins. These
macromolecules undergo various changes to achieve
different levels of compaction corresponding to the stages
of spermatogenesis and spermiogenesis. The complexity
of sperm chromatin lends itself to an eminent potential for
abnormal chromatin formation and damage to DNA
and/or nuclear proteins. Sperm DNA fragmentation may
occur as a result of six main mechanisms: (1) apoptosis
during spermatogenesis; (2) strand breaks during
chromatin remodeling stages of spermiogenesis;
(3) fragmentation produced by oxygen radicals present in
the seminiferous tubules and the epididymis;
(4) fragmentation induced by sperm caspases and
endonucleases; (5) damage resulting from chemotherapy
and radiotherapy; and (6) damage induced by
environmental toxicants (Sakkas and Alvarez, 2010).
Normal morphology, count and motility may not
necessarily indicate the genomic quality of a fertilizing
sperm. Many mechanisms function to allow only those
sperm with no genetic aberrations to fertilize an oocyte
and initiate embryonic development. Assisted
reproductive technologies, including in-vitro fertilization
(IVF) and intracytoplasmic sperm injection (ICSI), bypass
these checkpoints, and sperm containing chromatin
structure anomalies may be introduced. Research over the
past fifteen years demonstrates a link between damaged
sperm DNA and male sub-fertility, and establishes the
following statistical categories: ≤15% DNA fragmentation
index (DFI) for excellent fertility; >15% to

ARTICLES
sperm with low levels of DNA fragmentation release DNA
loops in an area surrounding the sperm head forming
large halos (Fernandez et al., 2005a). The SCA ® software is
designed to generate DNA fragmentation data from a
specified particle area of stained halos within a captured
image. The diagnostic power of the DNA fragmentation
test is derived from classification of patient test results into
three statistical categories described above (Evenson et al.,
1999).
Our initial control sample was prepared on 11/2008
and was analyzed in parallel on each test day and it
consistently demonstrated DNA fragmentation
repeatability as illustrated in Figure 1. The mean of the
control sample was 50.8% with a coefficient of variation
(CV) of 5.1%. Of the 204 individual patients sampled, the
range of DNA fragmentation was 2 to 100% (Figure 2). The
distribution of patient test values into the three categories
showed 20.1% of patients analyzed with ≤15% DFI
indicating excellent fertility, 22.6% with >15% to

ARTICLES
CONTINUED FROM PAGE 67
References
Evenson DP, Josk LK, Marshall D, Zinaman MJ, Clegg E, Purvis
K, de Angelis P, Claussen OP. (1999) Utility of the sperm
chromatin structure assay (SCSA) as a diagnostic and
prognostic tool in the human fertility clinic. Hum Reprod
14:1039-1049.
Fernandez JL, Muriel L, Rivero MT, Goyanes V, Vazquez R,
Alvarez JG. (2003) The sperm chromatin dispersion test: a
simple method for the determination of sperm DNA
fragmentation. J Androl 24:59-66.
Fernandez JL, Muriel L, Goyanes V, Segrelles E, Gosalvez J,
Encisco M, LaFromboise M, De Jonge C. (2005a) Simple
determination of human sperm DNA fragmentation with an
improved sperm chromatin dispersion test. Fertil Steril
84:833-842.
Fernandez JL, Muriel L, Goyanes V, Segrelles E, Gosalvez J,
Enciso M, LaFromboise M, De Jonge C. (2005b) Halosperm
is an easy, available, and cost-effective alternatie for
determining sperm DNA fragmentation. Fertil Steril 84:860.
Sakkas D, Alvarez JG. (2010) Sperm DNA fragmentation:
mechanisms of origin, impact on reproductive outcome, and
analysis. Fertil Steril 93:1027-1036.
Schlegel PN, Paduch DA. (2005) Yet another test of sperm
chromatin structure. Fertil Steril 84:854-859.
You can contact Kellie Williams at: kwilliams@obgynmail.com
Figure 2: Individual patient results for DNA fragmentation testing.
100
90
80
Percent Fragmented DNA
70
60
50
40
30
20
10
0
1
6111621263136414651566166717681869196
101
106111
116121126131136
141146
151156161166171
176181186191196
201
Sequential Patient Results
Figure 3: Distribution of patient test results.
100
90
80
70
Pecent of Patients
60
50
40
30
20
10
0
30%
Percent Fragmented DNA
68 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

ARTICLES
A Prospective Randomized Comparison of global ® Medium with Sequential Media
for Culture of Human Embryos to the Blastocyst Stage
In a recent report, Sepulveda et al. (2009) compared Global medium with ECM/Multiblast
sequential media for the culture of human embryos from the zygote to the blastocyst stage.
Oocytes were collected from 47 oocyte donors and fertilized with sperm of the partners of
80 patients who had been randomly assigned to have their embryos cultured in either
Global (N = 287 zygotes) or ECM/Multiblast (N = 322 zygotes). The zygotes were cultured
from Day 1 to Day 3 in droplets of Global and then in fresh droplets of Global from Day 3
to Day 5 or 6, or in droplets of ECM from Day 1 to 3 and then in droplets of Multiblast from
Day 3 to Day 5 or 6.
On Day 2, cell number was significantly greater (P = 0.022) and multinucleation was
DON
significantly less (P = 0.020) for embryos cultured in Global than for those cultured in
RIEGER, PHD
ECM/Multiblast. As shown in Figure 1, the proportion of zygotes that developed to ≥ 6 cells on Day 3, compacted on Day
3, morula on Day 4, blastocyst on Day 5 and full to hatching blastocyst on Day 5 were significantly greater in Global than
in ECM/Multiblast. There were no other significant differences in developmental parameters between the culture medium
treatments.
On Day 5 or 6, the best (usually two) embryos in each cohort were transferred to 40 patients in the Global group and to
38 patients in the ECM/Multiblast group (mean of 2.0 embryos/transfer in both groups). As shown in Figure 2, there were
no significant differences in pregnancy rates, as measured by being positive for hCG or for fetal heart beats (FHB),
between the culture treatments. However, the implantation rate, as measured by FHB, was significantly greater for
embryos cultured in Global than for those cultured in ECM/Multiblast.
The authors conclude that “A single medium was as good as or better than a sequential media system for human embryo
culture from the zygote to blastocyst stage.” and that “The idea that a sequential media system is required for optimal
development of the human embryo to the blastocyst stage is highly questionable.”
Figure 1: Percent of zygotes developing to the given stages on Days
3, 4, and 5 of culture
Figure 2: Pregnancy and implantation rates
Reference: Sepulveda S, Garcia J, Arriaga E, Noriega PL, Noriega HL (2009) In vitro development and pregnancy
outcomes for human embryos cultured in either a single medium or in a sequential media system. Fertil
Steril 91, 1765-70.
(Text and figures by D. Rieger, LifeGlobal LLC)
WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
69

