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Age-2005-05
CONTENTS
Fertility Magazine
The First Magazine In FertilityTM
FEATURED ON THE COVER
Pictured on the front cover of Fertility Magazine, Volume 4 is Lauren Cecchi.
The Challenge and Promise of Blastocyst Cryopreservation by Marius Meintjes, PhD . . . . . . . . 11
The Effect of CO 2 Concentration and pH on the In-Vitro Development of Mouse Embryos
by Avner Hershlag, MD and Huai L. Feng, PhD, HCLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
An Overview of the Effects of Age on Fertility in Women by Don Rieger, PhD . . . . . . . . . . . . . . 31
23
Degree of Re-expansion of Thawed
Blastocysts is Predictive of Frozen
Embryo Transfer (FET) Success
41
FEATURES
8 Preimplantation Genetic Diagnosis: A Domestic
Animal Perspective by W. Allan King, PhD
15 Use of the Sperm Chromatin Structure Assay
(SCSA®) as a Diagnostic Tool in the Human
Infertility Clinic by Donald P. Evenson, PhD, HCLD
and Regina L. Wixon, PhD
23 Degree of Re-expansion of Thawed Blastocysts is
Predictive of Frozen Embryo Transfer (FET) Success
by Deborah C. Merryman, MSc, ELD/TS (ABB)
27 Advanced Maternal Age and Infertility by Alan B.
Copperman, MD
29 ART and Aging by Michael S. Neal, BSc, MSc, PhD
candidate
Patient’s Corner
41 Patient’s Corner by Charlene Alouf, PhD and Albert
El-Roeiy, MD, MBA
©2005 IVFonline
4
FERTILITY MAGAZINE • VOLUME 4 •
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45 Helping You Understand “What Happens Next?” by Liz Sanders
Helping You Understand
“What Happens Next?”
46 Feature Products
47 Hypnosis for Childbirth by Shawn Gallagher, BA, CHt
49 Human Resources
45
50 Resources
46 Oocyte Cryopreservation: A Common Sense Approach by James
Stachecki, PhD
CONTRIBUTORS
Marius Meintjes, PhD
Avner Hershlag, MD
Don Rieger, PhD
Huai L. Feng, PhD, HCLD
W. Allan King, PhD
Donald P. Evenson, PhD, HCLD
Regina L. Wixon, PhD
Deborah C. Merryman, MSc, ELD/TS (ABB)
Editor in Chief: Monica Mezezi, MBA
Design: Debbie Nagy
Editorial Office: IVFonline, 24 Norwich St. E.
Guelph, Ontario, Canada, N1H 2G6
US/Canada: 800-720-6375
International: 519-826-5800
Fax: 519-826-6947
Email: [email protected]
www.IVFonline.com
www.FertilityMagazine.org
Alan B. Copperman, MD
Michael S. Neal, BSc, MSc, PhD candidate
Charlene Alouf, PhD
Albert El-Roeiy, MD, MBA
Liz Sanders
Shawn Gallagher, BA, CHt
James Stachecki, PhD
Instructions to Contributors
To submit an Article/Abstract email us at: [email protected]
To submit an Ad email us at: [email protected]
Fertility Magazine and all its associates ©2004, All Rights Reserved. Covers, contents, images, ads in print or web form are copryight protected and reprinting or reproduction of any
kind is expressly prohibited without the written permission of Fertility Magazine. Fertility Magazine does not knowingly accept false or misleading advertising, articles, opinions or
editorial, nor does the publisher assume any responsibility for the consequences that occur should any such material appear, and assumes no responsibility for content, text, opinions
or artwork of advertisements appearing in Fertility Magazine in print or web form. Some of the views expressed by contributors may not be the representative views of the publisher.
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• VOLUME 4 • FERTILITY MAGAZINE
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INTERNATIONAL NEWS
Preimplantation Genetic Diagnosis:
A Domestic Animal Perspective
by W. Allan King, PhD
Professor and Canada Research Chair, Department of Biomedical Sciences
University of Guelph, Guelph, ON Canada, N1G 2W1
P
reimplantation
genetic
diagnosis (PGD) is a technique
born out of the desire of
livestock breeders to intensify the
accuracy of selection of their
livestock, and the desire of parents to
ensure the health of their children.
The technique is the culmination of
scientific discoveries over a period of
time spanning Mendel's postulations
on the principles of inheritance in
1866 and Mullis's development of
polymerase chair reaction (PCR) in
Research Institute in Ottawa
reported the successful application
of the approach to another species.
This time, 2 week-old cattle embryos
were sexed by chromosome analysis
of trophoblast cells. The procedure
had limitations, mainly based on the
number of metaphases required to
make an accurate diagnosis and the
need to use embryos of later stages
in order to provide enough cells
without compromising the longterm viability of the embryo. With
The application of PGD is
increasing on an almost daily basis.
1983.
Genetic
screening
of
preimplantation embryos was first
documented in a paper by Gardner
and
Edwards
at
Cambridge
University in the April 1968 issue of
Nature. The abstract of the paper
stated that “An important step
forward has been made in
controlling the sex of rabbits by
sexing pre-implantation embryos
and transferring the sexed embryos
into recipient females. In the
experiments, fetuses were surgically
delivered and their sex was found to
have been correctly predicted at the
blastocyst stage.” The procedure
consisted of piercing the mucin coat
and zona pellucida of day 5
blastocysts and removing 200 to 300
blastomeres, which were fixed and
stained to reveal the presence or
absence of the Barr body. It was
another 8 years before Hare and
colleagues at the Animal Disease
8
FERTILITY MAGAZINE • VOLUME 4 •
the potential and proof of the
principle clearly established, the
pressure was on to stream-line the
technique, and make it compatible
with the commercial practice of
using day-7 cattle blastocysts for
cryopreservation and transfer. The
ground-breaking work on embryo
bisection by Willadsen at Cambridge
in 1979 led the way to embryo
splitting and biopsy, which quickly
became a commercially viable option
for increasing the rates of pregnancy
and twinning in valuable cattle. It
soon became apparent that if a cattle
blastocyst could be split in half
without severely compromising the
developmental potential of either
half, one demi-embryo could be
subjected to chromosome analysis
and the other half frozen or
transferred. In 1985, Picard and
colleagues at the University of
Montreal used this approach and
WWW.FERTILITYMAGAZINE.ORG
W. ALLAN KING, PHD
reported the birth of calves from
frozen, sexed embryos. While
somewhat successful, the technique
was far from suitable for routine
application in commercial settings
because of the need for sophisticated
microscopes, trained technologists
and, more importantly, adequate
number of metaphase spreads for
analysis to rule out diagnostic errors
since blastocysts had been shown to
contain subpopulations of aneuploid
and polyploid cells. The advent and
commercialization of the analysis of
DNA by PCR technology created
new vistas in the genetic analysis of
single cells. By the end of the 1980's,
cattle embryos were being routinely
sexed, often on the farm, by PCR
amplification and analysis of sex
chromosome linked genes. Around
this time, reports on the analysis of
human embryo biopsies also started
to appear in the literature. The birth
of children subsequent to genetic
screening of embryos was reported
INTERNATIONAL NEWS
in 1994.
The application of PGD is
increasing on an almost daily basis.
In domestic animals, there are three
main areas of application: sex
selection, avoidance of genetic defects
and
selection
of
production
characteristics.
In
agricultural
practices, one sex is often favoured
over the other. For example, the
artificial insemination industry is
based on the dissemination of semen
from the elite sons of animals of high
genetic merit, while milk production
requires high producing females. In
addition, a number of genetic defects
including bovine leukocyte adhesion
deficiency (BLAD) and deficiency of
uridine monophosphate synthase
(DUMPS), have been identified in
cattle for which DNA probes are
commercially available. In a PGD
context this offers the breeder a
unique opportunity to pre-select the
offspring
rather
than
culling
individuals postnatally. Finally, since
most production traits in domestic
animals are quantitative genetic traits
controlled by numerous genes,
intensive research into developing
markers for such traits as milk yield
and composition, quality of meat and
disease resistance is in progress.
Several markers based on single
nucleotide polymorphisms and
micro-satellite technologies, have
already been identified in dairy cattle
for traits including milk production
and resistance to mastitis. The use of
such markers for marker assisted
selection (MAS), in combination with
embryo biopsy and embryo transfer
techniques, offers the means to
realizing the mission of ensuring the
accuracy of selection of breeding
livestock and maximizing the
efficiency of production.
PGD has come a long way since
its initiation and become an
important tool for agriculture
industries. The potential and practice
of this approach were barely
imagined 40 years ago when the first
commercial embryo transfers were
performed in cattle. One can only
wonder if Mendel had any inkling
that his musings on factorial
inheritance in peas would one day
lead scientists to predict the sex and
genetic status of the conceptus and
help breeders pre-select their animals
for production traits.
You can contact W. Allan King, PhD at:
[email protected]
WWW.FERTILITY MAGAZINE.ORG
• VOLUME 4 • FERTILITY MAGAZINE
9
global® culture media was scientifically
designed with the embryo in mind
global® is a single culture medium used to culture embryos from Day 1 to blastocyst stage.
global®’s development began with the work of John Biggers. John Biggers and his development
team used the principles of Simplex Optimization to develop the medium for mice, it was then
adapted into a human embryo culture media through the efforts of our scientific committee lead
by Klaus Wiemer, PhD.
global ® culture media has been proven in multiple independent centers
worldwide to help increase the number of embryos developing to either Day 3
culture or blastocyst culture , yielding more embryos available for transfers and
has shown significant increased pregnancy rates.
The Media
IVF patients have greatly benefited from the use of global®, as it has been shown
to increase pregnancy results. Fertility clinics who have adopted the use of
global® have reported improved embryonic development, blastocyst development
and an increase in the number of embryos available for use in transfer or for
cryopreservation.
The Patients
global ® has proven to be stable in its use, maintains its pH levels, Osmolality
and has shown to produce the lowest levels of ammonia build-up over time.
Superior stability, quality and superior performance.
The Embryos
LifeGlobal®, ‘The Art Media Company’ was established to meet the industry needs for high quality,
specially designed media products. All products are designed with the nutritional needs of the
human embryo in mind, and with the strictest quality controls, tested ingredients, each batch is
independently tested and are FDA compliant. Our research team of John Biggers, Jacques Cohen,
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www.LifeGlobal.com
Order Online at www.IVFonline.com
US/Canada: 800-720-6375 • International: 519-826-5800 • Fax: 519-826-6947 • email: [email protected]
©2005 LifeGlobal ®
LG-global-2005-10
ARTICLES
The Challenge and Promise of Blastocyst Cryopreservation
by Marius Meintjes, PhD
Presbyterian/Harris Methodist ARTS Program, 8160 Walnut Hill Lane, Perot Building, Suite 116, Dallas, TX 75231
Introduction
Blastocyst culture and transfer has been promoted as a
novel approach to reduce multiple pregnancies. With the
high implantation rates and the resultant high percentage
of twin pregnancies observed when transferring fresh
blastocysts, single blastocyst transfers should be
considered. With a reliable blastocyst cryopreservation
program in place providing consistent acceptable frozenthawed blastocyst transfer outcomes, single blastocyst
transfer should be a logical next step to curtail the current
epidemic of twin pregnancies experienced with assisted
reproduction. However, the frequent experience of less
than attractive success rates with frozen-thawed
blastocysts discourages many centers, as cumulative
fresh-frozen pregnancy rates may seem unacceptable.
(Graph 1).
Patients with only
poor-quality ICM’s at
the time of transfer
may expect a high
rate of biochemical
pregnancies.
However, ongoing
pregnancies and live
births are possible.
Several healthy postthaw
trophoblasts
with no discernable
ICM (ICM-grade F,
Figure
2)
were
MARIUS MEINTJES, PHD
transferred, which
Post-thaw inner cell mass (ICM) survival
resulted in clinical pregnancies followed by firstA detailed blastocyst-specific grading system has been
trimester miscarriages. Similarly, weak and/or damaged
developed over the years in our program for fresh and
ICM's in the poor-quality ICM group may explain the
frozen blastocyst evaluation (Figure 1). High biochemical
high rate of biochemical pregnancies and the clinical
pregnancy losses observed.
to improve blastocyst
It is tempting to speculate that assisted Efforts
cryopreservation should focus to
hatching may improve frozen-thawed a significant degree on ICM
blastocyst transfer outcomes. survival. ICM survival may be
improved by proper selection of
pregnancy rates and miscarriage rates are frequent
blastocysts for cryopreservation, pre-freeze cytoskeletal
observations
when
transferring
frozen-thawed
stabilization, optimisation of cryoprotectant solutions, or
blastocysts. A biochemical pregnancy is defined as a
the application of ultra-rapid vitrification procedures.
positive ßhCG result failing to progress to a clinical
pregnancy. It has been observed that ICM’s are
A place for assisted hatching of frozen-thawed
preferentially damaged by cryopreservation and that the
blastocysts?
observed clinical outcomes directly relate to ICM quality
It is documented that the composition and function of the
zona pellucida may change due to freezing and thawing.
It is tempting to speculate that assisted hatching may
improve frozen-thawed blastocyst transfer outcomes. In a
randomized prospective study comparing assistedhatched (n=76) and non-hatched (n=76) frozen-thawed
blastocyst transfer cycles, it was observed that the
implantation rate was improved with assisted hatching.
However, there was no effect on the ongoing
pregnancy/live birth rate (Graph 2).
CONTINUED
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ON
PAGE 12
• VOLUME 4 • FERTILITY MAGAZINE
11
ARTICLES
day 6 (n=301 patients – Graph 3). The male:female sex
ratio (57:43) of babies born from blastocysts frozen on day
5 was significantly skewed and similar to that reported for
fresh blastocyst transfers. Interestingly, the male:female
sex ratio (54:46) was not different for blastocysts frozen on
day 6.
C ONTINUED FROM PAGE 11
Day-5, day-6 cryopreserved blastocysts and sex ratio
The exact timing and blastocyst developmental stage at
the time of cryopreservation may be imperative to optimal
frozen-thawed
blastocyst
pregnancy
outcomes.
Embryologists frequently face decisions in freezing
expanding blastocysts with reduced cell numbers on day 5
or freezing the next day with increased cell numbers, but
risking ICM degeneration or in-vitro hatching. It has been
documented that fresh blastocyst transfers may result in
an increased male:female sex ratio. Freezing the slower
blastocysts (not selected for fresh transfer) may possibly
result in a normalization of the sex ratio in frozen-thawed
blastocyst pregnancies.
