Fertility Magazine • Volume 18

Transcripción

V O LUME 1 8
LifeGlobal® global® Blastocyst Fast Freeze®:
Optimal simplicity and effectiveness with
successful outcomes
by Toni Di Berardino, BSc, MSc
Guidelines for the design of an IVF Laboratory
by Gloria Calderón, PhD & Nuno Costa Borges, PhD
Review: Reproductive Microbiome
and its effect on IVF
by Aami Mezezi, MS
The Use of
global® for Time-lapse Videographic
Analysis of Human Embryo Development
by Don Rieger, PhD
The Patient's Corner
ESHRE 2015, Lisbon
IVFs First & Leading ‘One Solution Medium®’
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Fertility Magazine • Volume 18 • www.FertMag.com – Page 1
Contents
Fertility Magazine
The First Magazine In FertilityTM
The Featured Article
LifeGlobal global® Blastocyst Fast Freeze®: Optimal simplicity and
effectiveness with successful outcomes.
by Toni Di Berardino, BSc, MSc.........................................................................6
Featured On The Cover
Review: Reproductive Microbiome and its effect on IVF
by Aami Mezezi, MS.................................................................12
The Use of global® for Time-lapse Videographic
by Don Rieger, PhD..................................................................20
by Gloria Calderón, PhD & Nuno Costa Borges, PhD........52
Instructions to Contributors
To submit an Article, Abstract or Ad email us at:
[email protected]
Note: Articles and Abstracts must be
accompanied by a photo of the author(s).
Page 2 – Fertility Magazine • Volume 18 • www.FertMag.com
Toni Di Berardino, BSc, MSc
Our New LifeGlobal Group facility
and operation in Florida
Features
28
Advanced maternal age and assisted reproductive technologies: developmental competence,
clinical outcomes and future strategies by Lynne C. O’Shea, PhD
37
High-magnification selection of spermatozoa prior to oocyte injection: confirmed and
potential indications by F Boitrelle, B Guthauser, L Alter, M Bailly, M Bergere, R Wainer, F
Vialard, M Albert, and J Selva
45
The spatial arrangement of blastomeres at the 4-cell stage and IVF outcome by Goedele
Paternot, Sophie Debrock, Diane De Neubourg, Thomas M D’Hooghe, Carl Spiessens
54
Cassandra’s prophecy: why we need to tell the women of the future about age-related fertility
decline and ‘delayed’ childbearing by Jane Everywoman
62
New Products
64Conferences
ASRM 2014 – Hawaii, October 2014
ESHRE 2014 – Munich, July 2014
Editor in Chief: Monica Mezezi, MBA
Editor(s):
Don Rieger, PhD
Assistant Editor: Michael West
Design:
Debrah Frank
Editorial Office: LifeGlobal Group, LLC
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Guelph, ON, Canada
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The Consistency of global® Medium
Don Rieger, PhD
May 15, 2013
The following graphs show the results of the quality control measurements of the same 24 sequential
lots of global® medium manufactured between May, 2012 and April, 2013
0HDQ &29 &/ 0HDQ &29 &/ 2VPRODULW\P2V0
S+
Third-party measurements of the pH of 24 sequential lots
of global®. With 10 mg/ml protein, at 37°C and 5.5% CO2
,QGLYLGXDO/RWV
The osmolarity of 24 sequential lots of global® measured
by freezing-point depression
0HDQ &29 &/ $OOORWV(8PO
EHORZDVVD\VHQVLWLYLW\
(QGRWR[LQ(8PO
,QGLYLGXDO/RWV
,QGLYLGXDO/RWV
Third-party measurements of the endotoxin content of
24 sequential lots of global®
([SDQGHG%ODVWVDWKRI&XOWXUH
,QGLYLGXDO/RWV
The results of third-party 1-cell mouse embryo assays
of 24 sequential lots of global®
THE FEATURED ARTICLE
LifeGlobal global® Blastocyst Fast Freeze®:
Optimal simplicity and effectiveness with
successful outcomes.
by Toni Di Berardino, BSc, MSc
Director of Clinical & Technical Support
LifeGlobal® Group, Guelph, Ontario, Canada
You can contact Toni Di Berardino at [email protected]
T
he cryopreservation of human oocytes, zygotes,
cleavage stage embryos and blastocysts is an integral
part of every human IVF program. The starting point
can be found in the early seventies with the collaboration
of three scientists: David Whittingham, Stanley Leibo and
Peter Mazur. This collaboration led to the live birth of mice
after thawing and transferring mouse embryos previously
frozen at –196°C (Whittingham et al., 1972). Their work
soon became the model for the cryopreservation of cow
embryos (Wilmut et al., 1973), sheep embryos (Willadsen et
al., 1976) and rabbit embryos (Whittingham et al., 1976). For
human cryopreservation, the breakthrough came ten years
later with the first report of human pregnancy following
cryopreservation, thawing and transfer of an eight cell
embryo (Trounson et al., 1983) and a human blastocyst
(Cohen et al., 1985).
Embryo and blastocyst cryopreservation has numerous
applications in reproductive medicine. Cryopreservation
of supernumerary embryos and blastocysts has been
highlighted as a method to reduce multiple gestations
but also to have subsequent transfers
if the first attempt was to fail (Gerris
et al., 2003, Kolibianakis et al., 2007).
Cryopreservation has become one
of the most important techniques in
assisted reproductive technology (ART).
Approximately 40,000 cryopreserved
embryo transfer cycles were performed
in 2013 in the US alone (SART 2013).
Currently there are published data that show improved
pregnancy and implantation rates by transferring
blastocysts in non-stimulated cycles (Shapiro et al., 2011,
2014). In cases when the risk of ovarian hyperstimulation
syndrome becomes apparent after oocyte retrieval, freezing
all the embryos is certainly an option, as demonstrated by
Wada et al., (1992). With the increased application of preimplantation genetic screening, immediate cryopreservation
of biopsied blastocysts and transfer of euploid embryos is
now common in many laboratories (Scott et al., 2013, Roy et
al., 2014).
Two different methods have been established
successfully to cryopreserve oocytes, embryos and
blastocysts: slow rate freezing and vitrification. Although
Toni Di Berardino, BSc, MSc
the basic objective of slow freezing and vitrification by
rapid cooling is the same, that is to protect cells from
cooling effects, intracellular ice formation, dehydration
and the toxic effects at both high and low temperatures, the
procedures are somewhat different.
During slow rate freezing, embryos and blastocysts are
exposed to relatively low concentrations of permeable and
non-permeable cryoprotectants, loaded into a straw, sealed,
and placed into a controlled rate freezer in order to avoid
intracellular ice crystal formation. Although thousand of
babies have been born from this technique, results have
not always been consistent, especially when handling
blastocysts.
Vitrification, by rapid cooling, was first applied for
cryopreservation of mammalian embryos in 1985 (Rall et
al., 1985). Vitrification is a process by which cells can be
frozen in such a way that a glass-like or vitrified state is
obtained. The strategy of vitrification results in the total
elimination of ice crystal
formation both within
the cells being vitrified,
and in the surrounding
solution. The success rate
of recently developed
vitrification methods for
the cryopreservation of
human oocytes, embryos
and blastocysts are encouraging (Liebermann et al., 2006,
Kuwayama et al., 2007) and vitrification is now widely
applied in many IVF laboratories. Most of the vitrification
methods currently available in the market are, in principle,
the same. They involve the exposure of oocytes or embryos
to high concentrations of cryoprotectants (CPAs) for a
brief period of time, followed by loading into small carrier
devices, that may or may not be sealed, and then immediate
submersion and storage in liquid nitrogen. The majority of
the studies support the implementation of a vitrification
system by emphasizing its advantages such as its simple,
inexpensive and rapid procedure leading to a higher
survival pregnancy and implantation rates. However, two
major drawbacks of vitrification are: the technical skill of
Embryo and blastocyst
cryopreservation has
numerous applications in
reproductive medicine.
the individual performing vitrification and the possible risk
of bacterial or viral contamination of the biological sample
during cooling when using an open device. Successful
vitrification of oocytes and blastocysts critically depends
on the proficiency of the staff involved in the vitrification.
Therefore a vitrification methodology that is easy to learn
and implement is crucial.
The risks of (cross) contamination of the biological
sample (Bielanski, 2012; Morris, 2005) or presence of toxic
compounds during cooling as well as during long-term
storage (Yan et al., 2011) remain contentious. Modified
strategies have been developed in order to ensure protection
during cooling and storage. The use of sterilized LN (Cobo
et al., 2011; Parmegiani et al., 2010) and the storage ofthe
straws in vapor (Cobo et al., 2010a) are seen asoptions if
open carrier devices are used. Another alternative is the
use of closed carrier devices (Vanderzwalmen et al., 2009,
Whitney et al., 2012 Schiewe et al., 2013), which allows
protection of the sample during the cooling procedure and
subsequent storage.
As stated by Vajta et al., (2006), vitrification is used to
solidify a solution containing a sample and a cryoprotectant,
without any ice crystal formation, and then to return it to the
liquid state, again without ice formation. However, neither
high cryoprotectant concentrations nor high cooling rates
are absolutely required for vitrification. The key to survival
is whether the intracellular contents vitrify. The survival
of cryopreserved cells maybe more dependent on the rate
at which the specimenis warmed, rather than the rate at
which it is cooled (Leibo and Pool, 2011). Furthermore,
the fundamentalprinciples of cryobiology suggest that
the mechanisms leading to high survival of vitrified cells
are similar, ifnot identical to the mechanisms resulting in
survival of slow-frozen cells (Leibo 2012).
Stachecki and his colleagues (Stachecki et al., 2008,
Stachecki and Cohen 2008), described the S3 (‘safe, simple
and successful’) system for vitrification of blastocysts. Most
notably, the system uses ethylene glycol and glycerol as
cryoprotectants, and the blastocysts were loaded into
standard 0.25 mL freezing straws, rather than the small
volume devices used for DMSO-based vitrification systems.