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PATIENT’S CORNER
“Morning Sickness” – An overview
by Michele Brown, MD, FACOG
Michele Brown, M.D. FACOG A practicing obstetrician of 26 years and a founder of Beaute
de Maman, a line of personal care products for pregnant women.
You can contact Michele Brown, MD at: mbrownmd54@gmail.com
Nausea and vomiting often referred to as ‘morning
sickness’ are common and extremely distressing
symptoms of pregnancy. The term “morning
sickness” is a misnomer as most women suffer from theses
symptoms throughout the day.
Women often experience some nausea in the early
stages of pregnancy. In most cases the symptoms are mild
transient and easily managed by dietary modifications. In
some cases however, the symptoms may be severe.
Relentless vomiting can last the entire pregnancy and be
associated with dehydration and weight loss.
Even in the milder cases nausea and vomiting can
affect the pregnant woman’s outlook on the pregnancy,
lead to significant distress, and interfere with the
nutritional requirements.
Some important facts about nausea and vomiting in
pregnancy
• Incidence: Nausea and vomiting of pregnancy are
extremely common and occur in 50 to 80% of pregnant
women.
• Time of occurrence: In most cases symptoms usually
appear by the 5th week and disappear by the 13th
week of pregnancy. Severity peaks between 11 and 13
weeks. However, in 20% of women, nausea and
vomiting can persist throughout pregnancy.
• The Causes of nausea and vomiting in pregnancy are
largely unknown. However, the following
associations have been noted.
1. Conditions in which pregnancy hormone levels
are high (twin pregnancy, molar pregnancy) are
associated with a higher incidence of nausea and
vomiting. On the other hand, women whose
pregnancy hormones are low (smokers) have a
lower incidence of nausea and vomiting.
2. Women taking prenatal vitamins are less likely to
have severe nausea and vomiting.
3. Psychological causes and transformation of a
mental disorder into physical symptoms
(psychosomatic conditions) have little supporting
evidence as the cause of nausea and vomiting.
However, nausea and vomiting especially in their
severe forms can cause severe psychological
MICHELE BROWN, MD, FACOG
distress, which in turn may worsen the condition.
4. Genetic susceptibility: A history of hyperemesis is
found in other family members suggesting a
genetic link.
5. History of nausea and vomiting in a previous
pregnancy suggesting individual susceptibility.
6. History of migraine headaches is linked to nausea
and vomiting.
7. Women on their first pregnancy are less likely to
develop hyperemesis as compared to subsequent
pregnancies.
8. Nausea and vomiting are more commonly found
when the interval between pregnancies is short.
Hyperemesis Gravidarum:
An extreme form of this common symptom. In 1 to 3%
nausea and vomiting are severe leading to dehydration
and electrolyte imbalance and weight loss occasionally
requiring hospitalization.
In addition to the severe psychological impact of
hyperemesis, it can also lead to serious pregnancy
complications or death;
• Rupture of the esophagus and bleeding from ruptured
blood vessels.
• Wernicke’s encephalopathy-a rare but serious brain
disorder associated with Vitamin B1 deficiency and
leading to memory loss, visual disturbances, and gait
disturbances.
• Low birth weight as a result of malnutrition.
• Pregnancy termination due to severe psychological
distress and depression.
CONTINUED ON PAGE 74
72 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

PATIENT’S CORNER
CONTINUED FROM PAGE 72
Treatment of nausea and vomiting in pregnancy:
In most cases the purpose of the treatment is to
improve the pregnant woman’s quality of life. In cases of
hyperemesis treatment is essential and can be lifesaving.
No single specific treatment is effective in all women.
A carefully tailored multidisciplinary approach which
includes dietary modifications, herbal remedies and
medication should be adjusted to the woman's needs.
Emotional support is extremely valuable.
Dietary Suggestions
• Avoid foods and odors that might trigger the
symptoms.
• Eat bland high protein and carbohydrate foods and
snacks—avoid fatty, spicy foods, short frequent meals
help. Solid starches such as potatoes, rice, and pasta
are recommended.
• Stop all iron tablets.
• Avoid dehydration (1.0 to 1.5 liter) by drinking sports
drinks and bouillon which contain salt, glucose, and
potassium. Ginger ale is another traditional remedy.
Advance to brothy soups with noodles or rice. Avoid
cream based soups because of fat content.
are severely dehydrated. In addition to proper
hydration, intravenous medications are used (Reglan,
Phenergan, Dramamine, and Zofran).
Tigan suppositories can also be of value in women
who cannot tolerate oral medications.
Steroid therapy has been reported to be successful in
some resistant cases but should be avoided if possible
due to the associated risk of fetal malformation (cleft
palate) when given within the first 10 weeks of
pregnancy.
• Vitamin B1 therapy: All women who have been
vomiting for 3 weeks and require IV hydration should
be given supplemental vitamin B1 to prevent
Wernicke's encephalopathy.
In summary: Nausea and vomiting of pregnancy is
extremely common and a normal part of a healthy
pregnancy in most cases. In severe cases, hospitalization
may be necessary along with lost time from work, and
multiple visits to your obstetrician. There are multiple
theories and mechanisms that can cause this problem.
Herbal and natural remedies
• Vitamin B6 (pyridoxine 10-25 mg taken 3 or 4 times a
day) is used due to its anti-emetic properties. An
antihistamine (doxylamine sold as over the counter as
Unisom sleep tabs 1/2 tab) can be added to the
pyridoxine as first line therapy (10 mg of each).
• Ginger tablets (250 mg given 3 or 4 times a day or
powdered ginger extract 1 gram/day).
• Acupuncture, acupressure (wristbands), and
hypnosis.
Drug Therapy
In most cases the condition can be managed on an
outpatient basis with close follow up by the obstetrician to
ensure proper hydration and nutrition.
• Drugs that are administered on an outpatient basis fall
into the following categories 1. Antiemetic drugs. e.g.
Zofran, Tigan, Reglan, 2. Phenothiazines e.g.
Promethazine, 3. Anti-histamines and 4. Steroids.
• IV fluid therapy, intravenous nutrition and
hospitalization may be required for those women who
74 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