It was observed that blastocysts frozen late on day 5
resulted in higher implantation and ongoing
pregnancy/live birth rates than blastocysts frozen early on
12
FERTILITY MAGAZINE • VOLUME 4 •
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Expectations
from
an
optimized
blastocyst
cryopreservation program
Even with the limitations of slow-rate blastocyst freezing,
live birth rates close to that of fresh blastocyst transfers
can be achieved. The optimization of a blastocyst
cryopreservation program mandates a comprehensive
approach. A sequential embryo culture system with
attention to the protein supplement and a low oxygen
environment has been shown to increase the number of
blastocysts available for cryopreservation, improve the
ICM quality and blastocyst post-thaw survival. Critical
timing of cryopreservation seems essential as the success
of slow-rate blastocyst cryopreservation appears to be
developmental stage specific. Freezing and thawing
protocols/solutions are crucial, however, only a part of the
overall cryopreservation scheme. Table 1 summarizes agespecific results that can be expected with slow-rate
cryopreservation and thawing of blastocysts when
attention is given to the factors mentioned. Interestingly, it
seems that as long as quality blastocysts are frozen,
pregnancy rates are not significantly affected by the age of
the patient.
Patient age Number of Blastocysts/ Implantations
Ongoing/
(years)
transfers
Transfer
(%)
live births (%)
≤34
94
1.7
45.1
54
35 to 37
41
1.7
39.4
42
38 to 40
25
1.7
53.5
60
>40
6
1.7
50
67
Donor
47
1.9
40.9
60
oocytes
Total
213
1.8
44.2
54
T able 1. Ongoing pregnancy/Live birth outcomes for agespecific frozen-thawed blastocyst transfers
Conclusions
It appears that the success of blastocyst cryopreservation
and thawing depends on the ability to protect and recover
a viable ICM (Figure 3). Assisted hatching seems to be of
little value if optimum culture conditions are established
in the laboratory. Freezing of fully expanded, but not
hatched, blastocysts before 118 hours post insemination
results in the best clinical outcomes. Frozen-thawed
blastocyst live birth rates equivalent to or better than those
obtained with fresh blastocysts for patients of all ages
should now enable IVF programs to offer routine fresh
single-blastocyst transfers. Experience by embryologists,
ARTICLES
not only with the freezing and thawing procedure, but
also with selecting appropriate blastocysts for
cryopreservation, is essential for a quality blastocyst
cryopreservation program. However, biochemical and
miscarriage rates are still significantly higher than those
observed for fresh blastocyst transfers. Even though
blastocyst cryopreservation can be implemented
successfully, refinements to current protocols or
investigation of alternative techniques will be necessary to
further improve ICM survival after thawing.
Acknowlegements
The author wishes to recognize Drs Brian Barnett, David
Bookout, Samuel Chantilis, James Douglas, Rheza
Guerami, Robert Kaufmann, Karin Lee, James Madden
and Alfred Rodriguez as well as embryologists Hannalie
Adriaanse, Tonya Davidson, Annelize De Kock, Oscar
Perez, Mario Smuts and David Ward as the real reason for
a successful blastocyst cryopreservation program.
You can contact Marius Meintjes, PhD at:
[email protected]
Figure 2. An example of a hatching blastocyst 14
hours post thaw with a poor (F-grade) ICM and a
“B” trophoblast grade. This embryo resulted in a
biochemical pregnancy.
Figure 1. ICM and trophoblast grading scores used to select blastocysts for
cryopreservation and to identify good quality- and poor quality blastocysts
before transfer.
Figure 3. A hatching blastocyst 17 hours after
thawing with a “B” ICM and an “A” trophoblast
grade. This blastocyst resulted in a live birth.
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• VOLUME 4 • FERTILITY MAGAZINE
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14
FERTILITY MAGAZINE • VOLUME 4 •
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ARTICLES
Use of the Sperm Chromatin Structure Assay (SCSA ®)
as a Diagnostic Tool in the Human Infertility Clinic
by Donald P. Evenson, PhD, HCLD and Regina L. Wixon, PhD
SCSA Diagnostics, Inc., Brookings Research & Technology Center, 807 32nd Avenue, Brookings, SD 57006, USA
I
nfertility has been attributed
equally to men and women with
20% of infertility problems due to
unknown factors. However, men’s
infertility is rapidly surpassing
women’s, according to infertility
experts at the European Society for
Human
Reproduction
and
Embryology
Meeting
in
Copenhagen, 2005. The decline in
male fertility can be attributed to a
number of factors, including but not
limited to, environmental toxins,
medications, infections, diet, drug
use, high fever, elevated testicular
temperature, binge drinking, air
pollution, cigarette smoking and
advanced age. What perhaps is an
underlying effect of these insults on
the fertility of men is an increase in
sperm DNA fragmentation. The
sperm chromatin structure assay
(SCSA® ), (Evenson et al. 1980,
(Palus et al. 2005), chemotherapy
(Evenson et al. 1984a, Evenson et al.
1984b), and a
variety of other
chemicals that
target different
testicular germ
cell types.
Elevated
sperm
DNA
fragmentation as
measured by the
SCSA has been
shown to have a
significant
REGINA L. WIXON, PHD
negative effect
on
pregnancy
outcome. The % DONALD P. EVENSON, P HD, HCLD
D
N
A
below 30%. Data from this study
Fragmentation Index (DFI) threshold
were used to establish the statistical
for infertility was first established in
the comprehensive “Georgetown
thresholds for the SCSA analysis of
Male Factor Infertility Study”
>30% DFI for ‘significant lack of’,
>15-<30% DFI for ‘reasonable’ and
<15% DFI for ‘high’ fertility potential
status.
To evaluate the effectiveness of
the SCSA analysis, meta-analyses
were conducted on studies of
intercourse, IUI, routine IVF and
ICSI methods of fertilization. A
(Evenson et al. 1999). The study
meta-analysis of three studies
included 144 couples trying to
(n=1575)
was
conducted
to
conceive naturally. SCSA data from
male partners of 73 couples that
investigate the relationship of sperm
DNA fragmentation on pregnancy
achieved pregnancy during months
outcome using intercourse and IUI.
1-3 (Group 1) were used as the
The meta-analysis indicated that
standard of sperm DNA integrity
compatible with high fertility. Group
patients were 7.3 times more likely to
achieve a pregnancy/delivery if the
1 had significantly different DFI
DFI was < 30% (p=0.0001) (Evenson
values compared to 40 couples
et al. 1999, Spano et al. 2000, Bungum
achieving pregnancy in months 4-12
(Group 3, P<0.01) and 31 couples not
et al. 2004). When routine IVF alone
was considered, couples were 2.2
achieving pregnancy (Group 4,
P<0.001). The couples in this study
were 6.5 times more likely to achieve
CONTINUED ON PAGE 16
a pregnancy when their DFI was
The application of PGD is increasing
on an almost daily basis.
Evenson et al. 1985) now in use for
over two decades, has proved to be a
very sensitive and precise measure
of sperm DNA damage resulting
from environmental toxicants or
other damaging agents. Agents
studied have been radiation (Sailer et
al. 1995), heat (Sailer et al. 1997), air
pollution (Selevan et al. 2000),
smoking (Potts et al. 1999),
insecticides (Xia et al. 2005, Bian et al.
2004),
PCBs
and
persistent
organochlorine pollutants (RignellHydbom et al. 2004), industrial
chemicals (Sanchez-Pena et al. 2004),
phthalates (Duty et al. 2003), arsenic
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• VOLUME 4 • FERTILITY MAGAZINE
15
ARTICLES
C ONTINUED FROM PAGE 15
times more likely to become
pregnant if their DFI was <30%
(n=521, p=0.0008) (Bungum et al.
2004, Henkel et al. 2004, Larson-Cook
et al. 2003, Adams et al. 2004). A metaanalysis of five studies using ICSI
and/or IVF (n=216) showed a nonsignificant trend where patients were
1.7 times more likely to achieve a
pregnancy/delivery if the %DFI was
<30% (p=0.11) (Bungum et al. 2004,
Larson-Cook et al. 2003, Chohan et al.
2004, Morris et al. 2002, Larson et al.
2000). These meta-analyses show that
the
SCSA
infertility
test
is
significantly predictive for reduced
pregnancy success using intercourse,
IUI and routine IVF and, to a lesser
extent, ICSI.
While environmental insults are
a concern for men’s infertility, the
natural aging process is also
associated with increasing levels of
sperm DNA fragmentation. The
latest research indicate that while
some older men are highly fertile, the
age of 47 years intersects with the
30% statistical threshold associated
with a negative pregnancy outcome
(see Figure 1). As seen in Figure 1, for
the factor of sperm DNA integrity
alone, a 50 year old man has on the
average, a 30% decreased chance for
a successful pregnancy relative to a
man in his 20’s. Morris et al. (2002),
also found sperm DNA damage to be
strongly associated with age in men
29-44 years (r=0.669, p=0.032).
Infertile couples less than 35 years of
age (for both male and female) had a
53% pregnancy rate in comparison to
4% in infertile couples greater than 40
years of age (Klonoff-Cohen and
Natarajan 2004). Since a quarter of
men entering a fertility clinic are over
the age of 40, aging and men’s
fertility become a significant factor.
When
sperm
DNA
fragmentation is elevated, a trend for
spontaneous miscarriages has been
16
FERTILITY MAGAZINE • VOLUME 4 •
reported in several studies. This was
first found in the “Georgetown Male
Factor Infertility Study” where the
DFI statistical groups were first
identified. This study predicted
almost half of the miscarriages in
couples that were using intercourse
and had elevated sperm DNA
fragmentation values (Evenson et al.
1999). In two other studies, IVF/ICSI
couples with elevated DFI values also
found a trend in miscarriages (Virro
et al. 2004, Check et al. 2005). In
another study, twice as much sperm
DNA fragmentation was found in
couples with unexplained recurrent
pregnancy loss in comparison to the
general population (Carrell et al.
2003). Using a light microscope
method of analysis, Morris et al.
(2002) found that elevated sperm
DNA damage was predictive of
embryo development failure in a
group of IVF/ICSI men. Three of the
nine pregnancies (33%) in the
elevated sperm DNA damaged group
resulted in an early miscarriage while
all six of the pregnancies in the low
sperm DNA damaged group yielded
live births.
Who should have a sperm DNA
fragmentation test?
• All couples with unexplained
infertility
• Couples with a history of
spontaneous miscarriages
• Men over 40 years of age
• Men with above listed pathologies
• Men with exposure to toxic agents
• Men with a questionable varicocele
for repair
Many patients with elevated
sperm DNA fragmentation want to
know
how
to
“fix”
their
fragmentation.
Treatment
may
include taking antioxidants, an
improved healthy lifestyle, avoidance
of hot tubs and not using a laptop
computer on lap.
Many couples have depleted
financial resources and lost precious
WWW.FERTILITYMAGAZINE.ORG
time with ART procedures that were
perhaps not appropriate for their
infertility treatment. Physicians will
typically use the SCSA as part of
their patients’ initial infertility
workup. The results of the SCSA
analysis enables the physician to
better manage their patients’
infertility care, knowing that when
the sperm DNA fragmentation levels
are consistently elevated, IUI should
be skipped and the patient move
onto routine IVF or preferably ICSI.
This saves the patient emotional and
financial costs.
References
Adams C, Anderson L, Wood S (2004)
High, but not moderate, levels of
sperm DNA fragmentation are
predictive of poor outcome in egg
donation cycles [abstract no. O-110].
In: Abstracts of the Scientific Oral &
Poster Sessions. Philadelphia, PA:
Journal of the American Society for
Reproductive
Medicine,
(with
permission):S44.
Bian Q, Xu LC, Wang SL, Xia YK, Tan LF,
Chen JF, Song L, Chang HC, Wang
XR (2004) Study on the relation
between occupational fenvalerate
exposure and spermatozoa DNA
damage of pesticide factory workers.
Occup Environ Med, 61, 999-1005.
Bungum M, Humaidan P, Spano M,
Jepson K, Bungum L, Giwercman A
(2004) The predictive value of sperm
chromatin structure assay (SCSA®)
parameters for the outcome of
intrauterine insemination, IVF and
ICSI. Hum Reprod, 19, 1401-1408.
Carrell DT, Liu L, Peterson CM, Jones KP,
Hatasaka HH, Erickson L, Campbell
B (2005) Sperm DNA fragmentation
is increased in couples with
unexplained recurrent pregnancy
loss. Arch Androl, 49, 49-55.
Check JH, Graziano V, Cohen R, Krotec J,
Check ML (2005) Effect of an
abnormal
sperm
chromatin
structural assay (SCSA ®) on
pregnancy outcome following (IVF)
with ICSI in previous IVF failures.
Arch Androl, 51, 121-4.
Chohan KR, Fiffin JT, Lafromboise M,
DeJonge CJ, Carrell DT (2004) Sperm
DNA damage relationship with
ARTICLES
embryo quality and pregnancy
outcome in IVF patients [abstract no.
O-137]. In: Abstracts of the Scientific
Oral & Poster Sessions. Philadelphia,
PA: Journal of the American Society
for Reproductive Medicine, (with
permission):S55.
Duty SM, Singh NP, Silva MJ, Barr DB,
Brock JW, Ryan L, Herrick RF,
Christiani DC, Hauser R (2003) The
relationship between environmental
exposures to phthalates and DNA
damage in human sperm using the
neutral comet assay. Environ Health
Perspect, 111, 1164-9.
Evenson DP, Darzynkiewicz Z, Melamed
MR (1980) Relation of mammalian
sperm chromatin heterogeneity to
fertility. Science, 210, 1131-3.
Evenson DP, Higgins PJ, Grueneberg D,
Ballachey BE (1985) Flow cytometric
analysis of mouse spermatogenic
function following exposure to
ethylnitrosourea. Cytometry, 6, 23853.
Evenson DP, Jost LK, Zinaman MJ, Clegg
E, Purvis K, de Angelis P, et al (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.
Evenson DP, Klein FA, Whitmore WF,
Melamed MR (1984) Flow cytometric
evaluation of sperm from patients
with testicular carcinoma. J Urol, 132,
1220-5.
Evenson DP, Arlin Z, Welt S, Claps ML,
Melamed
MR
(1984)
Male
reproductive capacity may recover
following drug treatment with the L10 protocol for acute lymphocytic
leukemia. Cancer, 53, 30-6.
Klonoff-Cohen HS, Natarajan L (2004)
The effect of advancing paternal age
on pregnancy and live birth rates in
couples undergoing in vitro
fertilization or gamete intrafallopian
transfer. Am J Obstet Gynecol, 191,
507-14.
Henkel R, Hajimohammad M, Stalf T,
Hoogendijk C, Mehnert C, Menkveld
R, et al (2004) Influence of
deoxyribonucleic acid damage on
fertilization and pregnancy. Fertil
Steril, 81, 965-972.
Larson-Cook K, Brannian JD, Hansen KA,
Kasperson K, Aamold ET, Evenson
DP (2003) Relationship between the
outcomes of assisted reproductive
techniques
and
sperm
DNA
fragmentation as measured by the
sperm chromatin structure assay.
Fertil Steril, 80, 895-902.
Larson KL, DeJonge CJ, Barnes AM, Jost
LK, Evenson DP (2000) Sperm
Relationship between Age and DNA Fragmentation
Figure 1.