Stachecki et al., (2008) reported that across five participating
clinics, overall survival was 88.7%, and overall implantation
rate was 42.8%. This demonstrated that blastocysts can be
cryopreserved using a simple protocol while achieving
excellent blastocyst survival, and good implantation rates.
LifeGlobal developed the global® Blastocyst Fast Freeze®
and Fast Freeze® Thawing kits, based on the S3 system, using
global® w/ HEPES as the base medium. The blastocysts are
held in a standard 0.25 mL straw and thus do not require
specialized small holding devices. There is considerably
more time to perform all the steps with this method, making
it less stressful for the embryologist. Another benefit of this
method and media is that blastocoel reduction or collapse is
not necessary. The global® Blastocyst Fast Freeze® and Fast
Freeze® Thawing kits are manufactured in GMP-certified
facilities (ISO 9001: 2008 & ISO 13485: 2003) and are FDA
510k registered (K092963).
Lopes et al., (2015) cultured surplus cleavage-stage
embryos to the blastocyst stage and then compared global®
Blastocyst Fast Freeze® with small-device vitrification. There
were no differences between global® Blastocyst Fast Freeze®
and small-device vitrification for immediate survival
following thaw (88 vs. 90%), survival at 24 h post-thaw (76
vs. 72%), or re-expansion at 24 h post-thaw (67 vs. 55%).
The proportion of live cells was not different among global®
Blastocyst Fast Freeze® (91.6%), small-device vitrification
(90.1%), and control (unfrozen) (95.8%) blastocysts. The
study demonstrated that global® Blastocyst Fast Freeze® is
effective for the cryopreservation of blastocysts, thatethylene
glycol and glycerol are efficient cryoprotectants and that
blastocyst vitrification can be easily achieved using a large
volume device which is sealed, as demonstrated earlier by
Stachecki et al., (2008).
Reed et al., (2015) demonstrated that, in clinical
practice, the transition from a slow freezing protocol to
global® Blastocyst Fast Freeze® and Fast Freeze® Thawing
Kits for the cryopreservation of blastocysts was easy, and
less technically demanding than small-device vitrification.
Blastocyst survival rate was significantly greater (P<0.05)
with global® Blastocyst Fast Freeze® (96.9%) than for both
small-device vitrification (84.4%) and slow-rate freezing
(89.7%). The implantation rate for biopsied (euploid)
blastocysts was significantly greater for global® Blastocyst
Fast Freeze® (47.6%) than for small-device vitrification
(24.0%).
LifeGlobal has conducted many national and
international workshops to introduce the principle and
practical aspects of global® Blastocyst Fast Freeze® system.
The response of the attendees has always been positive and
it generally agreed that the blastocyst vitrification could
be effectively achieved using large volume device, using
longer loading and exposure times and without the collapse
of the blastocoel.
In conclusion, the global® Blastocyst Fast Freeze® system
is a simple and effective approach to the cryopreservation
of human blastocyst. Blastocyst survival and implantations
rates have been shown to be as good as those for smalldevice vitrification. The use of standard freezing straws
with global® Blastocyst Fast Freeze® is technically much
simpler, very much less costly, and, because of the larger
thermal mass, less susceptible to inadvertent warming.
References
Bielanski, A. A review of the risk of contamination of semen and
embryos during cryopreservation and measures to limit
cross-contamination during banking to prevent disease
transmission in ET practices. Theriogenology. 2012; 77: 467–
482
Cobo, A., Romero, J.L., Pérez, S., de los Santos, M.J., Meseguer, M.,
and Remohi, J. Storage of human oocytes in the vapor phase
of nitrogen. Fertil. Steril. 2010; 94: 1903–1907
Cobo, A., Castello, D., Weiss, B., Vivier, C., DeLa Macorra,
A., Kramp, F., 2011. Highest liquid nitrogen quality for
vitrification process: Micro bacteriological filtration of LN2.
In: 16th World Congress on In Vitro Fertilization, Tokyo,
P-052, pp. 289
Cohen, J., Simons, R.F., Fehilly, C.B., Fishel, S.B., Edwards, R.G.,
Hewitt, J., Rowlant, G.F., Steptoe, P.C., Webster, J.M. Birth
after replacement of hatching blastocyst cryopreserved at
expanded blastocyst stage.Lancet. 1985 Mar 16; 1(8429):647
Gerris, J., De Neubourg, D., De Sutter, P., Van Royen, E.,
Mangelschots, K., Vercruyssen, M. Cryopreservation as a
tool to reduce multiple birth. Reprod Biomed Online. 2003
Oct;7(3):286-94. Review
Kolibianakis, E.M., Venetis, C.A., Tarlatzis, and B.C.
Cryopreservation of human embryos by vitrification or slow
freezing: which one is better? Curr Opin Obstet Gynecol.
2009; 21: 270–4.
Kuwayama, M. Highly efficient vitrification for cryopreservation
of human oocytes and embryos: the cryotop method.
Theriogenology. 2007; 67: 73–80
Leibo, S.P. and Pool, T.B. The principal variables of
cryopreservation: solutions, temperatures, and rate changes.
Fertil. Steril. 2011; 96: 269–276
Leibo, S.P. The Alpha consensus meeting on cryopreservation key
performance indicators and benchmarks: proceedings of an
expert meeting. Reprod. Biomed. Online. 2012; 25: 146-167
Liebermann, J. and Tucker, M.J. Comparison of vitrification and
conventional cryopreservation of day 5 and day 6 blastocysts
during clinical application. Fertil. Steril. 2006; 86: 20–26
Lopes, A.S., Frederickx, V., Vankerkhoven, G., Campo, R.,
Puttermans P, Gordts, S. Survival, re-expansion and cell
survival of human blastocysts following vitrification and
warming using two vitrification systems. J. Assist. Reprod.
Genet. 2015; 32: 83-90
Morris, G. The origin, ultrastructure, and microbiology of the
sediment accumulating in liquid nitrogen storage vessels.
Cryobiology. 2005; 50: 231–238
Parmegiani, L., Cognigni, G.E., Bernardi, S., Cuomo, S.,
Ciampaglia, W., Infante, F.E., Tabarelli de Fatis, C., Arnone,
A., Maccarini, A.M., and Filicori, M. Efficiency of aseptic open
vitrification and hermetical cryostorage of human oocytes.
Reprod. Biomed. Online. 2011; 23: 505–512
Rall, W.F. and Fahy, G.M. Ice-free cryopreservation of mouse
embryos at −196 °C by vitrification. Nature. 1985; 313: 573–575
Reed, M.L., Said, A., Thompson, D.J., Caperton, C.L. Largevolume vitrification of human biopsied and non-biopsied
blastocysts: a simple, robust technique for cryopreservation.
J. Assist. Reprod. Genet. 2015; 32: 207-214
Roy, T.K., Bradley, C.K., Bowman, M.C., McCarthur, S.J., Singleembryo transfer of vitrified-warmed blastocysts yields
equivalent live-birth rates and improved neonatal outcomes
compared with fresh transfers. Fertil. Steril. 2014; 101 No 5:
1294-1301
Schiewe MC, Nugent N, Zozula S, Whitney J and Anderson RE
(2013) Prospective incorporation of vitrified embryo transfer
(VFET) cycles into standard patient care: time to re-educate
physicians & patients. Fertil Steril100, S282 (Abstract P-461)
Scott, R.T., Upham, K.M., Forman, E.J., Hong, K.H., Scott,
K.L., Taylor, D., Tao, X., Treff, N.R. Blastocyst biopsy
with comprehensive chromosome screening and fresh
embryo transfer significantly increases in vitro fertilization
implantation and delivery rates: a randomized controlled
trial. Fertil. Steril. 2013; 100 No 3: 697-703
Shapiro, B.S., Daneshmand, S.T., Garner, F.C., Aguirre, M,
Hudson, C., Thomas, S. Evidence of impaired a endometrial
receptivity after ovarian stimulation for in vitro fertilization:
a prospective randomized trial comparing fresh and frozenthawed embryo transfer in normal responders. Fertil. Steril.
2011; 96: 344-348
Shapiro, B.S., Daneshmand, S.T., Garner, F.C., Aguirre, M, Hudson,
C. Freeze all can be a superior therapy to another fresh cycle
in patients with prior fresh blastocyst implantation failure.
Reprod Biomed Online. 2014; 29: 286-290
Stachecki, J.J., Garrisi, J., Sabino, S., Caetano, J.P.J., Wiemer, K.E.,
Cohen, J. A new safe, simple and successful vitrification
method for bovine and human blastocysts. Reprod Biomed
Online. 2008; 17: 360–7
Stachecki JJ, Cohen J. S3 vitrification system: a novel approach to
blastocyst freezing. J Clin Embryol. 2008; 11: 5–14
Trounson, A. and Mohr, L. Human pregnancy following
cryopreservation, thawing and transfer of an eight-cell
embryo. Nature. 1983; 305: 707–709
Vajta, G. and Nagy, Z.P. Are programmable freezers still needed
in the embryo laboratory? Review on vitrification. Reprod.
Biomed. Online. 2006; 12: 779–796
Vanderzwalmen, P., Ectors, F., Grobet, L., Prapas, Y., Panagiotidis,
Y., Vanderzwalmen, S., Stecher, A., Frias, P., Liebermann,
J., and Zech, N.H. Aseptic vitrification of blastocysts from
infertile patients, egg donors and after IVM. Reprod. Biomed.