Vitamin D Deficiency and Pregnancy
by Michele Brown, MD, FACOG
PATIENT’S CORNER
What is Vitamin D
Vitamin D is a fat-soluble vitamin that plays a central
role in calcium and phosphorous metabolism, which is
critical for bone formation and maintenance.
Why is Vitamin D important
Years ago, deficiency of this vitamin resulted in
rickets. New studies have demonstrated a resurfacing of
Vitamin D deficiency worldwide. This deficiency is
especially critical in the pregnant and lactating woman.
Studies have shown how important Vitamin D is to
skeletal, cardiovascular, and neurological development in
the infant. Other studies have shown Vitamin D deficiency
to be linked to diabetes, asthma, and schizophrenia in
children. Infants born to mothers with Vitamin D
deficiency had poor growth, and defects in enamel
formation of teeth. A newborn’s Vitamin D level is
completely dependent on maternal levels.
Vitamin D is believed to be critical in placental
development and function, which may be associated with
other complications during pregnancy including
miscarriage, preeclampsia, and preterm birth. Low
Vitamin D levels have been linked to bacterial infections in
the vagina in the first trimester of pregnancy. This can
increase the risk of preterm birth and adverse pregnancy
outcomes. One study found a four-fold greater risk of
cesarean section for women with low Vitamin D levels.
This may be due to the fact that skeletal muscle contains
Vitamin D receptors and deficiency can result in muscle
weakness and poor strength in labor. Vitamin D also
regulates calcium levels and, with deficiency, muscle
strength is lowered in labor.
Where do we get Vitamin D
Exposure to sunlight – An inactive form of Vitamin D
is synthesized in the skin by exposure to ultraviolet light,
and the inactive form is converted to the active form by the
liver and the kidney.
Diet – Very few food sources naturally contain
vitamin D. Examples are oily fish such as salmon,
sardines, mackerel, and tuna along with egg yolks, and
fish liver oils. Foods such as milk, orange juice, some
cereals, yogurt, cheese, and butter are often fortified with
Vitamin D.
Supplements – Over-the-counter supplements are
precursors to Vitamin D, which get converted in the body.
What is considered to be a Vitamin D deficiency
Deficiency is defined as serum levels of less than 20
mg/ml of 25-hydroxy Vitamin D. Levels between 20 and
30 mg/ml indicate insufficiency, and anything above 30
mg/ml is considered normal. Toxic levels are over 150
mg;/ml and are exceedingly rare.
Deficiencies appear to be more common among
African-Americans, due to high levels of melanin which
blocks light from entering the skin. In addition, levels are
lower in the winter months (November through March)
when less sunlight reaches the earth. Levels are low in
people living above 30 degrees latitude, in cultures where
the skin is covered (e.g. Arab countries), and in cultures
where people avoid sunlight and use sunscreen.
Certain medical conditions make one more prone to
deficiency such as individuals with bowel absorption
problems, people with renal disease, obese individuals,
vegetarians, people with lactose intolerance, and people
on certain medications, including anticonvulsants.
What are the requirements for vitamin D in pregnant
and lactating women
Current research has shown that pregnant women are
at high risk of Vitamin D insufficiency and prenatal
vitamins are inadequate to meet these demands. Current
vitamin preparations have approximately 200 to 400 IU of
Vitamin D. Experts recommend 1400 to 2000 IU of Vitamin
D per day during pregnancy. This can be accomplished
with regular prenatal vitamins in addition to another
supplement. It has been suggested that breastfeeding
women, whose infants only get vitamin D from breast
milk, need to ingest 4000 to 6000 IU of Vitamin D per day.
Serum Vitamin D levels should be determined at the
first prenatal visit along with the other prenatal blood
work. If women are Vitamin D deficient, they should be
treated with 2000 IU of vitamin D in addition to their
prenatal vitamins for 1-2 months and the blood test
repeated to confirm that the serum level is above
30 mg/ml. With continued supplementation of 1000 IU of
Vitamin D per day, levels should be sufficient for the
remainder of the pregnancy. Borderline individuals might
need to be tested again during the third trimester.
Ultraviolet light exposure can also increase the
vitamin D content of human milk.
WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
75