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• VOLUME 4 • FERTILITY MAGAZINE
17
ARTICLES
C ONTINUED FROM PAGE 17
chromatin
structure
assay
parameters as predictors of failed
pregnancy
following
assisted
reproductive
techniques.
Hum
Reprod, 15, 1717-1722.
Morris ID, Ilott S, Dixon L, Brison DR
(2002) The spectrum of DNA damage
in human sperm assessed by single
cell gel electrophoresis (Comet assay)
and its relationship to fertilization
and embryo development. Hum
Reprod, 17, 990-8.
Palus J, Lewinska D, Dziubaltowska E,
Stepnik M, Beck J, Rydzynski K,
Nilsson R (2005) DNA damage in
leukocytes of workers occupationally
exposed to arsenic in copper
smelters. Environ Mol Mutagen, 2005
May 6; [Epub ahead of print]
Potts RJ, Newbury CJ, Smith G,
Notarianni LJ, Jefferies TM (1999)
Sperm chromatin damage associated
with male smoking. Mutat Res, 423,
18
FERTILITY MAGAZINE • VOLUME 4 •
103-11.
Rignell-Hydbom
A,
Rylander
L,
Giwercman A, Jonsson BA, Lindh C,
Eleuteri P, Rescia M, Leter G, Cordelli
E, Spano M, Hagmar L (2005)
Exposure to PCBs and p,p'-DDE and
human sperm chromatin integrity.
Environ Health Perspect, 113, 175-9.
Spano M, Bonde J, Hjollund HI, Kolstatd
HA, Cordelli E, Leter G (2000) Sperm
chromatin damage impairs human
fertility. Fertil Steril, 73, 43-50.
Sailer BL, Jost LK, Erickson KR, Tajiran
MA, Evenson DP (1995) Effects of Xirradiation on mouse testicular cells
and sperm chromatin structure.
Environ Mol Mutagen, 25, 23-30.
Sailer BL, Sarkar LJ, Bjordahl JA, Jost LK,
Evenson DP (1997) Effects of heat
stress on mouse testicular cells and
sperm chromatin structure. Androl,
18, 294-301.
Sanchez-Pena LC, Reyes BE, LopezCarrillo L, Recio R, Moran-Martinez
J, Cebrian ME, Quintanilla-Vega B
(2004) Organophosphorous pesticide
WWW.FERTILITYMAGAZINE.ORG
exposure alters sperm chromatin
structure in Mexican agricultural
workers. Toxicol Appl Pharmacol, 196,
108-13.
Selevan SG, Borkovec L, Slott VL, Zudova
Z, Rubes J, Evenson DP, Perreault SD
(2000)
Semen
quality
and
reproductive health of young Czech
men exposed to seasonal air
pollution. Environ Health Perspect,
108, 887-94.
Virro MR, Larson-Cook KL, Evenson DP
(2004) Sperm chromatin structure
assay (SCSA®) related to blastocyst
rate,
pregnancy
rate
and
spontaneous abortion in IVF and
ICSI cycles. Fertil Steril, 8, 1289-1295.
Xia Y, Cheng S, Bian Q, Xu L, Collins MD,
Chang HC, Song L, Liu J, Wang S,
Wang X (2005) Genotoxic effects on
spermatozoa of carbaryl-exposed
workers. Toxicol Sci, 85, 615-23.
You can contact Donald P. Evenson, PhD and
Regina L. Wixon, PhD at: [email protected]
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ARTICLES
The Effect of CO2 Concentration and pH on the
In-Vitro Development of Mouse Embryos
by Avner Hershlag, MD and Huai L. Feng, PhD, HCLD
Center for Human Reproduction, North Shore University Hospital, 300 Community Drive, Manhasset, NY 11030,. U.S.A.
Summary
This study was designed to assess the effect of CO2
concentration on the pH of the medium and the
developmental rates of mouse embryos. Pronuclear
stage zygotes were placed in microdrops of HTF
containing 10% SSS and cultured at 37°C, under 4.0,
4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or 7.5% CO2 in air for 120 h.
The embryos were scored for cleavage at 24, 48, 72
and 96 hours and for blastocoel formation at 120hours of culture. Cleavage to the 2-cell stage was
lower under 4.0% CO 2 (76.8%), but remained
unchanged at all other CO 2 concentrations.
Development to the blastocyst stage was 69.2% under
5.0% CO 2, and significantly lower at all other CO2
concentrations. The development of mouse embryos
could be arrested in both high and low pH culture
media.
Introduction
Exposure of gametes or embryos to potential toxins at any
point during the IVF cycle may have a detrimental effect
on embryogenesis and consequent pregnancy rates.
Testing of factors such as CO2 concentrations of the
incubator and pH of the culture medium is the backbone
HUAI L. FENG, PHD
AVNER HERSHLAG, MD
microdrops and cultured under each of the CO 2
concentrations for 120 h (three microdrops per CO2
concentration). The embryos were scored for cleavage at
24, 48, 72 and 96 h, and for blastocoel formation at 120 h.
The CO 2 concentration was
Testing of factors such as CO2 concentrations of the measured using a CO2 analyzer
(Mid Atlantic, Marlton, NJ), and pH
incubator and pH of the culture medium is the backbone was determined with a digital pH
of any stringent quality control program in IVF. microelectrode.
of any stringent quality control program in IVF.
Developmental rates of a one-cell (1) and two-cell (2)
mouse embryo system have evolved into the standard
bioassays for quality control in clinical IVF laboratories (3).
In this study, one-cell mouse embryos were used to
evaluate the effects of CO2 concentration and pH on invitro development.
Materials and Methods
Pronuclear stage zygotes (B6C3 F1 female/ B6D2F1 male
mice) were obtained from Conception Technology Inc.
(San Diego, CA). Microdrops (50µl) of HTF (LifeGlobal,
Guilford, CT) supplemented with 10% SSS (Irvine, Santa
Ana, CA), were set up and equilibrated at 37oC for 18
hours under 4.0%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0% or 7.5%
CO2 in air. A total of 30-45 zygotes were then placed in the
Results
As shown in Figure 1, the pH of the media varied from 7.4
under 4.0% CO 2 to 6.8 under 7.5% CO2. Cleavage to the 2cell stage was lower under 4.0% CO2 (76.8%), but
remained unchanged at all other CO2 concentrations.
Further development to the blastocyst stage was 69.2%
under 5.0% CO 2 and significantly less than that at all other
CO2 concentrations (Figure 1).
Discussion
Carbon dioxide is required not only to maintain the pH of
bicarbonate buffered medium, but is also readily
incorporated into protein and nucleic acids by mouse
embryo at all stages prior to implantation (4). Culture
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ARTICLES
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Figure 1. The effect of CO 2 concentration on the pH of the culture
medium and development of mouse zygotes to the blastocyst
stage. ** P<0.01 vs. 5% CO 2 (pH 7.2), * P< 0.05 vs. 5% CO2 (pH
7.2). Using Fisher’s Exact Test.
systems for the preimplantation mammalian embryo
routinely employ a carbon dioxide concentration of 5%
coupled with a bicarbonate concentration of
approximately 25 mM. However, the carbon dioxide
concentrations of the uterine environment vary from 5% to
10% in different animal species (5). The optimal
concentration of carbon dioxide for human embryo
development has yet to be determined. According to the
pH (7.1 to 8.0) of fluid collected from the reproductive
tract of animal species, external pH of culture media
formulated for preimplantation embryos is commonly
between 7.3 ~7.4 (depending on the CO 2 concentration
used) (5).
Studies on the mouse embryos determined that
development from the 2-cell stage to the blastocyst stage
could occur in media with a pH range from 5.9 to 7.8 (6).
The embryos at 8-cell stage from hamster could develop to
the blastocyst stage in medium with an external pH range
from 6.4 to 7.4 (7). Similarly in our studies, the mouse
embryos from the zygote stage to the
blastocyst stage could occur in HTF
medium with a pH range from 6.8 to
7.4. However, the cleavage rate to the
2-cell stage was lower at pH 7.4
(76.8%), but remained unchanged at all
other
pH
(6.8~7.3).
Further
development to the blastocyst stage
was significantly decreased in both
high pH 7.4 (at 4.0% CO2) and low pH
22
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6.8~7.0 (at 6.5-7.5% CO 2) culture environments.
Development of mouse embryos could be arrested in both
the high and low pH culture media. The prior reports
indicated that even small fluctuations in pHi can
significantly
retard
subsequent
developmental
competence. Fluctuations in either the acidic or the
alkaline range can drastically reduce embryo
development (5, 8, 9). Therefore, care should be taken to
avoid fluctuations in the pH of media during gametes,
embryo manipulation and culture. In most of laboratories,
the use of an oil overlay not only reduces the harmful
effects of increases in osmolarity, it also reduces changes
in pH caused by a loss of CO2 from the media when
culture dishes are taken out of the incubator for gamete or
embryo examination. This is especially relevant for
oocytes that are stripped of their cumulus before an ICSI
procedure. These oocytes and embryos immediately
following the procedure cannot regulate their ionic
homeostasis, which may result in decrease fertilization
rate and pregnancy rate.
Conclusions
The results show that proportion of of one-cell mouse
embryos that develop to the blastocyst stage is reduced at
both high and low pH. Optimal blastocyst development
was observed at a 5.0% CO 2 concentration, which
produced a pH of 7.2.
References
1. Quinn P, Warnes , Kerin J.F. et al. Culture factors in relation
to the success of human in vitro fertilization and embryo
transfer. Fert Steril, 41, 202-9, 1984.
2. Ackerman S, Swanson RJ, Stokes G, and Veeck L., Culture of
preimplantation mouse embryos as a quality control assay
for human in vitro fertilization. Gamete Res, 9, 145-152, 1984.
3. McDowell JS, Swanson RJ, Maloney M, Veeck L., Mouse
embryo quality control for toxicity determination in the
Norfolk in vitro fertilization program. J In Vitro Fert
Embryo Transfer, 5,144-148, 1988.
4. Graves C.N. and Biggers J.D., Carbon dioxide fixation by
mouse embryos prior to implantation, Science, 167, 1506,
1970.
5. Trounson A.O. and Gardner D.K. Handbook of Ferilization,
Second Edition, New York, CRC Press, 2000.
6. Ludwig T.E., Lane M. and Bavister B.D., Increased fetal
development after transfer of hamster embryos cultured
with glucose, Biol. Reprod., 58(suppl.1), 306, 1998.
7. Carney E.W. and Bavister B.D., Regulation of hamster
embryo development in vitro by carbon dioxide, Biol.
Reprod., 36, 1155, 1997.
8. John D.P. and Kiessling A .A., Improved pronuclear mouse
embryo development over an extended pH range in Ham's
F-10 medium without protein, Fertil. Steril., 49, 150-5, 1988.
9. Naaktgeboren N., Quality control of culture media for in
vitro fertilization, Ann Biol Clin., 45, 368-72, 1987.
You can contact Avner Hershalg, MD and Huai L. Feng, PhD, HCLD at:
[email protected]
ARTICLES
Degree of Re-expansion of Thawed Blastocysts is Predictive
of Frozen Embryo Transfer (FET) Success
By Deborah C. Merryman, MSc, ELD/TS (ABB), Embryology Laboratory Director
ART Fertility Program of Alabama, Birmingham, AL
(Presented in part at the Annual Meeting of the American Society of Reproductive Medicine, Philadelphia, Pennsylvania, October 16-20, 2004,
poster #P252, DC Merryman, SE Stringfellow, CA Yancey, VL Houserman, CA Long, and KL Honea.)
INTRODUCTION
Cryopreservation is an integral component of an in-vitro
fertilization (IVF) program. Benefits from a successful
cryopreservation program include cost effectiveness,
enhancement of the cumulative pregnancy rate and
reduction in the multiple birth rate with fresh IVF. A
successful cryopreservation program is critical to those
patients needing only a single blastocyst transferred with
fresh IVF 1. Cryopreservation of excess embryos is
routinely performed at the blastocyst stage at the ART
Fertility Program of Alabama. Clinical pregnancy rates for
cryopreserved embryos have been shown to be higher for
the blastocyst as compared to other cell stages2. Some of
the factors associated with frozen embryo transfer (FET)
success with blastocysts include incubation time between
thaw and transfer3 and the degree of blastocyst expansion
at transfer4.
OBJECTIVE
Frozen-thawed blastocysts immediately contract after
thawing and subsequently re-expand over time. The
objective of this study was to evaluate the relationship
between the re-expansion of frozen-thawed blastocysts
two hours after thawing and pregnancy outcome.
post thaw evaluation
of
frozen-thawed
blastocysts
was
available. Blastocysts
exhibiting at least a
full blastocoel with a
good inner cell mass
and trophectoderm
(grade 3BB, Gardner,
et al 5), were frozen on
day 5 or 6 of embryo
culture. A two-step
freeze and a two- or
multi-step
thaw
procedure
was
DEBORAH C. MERRYMAN , MSC
utilized with glycerol
and sucrose in both
the freeze and thaw solutions. After thawing, blastocysts
were cultured in microdroplets of culture medium with 20
mg/ml protein under oil for 2 to 6 hours prior to transfer.
Two hours after thawing, blastocysts were evaluated for
degree of re-expansion. Re-expansion was assessed by
observing the percentage viable/re-expanded per the total
volume of the frozen blastocyst and documented in 25%
increments (see figures). The
replacement cycle routinely included
estradiol, 2mg bid orally, for 10 to 14
days. Ideally, progesterone, 200mg
vaginal capsules tid and 50 mg IM
qd, was given when endometrium was 8mm or more and
continued until pregnancy test. Blastocysts were thawed
and transferred on day 6 of progesterone utilizing an
echotip catheter (Cook) under ultrasound guidance.
Fisher's exact test, Mann Whitney u-test or Student's t-test
was used for statistical analysis as indicated. Statistical
significance was defined as P <0.05.
Cryopreservation is an integral component
of an in-vitro fertilization (IVF) program
DESIGN
This study was designed to retrospectively review the
outcome of FET cycles in relation to blastocyst reexpansion two hours post thaw. Outcome was evaluated
by ongoing pregnancy and/or delivery rate (ODR),
defined as the number of ongoing pregnancies and/or
deliveries per embryo transfer, and implantation rate (IR),
defined as the number of ongoing fetal heartbeats and/or
live births per number of embryos transferred.
Pregnancies and fetal heartbeats were considered ongoing
at 12 or more weeks' gestation.
RESULTS
FETs were grouped according to the number of
blastocysts transferred with 50% or more re-expansion
MATERIAL AND METHODS
The data consisted of 75 FET cycles in which a two-hour
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ARTICLES
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two hours after thawing: 0 (N=18) and 1 (N=57). A
significant increase in ODR (0% vs. 33.3%, P=0.004) and IR
(0% vs. 16.2%, P=0.02) was shown when one or more of the
blastocysts transferred were 50% or more re-expanded two
hours after thawing (Table 1). No ongoing and/or
delivered pregnancies occurred when all blastocysts
transferred were <50% re-expanded two hours after
thawing. A significant increase in the total number of
embryos transferred (1.7 v. 2.4, p=0.001) and the number of
embryos transferred with 50% or more re-expansion two
hours after thawing (0.0 v. 1.6, p<0.0001, Table 2) was seen
when one or more of the blastocysts transferred were 50%
or more re-expanded two hours after thawing. All other
patient characteristics evaluated were not significantly
different between groups.