Online. 2009; 19: 700–707
Wada I, Matson PL, Troup SA, Hughes S, Buck P, Lieberman
BA. Outcome of treatment subsequent to the elective
cryopreservation of all embryos from women at risk of the
ovarian hyperstimulation syndrome. Hum Reprod. 1992 Aug;
7(7):962-6
Whitney J, Nugent N, Duggan K, S. Zozula, Anderson RE and
Schiewe MC (2012) Controlled ovarian hyperstimulation
(COH) cycles may benefit from vitrification of all Day 5 PGSbiopsied blastocysts.Fertil Steril98 Suppl., S185 (Abstract
P-249)
Whittingham, D.G., Leibo, S.P., Mazur, P. Survival of mouse
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Whittingham, D.G., Adam, C.E. Low temperature preservation of
rabbit embryos. J.Reprod.Fertil. 1976; 47(2): 269-274
Willadsen, S.M., Polge, C., Rowson, L.E., Moor, R.M. Deep freezing
of sheep embryos. J.Reprod.Fertil. 1976; 46(1): 151-154
Wilmut I and Rowson LE. Experiments on the low temperature
preservation of cow embryos. Veterinary Record. 1973; 92(26):
686-90
Yan, J., Suzuki, J., Yu, X.M., Qiao, J., Kan, F., and Chian, R.C.
Effects of duration of cryo-storage of mouse oocytes on cryosurvival, fertilization and embryonic development following
vitrification. J. Assist. Reprod. Genet. 2011; 28: 643–64
μDrop GPS® Dishware
Embryo Culture Dish
Safe, Effective and Easy to Use
New Design Features
• Precise 20 µl micro-wells with GPS® feature for rapid location and visualization
• Enhanced optics
• Better orientation and identification
•EmbryoAddressTM helps to quickly identify and track embryos
• 32 mm inner ‘oil ring’ for VOC protection
• Uses up to 75% less oil than a conventional 60 mm dish
• Improved safety (no droplet collapsing or mixing)
New Breathable Packaging for all GPS® Dishware
• Fill the outer wells with oil for better temperature stability
• Reduces off-gassing time
out of the incubator
• Less VOCs introduced into the laboratory
• Works well with both oil-overlay and oil-underlay methods
• Tested and validated to maintain sterility for the
• Designed to ensure safe embryo culturing
entire 5-year shelf life of the dish
• For use with both standard and mini incubators
4-Well GPS®
Universal GPS®
embryo GPS®
embryo corral®
The best dish for PGD cases.
Standardize ART and other sensitive culture by using GPS® Dishware
embryo GPS® Dish
Traditional Petri Dish
Úrunning droplets
Ú droplets mixing
Ú droplets flattening
Ú poor temperature control
Ú losing track of marked droplet
Ú difficulty locating embryos
Ú sealing of dish lid & increased pH
ü
ü
ü
ü
ü
ü
ü
ü
ü
ü
‘drop-less’ environment
micro wells designed to enhance embryo culture
no droplets collapsing and mixing
save time and money by reducing set-up
time, observation time, testing and handling
better heat conservation
enhanced safety
easily locate and observe embryos
new lid design for better gas exchange
standardization of culture, uniform volumes
1-cell MEA and LAL testing by independent company
global
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Based on Pure Science
The leading scientifically based and clinically proven
‘Uninterrupted Single Solution Culture Medium®’.
‘Uninterrupted Time-Lapse Culture Medium®’
v The First leading scientifically based ‘Single Solution Medium®’.
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continuous culture.
v Over 150 Independent Publications using global® medium.
v Over 15 years of Consistent Superior Results worldwide.
Let the Embryos Choose! ®
“We are extremely happy with Global medium combined with the Embryoscope. Nice blastocyst
formation and our pregnancy rates have been over 70% with Day 5 transfer. We use it as a continuous
culture medium from Day 1 onwards with no media exchange.”
“We transitioned from using it from D1 thru 6 with a half change on Day 3 to using it for “uninterrupted
culture” from Day 1-Day 6. Equivalent results both ways.”
Nina Desai, PhD, HCLD, Cleveland Clinic
(global® user since 2001. No commercial ties with the company.)
1. Uninterrupted culture of human embryos in global® or global® total®
•
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Campbell A, Fishel S, Bowman N, Duffy S, Sedler M and Hickman CFL (2013) Modelling a risk classification of aneuploidy in
human embryos using non-invasive morphokinetics. Reprod Biomed Online 26, 477-85.
Costa-Borges N, Bellés M, Herreros J, Teruel J, Ballesteros A, Pellicer A and Calderón G (2013) Single medium culture in a
time-lapse incubator until the blastocyst stage with or without medium renewal on Day-3: a prospective randomised study with
donor oocytes. Human Reprod. 28 Suppl. 1, i184 (Abstract P-167).
Semeniuk L, Mazur P, Mikitenko D, Nagorny V and Zukin V (2013) Time-lapse and aCGH, is there any connection between
ploidy and embryo cleavage timing on early stages of embryo development? Fertil Steril 99 Supplement, S6 (Abstract O-5).
Cruz M, Garrido N, Herrero J, Perez-Cano I, Munoz M and Meseguer M (2012) Timing of cell division in human cleavage-stage
embryos is linked with blastocyst formation and quality. Reprod Biomed Online 25, 371-381.
Silva MM, Llanos BA, David Gumbao, Marcos J, Sanchez A, Nicolas M, Olmedilla LF and Gutierrez JL (2012) Optimization of
clinical outcomes in an oocyte donation programme. Reprod Biomed Online 24 Suppl 1., S7 (Abstract PP-6).
Bellver J, Mifsud A, Grau N, Privitera L and Meseguer M (2013) Similar morphokinetic patterns in embryos derived from obese
and normoweight infertile women: a time-lapse study. Hum Reprod 28, 794-800.
Munoz M, Cruz M, Humaidan P, Garrido N, Perez-Cano I and Meseguer M (2013) The type of GnRH analogue used during
controlled ovarian stimulation influences early embryo developmental kinetics: a time-lapse study. Eur J Obstet Gynecol Reprod
Biol 168, 167-72.
2. Time-lapse culture of human embryos in global® or global® total®
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Basile N, Morbeck D, Garcia-Velasco J, Bronet F and Meseguer M (2013) Type of culture media does not affect embryo kinetics:
a time-lapse analysis of sibling oocytes. Hum Reprod 28, 634-641.
Bellver J, Mifsud A, Grau N, Privitera L and Meseguer M (2013) Similar morphokinetic patterns in embryos derived from obese
and normoweight infertile women: a time-lapse study. Hum Reprod 28, 794-800.
Campbell A, Fishel S, Bowman N, Duffy S, Sedler M and Hickman CFL (2013) Modelling a risk classification of aneuploidy in
human embryos using non-invasive morphokinetics. Reprod Biomed Online 26, 477-85.
Campbell A, Fishel S, Bowman N, Duffy S, Sedler M and Thornton S (2013) Retrospective analysis of outcomes after IVF using
an aneuploidy risk model derived from time-lapse imaging without PGS. Reprod Biomed Online 27, 140-6.
Costa-Borges N, Bellés M, Herreros J, Teruel J, Ballesteros A, Pellicer A and Calderón G (2013) Single medium culture in a
time-lapse incubator until the blastocyst stage with or without medium renewal on Day-3: a prospective randomised study with
donor oocytes. Human Reprod. 28 Suppl. 1, i185 (Abstract P-167).
Cruz M, Garrido N, Herrero J, Perez-Cano I, Munoz M and Meseguer M (2012) Timing of cell division in human cleavage-stage
embryos is linked with blastocyst formation and quality. Reprod Biomed Online 25, 371-381.
Desai NN, Ploskonka S, Goldberg J, Austin C and Falcone T (2013) Morphokinetic analysis of embryos from patients having a
day 5 transfer: preliminary results with the embryoscope. Fertil Steril 100, S120 (Abstract O-392).
Martinez-Burgos M, Losada C, Pareja S, Agudo D and Bronet F (2013) Effects of low O2 concentration in extended embryo
culture using benchtop incubators (Embryoscope and MINC). Fertil Steril 100, S251 (Abstract P-360).
Munoz M, Cruz M, Humaidan P, Garrido N, Perez-Cano I and Meseguer M (2013) The type of GnRH analogue used during
controlled ovarian stimulation influences early embryo developmental kinetics: a time-lapse study. Eur J Obstet Gynecol Reprod
Biol 168, 167-72.
Nakahara T, Iwase A, Goto M, Harata T, Suzuki M, Ienaga M, Kobayashi H, Takikawa S, Manabe S, Kikkawa F and Ando H
(2010) Evaluation of the safety of time-lapse observations for human embryos. J Assist Reprod Genet 27, 93-6.
Ramirez JM, Fernandez FG, Bueno AS, Brandt M, Fernandez JAG and Lopez EG (2012) Importance of multinucleation at 2-cell
stage: study in a time-lapse incubator. Fertil Steril 98 Suppl., S-169 (Abstract P-196).
Semeniuk L, Mazur P, Mikitenko D, Nagorny V and Zukin V (2013) Time-lapse and aCGH, is there any connection between
ploidy and embryo cleavage timing on early stages of embryo development? Fertil Steril 99 Suppl, S6 (Abstract O-5).
Silva MM, Llanos BA, David Gumbao, Marcos J, Sanchez A, Nicolas M, Olmedilla LF and Gutierrez JL (2012) Optimization of
clinical outcomes in an oocyte donation programme. Reprod Biomed Online 24 Suppl 1., S7 (Abstract PP-6).
ARTICLES
Review: Reproductive Microbiome and its effect
on IVF.
by Aami Mezezi, MSc
Post Graduate Student at Georgetown University
You can contact Aami Mezezi at [email protected]
M
ore than 5 million babies have been born from
IVF since the technique was first introduced in
1978 (www.eshre.eu). Many factors have been
evaluated in an attempt to increase the IVF success rate;
but little attention has been paid to the microbiome of the
reproductive tract and its role in fertility. This relationship
demands increased focus especially considering that it has
been reported that up to 40% of patients undergoing IVF
have abnormal flora somewhere along the reproductive
tract (Leitech et al., 2007).
Hyman et al. (2012) examined the correlation between
the vaginal microbiome and IVF success rates and found
that the microbiome on the day of embryo transfer was
a factor in pregnancy outcome. In particular, this study
focused on the transition of the microbiome through the
course of an IVF treatment. Thirty one (31) women with
no signs of vaginal infection were enrolled in the study.