PATIENT’S CORNER
Too Much of a Good Thing
by Courtney Sirotin
Atlanta, GA, USA
Have you ever wished your period would just
magically go away For some women in their
child-bearing years, it does. As good as that may
sound on the days that you’re doubled over in pain with a
heating pad, losing your period is a lonely, terrifying
experience with repercussions ranging from osteoporosis
to infertility. I speak from experience because it happened
to me.
My husband and I waited awhile before trying to have
a baby. I was thirty years old when he finally said the
magic words, “I think I'm ready.” I was overjoyed and
went off my birth control pills immediately. I had heard
that a woman is extra fertile the month after going off The
Pill, so I quickly adapted the habits of a pregnant woman,
popping prenatal vitamins and giving up caffeine, in
anticipation of a positive pregnancy test. When I didn’t get
pregnant that next month, and when I didn’t have a
period, I chalked it up to having just gone off the birth
control pills that I had been taking for seven years. I had
read that some women have irregular cycles for awhile
after going off the pill, so I wasn't overly concerned.
For the next few months, I continued to live my life as
usual. In the previous two years, my husband and I had
moved to a new state, and I stayed at home to pursue a
career in writing. I had a lot of time on my hands and
without even being conscious of it I had taken what was
once a healthy habit, and turned it into an unhealthy
extreme. I’m talking about exercise.
I've always worked out, but never more than an hour
a day. When we moved and my free time increased, I
naturally started filling it up with more activity. At first I
just added strength training to my regimen, something I
had done before but only half-heartedly. Then I got a
subscription to a fitness magazine and learned about
things like intervals and plyometrics. Every time I learned
something new about fitness, a new move or a new style
of exercise, I just added it onto what I was already doing
each day, rather than spreading things out over the course
of a week or a month. It got to the point where I was
working out hard for hours and hours each day and rarely,
if ever, giving myself a day off to recover.
Some days I would wake up and go for a seven mile
run, come home and lift weights for an hour or two,
shower, eat a bowl of cottage cheese with strawberries,
and then do some writing until my husband came home
from work, at which time we would work out together. At
night I might go for another run and do intervals, maybe
COURTNEY SIROTIN
plyometrics, maybe lift more weights, maybe all three. The
more I worked out, the more obsessed I became. I created
a website and blogged about fitness and even wrote a
book on the topic. At that point I had also become a fitness
trainer and spent the few hours I was not working out on
my own, working out along side my clients, at times
pushing them to do more than they were ready for
because I wanted to go faster or work harder alongside
them.
I never consciously restricted my eating. I’ve always
eaten two thousand calories or more each day, but even
three thousand or four thousand may not have been
enough to fuel my body when I was at my extreme.
Needless to say I was thin. This was a reward
considering I have never been svelte by nature. Growing
up I was a thick, chunky child and remained that way
most of my teenage years. Working out had been my trick
to staying at a healthy weight for many years, that is, until
my working out got out of control. As thin as I was, I
didn't take much time to appreciate my size zero jeans
because I was always in my workout clothes getting
sweaty. I was thin, hungry, tired, and not happy.
So why was I so confused when I didn’t get my period
back after going off The Pill It doesn’t make sense, does
it I should have realized it was because of my lifestyle.
But that’s not the way it works when you are in the throes
of something so extreme. Even my husband, who had
been by my side through the whole process, never
suspected my extreme exercising was having an effect on
my menstrual cycle. But in the dark, in that place where a
woman just knows, deep down, I knew.
And because I knew somewhere deep inside me, I
used the right keywords when I finally investigated my
missing periods on Google: ‘extreme exercise,’ ‘low-fat
diets,’ and ‘amenorrhea.’ That’s how I came across a
message board for women suffering from a condition
78 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

PATIENT’S CORNER
called hypothalamic amenorrhea. I read the first post and
immediately identified with the writer.
According to the Mayo Clinic, the definition of
hypothalamic amenorrhea (HA), or secondary
amenorrhea, is the absence of menstruation in women
who previously had regular periods. The term
hypothalamic refers to the hypothalamus, an area at the
base of the brain that acts as a hormone control center for
the body, regulating, among other things, a woman’s
menstrual cycle. In certain situations, such as anorexia,
excessive exercise and stress, the flow of hormones is
interrupted. This results in the failure of the body to
produce enough estrogen and progesterone, the
suppression of ovulation, and, ultimately, the loss of
menses.
Excessive exercise Check. Anorexia In a way,
because I wasn’t eating enough calories to sustain my
activity level. Check. Stress You think it’s relaxing to do
high-intensity interval training on a daily basis No, it’s
stressful! Check. I had the trifecta.
There were thousands of entries on that message
board and I read every last one. When I had finished,
about a week later, I knew everything there was to know
on the topic. What I learned was that there was only one
way for me to ever have a chance of having a baby: I had
to stop exercising, eat fat, and gain weight. It’s an
evolutionary thing. Back in the days of early humanity
there were times of feasts and times of famine. There were
times when a woman would be running from predators
and times she would be nesting and taking care of her
family. While I was exercising like my life depended on it,
my body was registering the fact that I was living in a time
of stress and famine, when it is not prudent to bring a child
into the world. As a result, my body was shutting down
my reproductive system in order to prevent me from
getting pregnant. The only way I could reverse the
damage was to convince my body the famine was over and
times were looking plentiful again.
My gynecologist confirmed my self-diagnosis through
simple blood work that revealed my estrogen, luteinizing
hormone, and follicle stimulating hormone levels were
basically nonexistent. She encouraged me to take the next
year to forget about getting pregnant and just focus on
getting my body to a safe place. Because of the risk of low
bone density surrounding low estrogen levels, I was also
instructed to have a bone density test done and,
thankfully, it came back normal.
I have been ready to be a mother for as long as I can
remember. In fact, I think the intensity of my exercise
regimen was, in part, a way to numb the desire I was
having, as I was entering my thirties, to have a baby. I was
filling up the days and nights of what should have been
my motherhood with a distraction. Facing the fact that I
was infertile was devastating, but once I had that
information in hand, I didn’t think twice about doing
everything in my power to reverse the damage. I stopped
working out immediately and started eating more than my
activity level required. This was by no means easy, as
fitness had become not only my obsession but my identity,
but I just kept reminding myself that I wanted to have a
baby more than I wanted to be thin.
A year later I went to see a reproductive
endocrinologist, a specialist in infertility. I had done
everything right: I gained twenty pounds, ate plentifully,
and the only activity I engaged in was light, gentle
walking. I had not resumed menstrual periods on my own
yet, but I felt healed. I wanted to know what more I could
do to bring a life into this world.
My blood work came back with wonderful news. All
of my hormones were back in normal ranges. I was
overjoyed but also curious as to why I had not resumed
menstruating. The doctor explained to me that with HA it
can be a long, slow road to recovery and that every woman
is different. Some women resume menstruating quickly,
other take years, some never menstruate again.
Because of my hormone levels, I was a candidate to
take a fertility drug called Clomid. It is one of the most
common, least expensive, treatments for infertility. Clomid
indirectly stimulates ovulation which may increase your
chance to conceive. I went through four cycles of Clomid
and was about to give up further treatments until I could
afford in-vitro fertilization when I received a positive
pregnancy test. Using Clomid only worked for me because
I was ready. If I had not taken that year to heal, I do not
think I would be pregnant today, twenty pounds heavier
and a million times happier.
Pregnancy has been known to “reset” some woman’s
menstrual cycles and I am hoping it does so for me. I have
learned many things through this experience but mostly
I’ve learned to never let an obsession take over my life
again. If I ever wear a size zero again I'll know I am doing
something very, very wrong. Avoiding extremes is a
challenge for someone with my personality type, but my
goal for my own future, and for that of the baby inside me,
is to live a life of moderation in all things and to pass that
value along.
If you are an excessive exerciser and missing your
period, please consider the possibility that you have
hypothalamic amenorrhea and see a gynecologist for a
diagnosis. There are many other reasons that a woman
might stop having her period and all of them are
important to diagnose because having a menstrual cycle
every month is a barometer of a woman’s health.
CONTINUED ON PAGE 80
WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
79