FETs were also grouped according to pregnancy
outcome, specifically, cycles with an ongoing and/or
delivered pregnancy (N=19) vs. cycles without an ongoing
and/or delivered pregnancy (N=56). Characteristics
showing a significant difference between groups included
the total number of embryos transferred (2.6 v. 2.1, p=0.02),
the number of embryos transferred with 50% or more reexpansion two hours after thawing (2.2 v. 1.0, p<0.0001),
and the number of transfers with 1 blastocyst transferred
at 50% or more re-expansion two hours after thawing
(100% v. 67.9%, p=0.004, Table 3), with ongoing and/or
delivered pregnancy vs. without ongoing and/or delivered
pregnancy, respectively.
from the transfer of three and one from the transfer of two
re-expanded blastocysts and no ongoing or delivered
triplet gestation within the data set. Of note, ongoing
pregnancies and deliveries occurred from the thaw of
blastocysts cryopreserved on day five, day six and from a
combination of day five and six of embryo culture (data
not shown).
CONCLUSIONS
1. The assessment of the re-expansion of frozen-thawed
blastocysts two hours post thaw is predictive of FET
success.
2. The presence of one or more frozen-thawed blastocyst
with 50% or more re-expansion two hours post thaw
is related to FET success. On the other hand, the
absence of one or more frozen-thawed blastocyst with
50% or more re-expansion two hours post thaw is
related to FET failure.
3. IR and ODR can be optimized for FET cycles by
evaluating the re-expansion of thawed blastocysts
two hours post thaw.
4. Evaluation of the viability of frozen-thawed
blastocysts can be performed two hours post thaw;
extended incubation beyond two hours post thaw is
not necessary.
5. Thawing of excess frozen blastocysts may be avoided
by initially thawing the minimum number to be
transferred followed by a two hour assessment and
only then thawing more if needed.
DISCUSSION
For FET cycles, the ODR and IR increases with the number
of re-expanded blastocysts transferred. While an ODR of
33.3% and an IR of 16.2% was achieved in FETs with one or
more blastocyst transferred with 50% or more reexpansion (N=57), an ODR of 48.1% and an IR of 25.0%
was achieved in FETs with two or more blastocysts
transferred with 50% or more re-expansion (N=27) two
hours after thawing. Of the 10 FETs with three, 17 FETs
with two and 30 FETs with one re-expanded blastocyst
transferred, there were three sets of twins (15.8%), two
Table 1. Treatment outcome as a function of blastocyst re-expansion in a group of 75 FET cycles.
Number of blastocysts transferred
with ≥50% re-expansion 2 h post thaw
0
Frozen embryo transfers
24
≥1
P
18
57
–
Ongoing fetal heart beats and/or live births (IR)
0 (0)
22 (16.2)
0.02
Ongoing and/or delivered pregnancies (ODR)
0 (0)
19 (33.3)
0.004
FERTILITY MAGAZINE • VOLUME 4 •
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ARTICLES
Table 2. Summary of patient characteristics in relation to blastocyst re-expansion in a group of 375 FET cycles.
Number of blastocysts transferred
with ≥50% re-expansion 2 h post thaw
0
≥1
P
18
57
–
Age
33.7 ± 4.4
31.7 ± 4.8
NS
Embryos transferred
1.7 ± 0.8
2.4 ± 0.8
0.001
Embryos transferred with 50% re-expansion 2 h post thaw
0.0 ± 0.0
1.6 ± 0.8
<0.0001
Embryos thawed
2.3 ± 1.7
2.7 ± 1.1
NS
FSH, mIU/mL, highest basal or day 10
9.3 ± 1.6
9.5 ± 2.4
NS
Body mass index
24.4 ± 5.5
26.2 ± 5.9
NS
Endometrium, mm
9.4 ± 1.8
9.7 ± 1.3
NS
Previous live birth
10 (55.6)
26 (45.6)
NS
11b (20.4)
NS
Frozen embryo transfers
a
Previous live birth with IVF
5 (29.4)
Values are average with standard deviation or number with percentage in parentheses.
a
1 of 18 patients without previous fresh transfer attempt; b 3 of 57 patients without previous fresh transfer attempt.
Table 3. Summary of patient characteristics in relation to pregnancy outcome in a group of 75 FET cycles.
Pregnancy outcome
Ongoing and/
or delivered
Not Pregnant
P
19
56
–
FETs with 1 blastocyst transferred with 50% re-expansion 2 h post thaw
19 (100.0)
38 (67.9)
0.004
Age
32.5 ± 4.5
32.2 ± 4.9
NS
Embryos transferred
2.6 ± 1.1
2.1 ± 0.7
0.02
Embryos transferred with 50% re-expansion two hours post thaw
2.2 ± 1.0
1.0 ± 0.8
<0.0001
Embryos thawed
3.1 ± 1.3
2.6 ± 1.3
NS
FSH, mIU/mL, highest basal or day 10
10.3 ± 2.5
9.3 ± 2.2
NS
Body mass index
25.9 ± 4.3
25.6 ± 6.3
NS
Endometrium, mm
9.7 ± 1.4
9.6 ± 1.4
NS
Previous live birth (%)
8 (42.1)
26 (46.4)
NS
18b
NS
Frozen embryo transfers
a
Previous live birth with IVF (%)
6 (35.3)
(33.3)
Values are average with standard deviation or number with percentage in parentheses.
a
2 of 19 patients without previous fresh transfer attempt; b 2 of 56 patients without previous fresh transfer attempt.
6.
Multiple gestations can be reduced during FET cycles
by limiting the number of blastocysts transferred with
50% or more re-expansion two hours post thaw.
References
1. Tiitinen A, Halttunen M, Harkki P, et al. Elective single
embryo transfer: the value of cryopreservation. Hum Reprod
2001:1140-44.
2. Veeck LL. Does the developmental stage at freeze impact on
clinical results post-thaw? Reprod BioMed Online 2003:36774.
3. Guerif F, Cadoret V, Poindron J, Lansac J, Royere D.
4.
5.
Overnight incubation improves selection of frozen-thawed
blastocysts for transfer: preliminary study using
supernumerary embryos. Theriogenology 2003:1457-66.
Behr B, Gebhardt , Lyon J, Milki A. Factors relating to a
successful cryopreserved blastocyst transfer program. Fertil
Steril 2002:697-99.
Gardner DK, Schoolcraft WB. In vitro culture of human
blastocyst. In: Jansen R and Mortimer D, eds. Towards
Reproductive Certainty: Infertility and Genetics Beyond
1999. Carnforth, Parthenon Press, 1999:378-88.
You can contact Debbie Merryman at: [email protected]
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AGE & FERTILITY
Advanced Maternal Age and Infertility
by Alan B. Copperman, MD
Director of Reproductive Endocrinology, Vice-Chairman of Obstetrics and Gynecology, Mount Sinai School of Medicine
Co-Director, Reproductive Medicine Associates of New York, www.rmany.com
A
pervasive issue in the field of infertility today is
advancing maternal age and its impact on
conception. Though males experience some
impairment in their fertility around age 45 plus, females
begin to incur decline even prior to age 35. Because the
world is moving towards social equality, women are
waiting until a later age to start a family in order to
mature and attain a stronghold in the workplace, and
reproductive specialists are finding an increasingly larger
population of women requesting treatment due to
advanced age.
Infertility is defined as the inability to conceive after a
year of unprotected intercourse in women under 35 years
of age, after six months in women 30 or older, or the
inability to carry a pregnancy to term. Why does this age
disparity exist between women under 35 and women over
35? Although 35 should not be considered as a absolute
cut-off, many studies show that the chances of conceiving
rapidly deteriorate, and the incidence of miscarriage
increases. After age 40, the chances of a woman
conceiving using her own eggs declines at an even greater
rates. There are a number of interacting causalities
associated with age advancement that engender difficulty
in conception.
Women are born into this
world possessing a fixed egg
pool – the number of eggs in the
ovaries being about 2 million
eggs. By adolescence, most of
the eggs are depleted and only
about 500,000 are left. With the
onset of menstruation, follicle
stimulating hormone (FSH) is released by the pituitary
gland each month to stimulate egg maturation. A number
of eggs will start to mature but, in general, only one egg
will progress fully and be ovulated. Eventually, a woman
will exhaust her ovarian reserve and will experience
diminished ovarian reserve or even ovarian failure, and
the production of eggs, estrogen and progesterone ceases.
At this point, her only realistic chance of conception will
be through egg donation, where she uses another
woman’s eggs for conception and has the embryo
implanted following in-vitro fertilization.
exposure to radiation,
and other external
contaminants have
been implicated in
this decline, much is
predetermined
by
genetics, leaving little
for a patient to
control.
How to Test for
Ovarian
Reserve
Quality
Assessing the quality
of ovarian reserve is
ALAN B. COPPERMAN , MD
the most effective
way to test for how healthy and abundant the eggs are.
Although there is no absolutely accurate and reliable test
for ovarian reserve quality, a few exist that can provide
the physician an initial direction of which treatment
should be pursued.
One such test is the Day 3 FSH test, is a simple blood
test administered on the 3rd day of a woman’s menstrual
cycle. A woman’s FSH level is the most reliable indicator
of egg quality, because FSH is
produced in greater quantities
to stimulate ovarian growth as
the eggs are depleted. Therefore,
a high FSH suggests that the
woman’s ovarian reserve is
poor; while a low FSH suggests
that the woman's eggs are
healthy and she has a better prognosis for fertility.
Estradiol (E2) levels can also indicate the health of the
ovarian reserve, and most times is measured in
conjunction with Day 3 FSH. Estradiol is a form of
estrogen that also reaches elevated levels prior to ovarian
failure in order to stimulate ovulation. Estradiol levels
can be measured in the same blood sample that is taken
for the measurement of FSH.
The clomiphene citrate challenge test (CCCT)
requires a slightly greater effort on the woman’s part. A
woman taking this blood test initially has her baseline
FSH levels drawn on day 3 through 5 of her menstrual
With increasing age,
women become more prone
to infertility because of a
decline in egg quality
Age Factors Affecting Fertility
With increasing age, women become more prone to
infertility because of a decline in egg quality (often
referred to as ovarian reserve). While smoking, pollution,
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AGE & FERTILITY
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cycle. She then takes Clomid for 5 days and has her FSH
levels drawn again on the 10th day. FSH levels that have
not reverted to normal baseline level by the 10th day
indicate a poor ovarian reserve. The CCCT is a more
effective test but not as commonly used due to the
inconvenience of having to make multiple trips to the
medical office.
Basal antral follicle count is now considered a routine
part of an ultrasound evaluation. By counting the number
of immature follicles, the physician gets valuable
information about the ovaries potential to produce eggs.
Treatment Options
Although a woman can undergo hormonal treatment such
as clomiphene citrate therapy to overcome fertility
complications due to advanced maternal age, the
probability of conception will remain relatively low if she
exhibits poor preliminary testing. In fact, in patients with
early evidence of diminishing ovarian reserve, many
fertility specialists advocate a rapid progression to IVF as
the most effective treatment for these women. In IVF, a
woman’s eggs and male’s sperm are retrieved and
combined by an embryologist in an IVF laboratory. The
embryos are examined as they grow for 3-5 days, and the
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FERTILITY MAGAZINE • VOLUME 4 •
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strongest and most viable embryos are implanted back
into the woman’s uterus. Advancements in technology
have increased success rates for IVF; but the chances for
conception do decrease the older a woman gets. For
women who suffer from ovarian failure, donor egg IVF
offers a greater chance for fertility. In patients with
contraindications to pregnancy or with a damaged or
absent uterus, a gestational carrier (surrogate) can have a
couple's embryo implanted and carry the baby to term.
Conclusion
The struggle between career and family is an ongoing
struggle and, coupled with the financial pressures, many
couples do not attempt to start a family until they are in
their 30’s or even 40’s. Hundreds of thousands of years
would have to pass before evolution could accommodate
our changing human race, and medical technologies are
advanced but there is a threshold of what it can do for
conception. Though women should not feel pressured to
procreate before they are financially and emotionally
ready, but they need to be aware of the limitations in
fertility and the impact of the biological clock on the
reproductive system.
You can contact Alan B. Copperman, MD at:
[email protected]
AGE & FERTILITY
ART and Aging
by Michael S. Neal, BSc, MSc, PhD candidate
Centre for Reproductive Care, Hamilton Health Sciences, & Reproductive Biology Division, Department of Obstetrics & Gynecology,
McMaster University, Hamilton, Ontario, Canada
D
emographic
and
epidemiological
studies
consistently
show
that
infertility is closely related to female
age. This has become apparent in our
society as more women are delaying
childbirth and turning to assisted
reproductive technologies in an
attempt to beat their reproductive
biological clocks [1]. The basis of the
correlation between age and
infertility involves several factors.
From a biological standpoint, the
most important of these is egg
quantity and quality.
Oocyte quantity
The ovary has been eloquently
likened to an hourglass, with the
female germ cells considered
analogous to the grains of sand that
trickle through the narrow opening
depletion of these cells varies from
person to person, with dramatic
consequences on the fertility of
women as they age.
Poor response to ovarian
stimulation during IVF treatment, is
an indicator of diminishing ovarian
reserve, which in turn has been
shown to lead to reduced fecundity
and earlier onset of menopause [4].
Since a woman’s endowment of
oocytes is defined while she herself
was an embryo, the influence of inutero exposure(s) can have a
dramatic effect. As a result, recent
concerns are focused on the impact
of environmental contaminants on
fetuses, as chemical insults in utero
have been shown to result in
impaired postnatal fertility [5]. Part
of this effect may be due to reducing
the initial pool of germ cells
The basis of the correlation between age
and infertility involves several factors.
between the upper and lower
chamber to account for the passage
of time [2]. At birth, the finite
number of female germ cells have
already been established, although
recent work published by Johnson et
al [3] has challenged this dogma by
showing that postnatal mouse
ovaries possess mitotically active
germ cells. However, this very small
population of cells would not be able
to turn around the effects of aging
over the individual's lifespan. With
each cycle the initial endowment
provided at birth is depleted. Once
the complete oocyte reserve has been
exhausted, ovarian senescence
occurs and women experience
menopause. But, the size of follicle
reserve at birth, and the rate of
available at birth. Evidence to
support this notion comes from a
study showing that offspring from
in-utero nicotine exposed rats had
fewer follicles in the ovary, and
significantly smaller litter sizes
compared to saline controls [6].
Furthermore,
constituents
of
cigarette smoke has been shown to
be cytotoxic to developing germ cells
[7].