Vaginal swabs, followed by 16rRNA pyrosequencing of
microbial cultures, were performed at various times during
IVF treatment, including on the day of embryo transfer.
Interestingly, a transition in the microbial flora occurred in
most of the women during the course of IVF treatments.
The study concluded that a vaginal microbiome, consisting
predominantly of the lactobacilli species, L. crispatus, L.
gasseri, and L. jensenii at the time of embryo transfer, had
a statistically significant relationship to the live birth rate.
Pyrosequencing of the V1 and V3 regions of the 16s rRNA
on Lactobacilli species in vaginas with normal flora, has
shown that L. crispatus, L. gasseri, and L. jensenii are the three
predominant lactobacilli species (Jakobbson and Forsum,
2008). The study also concluded that all of the women who
did conceive had a substantial decrease in their E2 levels
in-between the time of hCG administration and embryo
transfer. This particular observation has been previously
observed and is now considered a necessary event for
successful IVF treatments (Kim et al., 2010).
How is this related to the microbiome?
Due to the small sample size, the conclusions of this
study are preliminary and must be further investigated
through future research. However, the observations were
consistent with previous findings and the same time raised
a number of key questions. Of particular interest is the fact
that a transition of the vaginal microbiome was observed
along the course of an IVF treatment and that the transitions
were seemingly related to hormonal changes, particularly
with respect to variations in estradiol.
Fluctuations in estradiol levels are seen in women
Aami Mezezi, MSc
undergoing IVF. In fact, women undergoing IVF may have
estradiol (E2) levels eight times higher than normal (Kushnir
et al., 2009). In a study performed on the vaginal microbiome
in rats it was shown that following an ovariectomy the
subsequent decrease in E2 led to a drastic decrease in the
number of Lactobacilli species in the lower genital tract.
However, a normal colony of Lactobacilli species was reestablished following exogenous replacement of E2 thus
establishing the link between estrogen status and Lactobacilli
colonization in the vaginal microbiome (Bezirtzoglou et
al., 2004). Jakobsson and Forsum (2008) were the first to
note changes in the human vaginal microbiome related to
changes in E2 levels. Their study showed that women who
had vagina flora dominated by the three normal Lactobacilli
species underwent very little transition through the course
of IVF treatment; while women whose flora was dominated
by L. delbrueckii, L. rhamnosus or L. vaginalis transitioned to a
normal lactobacilli flora as their E2 levels rose.
These studies all point to E2 levels as having a positive
effect on establishing and maintaining a normal and
healthy vaginal microbiome dominated by L. crispatus, L.
gasseri and/or L. jensenii. The most likely explanation for
this observation is the increase of vaginal glycogen content
in direct response to an increase in E2 (Mirmonsef et al.,
2014). Glycogen is a preferred energy source by Lactobacilli.
Therefore, there are known fluctuations in the numbers of
lactobacilli and in turn the pH of the vagina as estrogen
levels fluctuate naturally over the reproductive life cycle of
a woman or more artificially during the course of an IVF
treatment.
Another possible consideration that must be addressed
is the potential association between progesterone
resistance and the vaginal microbiome. Progesterone is
critical for endometrial function, blastocyst implantation
and prevention of preterm birth and administration of
progesterone is commonly provided to infertile patients
to increase pregnancy rates during IVF (Usadi et al., 2008).
It is also the only pharmacological prevention for preterm
birth (Stout et al., 2014). However, some patients display
progesterone resistance and it has been hypothesized that
the vaginal microbiome could determine the efficacy of
progesterone treatment. A recent study involving 10 patients
ARTICLES
at risk of pre-term birth and on progesterone therapy
suggested that the vaginal microbiome may help determine
the efficacy of progesterone treatment (Stout et al., 2014).
A more elaborate study is needed to further establish the
microbiome as a causative factor in progesterone resistance
and to further determine the possible mechanisms at play.
However, a possible mechanism linking the status of the
vaginal microbiome to progesterone resistance may reside
in the role of the vaginal microbiome in the inflammatory
response. It has been demonstrated that cytokine levels
change with respect to
the vaginal microbiome
(Hodges et al., 2005), and
inflammation has been
hypothesized as a cause
for progesterone resistance
(Sirota et al., 2014). Eckert et
al. (2003) showed that IVF
patients with a decreased
population of normal
Lactobacilli species in their
vaginal flora had increased vaginal inflammation, decreased
conception rates, and increased early pregnancy loss. A
more comprehensive study is needed to better understand
the mechanisms involved in a possible relationship between
the vaginal microbiome, inflammation and progesterone
resistance.
Prophylactic antibiotics and glucocorticoids are
commonly administered during IVF procedures in order
to prevent infections and control inflammation. The role
of these pharmacological interventions on the vaginal
microbiome and IVF success rates is poorly understood.
Although it has been shown that commonly used
antibiotics such metronidazole reduce virulent bacteria,
their effects on lactobacilli species in the normal vaginal
flora can be detrimental (Egbase et al., 1996). Importantly,
the Lactobacilli species L. iners, once believed to be part of
the normal vaginal flora, is now thought be an indicator of
a transition from a normal to an abnormal flora (Vasquez et
al., 2002)., Jokobsson and Forsum (2008) found an increased
presence of L. iners during times of artificially high estrogen
levels during IVF treatments, and following administration
of metronidazole (Jokobsson and Forsum, 2012). This
is a critically important finding that must be further
investigated as it could a) help explain why a decrease in
estrogen levels between the time of hCG administration and
embryo transfer has been shown to be a significant factor for
conception following IVF and b) lead us to re-visit which
antibiotics are being prescribed during IVF treatments due
to their possibly negative effect on normal flora.
It has also been demonstrated that the microbial flora
of the uterine cervical canal could also be a determinant
in IVF success rates. In a study of 204 ART patients, Salim
et al., (2002) found that either a sterile cervical culture
or Lactobacilli only positive culture had pregnancy
rate of 30.7% compared to a pregnancy rate of 16.3% in
women who had a cervical culture containing pathogenic
microorganisms.
Research findings have highlighted the importance
of a normal vaginal and cervical flora on IVF success
rates, particularly at the time of embryo transfer. These
observations may suggest the clinical use of probiotics as
a treatment to improve IVF success rates. At present, the
majority of IVF clinics do not have procedures in place to
screen for vagina flora prior to IVF. However, Gliboa (2005)
concluded that the probiotic treatment they administered
had no effect on either
vaginal
colonization
or pregnancy rates.
This particular study
included 107 women
into either a treatment
group that received 2
probiotic supplements
or a control group
that
received
no
supplementation.
Intravaginal probiotic supplementation with 3 billion live L.
acidophilus, Bifidobacterium bifidum and Bifidobacterium longum
bacteria was administered immediately following oocyte
retrieval. Day 3 embryo transfers were then performed,
allowing only 3 days for probiotic supplementation to have
an effect on colonization. Vaginal swabs and gram staining
was performed to determine the microflora composition
before and after probiotic treatment.
Although Gliboa (2005) did not show any positive
effect of intravaginal probiotic therapy, the sample size of
the study was small and the number and types of probiotic
species administered could be altered to produce a more
beneficial effect. For example, it was found that colonizing
the transfer tip catheter with L. crispatus during embryo
transfer in healthy women of reproductive age led to
increased implantation and live birth rates (Sirota et al.,
2014). Therefore, the use of probiotics as a clinical therapy
during IVF has promise and must be further evaluated.
The microbiome of the female reproductive tract should
become an important consideration in IVF treatments.
Recent preliminary research findings have illustrated the
importance of a normal vaginal and cervical flora composed
primarily of lactic acid producing Lactobacilli species
L. crispatus, L. gasseri, and L. jensenii, in successful IVF
outcomes. The presence of a normal flora has been shown
to have a significant and positive effect on implantation
and , live birth rates, reduced inflammation and prevention
of preterm birth. The composition of the reproductive
tract microbiome is hormone dependent with increased
E2 and P4 levels showing a positive effect on establishing
and maintaining a normal Lactobacilli flora. However,
artificially high E2 levels following hCG administration and
before embryo transfer have a seemingly negative effect on
the microbial flora and IVF success rate. Therefore, there
appears to be time dependence to the hormonal effect on
Research findings have
highlighted the importance of a
normal vaginal and cervical flora
on IVF success rates, particularly
at the time of embryo transfer.
ARTICLES
microbial status which could be due to other hormones and
interacting factors in play at this particular physiological
time-point.
It also appears as if the microbiome itself could influence
hormone status. Recent findings have given credence to the
hypothesis that abnormal vaginal flora could be a causative
factor in progesterone resistance. The possibility that the
vaginal microbiome could help determine progesterone
therapy efficacy could be of great interest to the field of
IVF and is an avenue that should be further researched.
Although significant correlations appear to exist between;
the reproductive tract microbiome, hormone status and IVF
success rate, the mechanisms underlying these relationships
have yet to be determined., one possible consideration to
further investigate is the role of the microbiome on the local
and systemic inflammatory response. Cytokine levels have
been proven to change with respect to transitions in the
vaginal microflora and abnormal flora in the reproductive
tract has been shown to lead to an increase in proinflammatory cytokines.
The use of prophylactic antibiotics during IVF could
also have a potentially detrimental effect on the female
vaginal microbiome.. Antibiotics could also help select for
and increase the amount of resistant pathogenic microbes.
Screening of the vaginal microbiome of women
undergoing IVF should become a common clinical practice,
particularly on or near the day of embryo transfer. Research
should focus on the possible solutions to mitigate the
various factors effecting microbiome flora, implantation
and delivery rates during an IVF cycle.
References
Bezirtzoglou E, Voidarou Ch, Papadaki A, Tsiotsias A, Kotsovolou
O, et al. Hormone therapy alters the composition of the vaginal
flora in ovariectomized rats. Microb Ecol. 2008; 55:751–9.