PATIENT’S CORNER
CONTINUED FROM PAGE 79
Remember that birth control creates a synthetic menstrual
cycle and can mask amenorrhea, so if you are concerned
you might have it consider stopping the pill temporarily
and using a back up form of birth control until you know
for sure that you are cycling normally. And if you ever
want to have kids, please don't take your womanhood for
granted. Not having hormones is a major drag.
Note from the editor:
As the author notes, excessive exercise can be a cause
of secondary amenorrhea, and thereby loss of fertility.
However, it is extremely important to know that there are
many other causes of secondary amenorrhea, including
physiological disturbances and pharmaceuticals. Many of
these can have serious and long-term effects if not treated
promptly. Medline Plus, a service of the U.S. National
Library of Medicine and the National Institutes of Health,
contains the following advisory (my emphasis):
“Call for an appointment with your primary health care
provider or OB/GYN provider if you are a woman and have
missed more than one period so that the cause, and
appropriate treatment, can be determined.”
(http://www.nlm.nih.gov/medlineplus/ency/article/00
1219.htm, accessed May 20, 2010)
You can contact Courtney Sirotin at: courtney.sirotin@gmail.com
80 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

• Designed for IVF
• Prewashed extensively with ultra-pure water
• Pharmaceutical grade quality mineral oil
• Sterile Filtered (SAL 10–3)
• 1 cell MEA and Endotoxin Tested
• Optimal viscosity for embryo culture
• 24 Month minimum shelf life
• Prewashed extensively with ultra-pure water
• Pharmaceutical grade, light paraffin oil
• Sterile Filtered (SAL 10–3)
• 1 cell MEA and Endotoxin Tested
• 24 Month minimum shelf life
www.LifeGlobal.com
• Proven in IVF Labs Worldwide
• Maximum Air Flow of 530 Cubic Feet/Minute
• 1 Year Warranty
• Effective Coverage of up to 400 Square Feet
The Coda ® Aero is a technology that is designed to improve the air health conditions in your IVF laboratories
and workspace.
Our Coda ® Technology reduces particulates, harmful volatile organic compounds (VOCs) and chemical
airborne contaminants (CACs).
Our specially designed HEPA filters remove 99.97% of contaminants and is combined with our unique blend
of activated carbon and alumina impregnated with potassium permanganate for optimum absorption and
oxidation of a wide variety of gases and particulate contaminants.
Coda ® is the ‘First’ product in laboratory air purification and has been the most successful for the last 7 years.

Traditional Petri Dish
Embryo GPS ® Dish
→ running droplets
→ droplets mixing
→ droplets flattening
→ poor temperature control
→ losing track of marked droplet
→ difficulty locating embryos
→ sealing of dish lid & increased pH
‘drop-less’ environment
micro wells designed to enhance embryo culture
no droplets collapsing and mixing
save time and money by reducing set up time,
observation time, testing and handling
better heat conservation
enhance safety
easily locate & observe embryos
new designed lid for better gas exchange
standardization of culture, uniform volumes
1-cell MEA and LAL testing by independent
company
Standardize ART and other sensitive culture by using GPS Dishware TM
Eliminate these concerns and enhance safety by using GPS Dishware TM : embryo GPS ® , embryo
corral ® , and Universal GPS TM .
embryo GPS ® – The best dish for PGD Cases
Introducing a new dish design for larger volume culture.
Patented & Patent Pending
US/Canada: 1-800-720-6375
International: 1-519-826-5800
Fax: 1-519-826-6947
email: gps@IVFonline.com
www.SunIVF.com
www.IVFonline.com

NEW PRODUCTS
Powder Free Surgical Gloves
Pristine gloves are completely free of
talcs, starches, calcium carbonates or
powdered substances or lubricants of any
kind. Developed to protect the patient
from granuloma, peritonitis, adhesions
and other powder-related complications,
Pristine gloves are also designed to
protect against powder-related dermatitis
and other powder-associated irritations of
the hands and skin.
US/Canada: 1-800-720-6375
International: 1-519-826-5800
Fax: 1-519-826-6947
sales@IVFonline.com
www.IVFonline.com
84 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

NEW PRODUCTS
LifeGlobal® Protein Supplement
MatriPlus TM liver biomatrix and our
StemQ® Media to grow differentiate
and maintain hepatocytes…
StemQ® HepatoGro TM
StemQ® HepatoDiff TM
StemQ® HepatoMainDiff TM
LifeGlobal® Protein Supplement with
the enhanced performance provided by
α- & β-globulins.
• Tested lot-to-lot
• Stringent quality control and testing
• One-cell Mouse Embryo Assay Testing
• 12-month shelf life
• pH, Osmolality and Endotoxin Tested
• High quality of manufacturing
• ISO 13485:2003 & 9001:2000 Certified
StemQ® Scalable Human Neural
Model System
An In Vitro Regenerative Model
global® Blastocyst Vitrification System
Based on S 3
DMSO-free and regular sealable straws
global® Blastocyst Vitrification Kit
– Based on S3
High quality lite mineral
oil is sterile-filtered and
QC tested. MEA and
endotoxin tested.
• Novel serum-free media formulations
– StemQ® NeuroGro Growth Media
– StemQ® NeuroDiff Differentiation Media
• Human Neural Progenitor Cells (NPCs)
– Isolated from human brain tissue
– 11 Donor Lots available (genetic diversity)
– Low passage number
– Average doubling time of 45 hours
– Differentiates into multiple neuronal subtypes,
astrocytes and oligodendrocytes
– Scalable (109 cells)
– Consistent Reproducible Results (single batch)
• Can be used in a High Throughput Screening
Platform
global® Blastocyst Vitrification Thawing Kit
– Based on S3
Paraffin Oil is
sterile-filtered and
QC tested. MEA
and endotoxin
tested.
WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
85