Follicular Atresia
Aside from the beginning
number of follicles in the ovarian
pool, the rate at which these follicles
are recruited and released is critical
in
determining
reproductive
lifespan. Postnatal germ cell loss
resulting from follicle atresia occurs
MICHAEL S. NEAL, BS C, MS C,
PHD CANDIDATE
through
a
mechanism
of
programmed cell death called
apoptosis. Oocyte loss occurs either
directly from oocyte apoptosis or
indirectly from apoptosis of the
supporting (granulosa) cells that
nourish and mature the oocyte
through postnatal life. The rate at
which the female biological clock
ticks can be dramatically accelerated
by apoptosis. For example, sterility
and premature menopause are
known to occur in young women
following chemotherapy or radiation
treatment for cancer [8]. A more
subtle effect is seen in women who
smoke. Female smokers take longer
to conceive naturally [9], and have
poorer pregnancy rates using IVF
[10], are 2.5 time more likely to have
diminished oocyte reserves [11, 12],
and reach menopause 2-4 years
earlier than their nonsmoking
counterparts [13]. Taken together,
this data strongly suggests that
avoiding environmental tobacco
smoke is one way to prevent
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• VOLUME 4 • FERTILITY MAGAZINE
29
AGE & FERTILITY
oocytes from women of advancing
age.
C ONTINUED FROM PAGE 29
shortening
one's
reproductive
lifespan.
Although, the reasoning behind
the occurrence and regulation of
apoptosis in the germ line is poorly
understood, there is great interest in
unraveling
this
mystery.
Understanding the cellular and
molecular mechanisms that activate
and execute programmed cell death
in the ovary has great implications for
the management of infertility and
also, perhaps, of the aging process
itself.
Oocyte quality
Even staving off follicular atresia will
not solve the parallel problem of
chromosomal damage in ageing eggs.
It is well established that the
considerable increase in anueploidy
among embryos from older women
contributes to reduced fertility by
increasing both implantation failure
and pregnancy loss [14]. Almost 80%
of the eggs from women aged 40-45,
undergoing IVF showed chromosome
deficiencies that likely accounts for
the higher rates of miscarriage and
birth defects seen in these women
[15]. Oocyte quality is further
implicated by the fact that women
who use the eggs of younger ovum
donors have conception rates
expected for the age of the donor and
not that of the recipient.
Successful reproduction hinges
on oocyte quality, which is largely
determined
by
the
genetic
composition of the oocyte. Many key
components of the egg contribute to
normal embryogenesis including
cytoplasmic maturation, spindle
formation, energy supply, pronuclear
formation,
oocyte
activation,
syngamy, early cleavage events and
embryonic development. Disruption
or alteration of one or several of these
factors will lead to embryonic demise
and are more likely to occur in
30
FERTILITY MAGAZINE • VOLUME 4 •
Summary
For many women of reproductive
age, controlling fertility is the main
concern and in contrast being able to
conceive when desired is considered
easy. However, putting off child
bearing to an older age, when fertility
is in decline, has brought home the
reality, and placed high expectations
on what can be achieved with
assisted reproductive technologies.
Even with the transfer of multiple
embryos, there is a marked agerelated decline in female fertility [16].
An admirable goal of all medical
caregivers should be increased
awareness to the general public of the
link between fertility and aging.
References
1. Leridon
H.
Can
assisted
reproduction technology compensate
for the natural decline in fertility
with age? A model assessment. Hum
Reprod 2004; 19: 1548-1553.
2. Morita Y, Tilly JL. Oocyte apoptosis:
like sand through an hourglass. Dev
Biol 1999; 213: 1-17.
3. Johnson J, Canning J, Kaneko T, Pru
JK, Tilly JL. Germline stem cells and
follicular renewal in the postnatal
mammalian ovary. Nature 2004; 428:
145-150.
4. Lawson R, El-Toukhy T, Kassab A,
Taylor A, Braude P, Parsons J, Seed P.
Poor response to ovulation induction
is a stronger predictor of early
menopause than elevated basal FSH:
a life table analysis. Hum Reprod
2003; 18: 527-533.
5. Vidaeff AC, Sever LE. In utero
exposure to environmental estrogens
and male reproductive health: a
systematic review of biological and
epidemiologic evidence. Reprod
Toxicol 2005; 20: 5-20.
6. Pettric JJ, Neal MS, Foster WG,
Holloway AC. In utero and
lactational exposure to nicotine:
ovarian effects. Annual FL Johnson
Research Symposia, Dept of
Obstetrics
and
Gynecology,
McMaster University. 2005.
7. MacKenzie KM, Angevine DM.
WWW.FERTILITYMAGAZINE.ORG
8.
9.
10.
11.
12.
13.
14.
15.
16.
Infertility in mice exposed in utero to
benzo(a)pyrene. Biol Reprod 1981;
24: 183-191.
Familiari G, Caggiati A, Nottola SA,
Ermini M, Di Benedetto MR, Motta
PM. Ultrastructure of human ovarian
primordial follicles after combination
chemotherapy for Hodgkin's disease.
Hum Reprod 1993; 8: 2080-2087.
Baird DD, Wilcox AJ. Cigarette
smoking associated with delayed
conception. Jama 1985; 253: 29792983.
Neal MS, Hughes EG, Holloway AC,
Foster WG. Sidestream smoking is
equally as damaging as mainstream
smoking on IVF outcomes. Hum
Reprod 2005; 20: 2531-2535.
Sharara FI, Beatse SN, Leonardi MR,
Navot D, Scott RT, Jr. Cigarette
smoking accelerates the development
of diminished ovarian reserve as
evidenced by the clomiphene citrate
challenge test. Fertil Steril 1994; 62:
257-262.
El-Nemr A, Al-Shawaf T, Sabatini L,
Wilson C, Lower AM, Grudzinskas
JG. Effect of smoking on ovarian
reserve and ovarian stimulation in invitro fertilization and embryo
transfer. Hum Reprod 1998; 13: 21922198.
Jick H, Porter J. Relation between
smoking and age of natural
menopause. Report from the Boston
Collaborative Drug Surveillance
Program, Boston University Medical
Center. Lancet 1977; 1: 1354-1355.
Munne S, Alikani M, Tomkin G, Grifo
J, Cohen J. Embryo morphology,
developmental rates, and maternal
age are correlated with chromosome
abnormalities. Fertil Steril 1995; 64:
382-391.
Battaglia DE, Goodwin P, Klein NA,
Soules MR. Influence of maternal age
on meiotic spindle assembly in
oocytes from naturally cycling
women. Hum Reprod 1996; 11: 22172222.
Templeton A, Morris JK, Parslow W.
Factors that affect outcome of in-vitro
fertilisation treatment. Lancet 1996;
348: 1402-1406.
You can contact Michael S. Neal, BSc, MSc,
PhD candidate at: [email protected]
AGE & FERTILITY
An Overview of the Effects of Age on Fertility in Women
by Don Rieger, PhD
LifeGlobal, LLC, Guelph, Ontario, Canada
I. INTRODUCTION
For a variety of social and economic reasons, women (and
couples) are putting off having babies until they are in
their mid-thirties, or later. As shown in Figure 1, this
trend is common to most industrialized countries. In the
United States, the average maternal age at first birth
increased from 21.4 to 24.9 years of age from 1970 to 2000.
Overall, birth rates in the United States decreased from
1970 to 1980, but since then, the birth rates for women
over 30 years of age have increased significantly (Figure
2).
Figure 1. The average maternal age at first birth in 1970 and
2000 for ten industrialized countries. (Mathews and Hamilton
2002)
infertile
is
approximately twice
as great for women
40-44, compared with
women 30-34. It is
important to note
that the onset of agerelated
infertility
o c c u r s
approximately
10
years
before
menopause (te Velde
and Pearson, 2002).
The
decrease
of
fertility with age,
DON RIEGER , PHD
coupled with the
tendency toward later child-bearing has led to the
suggestion that “female ageing … is now the main
limiting factor in the treatment of infertility” (Ford et al.
2000).
Figure 3. The effect of a woman's age on infertility and the
chance of remaining childless. Adapted from Menken et al.
(1986).
Figure 2. The birth rate for women 30-34, 35-39 and 40-44
years of age in the United States from 1970 to 2000. (U.S.
National Center for Health Statistics 2003)
* Historical data based upon the age at which a woman marries.
This paper is intended as an overview of the information
available on the relationships between age and fertility in
women. It is by no means an extensive review of the
literature, but rather, the data have been selected to
illustrate general principles. Moreover, it is becoming
increasingly evident that the fertility of a couple
decreases with age of the male, as has been recently
reviewed by Fisch (2005). The effect of male age is beyond
Although some women can, and do, have babies into their
forties and even fifties, in general, fertility decreases
markedly with a woman's age, particularly from 35 years
onward. As shown in Figure 3, the chance of being
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AGE & FERTILITY
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the scope of this paper, but where relevant and whenever
possible, studies have been chosen that have been
controlled or adjust ed for male age as well as frequency of
intercourse and lifestyle factors.
II. THE EFFECT OF AGE ON NATURAL CONCEPTION
The birth of a normal healthy baby requires that a woman
be able to ovulate a mature, normal oocyte (egg) at the
appropriate time, that a fertile sperm be present within the
oviduct (Fallopian tube) for fertilization, that the oviduct
and uterus be capable of supporting the development of
the embryo, and that the embryo (later the fetus) reside in
uterus until fully developed and delivered without major
complications. Many of these processes have been shown
to be affected by the woman's age.
The development of the oocytes and the follicles in
which they reside begins when the woman is herself a
fetus. Primordial germ cells develop into oogonia which
divide and differentiate into primary oocytes. The primary
oocytes are enclosed in a single layer of granulosa cells to
form primordial follicles, numbering approximately
7,000,000 in both ovaries at 4-5 months of gestation. From
there onward, the oogonia stop dividing, and the primary
oocytes are arrested in development until puberty. Most of
the primordial follicles are lost to atresia such that only
approximately 1,000,000 are left at birth, 40,000 at the time
of puberty and 1,000 at menopause. Throughout
reproductive life, groups of the primordial follicles
spontaneously grow and develop into early antral follicles.
Most of these are also lost, except for (usually) one follicle
per menstrual cycle that continues to develop into a large
antral (dominant) follicle in response to gonadotrophic
hormones (FSH and LH) secreted by the anterior pituitary
gland. A surge of LH at mid-cycle induces the final
maturation of the oocyte in the dominant follicle and its
release (ovulation) into the oviduct where it can be
fertilized. (See Piñón, 2002)
The ability to produce and ovulate that one oocyte is
directly related to the number of antral follicles on the
ovaries at that time. As shown in Figure 4, the number of
antral follicles present on the ovaries declines with age.
In addition to the significant decline in the number of
antral follicles, increasing age is also associated with
chromosomal and functional aberrations in the oocytes.
Most notably, as shown in Figure 5, the frequency of
aneuploidy (abnormal numbers of chromosomes) in
human oocytes increases exponentially after 35 years of
age.
Although the sperm can also contribute to
chromosomal defects in the embryo, defects in the aged
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FERTILITY MAGAZINE • VOLUME 4 •
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oocyte are thought to be the major cause of Downs
Syndrome and other chromosomal abnormalities in
newborns (Figure 6). Increased maternal age is also
associated defects in oocyte mitochondria, a structure
responsible for energy production and many other
important functions. These include an increased rate of
point mutations in oocyte mitochondrial DNA (Barritt et
al. 2000), and with a decreased ability of the mitochondria
to produce energy (Wilding et al. 2002).
Figure 4. The mean number of antral follicles detected by
ovarian ultrasound in women. The values are derived from the
regression lines presented in Scheffer et al. (1999).
Figure 5. The effect of a woman's age on the frequency of
aneuploidy in human oocytes (Pellestor 2004).
Figure 6. The effect of maternal age on the risk of Downs
Syndrome and other chromosomal abnormalities in newborns
(Simpson and Elias 1994).
AGE & FERTILITY
As discussed in the following section on assisted
reproduction, there is evidence that maternal age can have
effects on fertilization and on development of the embryo.
In natural conception, it is difficult to be certain whether
problems of fertility arise from deficiencies in the embryo
or deficiencies in the reproductive tract. However, it is
very clear that the ability to get pregnant, to maintain the
pregnancy, and to deliver a healthy baby decreases with
maternal age.
Figure 7 shows that the probability of establishing a
clinical pregnancy following intercourse on the most
fertile day of the cycle decreases steadily with increasing
maternal age. Even for women who do eventually become
pregnant, the likelihood of achieving conception within
six months is dramatically reduced for women 35 years of
wide variety of perinatal complications in the mother and
the baby are significantly greater in women older than 35
years of age compared with women from 18-34 years of
age (Figure 11). For example, compared with women 1834 years old, the risk of an emergency Caesarian section
was 1.5 times as great for women 35-40 years old and
more than twice as great for women older than 40. Based
on historic data and on animal studies, Tarin et al. (2005)
have suggested that delayed motherhood may also have
long-term effects on the health of the children, including
impaired fertility and reduced lifespan.
Figure 9. The effect of maternal age on the incidence of
miscarriage (Gindoff and Jewelewicz 1986).
age and older (Figure 8).
Figure 7. The effect of a woman's age on the probability of
establishing a clinical pregnancy by intercourse on the most
fertile day of the cycle. The data have been adjusted for paternal
age and controlled for frequency of intercourse and lifestyle
factors (Dunson et al. 2002).
Figure 10. The effect of maternal age on the frequency of
preterm delivery (Astolfi and Zonta 1999).
Figure 8. The effect of maternal age on the likelihood of
achieving conception within six months (Ford et al. 2000). The
data are adjusted for lifestyle factors and paternal age.
When pregnancy is established, increasing maternal
age has severely detrimental effects on the outcome. Many
of these effects probably result from the effects of maternal
age on the oocyte/embryo/fetus or the embryonic
contribution to the placenta. Both the miscarriage rate
(Figure 9) and the frequency of preterm delivery (Figure
10) increase with increased maternal age. The risks of a
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Figure 11. The risk of perinatal complications for women 35-40
or >40 years of age compared with women 18-34 years of age
(Jolly et al. 2000).
significant effect on the clinical pregnancy rate following
IUI, but for any given age of the man, the clinical
pregnancy rate decreases markedly with increasing age of
the woman. Similarly, the pregnancy rate resulting from
IUI with sperm from fertile donors is also significantly
reduced with increasing age of the woman (Figure 13).
Figure 12. The effect of the woman's age on clinical pregnancy
rate following intrauterine insemination with the partner's sperm
(Brzechffa et al. 1998).
III. THE EFFECT OF AGE ON THE SUCCESS OF
ASSISTED REPRODUCTION
Infertility can result from lack of ovulation, poor quality
oocytes, blocked oviducts (Fallopian tubes), impotence in
the man, inadequate sperm numbers, poor quality sperm,
or a poor interaction between the sperm and the cervical
mucus. Many of these problems (and infertility due to
unknown causes) can be treated, or at least circumvented,
by assisted reproductive technologies (ART). However,
although ART procedures can improve the chances of a
having a baby, the success rate decreases markedly with
increasing age of the woman.