Eckert LO, Moore DE, Patton DL, Agnew KJ, Eschenbach DA.
Relationship of vaginal bacteria and inflammation with
conception and early pregnancy loss following in-vitro
fertilization. Infect Dis Obstet Gynecol. 2003; 11(1):11–17.
[PubMed: 12839628].
Egbase PE, al-Sharhan M, al-Othman S, al-Mutawa M, Udo EE,
Grudzinskas JG. Incidence of microbial growth from the tip of
the embryo transfer catheter after embryo transfer in relation
to clinical pregnancy rate following in-vitro fertilization and
embryo transfer. Hum Reprod 1996;11(8):1687–1689.
Hodges S, Barrientes F, Desmond R, Schwebke J: Local and
Systemic Styokine Levels in Relation to Changes in Vaginal
Flora. Journal of Infectious Disease 2006: 193; 556-562.
Hyman RW, Herndon CN, Jiang H, et al. The dynamics of the
vaginal microbiome during infertility therapy with in
vitro fertilization- embryo transfer. J Assist Reprod Genet
2012;29(2):105–115.
Jakobsson T, Forsum U. Changes in the predominant human
Lactobacillus flora during in vitro fertilisation. Ann Clin
Microbiol Antimicrob. 2008;7:14–21.
Kim YJ, Ku SY, Jee BC, et al. Dynamics of early estradiol production
may be associated with outcomes of in vitro fertilization.
Fertil Steril 2010;94(7):2868–2870.
Kushnir, M. M., Naessen, T., Kirilovas, D., Chaika, A., Nosenko,
J., Mogilevkina, I., Rockwood, A. L., Carlstrom, K. and
Bergquist, J. (2009) Steroid profiles in ovarian follicular fluid
from regularlymenstruating women and women after ovarian
stimulation. Clin Chem, 55, 519-526.
Leitich H, Kiss H. Asymptomatic bacterial vaginosis and intermediate flora as risk factors for adverse pregnancy outcome. Best
Pract Res Clin Obstet Gynaecol 2007;21(3):375–390.
Mirmonsef P, Hotton AL, Gilbert D, Burgad D, Landay A, et al.
(2014) Free Glycogen in Vaginal Fluids Is Associated with
Lactobacillus Colonization and Low Vaginal pH. PLoS ONE
9(7).
Stout M, LaRosa P, Shannon W, Weinstock G, Sodergen E, Macones
G, Tuuli M: Role of the vaginal microbiome in the efficacy
of progesterone for prevention of preterm birth. American
Jounral of Obstetrics & Gynecology, 2014.
Sirota I, Zarek S, Segars J: Potential Influence of the Microbiome
on Infertility and Assisted Reproductive Technolgy. Semin
Reprod Med 2014; 32; 35-42.
Usadi RS, Groll JM, Lessey BA, et al. Endometrial development and
function in experimentally induced luteal phase deficiency. J
Clin Endocrinol Metab 2008;93(10):4058–4064.
Vasquez, A., T. Jakobsson, S. Ahrne, U. Forsum, and G. Molin.
2002. Vaginal lactobacillus flora of healthy Swedish women.
J. Clin. Microbiol. 40:2746–2749.
LifeGuard® Oil
LifeGuard® Oil is a high viscosity,
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Processed and sourced specifically
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24 month minimum shelf life
A unique LG PGD Biopsy Medium
formulation based on global® medium
LG PGD Biopsy Medium is ready-to-use and designed to maintain embryos during
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LG PGD Biopsy Medium Advantages
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Shelf Life:
No more than 10 weeks from the date of production when stored unopened at 2-8°C
and protected from light.
Composition:
Sodium Chloride, Amino Acids, Potassium Chloride, EDTA, Potassium Phosphate,
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Recommended Product For Culture:
LG PGD Biopsy Medium is based on global® medium. For optimal embryo
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before (Day 1-3), and after (Day 3-5), the biopsy procedure.
Ordering Information:
LPGG-020........ LG PGD Biopsy Medium, 20 ml
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Q1-3 (Low Air Pollution)
Q4 (High Air Pollution)
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Carbon-activated gas filtration during in vitro
culture increased pregnancy rate following
transfer of in vitro-produced bovine embryos
50
Control
40
Coda
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Rate
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ARTICLES
The Use of global® for Time-lapse Videographic
by Don Rieger, PhD
Vice President, Research and Development, LifeGlobal, LLC
You can contact Don Rieger at [email protected]
1. The Early History of Cinematography in Biomedical
Research
“As a matter of fact we can safely say that the motion picture
originated in the biological laboratory.” (Rosenberger 1929)
Most people would tend to think of motion pictures
as primarily an entertainment medium that is occasionally
used for educational purposes and, even less often, for
scientific investigations. However, as noted by Rosenberger
(1929), motion pictures were first used as a tool for the
analysis of movement of humans and other animals, in
particular the work of Eadweard J. Muybridge and ÉtienneJules Marey. (See also, Ruddock 2001)
Muybridge was a photographer and a bona fide
eccentric. His interest in biology was sparked (and
financially supported) by A. Leland Stanford, a railroad
tycoon and, later, governor of the state of California, who
wanted to know whether the four feet of the running horse
were ever off the ground at the same time. Muybridge
invented a multi-camera apparatus to photograph
successive images of Stanford’s prize race horse running,
and showed that all four feet were, in fact, off the ground at
the same time (Muybridge 1882). The history of this work
has more the flavour of a way to settle a bar bet rather than
a serious scientific study. Nonetheless, Muybridge went on
to apply his technique to a wide variety of studies of the
movement of humans and other animals at the University
of Pennsylvania (Marks et al. 1888), and is recognized as the
“Father of the Motion Picture.” It is perhaps interesting to
note that Stanford later established Stanford University,
the site of recent videographic studies of human embryo
development (Wong et al. 2010; Chavez et al. 2012).
Marey, by contrast, was a highly-accomplished
physiologist and educator at the Collège de France, in
Paris. The major focus of his career was in developing
techniques and devices to make objective and graphic
measurements of a wide variety of physiological processes
(Marey 1876; 1886). Marey (1879) used his “graphic
method” to demonstrate that all four feet of a horse were
off the ground at the same time, which inspired Muybridge
to develop his photographic method to confirm it. Marey,
in turn, was inspired by Muybridge’s work to develop his
own photographic equipment for the study of movement
(Marey 1882; 1884).
Following these early studies, cinematography was
applied to numerous aspects of animal biology and
medicine including x-ray cinematography of the heart,
diaphragm, stomach and joints (Groedel 1909), nystagmus
Don Rieger, PhD
(Lucchetti 1912), mental and nervous diseases (Weisenburg
1912), and medical (Anonymous (BMJ) 1910;Taylor 1918)
and public health (Moree 1916) education.
Ruddock (2001) notes that photography was developed
in 1839, and was quickly applied to medical science,
especially for photomicrography (within the same year).
It is therefore not at all surprising that medical scientists
similarly attached cinematographic cameras to microscopes
for the study of living specimens (micro-cinematography).
Early among these was Comandon (1909), who combined
a Zeiss “ultra-microscope” with a Pathé motion picture
camera to film blood-borne trypanosomes and spirocytes,
and polynuclear blood cells. Comandon was concerned
with the effects of heat from the lights on the specimens
and of ambient vibration on the images, which will strike a
chord with modern embryologists. Micro-cinematography
was subsequently applied to the study of ciliary motion
(Buytendijk 1912; Gray 1930), bacterial penetration through
capillary spaces (Mudd & Mudd 1924), capillary blood flow
(Crawford & Rosenberger 1926a; b), and bacterial growth
(Bayne-Jones & Tuttle 1927; Rogers & Greenbank 1930).
Alexis Carrel, a pioneer of mammalian cell culture (Carrel &
Burrows 1911; Carrel 1912) used micro-cinematography to
study the locomotion of the macrophage (Carrel & Ebeling
1926).
Comandon and Jolly (1917) filmed the division of newt
red blood cells. They took exposures of one frame every 2
3/10 seconds or every 5 seconds, and then viewed the films
at the normal rate of 16 frames/second, resulting in an
acceleration of 36.8 or 80 times normal speed. They referred
to this as “chronophotographie,” what we call time-lapse
photography. They described the advantages of time-lapse
cinematography as follows (my translation):
“Marey also indicated the use of time-lapse photography in
the study of phenomena which, because of their extreme
slowness, are difficult to appreciate by direct observation:
attention wearies, the eye tires, and the changes are
imperceptible. …Sometimes the movement is too rapid,
sometimes it is too slow. …With cinematographic projection,
the movement can be accelerated and rendered
perceptible to the eye.”
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2. Time-lapse Studies of Pre-implantation Embryo
Development
In essence, time-lapse photography serves two functions,
to capture and determine the timing of discrete events that
would not likely be seen by intermittent observation, and to
allow the visualization of processes that would otherwise
seem to be unconnected events. Both of these functions are
highly useful for the study of early embryonic development,
as was shown by Lewis and Gregory (1929) in their seminal
time-lapse study of development of the rabbit embryo.
The rabbit was a good choice for this work because it is an
induced ovulator, and therefore the time of fertilization is
closely related to the time of mating. They collected zygotes
at 21⅔-23½ hours, and late morulae at 67-71 hours, after
mating. The embryos were cultured in blood plasma or
modified plasma on microscope slides on the microscope
stage in a warm box. The zygotes divided to 2 cells by
24½-24¾ hours, to 4 cells by 28-30 hours, and to 8 cells by
39-42 hours, after mating. The very narrow spans of time
for the various cleavages are remarkably similar to those
seen in modern-day time-lapse micro-videographic studies
of human embryo development (see below). The embryos
collected at the morula stage survived for as long as 10
days in culture and developed into blastocysts, expanded,
and herniated through the zona pellucida. The blastocysts
went through (a process of) repeated cycles of expansion
and contraction prior to and during herniation through the
zona pellucida, as was later shown for hatching of cattle
(Massip & Mulnard 1980) , mouse (Bin & Mulnard 1980),
hamster (Gonzales & Bavister 1995), and horse and human
(Gonzales et al. 1996) blastocysts. The pioneering study of
Lewis and Gregory (1929) was followed by numerous timelapse studies of embryo development in various species.