BOOKS
DEBRAH FRANK, CH, CI
About the Author
Debrah Frank has been
writing short stories, poetry
and articles for most of her
life. She is a Reiki Master,
Certified Reflexologist and a
Certified Hypnotherapist as
well as instructor and has
been working in the field of
alternative healing for quite
some time.
You can be, do and have anything imaginable. All you
need is a little bit of self awareness. Awareness is the
starting point of all personal growth. Once you become
aware of something it is then that you can change it.
Without awareness nothing changes.
Your Personalized copy of “Inner Metamorphosis”
can be purchased at: www.PersonalDynamix.com
About the Author
Harry Fisch, MD, Columbia
University Medical Center, NY,
author of “The Male Biological
Clock”, is one of the nation’s
leaders in the diagnosis and
treatment of male infertility.
HARRY FISCH, MD
The Male Biological Clock can be purchased at:
www.malebiologicalclock.com
86 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

RESOURCES
WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
87

RESOURCES
The New England Fertility Institute is a patient oriented, full service treatment
center, providing state-of-the-art, caring, fertility services.
Dr. Gad Lavy and his trained team of fertility specialists are here to help you, by
treating both female and male infertility with the most up-to-date techniques, in
a modern facility.
The New England Fertility Institute is consistently ranked among the top fertility
clinics in the United States for the past 15 years. Come share in our success.
New England Fertility Institute
1275 Summer Street, Stamford, CT, USA 06905 • 203-325-3200
9 Washington Avenue, Hamden, CT, USA 06518 • 203-248-2353
Visit our website at www.nefertility.com to see our success. Contact Dr. Lavy at
glavy@nefertility.com. We are conviently located close to New York City and major airports.
88 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

RESOURCES
WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
89

RESOURCES
Our Experts:
Megan Freebury Karnis, BA/Sc, MD, FRCSC
Shilpa Amin, BSc, MD, FRCSC
Michael Neal, BSc (Hons), MSc, PhDc
Edward G. Hughes, MBChB, MSc, FRCSC
Caroline Beliveau, MD, FRCSC, FACOG
Mehrnoosh Faghih, MD, FRCSC, FACOG
Marc Anthony Fischer, BSc (Hons), MD, CFP, FRCSC
D. Ray Freebury, MBChB, D(Obst)RCOG, FRCPC, DFAPA
H. A. Pattinson, MBChB, FRCOG, FRCSC (Windsor)
Our patient referral base includes Burlington, Hamilton, Guelph, Kitchener, Cambridge, Windsor,
Niagara, Oakville, and Mississauaga.
90 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

RESOURCES
American College of Embryology (ACE) transforms a diverse group of embryology
practitioners into a uniformly trained group of professionals. ACE is your organization
and your voice. Please join our effort to advance the practice of clinical embryology and
establish standards for embryology care in the United States.
American College of Embryology is offering certification for Embryology
practitioners at the following levels:
Embryologist (EMB)
Embryology Associate (EMA)
Embryology Technologist (EMT)
Certification requires theoretical and practical examinations.
The College also offers grandfathering on a limited basis for qualified candidates.
Please join the College and become certified Embryology practitioner!
WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
91

CONFERENCES
IVFonline Workshops on the Use of the
global ® Blastocyst Vitrification and Thawing Kits – Based on S 3
The success rates of recently developed vitrification methods for the
cryopreservation of human oocytes and embryos have been highly
encouraging, and vitrification is now widely applied in human IVF
clinics. Vitrification of embryos increases the change of a pregnancy
following cryopreservation, post-warming survival rates are shown to
be as high as 90% which are significantly better than those achieved
with slow freezing methods (Vajta et al., 2009, Stachecki et al., 2008).
These long-waited results allow clinicians and embryologists to opt
for other strategies such as single embryo transfer and vitrification of
the remaining embryos, hence reducing multiple pregnancies and
their associated consequences.
LifeGlobal has recently developed kits for blastocyst vitrification and
thawing based on the S 3 system developed by James Stachecki
(Stachecki et al., 2008; Stachecki and Cohen, 2008) The S 3 system uses
a combination of ethylene glycol and glycerol as the cryoprotectants,
avoiding the toxic effect of DMSO. The embryos are held in standard
0.25-0.30 cc straws and thus not require any specific device. But
undoubtedly, its most important features are that no special skills are
required and that there is considerably more time to perform all steps
of this method, making it a less stressful procedure that can be
performed by all laboratory staff. Using this method, a pregnancy rate
per transfer of 56 % has been reported (Stachecki et al. 2008).
In order to demonstrate this technique, IVFonline has conducted
several demonstrations and workshops in Europe in 2010. Nathalie
Boonen, IVFonline Product Manager, and Dr. Ana Sousa Lopes,
clinical and research adviser, coordinated the organization of these
workshops, in conjunction with the meetings of the British Fertility
Society, Bristol, UK., the Mediterranean Society for Reproductive
Medicine, Budva, Montenegro, and The 1st International Congress on
Controversies in Cryopreservation of Stem Cells, Reproductive Cells,
Tissue and Organs, Valencia, Spain.
Montenegro Workshop – May 6, 2010
Danilo I Hospital Cetinje
Montenegro Workshop – May 6, 2010
Danilo I Hospital Cetinje
The Montenegro workshop was held in the IVF unit of the Hospital of
Cetinje and attracted 19 participants from the Balkan countries. In the
morning session, Dr. Lopes presented an overview of the use of the
global ® Blastocyst Vitrification and Thawing Kits – Based on S 3 ,
followed by a practical demonstration to introduce all of the steps in
detail. In the afternoon, the attendees were divided into four working
groups for hands-on training. The interest of the attendees for this
technique was clearly demonstrated by the questions raised
throughout the day.
The workshops in Spain were organized with the Fundación Jimenez
Díaz, Madrid, the IVF unit of the Hospital De la Fé, Valencia, and the
Clinic El Pilar, San Sebastian. Dr. Lopes gave the presentations and
Madrid Workshop – May 26, 2010
Fundación Jiménez Diaz
92 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