Figure 13. The effect of maternal age on clinical pregnancy rate
following intrauterine insemination with frozen donor sperm
(Ferrara et al. 2002).
1. Intrauterine Insemination and Donor Insemination
Intrauterine insemination (IUI) is the simplest form of
ART. Semen is collected from the male partner by
masturbation and then the sperm are usually washed to
remove dead cells and other possible deleterious
components of the seminal plasma. The washed sperm are
then placed directly into the uterus via a catheter which
has been passed through the cervix. This serves to avoid
any problems of passage of sperm through the cervix or
cervical mucus, and provides a greater number of sperm
within the uterus to increase the chances of fertilization.
Intrauterine insemination is also commonly used in
conjunction with ovulation induction, in order to ensure
optimal timing of insemination.
Figure 12 shows that the age of the man can have a
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FERTILITY MAGAZINE • VOLUME 4 •
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2. In-Vitro Fertilization
In-vitro fertilization (IVF) was originally developed to
overcome the problem of blocked oviducts but is now also
used to treat male-factor infertility (low numbers or
quality of sperm) and infertility for which there is no
apparent cause. In general, the woman is treated with
gonadotrophins to increase the number of antral follicles
that fully develop. It is important to note that
gonadotrophin treatment has no effect on the numbers of
AGE & FERTILITY
primordial follicles that develop to the antral stage – it
only acts to rescue the follicles that have already
developed to the antral stage and would normally be lost
to atresia. When the follicles have reached the appropriate
size, the woman is given human chorionic gonadotrophin
to mimic the normal ovulatory LH surge, and induce final
oocyte maturation. A needle is used to recover the oocytes
from the mature antral follicles. For standard IVF, the
oocytes are placed together with sperm from the partner
or a donor and the sperm penetrate the oocyte naturally.
In cases where only small numbers or immotile sperm are
available, fertilization can be achieved by injection of a
single sperm into each oocyte (ICSI). After fertilization, the
resultant embryos are cultured for 2 to 6 days and then
transferred back into the uterus of the woman.
As shown in Figure 4, the number of antral follicles
present on a woman's ovaries decreases with age and this
results in a decreased number of oocytes that can be
retrieved for following gonadotrophin treatment for IVF
(Figure 14a). Moreover, the quality of the oocytes also
decreases with increasing age (Figure 14b), resulting in a
decreasing proportion of the oocytes that can be
successfully fertilized in vitro (Figure 14c).
none were suitable for transfer) more than doubled for
women 41-42 years old compared with women younger
than 35. This reflects the decrease in the numbers and
quality of oocytes retrieved with increasing age. For cycles
in which embryos were produced, the proportion of those
embryos that developed to the fetal stage (the
implantation rate) decreased from 28% in women less than
35 years old to only 8% in women 41-42 (Figure 16b). Of
the embryos that did implant, 38% were subsequently lost
(miscarried) in women 41-42 years old, compared with
only 14% in women younger than 35 (Figure 16b).
Figure 15. The effect of a woman’s age on the development of
fertilized oocytes to the blastocyst stage by Day 5 of culture
(Wiemer et al. 2002).
Figure 14. The effect of a woman's age on a) the number of
oocytes retrieved, b) chromatid separation rate of the oocytes,
and c) in-vitro fertilization rate (Lim and Tsakok 1997).
Embryo development in culture is also affected by the
age of the woman. In the example shown in Figure 15, the
proportion of fertilized oocytes that developed to the
blastocyst stage by Day 5 was significantly reduced with
increasing age of the woman.
The effects of age of the woman on the outcome of
ART using her own oocytes are strikingly demonstrated
by the statistics for ART procedures for 2002 reported to
the U.S. Centers for Disease Control (U.S. Department of
Health and Human Services – Centers for Disease Control
and Prevention 2004) that are shown in Figure 16. The data
include a total of 81,888 treatment cycles which resulted in
approximately 24,100 live births.
First, as shown in Figure 16a, the cancellation rate
(treatment cycles in which no embryos were created or
Overall the reduced implantation rate and increased
fetal loss rate in older women resulted in only 5% of
embryos transferred developing to a live baby in women
41-42 years old compared with 23% in women younger
than 35 (Figure 16d). For women 41-42 years old, only 11%
of cycles started yielded a live birth compared with 37%
for women younger than 35 (Figure 16e). This would mean
that on average, a woman 41-42 years old would need 12
IVF treatment cycles to have a 75% chance of one live birth,
compared with only 3 treatment cycles for a woman
younger than 35.
In addition to the decrease in live-birth rate with
increasing age, pregnancies and babies resulting from
ART in older women using their own oocytes are subject
to the same problems of pre-term delivery, perinatal
complications and chromosomal abnormalities seen with
natural conception.
3. The Use of Donor Oocytes
In cases where a woman has no ovaries or is otherwise
unable to produce her own viable oocytes, oocytes may be
obtained from other women. The donors are most often
anonymous fertile, young women but may be a relative or
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• VOLUME 4 • FERTILITY MAGAZINE
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AGE & FERTILITY
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friend of the patient. The donor is treated with
gonadotrophins and oocytes collected as described in the
preceding section. Sperm from the patient's male partner
is usually used for fertilization and the resulting embryos
cultured and then transferred into the patient.
Interestingly, it appears that the patient's age has no
appreciable effect on the ability to support a pregnancy.
The live-birth rate for women receiving embryos created
from donor oocytes is approximately 50% at all ages from
25 to 45 (U.S. Department of Health and Human Services
– Centers for Disease Control and Prevention 2004). Of
course, the babies born from donated oocytes have no
direct genetic relationship to the patient.
Figure 16. The effect of a woman's age on the a) cancellation
rate, b) implantation rate, c) fetal loss, d) approximate babies
born per embryo transferred, and e) live birth rate with in-vitro
fertilization of non-donor oocytes in the United States in 2002.
(Taken or derived from: U.S. Department of Health and Human
Services – Centers for Disease Control and Prevention 2004).
Clearly, common ART procedures can improve the
chances of pregnancy but cannot overcome the deleterious
effects of aging on numbers and quality of the oocytes.
Based on a computer model, Leridon (2004) has calculated
that ART can make up for only half of the births lost by
postponing an attempt to become pregnant from 30 to 35
years, and less than 30% of the births lost by postponing
from 35 to 40 years. Based on a literature review and their
own data, Broekmans and Klinkert (2004) conclude that
the prognosis for a successful pregnancy with IUI or IVF
for women 44 or older “is flat zero.”
There are, however, two specialized ART procedures,
pre-implantation genetic diagnosis and oocyte
cryopreservation, that can, or have the potential to,
circumvent the effects of aging on fertility.
IV. APPROACHES TO CIRCUMVENTING
EFFECT OF AGE ON FERTILITY
THE
1. Pre-Implantation Genetic Diagnosis
As noted above, the frequency of chromosomal
abnormalities in oocytes increases with age in women, and
this results in increased frequencies of chromosomal
abnormalities in the embryos, fetuses, and babies born. An
early approach to this was to obtain cells from the fetus by
amniocentesis or chorionic villus sampling for evaluation
of the chromosomes. Fetuses with abnormal numbers of
chromosomes were then aborted, in order to prevent the
birth of chromosomally abnormal babies. More recently, it
has become possible to determine the chromosome status
of early embryos produced by ART, before they are
transferred back into the patient (preimplantation genetic
diagnosis, PGD). In this case, only embryos with normal
chromosome numbers are transferred.
A positive side effect of embryo selection following
PGD is that the implantation and birth rates are increased
because chromosomally abnormal embryos are often also
developmentally compromised. An example is shown in
Figure 17, where the implantation rate for embryos that
had been tested and judged to chromosomally normal was
17.6% compared with 10.6% for embryos that had not been
tested (and presumed to be a mixture of normal and
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AGE & FERTILITY
abnormal embryos). Pre-implantation genetic diagnosis is
usually used for couples with some history of
chromosomal or other genetic defects, recurrent
miscarriage, or in older women. Based on the improved
rates of development following PGD, it has been suggested
that all embryos should be tested.
Figure 17. Implantation rates for unselected embryos and for
embryos judged as chromosomally normal by pre-implantation
genetic diagnosis (Munné et al. 2003).
so, but it is imperative that women be aware that fertility
decreases significantly with age, particularly after 35 years
of age. From a purely biological perspective, the best
approach to ensuring fertility is for women to have their
babies before they have reached their mid-thirties, but for
many women, this is not a desirable or even practical
option. At any given age, assisted reproduction techniques
may improve the chances of becoming pregnant, but
cannot make up for the loss of fertility due to the effects of
aging on the numbers and quality of oocytes.
References
2. Oocyte Cryopreservation
When living tissues are deep-frozen (cryopreserved)
under the appropriate conditions, all biological processes
are arrested and aging of the tissue stops until it is thawed.
This approach has been long used for the storage of sperm
and embryos, and has recently been extended to oocytes.
A major interest in oocyte cryopreservation is to preserve
the possibility of fertility for young women that are due to
undergo radiotherapy and chemotherapy for the
treatment of cancer. Such treatments can have severely
deleterious effects on the oocytes. By removing and
freezing the oocytes, they are not exposed to the cancer
treatments. After the patient has recovered from the cancer
treatments and wants to start a family, the oocytes can be
thawed and fertilized, and the embryos transferred back
into her uterus.
In the same way that cryopreservation can protect
oocytes from cancer treatments, it could also be used to
protect oocytes from natural loss and degeneration due to
aging. Stachecki and Cohen (2004) have suggested that this
may offer an approach to preserving fertility for women
wishing to delay reproduction. Oocytes would be
collected from young women and then cryopreserved
until they are ready to begin their families. Although as yet
largely experimental, the pregnancy rates from
cryopreserved oocytes are improving.
V. CONCLUSIONS
There is a tendency for women in industrialized countries
to delay having babies until their mid-thirties or later.
There are important social and economic reasons for doing
Astolfi P, Zonta LA (1999) Risks of preterm delivery and
association with maternal age, birth order, and fetal gender.
Hum Reprod 14, 2891-4.
Barritt JA, Cohen J, Brenner CA (2000) Mitochondrial DNA point
mutation in human oocytes is associated with maternal age.
Reprod Biomed Online 1, 96-100.
Broekmans FJ, Klinkert ER (2004) Female age in ART: when to
stop? Gynecol Obstet Invest 58, 225-34.
Brzechffa PR, Daneshmand S, Buyalos RP (1998) Sequential
clomiphene citrate and human menopausal gonadotrophin
with intrauterine insemination: the effect of patient age on
clinical outcome. Hum Reprod 13, 2110-4.
Dunson DB, Colombo B, Baird DD (2002) Changes with age in
the level and duration of fertility in the menstrual cycle. Hum
Reprod 17, 1399-403.
Ferrara I, Balet R, Grudzinskas JG (2002) Intrauterine
insemination with frozen donor sperm. Pregnancy outcome
in relation to age and ovarian stimulation regime. Hum
Reprod 17, 2320-4.
Fisch H (2005) ‘The Male Biological Clock.’ (Free Press: New
York)
Ford WC, North K, Taylor H, Farrow A, Hull MG, Golding J
(2000) Increasing paternal age is associated with delayed
conception in a large population of fertile couples: evidence
for declining fecundity in older men. The ALSPAC Study
Team (Avon Longitudinal Study of Pregnancy and
Childhood). Hum Reprod 15, 1703-8.
Gindoff PR, Jewelewicz R (1986) Reproductive potential in the
older woman. Fertil Steril 46, 989-1001.
Hassan MA, Killick SR (2004) Negative lifestyle is associated
with a significant reduction in fecundity. Fertil Steril 81, 38492.
Jolly M, Sebire N, Harris J, Robinson S, Regan L (2000) The risks
associated with pregnancy in women aged 35 years or older.
Hum Reprod 15, 2433-7.
Leridon H (2004) Can assisted reproduction technology
compensate for the natural decline in fertility with age? A
model assessment. Hum Reprod 19, 1548-53.
Lim AS, Tsakok MF (1997) Age-related decline in fertility: a link
to degenerative oocytes? Fertil Steril 68, 265-71.
Mathews T, Hamilton B (2002) 'Mean age of mother, 1970-2000.'
National Center for Health Statistics, Hyattsville, Maryland.
CONTINUED
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ON
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• VOLUME 4 • FERTILITY MAGAZINE
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AGE & FERTILITY
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Menken J, Trussell J, Larsen U (1986) Age and infertility. Science
233, 1389-94.
Munné S, Sandalinas M, Escudero T, Velilla E, Walmsley R,
Sadowy S, Cohen J, Sable D (2003) Improved implantation
after preimplantation genetic diagnosis of aneuploidy.
Reprod Biomed Online 7, 91-7.
Pellestor F (2004) Âge maternel et anomalies chromosomiques
dans les ovocytes humains. Med Sci (Paris) 20, 691-6.
Piñón R (2002) ‘Biology of Human Reproduction.’ (University
Science Books: Sausalito, CA, USA)
Scheffer GJ, Broekmans FJ, Dorland M, Habbema JD, Looman
CW, te Velde ER (1999) Antral follicle counts by transvaginal
ultrasonography are related to age in women with proven
natural fertility. Fertil Steril 72, 845-51.
Simpson JL, Elias S (1994) Prenatal diagnosis of genetic disorders.
In ‘Maternal-fetal Medicine : Principles and Practice’. (Eds
RK Robert K. Creasy and R Resnik) pp. 61-87. (W.B.
Saunders: Philadelphia)
Stachecki J, Cohen J (2004) An overview of oocyte
cryopreservation. Reprod BioMed. Online 9, 152-163.
Tarin JJ, Gomez-Piquer V, Rausell F, Navarro S, Hermenegildo C,
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Cano A (2005) Delayed motherhood decreases life
expectancy of mouse offspring. Biol Reprod 72, 1336-43.
te Velde ER, Pearson PL (2002) The variability of female
reproductive ageing. Hum Reprod Update 8, 141-54.
U.S. Department of Health and Human Services - Centers for
Disease Control and Prevention (2004) ‘2002 Assisted
Reproductive Technology Success Rates: National Summary
and Fertility Clinic Reports.’ Atlanta, GA, USA.
U.S. National Center for Health Statistics (2003) Crude birth
rates, fertility rates, and birth rates by age of mother,
according to race and Hispanic origin: United States,
selected years 1950-2002 .ftp://ftp.cdc.gov/pub/Health_
Statistics/NCHS/Publications/Health_US/hus04tables/
Table003.xls Date of access, July 2005.
Wiemer KE, Anderson AR, Kyslinger ML, Weikert ML (2002)
Embryonic development and pregnancies following
sequential culture in human tubal fluid and a modified
simplex optimized medium containing amino acids. Reprod
Biomed Online 5, 323-7.