An extensive review of the literature is beyond the scope
of this paper, but a number of salient studies are listed in
Table 1.
Table 1. Time-lapse studies of in-vitro embryo development.
Reference
Species
Observation(s)
Cassini (1961)
Mouse
Development from 2-cell to blastocyst
Cole (1967)
Mouse
Blastocyst hatching
Brackett (1972)
Rabbit
Fertilization and development to morula
Massip et al. (1982)
Cattle
Blastocyst expansion and hatching
Massip et al. (1983)
Cattle
Atypical blastocyst hatching resulting in twin half-blastocysts
Cohen et al. (1988)
Human
Morphological assessment of cleavage-stage embryos
Selwood and Smith (1990)
Marsupials
Cleavage and development to blastocyst
Gonzales et al. (1995)
Hamster
Cleavage, and timing of cleavage
Gonzales and Bavister (1995)
Hamster
Blastocyst hatching
Gonzales et al. (1996)
Cattle, horse, human Blastocyst expansion, contraction and hatching, trophectoderm projections
and locomotion of hatched blastocysts
Payne et al. (1997)
Human
Polar body extrusion and pronuclear formation
Holm et al. (1998)
Cattle
Developmental kinetics of early cleavage with respect to viability
Van Blerkom et al. (2001)
Human
Fragmentation and subsequent development
Peippo et al. (2001)
Cattle
Effect of sex and glucose on developmental kinetics
Holm et al. (2002)
Cattle
Effect of serum on developmental kinetics
Lequarre et al. (2003)
Cattle
Effect of oxygen on cell-cycle duration
Mateusen et al. (2005)
Pig
Relationships among developmental kinetics, fragmentation and apoptosis
Zaninovic et al. (2005)
Human
Development from zygote to hatched blastocyst
Mio and Maeda (2008)
Human
Fertilization, development to blastocyst and hatching
Gendelman et al. (2010)
Cattle
Effect of season on developmental kinetics
Lopes et al. (2010)
Cattle
Relationship of cleavage to oxygen consumption
Wale and Gardner (2010)
Mouse
Effect of oxygen on cell-cycle duration
3. Time-lapse Evaluation of Human Embryo Development
Comparisons of culture of human embryos under timelapse videography with conventional culture have shown
no differences in fertilization rate, embryo development,
development to blastocyst, blastocyst quality or ongoing
pregnancy rate (Cruz et al. 2011; Barrie et al. 2012; Kirkegaard
et al. 2012b). These reports indicate that time-lapse
videographic monitoring of human embryo development is
a safe and effective technique. Most notably, Meseguer et al.
(2012b) found that clinical pregnancy rates were better for
embryos cultured in a time-lapse incubator than for those
cultured in a conventional incubator. They attributed this
to both a more stable culture environment and the use of
ARTICLES
morphokinetic parameters for embryo selection for transfer.
Morphokinetic is defined as “a temporally demonstrable change
in shape or form” (Daneo-Moore & Higgins 1972).
Much of the interest in time-lapse videography of human
embryos is as an approach to predicting development in
vitro, and, ultimately, after transfer. A number of studies
have shown that various morphokinetic measurements are
related to subsequent in-vitro development. Development
to the blastocyst stage and blastocyst quality have been
shown to be related to the time of syngamy and timing of
the early cleavage divisions (Wong et al. 2010; Cruz et al.
2012; Dal Canto et al. 2012; Hashimoto et al. 2012; McEvoy
et al. 2012). Iwata et al. (2010) found that compaction before
the 8-cell stage was associated with developmental arrest
and multinucleation. Yumoto et al. (2012) observed that, for
previously frozen-thawed embryos, blastocyst collapse was
detrimental to hatching.
Clinical outcome following transfer has also been
related to morphokinetic measurements. Azzarello et al.
(2012) observed that no live births resulted from embryos
that experienced early pronuclear breakdown. Ramirez et
al. (2012) found that early appearance of two pronuclei was
associated with multinucleation and reduced implantation.
Implantation has also been shown to be related to the timing
of early cleavage events (Rubio et al. 2012; Chamayou et
al. 2013). It is not at all surprising that successful clinical
outcome can be related to the timing and other characteristics
of early cleavage because they set the stage for subsequent
development. However, in view of the fact that the major
onset of activation of the human embryonic genome does
not occur until the 4-8-cell stage (Telford et al. 1990), it
would seem unwise to suggest that early cleavage events,
alone, should be used for embryo selection for transfer.
Evaluation of morphokinetics to the blastocyst stage would
include evaluation of events controlled by expression of the
embryonic genome, and thus be a much better indicator of
viability.
Time-lapse videography also offers much promise
for fundamental studies of the early human embryo. For
example, both the dose of FSH (Munoz et al. 2012) and
the type of GnRH analogue used (Munoz et al. 2013) have
been related to the morphokinetics of the early embryo,
pointing out the significance of ovarian stimulation to early
development. Mio et al. (2012) )have proposed a novel
mechanism for the block to polyspermy, based on time-lapse
observations of fertilization. Freour et al. (2013) have shown
that maternal smoking is related to delays in early cleavage.
Conversely, Bellver et al. (2013) found no effect of maternal
obesity on morphokinetic patterns. Kirkegaard et al. (2012c)
showed that cleavage-stage blastomere biopsy resulted
in delayed compaction and a change in the mechanism of
hatching. Chavez et al. (2012) found that the timing of early
cleavage was disturbed in aneuploid embryos. Conversely,
Semeniuk et al. (2013) found no relationship between ploidy
and the timing of early cleavage events. Campbell et al.
(2013) similarly found that ploidy was not related to early
cleavage events, but aneuploidy was associated with delayed
compaction and blastocyst formation.
The demonstration of the effects of culture under
atmospheric oxygen concentration (20%) on cell-cycle
kinetics in cattle (Lequarre et al. 2003), mouse (Wale and
Gardner 2010), and human (Kirkegaard et al. 2013) embryos
is a particularly notable example of the potential value of
time-lapse videography. Despite overwhelming evidence to
show the deleterious effects of culture under 20% oxygen on
embryo development and viability, culture under reduced
oxygen is not yet universally practiced in human ART
(Gardner 2005; Bontekoe et al. 2012). Perhaps the highly
objective and precise observations from time-lapse studies
will serve to finally put to rest the unphysiological practice
of culture of human embryos under 20% oxygen.
More extensive reviews of the literature are provided by
Meseguer et al. (2012a), Kirkegaard et al. (2012a), Herrero and
Meseguer (2013) and Wong et al. (2013).
4. The Use of
global® for Time-lapse Evaluation of
Human Embryo Development
As noted by Herrero and Meseguer (2013), timelapse imaging of embryo development offers a significant
advantage over standard culture because the embryos can
be monitored without removing them from the stable gas
and temperature conditions. This, in essence, is consistent
global® medium
with the philosophy of the use of
®
and the
global family of media in which stress on the
embryo is minimized by maintaining it in the same chemical
background throughout culture and other ART procedures
(see Biggers and Summers 2008). Given the extensive history
of the success of global® for human embryo culture from
the zygote to the blastocyst stage, time-lapse imaging of
embryos in global® is a natural fit.
The results of a number of time-lapse imaging studies
are described below in which the embryos were cultured
in global®. Unless otherwise indicated, the medium was
renewed or refreshed on Day 3. As previously discussed
(Rieger 2012), our general recommendation is that embryos
be moved to fresh medium under fresh oil on Day 3 (2step culture), in order minimize the possibility of exposure
to volatile organic contaminants. It is certainly possible to
culture human embryos from the zygote to the blastocyst
stage without renewing the medium (1-step culture),
providing that the environmental and other conditions in the
laboratory are appropriate. (Reed et al. 2009; 2010; Keskintepe
2012; Singh et al. 2012) In this regard, time-lapse culture may
be particularly suitable for 1-step culture because there is
a significant reduction in the possibility of exposure of the
embryos to environmental VOCs compared with culture in
conventional incubators.
Cruz et al. (2012) cultured embryos from the zygote stage
in global® in a time-lapse incubator for 5 days, and then
compared the morphokinetic data between embryos that
developed to the blastocyst stage (66.2%) with those that did
not. 33.8%). Development to 2, 3, 4 and 5-cells, and to morula
was significantly longer for embryos that did not develop to
blastocyst. Of the 247 blastocysts that were transferred, 136
(49.6%) implanted (Figure 1).
Silva et al. (2012) incubated embryos from donor
oocytes in
global® in a time-lapse incubator up to the
blastocyst stage, and then performed laser-assisted hatching
ARTICLES
before transfer. As shown in Figure 2, this resulted in an
implantation rate of 52.4%.
Bellver et al. (2013) compared the development of
embryos from obese infertile, normal weight infertile, and
normal weight fertile women during culture in global® in
a time-lapse incubator over 5 days. The timing of cleavage of
the embryos was not different between embryos from obese
and normal weight infertile women, but was slower than
cleavage of those from normal weight fertile women (Figure
3).
Munoz et al. (2013) compared the development of
embryos derived from donor cycles after ovarian stimulation
using GnRH agonists with hCG triggering, or GnRH
antagonists with GnRH agonist triggering. The embryos
were cultured to Day 3 or Day 5 before transfer. As shown
in Figure 4, early cleavage events were delayed in the GnRH
agonists/hCG triggering group. The implantation rate was
greater in the GnRH antagonists/GnRH agonist triggering
group, and approached statistical significance (P = 0.084).
There was no difference in miscarriage rate between the
treatment groups (P = 0.524).