CONFERENCES
If you are interested to know when the next vitrification S 3 workshop
will take place in or around your country, please
contact Nathalie Boonen: nathalie@IVFonline.com
demonstrations in Madrid and Valencia. Dr. James Stachecki gave the
presentation and demonstration in San Sebastian. A total of 39
participants was present in the 3 workshops, representing 22 Spanish
IVF clinics.
The objective of these workshops is to introduce the participants to the
principle behind S3 blastocyst vitrification system, and to give them
the opportunity to see all steps of the procedure. With this background,
they are able to proceed with the testing of the global ® Blastocyst
Vitrification and Thawing Kits – Based on S 3 in their own IVF units.
Valencia Workshop – May 27, 2010
Hospital Universitario La Fe de Valencia
The overall response of the attendees was very positive and it was
generally agreed that the methods for use of the global ® Blastocyst
Vitrification and Thawing Kits – Based on S 3 are simple and easy to
perform. Vitrification offers a solution for embryo cryopreservation
and is about to become a common part of the everyday routine in a
human embryo laboratory, IVFonline encourages anyone interested in
receiving training with the global ® Blastocyst Vitrification and
Thawing Kits – Based on S 3 to contact us about joining one of our
future workshops.
References
Valencia Workshop – May 27, 2010
Hospital Universitario La Fe de Valencia
Nathalie Boonen, Dr Ana S Lopes-Ana Ortiz,
Dr Gloria Calderon, Dr Pedro Fernandez
Stachecki, J.J., Cohen, J., (2008) S 3 Vitrifcation System: A novel approach to
blastocyst freezing. J. Clin. Embryol. 11, 5-14.
Stachecki, J.J., Garrisi, J., Sabino, S., Caetano, J.P., Wiemer, K.E., Cohen, J.,
(2008) A new safe, simple and successful vitrification method for bovine
and human blastocysts. Reprod Biomed Online 17, 360-367.
Vajta, G., Nagy, Z.P., Cobo, A., Conceicao, J., Yovich, J., (2009) Vitrification in
assisted reproduction: myths, mistakes, disbeliefs and confusion. Reprod
Biomed Online 19 Suppl 3, 1-7.
San Sebastian Workshop – June 1, 2010
Centro Sanitario Virgen del Pilar
San Sebastian Workshop – June 1, 2010
Centro Sanitario Virgen del Pilar
Dr James Stachecki
WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
93

CONFERENCES
A Successful Workshop presented by IVFonline/LifeGlobal
West Coast Workshop held on January 23, 2010
global Blastocyst Vitrification System – Based on S3
On Saturday January 23, 2010 IVFonline held its first hands on S3
Vitrification workshop at the Pacific Fertility Center in San Francisco. It
was a one day workshop where participants were able to gain hands on
experience working with the global® Blastocyst Vitrification System –
Based on S3 and gain CEU’s in the process. We had a fantastic turn out at
the event, with 21 West Coast participants. The response to the workshop
invitation was so positive; it necessitated a waiting list be started to
accommodate potential attendees.
Speaking at this workshop were Jacques Cohen, PhD, Klaus Wiemer, PhD
and Don Rieger, PhD. Dr. Cohen gave an overview and spoke about the
principals of the global® Blastocyst Vitrification System – Based on S3. Dr. Wiemer shared his own experience with
the global® Blastocyst Vitrification System – Based on S3 and provided some of his data. Afterwards Dr. Wiemer
demonstrated a “vitrification and devitrification” procedure. Participants then broke into small groups and went to one
of the five stations to practice vitrifying and devitrifying mouse blastocysts with the global® Blastocyst Vitrification
System – Based on S3. After the workshop IVFonline/LifeGlobal offered to send practice kits to all participating centers.
The response and feedback from the workshop was tremendous.
“I was very happy with the way the course went. I thought it was really excellent.”
“Thank you for providing the educational activity for us. We look forward to the next one.”
“Thanks for the workshop! You all did a stellar job and I learned a lot.”
“I had a wonderful time and learned a lot! I especially loved the small, intimate setting and the one-on-one time.”
A special thank you goes to Joe Conaghan PhD. and the Pacific Fertility Center who hosted the workshop at their
beautiful education center. IVFonline provided breakfast goodies and lunch. Also, this workshop was an AAB-PEER
approved event allowing participants to earn 0.5 CEU’s for attending.
IVFonline has plans to hold more of these workshops this year. If you are interested in attending please contact us at
sales@IVFonline.com and place your name on a list for one of our future workshops; no registration fee required.
94 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

CONFERENCES
Welcome to PCRS
2011 Annual Meeting, April 13 to 17
Rancho Las Palmas
41-000 Bob Hope Drive
Rancho Mirage, CA 92270
www.pcrsonline.org
ASRM 2010
American Society for Reproductive Medicine
66th Annual Meeting
"“Taking Reproduction to New Heights"
October 23-27, 2010
Colorado Convention Center
Denver, Colorado
www.asrm.org
WWW.FERTMAG.COM • VOLUME 12 • FERTILITY MAGAZINE
95

CONFERENCES
AAB’s 2011 Annual Meeting and Educational
Conference/CRB Symposium/NILA Meeting
May 12-14, 2011
Hyatt Regency Austin
Austin,Texas
www.aab.org
96 FERTILITY MAGAZINE • VOLUME 12 • WWW.FERTMAG.COM