Wilding M, Fiorentino A, De Simone ML, Infante V, De Matteo L,
Marino M, Dale B (2002) Energy substrates, mitochondrial
membrane potential and human preimplantation embryo
division. Reprod Biomed Online 5, 39-42.
You can contact Don Rieger, PhD at: [email protected]
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PATIENT’S CORNER
Exploration of Clinical and Laboratory
Aspects of Infertility
Series: Part 3 of 4
by Charlene Alouf, PhD, Laboratory Director, and Albert
El-Roeiy, MD, MBA, Medical Director Crozer Chester Medical
Center Fertility Center
T
he current topic for The Patient’s Corner is recurrent
pregnancy loss or recurrent miscarriage.
Nicole and her husband Jason have been married for
4 years and have been trying to have a child for the past
CHARLENE A LOUF, PHD
two. Nicole is a 35-year-old with a history of two first
trimester miscarriages that occurred at 8 and 10 weeks of
gestation. Nicole and Jason did not experience difficulty
in conceiving either pregnancy. For the most part, Nicole ALBERT EL-ROEIY, MD, MBA
has regular monthly menstrual cycles and there is no
What are the causative factors?
component of male factor infertility.
A D&E was performed by Nicole’s gynecologist
Factors attributed to habitual spontaneous abortions can
following the second loss. During the procedure, Nicole’s
be subdivided into genetic and non-genetic causes. The
surgeon observed an abnormal structure, a wall dividing
non-genetic factors include deviations in the uterine
the uterine cavity called a septate uterus. Following the
anatomy, infection of the reproductive tract, or
surgery, the tissue from the pregnancy, or products of
immunological or endocrine disturbances. Additionally,
conception, was sent to a cytogenetics laboratory for a diseases associated with thrombotic complications may be
chromosomal analysis (karyotype). Unfortunately, the
responsible for recurrent loss.
karyotype was inconclusive as the cells failed to grow in
As a genetic basis for habitual abortions, numerical
culture prohibiting a complete analysis. Therefore, the role
chromosome abnormalities (aneuploidy) or structural
of a possible contributory genetic abnormality could not
aberrations (inversions, translocations, duplications
be determined. The gynecologist referred Nicole and Jason
and/or deletions) may be involved. Gonadal aneuploid
to a reproductive endocrinologist for a second opinion on
mosaicism (genetically unbalanced gametes in couples
the uterine septum and to discuss the repeated pregnancy
with a normal karyotype) must be considered. Single gene
losses. At their consultation Nicole and Jason had many
disorders may also contribute to recurrent losses. Most
questions regarding their reproductive history and
certainly, multiple etiologies may play a role in recurrent
desired a plan of treatment to assist in achieving an
loss as well as a host of unknown factors not currently
ongoing pregnancy and live birth.
established, as this list is non-exhaustive (reviewed in
Simon et al., 1998, and Kearns et al., 2005).
What defines recurrent pregnancy loss?
The Inherited Pregnancy Loss Working Group defines
What diagnostic tools are available to determine the
recurrent pregnancy loss as 3 or more spontaneous losses
cause of my repeated losses?
occurring prior to fetal viability (<24 weeks gestation)
Nicole and Jason were counseled regarding Nicole's
(Laurino et al., 2005). However, it is not uncommon for history of subsequent pregnancy losses. As many factors
medical professionals to commence evaluation of the
impact reproductive wastage, Nicole and Jason
couple after 2 such losses. Pregnancy loss is subunderwent a comprehensive baseline evaluation for
characterized by gestational age, with loss occurring prior recurrent spontaneous abortion. The assessment included
to 10 weeks defined as embryonic, while fetal loss defines
that occurring at 10 weeks gestation and beyond.
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41
PATIENT’S CORNER
C ONTINUED FROM PAGE 41
a thrombophilia workup that would identify factors that
can cause clotting abnormalities. These include
methyltetrahydrofolate deficiency, prothrombin and factor
V Leiden gene mutations. These factors are involved with
an increased frequency of venous thrombosis, or clotting
events within the placenta, which result in pregnancy loss.
Autoimmune studies, including anti-cardiolipin and antiphospholipid antibodies, are used to diagnose any
immune reaction mounted against “self” tissue.
Autoimmune disorders, or low titer antibodies which
mimic autoimmune syndromes, may also contribute to
pregnancy loss. Nicole’s thrombophilia workup and
autoimmune study were both unremarkable.
The impact of the endocrine system was also assessed
during the baseline study. Abnormal hormonal levels
could significantly impact the uterine environment and
therefore, its function. Nicole’s androgen, progesterone,
and prolactin levels were found to be within normal limits.
Additionally, her thyroid function was normal, and she
had no history of insulin-dependent diabetes.
Although microbial infection as a causative agent in
miscarriage is relatively low and controversial,
mycoplasma and ureaplasma cultures were performed to
exclude this as a factor. The results of the cultures were
both negative.
Confirming the gynecologist's suspicion, a septate
uterus was identified by appropriate tests including a
hysterosalpingogram and hysteroscopy. Additionally
Nicole’s specialist recommended that a karyotype be
performed on both her and Jason, in order to determine
whether they were carriers of any chromosomal structural
rearrangements, such as translocations, duplications and
inversions. Healthy individuals can display balanced
chromosomal structural rearrangements on karyotype
analysis, meaning they contain the correct amount of
genetic material. Balanced rearrangements can be passed
on to healthy offspring or can become unbalanced during
sperm or oocyte production. Embryos arising from the
unbalanced gametes are at an elevated risk of growth
arrest, implantation failure, and pregnancy wastage or the
birth of a baby with a genetic syndrome.
The karyotype analysis revealed that Nicole carries a
balanced reciprocal translocation between chromosomes 9
and 10. She and Jason were referred to a geneticist for
consultation to discuss the impact of these findings on past
and future pregnancies.
Does one factor outweigh the other with regards to the
losses and what are our options for treatment?
As habitual abortion represents a complex scenario
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FERTILITY MAGAZINE • VOLUME 4 •
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influenced by
m u l t i p l e
factors, it is
difficult
to
discriminate
between
the
contribution of
either
the
u t e r i n e
anomaly or the
translocation
per se, on an
individual
basis. However,
due to Nicole’s
translocation,
she has at least
a 50% chance of
producing
oocytes with a
g e n e t i c
imbalance.
Because Nicole’s repeated losses might be the result of
multiple etiologies, it was clear that the anatomical
abnormality had to be addressed first. Nicole was in
agreement and therefore a surgical procedure
(hysteroscopy) with resection of the uterine wall was
scheduled to correct the uterine anomaly.
Nicole’s reproductive endocrinologist discussed
additional technology available for her treatment – IVF
with preimplantation genetic diagnosis (PGD). Recurrent
miscarriage is just one diagnosis that appears to
significantly benefit from IVF with PGD. The PGD process
permits the identification of embryos that are affected with
unbalanced chromosomal structural abnormalities or
those which have an abnormal number of chromosomes
(aneuploidy). The PGD procedure has an international
misdiagnosis rate of approximately 5%. PGD offers the
technology to transfer genetically balanced, normal
embryos (for the probes tested) and thus increase the
implantation rate and reduce the miscarriage rate.
Nicole and Jason were awaiting a benefits election in
her new employment position so they opted to delay entry
into the IVF program for several months. Nicole
proceeded with the hysteroscopy and within three months
of the surgery she conceived again naturally.
Unfortunately Nicole miscarried at 11 weeks of gestation.
A karyotype was performed on the products of conception
which revealed an unbalanced translocation (between
chromosomes 9 and10) and also an XO karyotype
(Turner’s Syndrome).
After taking 5 months off from trying to conceive, the
couple scheduled a return consult with the reproductive
PATIENT’S CORNER
endocrinologist to discuss starting an IVF/PGD cycle.
Nicole and Jason were still in a good prognosis category as
an IVF/PGD candidate despite their history of losses. They
discussed aneuploidy screening in addition to that of the
translocation due to the genetic evaluation of the previous
pregnancy. Additionally, patients with a history of
recurrent loss may have a higher frequency of aneuploid
embryos during their IVF cycles. With this knowledge,
Nicole and Jason elected to include aneuploidy screening
in their PGD cycle.
Nicole completed her IVF baseline testing and began
stimulation. She demonstrated a very good response to her
stimulation regimen; her follicular growth was well
synchronized, and her estradiol level was appropriate for
the number of mature follicles viewed on ultrasound. At
the time of the retrieval, sixteen oocytes were identified.
Fourteen oocytes were fertilized by conventional IVF, out
of fifteen mature. The embryos developed well and were of
good quality and by the third day of culture eleven
embryos were available for biopsy. Two embryos were
identified as genetically balanced and normal, based upon
Nicole's translocation chromosome. These embryos were
transferred and resulted in a single intrauterine pregnancy.
Although Nicole and Jason were cautious throughout the
entire pregnancy, Nicole gave birth to a full term healthy
baby girl.
In our next issue, we will address the treatment
modalities available for couples diagnosed with severe
male factor. Compromised sperm parameters not only
affect fertilization, but also impact embryo development
and implantation potential, and can contribute to
pregnancy loss.
References
Laurino, MY, et al. Genetic evaluation and counseling of couples
with recurrent miscarriage: recommendations of the
national society of genetic couselors. J Genet. Couns. 14:165181, 2005.
Simon, C, et al. Increased chromosomal abnormalities in human
preimplantation embryos after in vitro fertilization in
patients with recurrent miscarriage. Reprod. Fertil. Dev.
10:87-92, 1998.
Kearns, WG, et al. Preimplantation genetic diagnosis and
screening. Semin. Reprod. Med., in press. 2005.
You can contact Charlene Alouf, PhD at: [email protected] and
Albert El-Roeiy, MD, MBA at: [email protected]
WWW.FERTILITY MAGAZINE.ORG
• VOLUME 4 • FERTILITY MAGAZINE
43
PATIENT’S CORNER
Helping You Understand “What Happens Next?”
by Liz Sanders, Chief Embryologist
Mississippi Fertility Institute, Jackson, Mississippi
O
ne of the key factors in
resolving “uncertainty and
fear: is communication. The
IVF
Team
consists
of
the
Reproductive Endocrinologist (R.E.),
the Nurse Coordinator, and the
Embryologist. It is imperative that
you and the IVF Team communicate
to get a better understanding of
“what happens next” and what to
expect.
By the time you go to the R.E.’s
office for your first visit, you have
overcome many hurdles and crossed
retrieval procedures, how to prepare
for it, and what to expect. Also, the
nurse will explain what happens
next and how to prepare for the
Embryo Transfer. They can also
show you the room where
everything will take place and the
equipment used. This will help to
resolve some of your anxiety about
the procedures.
Another member of the IVF
Team is the Embryologist. The
Embryologist can meet with you and
explain everything that will take
The basis of the correlation between age
and infertility involves several factors.
many bridges concerning infertility.
But what lies ahead, and what can
you expect?
First, have all your questions
written down, be brief and concise.
You and the doctor will discuss what
you have already been through and
the tests that you have had done
until now. At this point, the doctor
may order more tests for you and
your husband. One of the first tests
he may order is a semen analysis for
your husband, to be run in the clinic
lab. You should receive a collection
kit including written instructions
concerning collection, transporting,
and where to deliver the sample.
Next, you will meet with another
member of the IVF Team, the Nurse
Coordinator. The nurse will sit down
with you and explain your fertility
plan in detail and go over
instructions about your medications,
when and how to take them, and
when and where to have lab tests
drawn and run. The nurse will
explain to you about the egg
place after your egg retrieval and
leading up to the day of Embryo
Transfer. The Embryologist will
discuss with you the best procedure
to use to achieve fertilization of your
eggs. If the semen sample is
considered below normal values, a
procedure called Intracytoplasmic
Sperm Injection (ICSI) will be
performed. The Embryologist will
actually select and pick up the best
sperm and inject it into the
cytoplasm of the egg.
When lab tests are ordered you
might hear acronyms used like P4,
hCG, E2, and LH. Ask to have these
test explained to you, what they
stand for, and what YOUR results
should be. Be sure the nurses have
accurate information about how to
get in touch with you concerning
your lab results. Further, discuss if
they can leave results and messages
on your answering machine. This
will save valuable time as the nurses
make their calls each day.
It is our goal as an IVF Team to
LIZ SANDERS, CHIEF EMBRYOLOGIST
answer all your questions and
explain to you about In-Vitro
Fertilization (IVF). We strive to treat
our patients like people and not as
numbers. We will help you
understand “what happens next” in
the process of achieving a pregnancy
and experiencing the joys of
parenthood.
You can contact Liz Sanders, Chief
Embryologist at: [email protected]
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46
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Tyho-Galileo Research Laboratories is an
organization created by world-renowned
specialists in reproductive science and
molecular genetics. In founding the TyhoGalileo Laboratories, these individuals
have joined together to stimulate progress
in medical research, particularly in the
areas of reproductive medicine,
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embryology and genetics. The research
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the understanding of human reproduction
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ALTERNATIVE MEDICINE
Hypnosis for Childbirth
by Shawn Gallagher, BA, CHt, Certified Hypnotherapist
Midwifery Consulting Services
T
he staff in Labour and Delivery
were stunned when they
realized that the woman who
presented was nine centimeters
dilated and almost ready to push. It
was her first baby and she had calmly
laboured for a few hours at home
before casually walking into the birth
unit of the central Florida hospital
her obstetrician had privileges in.
Anna* was 29 years old and had
seen me for a few hypnosis sessions
in mid pregnancy to prepare for her
baby’s birth. I first met her as a client
of the Ontario Hypnosis Centre in
association between hypnosis and
better birth. Statistically significant
outcomes of hypnosis include: faster
labour, reduction in surgical delivery,
less pain medication and shorter
hospital stay. i-ix
While individual response to
pain in birth varies, women report a
significant ability to manage the
intensity of contractions without
epidural or narcotic use. Additional
research
shows
beneficial
applications for nausea and vomiting
in pregnancy (NVP), turning breech
babies,
reducing
hypertension,
…use the principles of hypnosis to promote a
calmer, faster and more comfortable birth
downtown Toronto where she had
come for hypnosis to assist fertility.
She had previously had an
unsuccessful IVF cycle and was now
looking at potentially reduced
chances of pregnancy success with
the use of frozen embryos. She had
never been pregnant and now she
wanted to improve her chances in
any way she could. After four
hypnosis sessions, she conceived a
healthy pregnancy with her first
frozen embryo transfer at a wellknown Toronto fertility clinic. Not
long after conceiving, her husband
received a work transfer to Florida
and they moved there. She decided in
mid pregnancy to return to Toronto
for hypnosis as a way to encourage a
natural birth.