Costa-Borges et al. (2013) evaluated embryo development
in a time-lapse incubator during culture in global total®
120
Developed to blast (N=552)
Mean time (h)
72
24
0
80
% of Embryos
Did not develop to blast (N=282)
96
48
for 5 days. The medium was either renewed on Day 3 (2-step
culture) or not (1-step culture). There was no difference in
any of the morphokinetic parameters measured, blastocyst
development or quality, or implantation rate between 2-step
and 1-step culture (Figure 5).
Campbell et al. (2013) cultured embryos from the zygote
stage until the blastocyst stage in global® in a time-lapse
incubator and then performed trophectoderm biopsy
in order to determine ploidy by comparative genomic
hybridization. Morphokinetic parameters were compared
between embryos determined to be euploid, or to have
single or multiple aneuploidy. There were no differences
in the timing of the early cleavage events among the three
groups. The first indication of compaction and/or blastocyst
formation was delayed in the single or multiple aneuploidy
groups compared with the euploid group (Figure 6).
Semeniuk et al. (2013) cultured embryos from the zygote
stage until the blastocyst stage in global® in a 1-step protocol
in a time-lapse incubator, and then performed trophectoderm
biopsy in order to determine ploidy by comparative genomic
hybridization. There were no differences in the timing of
the early cleavage events between euploid and aneuploid
embryos (Figure 7).
16
60
40
20
80
Obese Infertile
60
40
20
0
Clin. Preg. Rate
Impl. Rate
Miscarriage Rate
Figure 2. Clinical outcomes for embryos cultured in
a time-lapse incubator (Silva et al. 2012).
global® in
Normal Wt Fertile
Normal Wt Infertile
60
Mean time (h)
% of Embryos or Transfers
0
t3
t4
t5
tM
Blastocysts
Impl. Rate
Morphokinetic Event
Figure 1. Comparison of the times of morphokinetic events between embryos that did or did not reach the blastocyst stage, and
blastocyst development and implantation rates for embryos cultured in global® in a time-lapse incubator (Cruz et al. 2012).
t2
40
20
0
t2
t3
t4
t5
s2
Cc2
Cc3
Figure 3. Comparison of the times of morphokinetic events
between embryos derived from oocytes from obese infertile,
normal-weight infertile, and normal-weight fertile women. The
embryos were cultured in global® in a time-lapse incubator
(Bellver et al. 2013).
ARTICLES
96
Mean time (h)
16
GnRH Agonist
GnRH Antagonist
72
48
24
0
t2
t3
t4
16
16
16 16 16
16
% of Patients or embryos
120
t5 t6 t7 t8 t9+ tM
Morphokinetic Event
30
20
16
10
0
tB tEB
16
40
Implantation
Rate
Miscarriage
Rate
Figure 4. The effect of ovarian stimulation protocol on the times of morphokinetic events and clinical outcomes for embryos cultured
in global® in a time-lapse incubator (Munoz et al. 2013).
2-step
96
1-step
16
72
48
16 16
16
16
16
16
16 16
80
16
16
60
16
40
20
24
0
16
% of Embryos
Mean time (h)
120
t2
t3
t4
0
t5 t9+ tM tCM tB tEB tHB
Morphokinetic Event
Blastocysts
G.Q. Blasts
Impl Rate
Figure 5. Comparison of the times of morphokinetic events, blastocyst development, and implantation rates between embryos
cultured in global total® with either medium renewal on Day 3 (2-step culture) or not (1-step culture) (Costa-Borges et al. 2013).
Mult. Aneuploid
Euploid
96
72
16
48
24
0
16
t2
16
16
60
16
Euploid
48
Mean time (h)
Median time (h)
120
16
16
36
Aneuploid
16
16
16
16
24
12
t3
t5
t8 tSC tM tSB tB
Morpholokinetic Event
tEB tHB
Figure 6. Effect of ploidy on the times of morphokinetic events
global® in a time-lapse incubator
for embryos cultured in
(Campbell et al. 2013).
0
t2
t3
t4
Morphokinetic Event
t5
Figure 7. Effect of ploidy on the times of morphokinetic events
for embryos cultured in
global® in a time-lapse incubator
(Semeniuk et al. 2013).
ARTICLES
5. Discussion and Conclusions
1.
Time-lapse videography of embryos throughout
early development has been shown to have no
deleterious effects on the in-vitro development
of the embryos or on clinical outcomes after
transfer. Conversely, time-lapse culture may, in
itself, be advantageous because the embryos can
be monitored without removing them from the
incubator.
2.
The measurement of various morphological
parameters throughout culture offers the promise
of an effective addition to the armament of
techniques for selection of a single embryo for
transfer with the maximum potential to produce a
healthy baby. However, this will almost certainly
require morphokinetic analysis up to and including
the blastocyst stage.
3.
Time-lapse videography of embryo development
is also potentially an important tool for the study
of more fundamental aspects of ART, including
ovarian stimulation, fertilization, culture, and
embryo manipulation. The deleterious effect of
environmental oxygen concentrations (20%) on the
morphokinetics of the early embryo is one notable
example.
4. global® medium has been shown to be safe and
effective for time-lapse culture of human embryos.
Given appropriate air quality, 1-step culture in
global® has been shown to be highly effective for
human embryos.
5. global® is particularly suitable for time-lapse
embryo culture.
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Advanced maternal age and assisted reproductive
technologies: developmental competence, clinical
outcomes and future strategies.
By Lynne C. O’Shea, PhD
Rotunda IVF, Dublin, Ireland.
University College Dublin, Belfield, Dublin, Ireland.
You can contact Lynne O’Shea at [email protected] or [email protected]
Lynne C. O’Shea, PhD
Abstract
Background: In recent decades the number of women over 40 seeking assisted reproductive technology (ART)
interventions in order to become pregnant has increased dramatically. This is due to an increase in the average age
at which women are choosing to have their first child while additionally, many couples are choosing to have a second
family later in life. However, as with natural conception, ART success rates decrease with maternal age. The decrease
in developmental competence of aged oocytes has pushed the field of assisted reproduction to develop additional
management strategies and new techniques for optimising fertility outcomes in older women.
Methods: In the present study, detailed analysis was performed on our clinical data of women between 40 and 45
years of age, who have undergone ART (16 year retrospective analysis) at our tertiary referral ART clinic. The percentage
of patients in this age group was analysed over time, in addition to follicle recruitment, % oocyte yield (and oocyte
numbers), embryonic quality, positive hCG (pregnancy rate), clinical pregnancy rate (presence of intrauterine sac),
and rate of preclinical pregnancy loss. Published literature to date was also analysed, in conjunction with our clinic’s
experience in treating patients of advanced maternal age, in order to identify both current and emerging treatment
strategies.
Results: Results from our clinic show that women greater than 43 years of age have a significantly reduced reproductive
potential, compared even to women in the 40 to 42 years age group. Woman in the 43-45 age group showed reduced
fertilization rates (53.73 vs. 58.82%), reduced positive hCG rates (11.51 vs.19.03%) and clinical pregnancy rates (5.04
vs.12.52%) and increased rates of preclinical pregnancy loss (56.23 vs.34.23%), compared to women in the 40-42 age
group. Previously published reports showed that the chance of achieving a clinical pregnancy in women greater than
43 years of age is less than 2%.
Conclusions: With the age at which couples are choosing to have children constantly increasing, novel patient
management and treatment strategies need to be developed for women of increased reproductive age. Several
emerging ART techniques, including oocyte/embryo donation, ‘social’ oocyte freezing, preimplantation genetic
screening and ovarian tissue freezing, are being established to enhance clinical outcomes in patients of increased
maternal age.
Keywords: infertility, advanced age, ART, maternal age, over 40.
Background
There is a well-established link between advanced
maternal age and reduced reproductive potential. This
natural decrease in fertility in women is caused by several
factors including reduced oocyte numbers, diminished
oocyte and embryo quality, and an increase in miscarriage
rate [1-3]. In addition, pregnancy at this later stage in life
involves increased maternal and fetal risks including:
miscarriage, hypertension, preeclampsia, gestational
diabetes, placenta praevia, placental abruption, caesarean
section, genomic disorders, premature birth, low foetal
birth weight and neonatal morbidity [4].
In recent decades, the number of women over 40 seeking
assisted reproductive technology (ART) interventions,
in order to become pregnant, has dramatically increased.
This is due to an increase in the average age at which
women are choosing to have children; while additionally,
many couples are choosing to have a second family later
in life [5]. However, as with natural conception, ART
success rates decrease with maternal age. In addition to a
reduction in oocyte numbers retrieved during a standard
ART cycle, maternal age has a detrimental effect on oocyte
competence [6-8]. One major factor evident in aged oocytes
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is an increase in aneuploidy [9, 10]; with chromosomal
abnormalities being a major determinant of subsequent
embryonic development. In addition to morphological and
structural anomalies evident in aged oocytes, they also
possess altered gene expression profiles, and an increased
potential to undergo apoptosis [11, 12]. Studies have shown
alterations in the ability of aged oocytes to stockpile the
maternal RNAs that are required to sustain both oocyte and
embryo development prior to embryonic genome activation
[13, 14].
The decrease in reproductive potential observed in
couples of increased age has pushed the field of assisted
reproduction to develop additional management strategies
and new techniques for optimising fertility outcome. This
has led to the establishment of oocyte donor programmes
and ‘social’ oocyte-freezing in younger women, in addition
to improved genetic testing (preimplantation genetic
diagnosis) and advanced methods for determining ovarian
reserved (AMH). However, in conjunction with the medical
implications of increased reproductive age, societal and
ethical implications need to also be considered when
employing ART. Most ART clinics have self-imposed limits
on the age of patients they are willing to provide with
treatment; with vast variation between clinics and countries,
and limited regulation in place.
The aim of the present study was to perform a detailed
analysis of our clinical data on women greater than 40 years
of age, who have undergone ART at our tertiary referral
ART clinic, between 1997 and 2013. We analysed our own
data in the context of published data, in order to determine
specific outcomes and review both current and emerging
strategies for improving fertility in women greater than 40
years of age.