Introducing the Coda ® 2
Proven Filtration Technology to remove contaminants such as Volatile Organic
Contaminants (VOCs) inside incubators
…Common Sense Safety Devices to Protect Embryos, Stem Cells or any other Cell Culture
The Coda ® Incubator Unit has proven itself in human IVF for the past two decades to remove Volatile Organic
Contaminants (VOCs) presents in any incubator or laboratory.
Coda ® 2 Kit
Placed on the top shelf of the incubator
Coda ® Unit
Placed on the top shelf of
the incubator
Coda ® SP
Mounted into the access
hole of your incubator
Coda ® 2 Kit
C2KT-107
C2KT-106 (w/o Coda® Xtra Inline® Filter)
Available as an economical start up Kit for incubator
and gas line: with 6 months supply of Coda ® Filters,
1 Coda ® Xtra Inline ® Filter and accessories.
CodaInside
Coda ® Inside your incubator
has proven to remove VOCs
and CACs. Particulates that can
potentially inhibit any cell
culture development. Testing in
certain laboratories has shown
that in the presence of
formaldahydes even the mouse cell culture was inhibited. Coda ® is a
patented technology, manufactured by a certified ISO medical company and each building material is tested
for any off-gassing and safe use in human embryos. Coda ® includes activated carbon filtration and a 99.997
% HEPA filter to remove VOCs and CACs and particulates completely from the air inside your environment.
This will improve your embryo and stem cell quality and development, along with giving you better overall
performance and results. Our Coda ® 2 is ‘greener’, saves energy and is made of 100% recyclable materials.
Do you know what VOCs reside inside your incubators
styrene, methylcyclohexane, acetone, benzene, n-Decane, octane, etc.
Coda …the only solution introduced since 1997
US/Canada: 1-800-720-6375
International: 1-519-826-5800
Fax: 1-519-826-6947
email: sales@IVFonline.com
FDA 510(k) Cleared

Introducing LGPS
FDA 510(k) Cleared
LifeGlobal ® Protein Supplement
LifeGlobal ® Protein Supplement
with the enhanced performance
provided by α- & β-globulins.
• Tested lot-to-lot
• Stringent quality control and testing
• One-cell Mouse Embryo Assay Testing
• 12-month shelf life
• pH, Osmolality and Endotoxin Tested
• High quality of manufacturing
• ISO 13485:2003 & 9001:2000 Certified
Product Description Features and Highlights Size Cat. #
LifeGlobal ® Protein Supplement with the enhanced 20 ml LGPS-020
LifeGlobal ® Protein Supplement (LGPS) performance provided by α- & β-globulins. Protein 50 ml LGPS-050
supplement for in-vitro culture media.
250 ml LGPS-250
US/Canada: 1-800-720-6375
International: 1-519-826-5800
Fax: 1-519-826-6947
email: sales@LifeGlobal.com

Coda ® Xtra Inline ® Filters
Improved, more effective and economical due to an
increased size and amount of pure filtering materials, with
more amplified residence contact time and absorption
capacity.
Your first line of defense in removing contaminants such
as Volatile Organic Contaminants (VOCs) and Chemical
Air Contaminants (CACs) from incoming gas sources.
Coda ® Inline ® Filters have proven over 10 years to be safe and
effective in Human IVF in purifying the gas for incubators,
reducing contaminants and helping to improve results.
The Coda ® Xtra Inline ® Filter protects the air and environment inside your incubator
from harmful contaminants present in your gas sources ending up in your incubator,
by removing all Volatile Organic Contaminants (VOCs), Chemical Air Contaminants
(CACs) and particulates which may be in the gas or reside in the old tanks and
tubing.
Coda ® Inline ® Filters are the ‘only’ filter proven Safe and Effective in IVF and Human
Reproduction.
Coda ® Xtra Inline ® Filters are a proven quality product.
• Your first line of defense in removing contaminants such as Volatile Organic
Contaminants (VOCs) and Chemical Air Contaminants (CACs) from incoming
gas sources.
• Contains 100% clean activated carbon and a 99.997% HEPA filter system.
• Made in the USA with tested proven materials.
• Proven in over 600 labs worldwide for 15+ years in stem cell research, hES
cells, embryonic development and human reproduction and IVF.
• Human IVF Laboratories that use our Coda ® Inline ® Filters have reported
better and more consistant overall pregnancy rates and increases in live birth
rates.
Coda ® Xtra Inline ® Filters – Small Investments – Big Savings
• Attaches to your incoming gas lines supplying cleaner gas due to an
increased amount of pure filtering materials, with an amplified residual contact
time and absorption capacity.
• Manufactured in a clean room environment with stringent quality control.
• Delivered to you in an air tight sealed, sterilized packages.
• Economical and easy to install.
• Our Patented Technology makes Coda ® Filters a superior product and is
Patented Protected in the United States, European Union, Australia and other
countries worldwide. Coda ® Inline ® Filters are covered by the following
patents: U.S. Patent No. 6,013,119, No. 6,843,818, No. 6,225,110 and No.
6,200,362. The European Community Patent No: 98 923 448.9.
N.S.
Control
Coda
N.S.
P < 0.05
P < 0.05
FDA 510(k) Cleared
Be aware of counterfeit Inline ® Filters not safe for use in Human IVF.
You may introduce more contaminants as filters may not be manufactured
in a clean room environment and filtering materials may not be cleaned,
tested and properly qualified for safe use in Human IVF.
Grade 1
Day 7
Blasts
Fresh Frozen
Day 8 Pregnant (Day 90+)
Effect of Coda Filtration on Bovine Embryo Qualuty and Pregancy Rates
(Merton et al., Theriogenology 67, 1233-1238, 2007)

A Unified Approach to Human Embryo Culture
More than 20 independent studies with published results on
global ® medium.
• Based on global ® success
• Minimizes stress to the embryo
• Same chemical environment
throughtout all stages of oocyte
and embryo handling and culture
• Better embryo development
• Easy to use
• High Quality of Manufacturing
• Stringent Quality Control
• Fresh Delivery
• ISO 13485:2003 & 9001:2000 Certified
US/Canada: 1-800-720-6375
International: 1-519-826-5800
Fax: 1-519-826-6947
email: sales@LifeGlobal.com