As a clinical hypnotherapist, I
have trained over four hundred
couples just like Anna and her
partner to use the principles of
hypnosis to promote a calmer, faster
and more comfortable birth. Since
hypnosis was accepted in 1955 by the
American Medical Association,
research in the medical uses of
hypnosis
clearly
shows
an
insomnia, restless leg syndrome,
smoking
cessation
and
even
threatened preterm delivery. High
risk patients use hypnosis for preand post operative purposes with
significant reductions in blood loss,
infection rates and length of surgical
time.
The concern of potential negative
effects of drugs on the fetus can result
in some women avoiding physicianprescribed medication – hypnosis can
be employed to encourage women to
take their essential medication.
Alternatively, women can also use
hypnosis to calm themselves and
even reduce their use of such prn
prescriptions
as
anti-anxiety
medication.
Hypnosis encourages a very deep
level of relaxation (not sleep) in even
the most anxious, thus encouraging
patient compliance and resulting in
high satisfaction levels on the part of
the woman, her partner and L&D
staff. Women are always able to
respond to the request of their
attending
nurse,
midwife
or
physician while using hypnosis. In
fact, women are in greater control of
SHAWN G ALLAGHER, BA, CHT
their responses and are significantly
less likely to have a panic attack that
can distract staff from their other
important medical duties. “We have a
simple method that decreases pain
and anxiety and makes the invasive
procedure safer and faster,” says
Elvira Lang, M.D., director of
cardiovascular and interventional
radiology at Beth Israel Deaconess,
Harvard Medical School in Boston,
commenting on her study of the use
of hypnosis as an adjunct in surgery.
Nulliparous clients of the
Hypnosis for Childbirth prenatal
series report about half the length of
labour as compared to the average
population. In addition, these first
time mothers have a caesarian section
rate of 6.7 percent (vs an average of
20-25 percent) and an epidural rate of
18 percent (vs an average of 70-95
percent). Other reported effects
include calmer babies postpartum
and high rates of successful
breastfeeding.
Most women attending the four
class Hypnosis for Childbirth series
are highly anxious and list needles,
hospitals, epidurals, caesarians and
even doctors as among their worst
fears. Stated goals at the first class
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47
ALTERNATIVE MEDICINE
C ONTINUED FROM PAGE 47
include: being in control of one’s
responses, being able to relax in a
busy hospital setting, natural
childbirth and feeling confident.
Women and their partners are taught
self-hypnosis, a variety of pain
management tools and methods of
self-induced deep relaxation.
In today’s stress-filled culture,
many are looking for alternatives for
birth that are safe for them and safe
for their babies. The safest method of
pain relief is that achieved with
hypnosis; the safest birth is a birth
with a minimum of intervention.
Anna’s birth proceeded
smoothly. She was quickly ushered
into the birth room and the attending
physician was paged. As the birth
equipment was laid out, she squatted
with each push and rested between
the pushes. She was able to relax
deeply to her husband’s touch and
voice as she was taught in the
hypnosis sessions. As her doctor
guided her, she was able to slow the
delivery of the baby’s head and her
alert baby was gently placed in her
arms moments after the birth. The
baby’s Apgar scores of 9/10 and 9/10
testified to the baby’s smooth
transition into the world. The staff all
commented on how smoothly the
birth went and how unusual it was to
see a birth with such a calm and
relaxed patient.
relaxation.
Regardless of risk status,
hypnosis has many other applications
in obstetrics by reducing patient
fears, encouraging comfort and
improving the outcome of medical
procedures. Medical professionals
who wish to enhance patient comfort
and satisfaction, contain health care
costs and increase the incidence of
natural
childbirth
can,
with
confidence, encourage their patients
to use hypnosis.
*a pseudonym
References
i
Abramson, M., & Heron, W.T. An
objective evaluation of hypnosis in
obstetrics:
Preliminary
report.
American Journal of Obstetrics and
Gynecology, 59, 1069-1074, 1950.
ii
August, R.V. Obstetric hypnoanesthesia.
American Journal of Obstetrics and
Gynecology, 79, 1131-1137, 1960, and
August, R.V. Hypnosis in obstetrics.
New York: McGraw Hill, 1961.
iii
Brann LR, Guzvica SA. Comparison of
hypnosis
with
conventional
relaxation
for
antenatal
and
intrapartum use: A feasibility study
in general practice. J R Coll Gen Pract
1987; 37:437-440.
iv
Hao TY, Li YH, Yao SF. Clinical study on
shortening the birth process using
psychological suggestion therapy.
Zhonghua Hu Li Za Zhi. 1997 Oct;
32(10):568-70. (General Military
Hospital of Jinan, P.R. China.)
Summary
Hypnosis is a focused form of
concentration. Self-hypnosis is one
form of hypnosis in which a certified
practitioner teaches an individual to
induce his or her own state of altered
consciousness.
Hypnotic analgesia is a safe and
effective alternative and/or adjunct to
pain medication. When used for
childbirth pain, the primary aim of
self-hypnosis is to help women
maintain control by managing
anxiety and discomfort through
inducing a focused state of
48
FERTILITY MAGAZINE • VOLUME 4 •
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v
Harmon, T.M., Hynan, M., & Tyre, T.E.
Improved obstetric outcomes using
hypnotic analgesia and skill mastery
combined with childbirth education.
Journal of Consulting and Clinical
Psychology, 58, 525-530, 1990.
vi
Hornyak, Lynne M. and Joseph P. Green.
Healing From Within: The use of
hypnosis in women's health care.
Washington,
DC:
American
Psychological Association, 2000.
vii
Jenkins, M.W., & Pritchard, M.H.
Hypnosis: Practical applications and
theoretical considerations in normal
labour. British Journal of Obstetrics
and Gynecology, 100(3), 221-226,
1993.
viii
Martin, Alice A., PhD; Paul G. Schauble,
PhD; Surekha H. Rai, PhD; and R.
Whit Curry, Jr, MD The Effects of
Hypnosis on the Labor Processes and
Birth
Outcomes
of
Pregnant
Adolescents. The Journal of Family
Practice, May 2001, 50(5): 441-443.
ix
Mellegren, A. Practical experiences with
a
modified
hypnosis-delivery.
Psychotherapy and Psychosomatics,
14, 425-428, 1966.
x
Lang, Elvira et al. Adjunctive nonpharmacological
analgesia
for
invasive medical procedures: a
randomised trial. The Lancet, Vol
355, April 29, 2000, pages 1486-1490.
xi
Gallagher, Shawn. Hypnosis for
Childbirth: A retrospective survey of
birth outcome using prenatal selfhypnosis. Unpublished 2001 June;
Toronto.
You can contact Shawn Gallagher, CHt at:
[email protected]
HUMAN RESOURCES
Full Time Embryologist Required
The Fertility Center at Crozer-Chester Medical Center has a position available for a full
time embryologist. The candidate must possess a B.S. with a minimum of 3 years
experience in all facets of andrology and embryology. Preference will be given to those
individuals with documented micromanipulation training. Alternating weekend
coverage is required.
CCMC is located ~20 minutes southwest of Philadelphia. US citizenship or permanent
residency is required.
Please contact Charlene A. Alouf, PhD, Laboratory Director, for additional information
via email at [email protected] or call 610-447-2727. Visit our website at
www.crozerfertility.com for additional information and our most recent CDC statistics.
WWW.FERTILITY MAGAZINE.ORG
• VOLUME 4 • FERTILITY MAGAZINE
49
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
[email protected]. We are conviently located close to New York City and major airports.
50
FERTILITY MAGAZINE • VOLUME 4 •
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RESOURCES
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• VOLUME 4 • FERTILITY MAGAZINE
51
CONFERENCES
Delaware Valley Reproductive Biology Group
Fall Dinner Lecture, September 7th, 2005
T
he Delaware Valley Reproductive Biology Group held its fall
dinner lecture on September 7th at the Red Sky Restaurant in
Philadelphia. Members from Fertility Centers in Delaware,
New Jersey and Pennsylvania were present to hear Dr. James
Stachecki, Research Associate for
Tyho-Galileo/Via-Cell Laboratories,
present results from his ongoing
oocyte cryopreservation project.
The DVRBG members would like
to express their sincere gratitude
towards the generous sponsors of the
evening’s
event,
Serono
and
Rosemont Pharmacy. I would like to
thank everyone who attended, it was
truly a wonderful evening!
Charlene A. Alouf, PhD
President, DVRBG
Oocyte Cryopreservation: A Common
Sense Approach
James Stachecki, PhD
O
ocyte cryopreservation will be beneficial to women wanting to prolong reproduction in order to start a career,
finish school, etc.; for egg donation purposes which could eliminate problems associated with donor-recipient
synchronization and egg numbers; and for patients needing to undergo chemo- or radio-therapy.
To date there is no reproducible method for freezing mature human eggs, although numerous children have been
born (>100 children born worldwide, <5% pregnancy rate). Investigations over the past 20 years have failed to surpass,
on a consistent basis, Chen’s 1986 report of the first child born from a frozen egg. This suggests that the current
methodology is not adequate, and major changes need to be made in order to develop a successful, highly reproducible
method for egg storage.
Cryopreservation involves removing water from the cell by slow cooling in the presence of extracellular ice and
disrupting the structure of the water that remains using a cryoprotectant(s) like PrOH, DMSO, EG, etc. For thawing, eggs
are rewarmed at a rate slow enough to prevent thermal shock and rapid enough to prevent or minimize lethal ice crystal
formation.
Embryo storage has met with much greater success than egg storage and is used in IVF clinics throughout the world.
Unfortunately, methods used for embryo storage do not work well for storing unfertilized eggs. In order to find a
method that would allow successful egg storage, investigators have looked at the differences between eggs and
embryos. Among these are microtubule and nuclear organization and membrane structure. There are numerous articles
regarding spindle depolymerization and reformation upon cooling and rewarming, and as many that discuss
differences in DNA structure (chromosomes vs. interphase nuclei), however these differences, although they could be
responsible for possible post fertilization genetic errors, do not have a logical link with membrane intactness after
thawing. In other words, survival rates should be near 100% even with spindle and/or chromosome disruption. On the
other hand, membrane differences between eggs and embryos would explain the differences in survival rates after
freezing. But do we really know why eggs survive cryopreservation poorly and what exactly are the causes of damage?
Of the damage that could occur during cryopreservation, Intercellular Ice Formation
(IIF) and osmotic effects are over-described in the literature as the major causes of damage.
CONTINUED ON PAGE 54
However, there are other potential problems often overlooked, such as ion loading in
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• VOLUME 4 • FERTILITY MAGAZINE
53
CONFERENCES
response to cooling. As cooling
progresses, energy driven processes
will shut down. The Na/K antiporter is
one of these processes driven by ATP and its functioning is responsible
for maintaining a electrochemical balance inside the cell, necessary for
normal cellular function. When this antiporter shuts down, sodium
ions can leak inside the cell and load the cell with sodium, a condition
that will exist upon thawing. The cell upon thawing “wakes up” to
find that there is an overabundance of sodium ions that present a
potentially toxic situation for the cell. Other reports only hint at the
fact that IIF and solution effects may not present a problem when
conventional cryopreservation procedures are used. We hypothesize
that sodium loading along with freezing medium, cryoprotectant
exposure, cooling, dehydration, thawing, and rehydration all present
potential problems and need to be optimized in order to obtain 100% oocyte survival. We feel that IIF and solution effects,
although potential problems to be aware of, are not the main cause for oocyte demise during cryopreservation.
Early reports by our group demonstrated the effectiveness of removing sodium ions from the freezing medium and
substituting these with choline ions. With mouse oocytes we achieved
over 90% survival and 60% blastocyst formation rates in vitro, as well
as pregnancy rates similar to control non-frozen oocytes. Preliminare
experiments with human oocytes revealed that a low-sodium cholinebased cryopreservation medium (CJ3) was far superior to
conventional sodium-based media. After protocol optimization along
with modifications to the choline-based freezing medium, we
obtained over 90% survival of fresh unfertilized human oocytes. These
rates are higher than the best rates obtained with a high sucrose
sodium-based media (Fabbri et al., 2001).
Choline-based medium is being widely tested for its effectiveness
in IVF clinics worldwide. CJ3 has significantly improved the already
good results obtained with CJ2 when freezing mouse and human
oocytes. At least 5 babies have been born worldwide using CJ3 and
over 15 babies using a low-sodium choline-based medium.
In addition to slow-cooling protocols, vitrification methods have
recently become popular, but they remain to be perfected for use with
oocytes. We present here preliminary results when vitrifying
blastocysts with a newly developed method called: S3-vitrification.
As more and more clinics convert to blastocyst transfer to reduce
multiple pregnancies, a safe, efficient, and reproducible method for
storing blastocysts will be needed. S3-vitrification technology fills this
need. This is a new method that avoids the problems associated with
other vitrification methods currently used and is an effective,
reproducible, and user-friendly method for storing human
blastocysts. S3-Vitrification does not use DMSO, a potentially toxic
cryoprotectant used in other vitrification procedures. Blastocysts are
frozen in conventional heat-sealed 0.25cc straws eliminating direct
contact with LN2 and thereby helping to avoid potential bacterial and viral contamination. There are no micro-sized
straws, loops, or grids to mess with in our system. No blastocoel collapsing or reducing is required for maximal survival,
and all blastocyst stages, from expanding to fully hatched, can be frozen with this method. S3-vitrification employs
longer exposure and loading times (up to 3 time longer) which leaves room for possible technician-related errors when
trying to expose, load, and freeze blastocysts in 45 seconds or less, the typical time constraint of other methods.
Preliminary results have provided over 90% survival and re-expansion of any stage blastocyst, including >90% survival
of ICM & trophectoderm cells. We have preliminary clinical reports of ongoing survival rates of over 90% and ongoing
pregnancy rates of over 60%.
For more information regarding S3-vitrification please contact Kelly at: [email protected] or (973) 322-6315
C ONTINUED FROM PAGE 53
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CONFERENCES
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• VOLUME 4 • FERTILITY MAGAZINE
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CONFERENCES
XVIII FIGO
Contact US
Tel: +60 4252 9100
Fax: +60 4257 1133
email: [email protected]
www.figo2006kl.com
56
FERTILITY MAGAZINE • VOLUME 4 •
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World Congress of Gynecology and Obstetrics
Kuala Lumpur, Malaysia
November 5-10, 2006
Kuala Lumpur Convention Centre
CONFERENCES
5th MSRM ANNUAL MEETING
Contact Information:
MSRM Coordinator
Dr. Ashraf Samir
[email protected]
PO Box 125, Ibrahimieh
Alexandria, Egypt 21321
Tel. & Fax: +20 3 359 50 43/44
Congress Secretariat
ERA Ltd.
8, Alex. Soutsou str.
106 71 Athens, Greece
Congress Website
www.msrmcrete2006.com
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• VOLUME 4 • FERTILITY MAGAZINE
57
I’ve planned for this my entire life.
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FDA Statement
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©2005 FertileAge.com
Infertility-2005-05
US/Canada: 800-720-6375
International: 519-826-5800
Fax: 519-826-6947
email: [email protected]