Methods
Study design
A retrospective analysis of clinical and laboratory data
was performed by collecting data from our tertiary referral
ART academic program. All IVF/ICSI cycles carried out
on women between 40 and 45 years of age, at the Human
Assisted Reproduction Ireland (HARI) clinic, Rotunda
Hospital, Ireland between January 1997 and January 2013
were identified. All patient treatment data is prospectively
collected in a custom made, FilemakerPro-based electronic
database. Our clinic has an age cut-off limit for ART of 45
years of age. Patients between 40 and 45 years of age at egg
collection were selected for analysis. The percentage of
patients in this age group were analysed over time. Due to
previous studies demonstrating that the chance of achieving
a clinical pregnancy in women greater than 43 years of age
is significantly diminished, we also split our patients into
two age groups for comparative analysis; 40-42 and 4345. The following parameters were then analysed: follicle
recruitment, % oocyte yield (oocyte/follicle ratio), positive
hCG, clinical pregnancy rate (ultrasound confirmation
of a gestational sac at 7 weeks) and preclinical pregnancy
loss (absence of intrauterine sac at 7 week ultrasound scan
following previously positive urinary hCG test).
Ovarian stimulation and IVF/ICSI treatment
Ovarian stimulation was carried out as previously
described [15, 16]. Briefly, female patients underwent one
of three different types of ovarian stimulation protocol
employed during the assessment period; namely using
urinary (Menopur, Ferring, Ireland) or recombinant FSH
(Puregon, MSD, Ireland) in combination with GnRH
antagonist, GnRH agonist or a buserelin nasal spray
(Suprecur, Sanofi-Aventis). Final oocyte maturation was
induced by injection of 10,000 IU hCG (Pregnyl, MSD,
Ireland), as soon as three follicles of 18 mm or more were
observed on ultrasound scan. Oocyte retrieval was carried
out under sedation using transvaginal ultrasound-guided
puncture of ovarian follicles 36 h after hCG administration.
IVF and ICSI treatments were carried out as previously
described [15].
Statistical analysis
Groups of interest were compared using MannWhitney U-test or Wilcoxon’s matched pairs rank sum
test was used to compare mean values and Pearson chisquare or McNemar chi-square analysis for comparison of
proportional values. All statistical analyses were carried out
using SPSS statistics, version 20 (IBM Corporation, 2011,
Armonk, New York, USA). Differences between groups
were considered significant when p-values were less than
0.05.
Systemic literature review: clinical data for ART in women
over 40 years of age
A systematic literature review was performed
by searching PubMed, MEDLINE, CINAHL and The
Cochrane Library for data relating to ART in women
of advanced reproductive age. Phrases and key
words searched for included: <fertility> and <assisted
reproductive technology> in <advanced maternal age>
or <older women>, including specific end points such as
<fertilization>, <pregnancy>, <clinical pregnancy>, <live
birth>, <in vitro fertilization>, <intracytoplasmic sperm
injection>, <miscarriage>, <pregnancy loss>, <aneuploidy>,
<pregnancy complications>, etc. Data was extracted from
literature sources and collated, in conjunction with annual
reports on ART success rates form the U.S. Centers for
Disease Control and Prevention (http://www.cdc.gov/
ART).
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Results
Our clinical data: ART in women between 40 and 45 years
of age
For the study period, we identified a total of 2068 egg
collections from women between 40 and 45 years of age.
The average age of the female patients on the date of egg
collection was 41.14 ± 1.28 years and the average age of the
male partner was 41.62 ± 5.63 years. Figure 1 demonstrates
the percentage of patients, in the 40 to 45 year old age
group, undergoing IVF/ICSI in our clinic from 1997 to 2013.
The proportion of patients in this age group has grown
significantly over this 17 year period; increasing from 7.2%
of patients in 1997 to 19.8% in 2013. Table 1 presents the
patient characteristics, including hormone profiles.
Table 2 presents the parameters for oocyte retrieval and
oocyte competence. The number of follicles aspirated and
oocytes retrieved was significantly lower in patients in the
43-45 age group compared to the 40-42 age group (Table 2).
The rate of mature (MII) oocytes was similar in both groups.
The percentage of normal fertilization (2PN zygotes)
was significantly lower in the 43-45 age group compared
to the 40-42 age group and this corresponded to a lower
fertilization rate for both ICSI and IVF fertilized oocytes.
Table 3 presents the embryo transfer parameters and
clinical outcomes. There was no difference in embryo
transfer rates between the two age groups, or the mean
number of embryos transferred per group. The pregnancy
rate (positive hCG) per cycle and per embryo transfer
was significantly lower in the 43-45 age group compared
to the 40-42 age group. Clinical pregnancy rate was also
significantly lower in the 43-45 age group, while the rate of
preclincial pregnancy loss was markedly increased (Table
3).
Systemic literature review: clinical data for ART in women
greater than 40 years of age
Table 4 presents the clinical data collated from the
published literature for female patients over 40 years of age
undergoing ART. These studies report a clinical pregnancy
rate of 0–29.15% in women over 40 undergoing ART and a
cut-off for ART efficiency after 43 years of age; with a less
than 2% chance of achieving a clinical pregnancy in women
beyond this age. Live birth rates observed in several of the
studies appear to be compounded by high miscarriage rates
(16.85 - 85.3%). The data also show that miscarriage rate
increases with age, with a miscarriage rate of 50-85.3 % in
women greater than 43 years of age.
Discussion
Women over 40 represent the fastest growing
population of patients seeking ART in our clinic, with
numbers increasing dramatically from 17 years ago. This
corresponds with a proportionate increase in numbers
worldwide [5]. With the age at which couples are choosing
to have children constantly increasing, novel patient
management and treatment strategies need to be developed
for women of increased reproductive age.
The ability to get pregnant following ART relies largely
on the quality of oocytes retrieved and the subsequent
development of high quality embryos for transfer.
Although oocyte maturation rate in our study was similar
between both groups, fertilization rate was greatly reduced
in women in the 43-45 age group, compared to 40-42.
This corresponds to data previous reported by Cabry
Figure 1. The percentage of patients between 40 and 45 years of age undergoing egg collection at our clinic between 1997 and 2013
inclusive.
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Table 1. Baseline characteristics of patient parameters, in women between 40 and 45 years of age, undergoing IVF/ICSI.
Parameter
40-45 years
Number of cycles
2068
Number of cycles where no eggs were retrieved
225
Average maternal age (years ± SD)
41.14 ± 1.28
Average paternal age (years ± SD)
41.62 ± 5.63
Infertility duration (year ± SD)
4±3
Previous failed cycle
1.73 ± 1.94
Day 3 AMH (IU/L)*
9.03 ± 11.35
Day 3 E2 (IU/L)
196.23 ± 203.39
Day 3 FSH (IU/L)
9.15 ± 4.28
Day 3 LH (IU/L)
6.23 ± 10.44
*AMH data from March 2010-January 2013
Table 2. Oocyte retrieval and competence, in women over 40 years of age undergoing IVF/ICSI.
Parameter
Follicles aspirated (mean ± SD)
40-42 years
a
9.44 ± 5.38
a
Oocytes retrieved (mean ± SD)
7.93 ± 4.82
Oocyte yield (%)
84.07a (10141/12062)
a
43-45 years
Total (40-45 years)
7.57 ± 4.62
b
8.51 ± 5.0
6.28 ± 4.27
b
7.11 ± 4.55
82.84a (1381/1667)
a
83.93 (11522/13729)
Mature (metaphase II) oocytes
(ICSI only) (%)
78.76 (2558/3248)
78.34 (416/531)
78.70 (2974/3779)
Normal fertilization (all) (%)
58.82a (5965/10141)
53.73b (742/1381)
58.21 (6707/11522)
a
b
Normal fertilization (IVF) (%)
60.18 (4148/6893)
55.53 (472/850)
59.67 (4620/7743)
Normal fertilization (ICSI) (%)
55.92a (1817/3248)
50.85b (270/531)
55.23 (2087/3779)
Number of fertilized oocytes/cycle
a,b,c
a
3.41 (5965/1750)
b
2.33 (742/318)
3.24 (6707/2068)
Superscripts of different alphabetic characters across rows indicate significant differences (P < 0.05) between groups, whereas presence of identical characters
indicates lack of significant differences.
Table 3. Clinical outcomes, in women over 40 years of age undergoing IVF/ICSI.
Parameter
40-42 years
43-45 years
Total (40-45 years)
Embryo transfer performed (%)
89.49a (1566/1750)
87.42a (278/318)
89.17 (1844/2068)
Transferred embryos (mean ± SD)
2.12 ± 0.93a
2.02 ± 0.92a
2.07 ± 0.93
a
b
Positive hCG/cycle (%)
17.03 (298/1750)
10.06 (32/318)
15.96 (330/2068)
Positive hCG/embryo transfer (%)
19.03a (298/1566)
11.51b (32/278)
17.90 (330/1844)
Clinical pregnancy rate/embryo
transfer (%)
12.52a (196/1566)
5.04b (14/278)
11.39 (210/1844)
Preclinical pregnancy loss (%)
34.23a (102/298)
56.23b (18/32)
36.36 (120/330)
a,b,c
Superscripts of different alphabetic characters across rows indicate significant differences (P < 0.05) between groups, whereas presence of identical characters
indicates lack of significant differences.
et al. [17] and demonstrates how the ability of an oocyte
to undergo nuclear maturation is not always a reliable
predictor of oocyte quality and subsequent developmental
potential. Maternal age is known to be the biggest factor
affecting clinical outcome following ART. Our data show
that female patients greater than 42 years of age have a
diminished prognosis following IVF/ICSI compared to
patients between 40 and 42 years of age. This corresponds
to previously published data which clearly shows a cut-off
for success following ART in women to be 44 years of age
(Table 4). The limitations of the studies performed to date
reside in their retrospective nature, in addition to changing
protocols over time. In addition, many variables associated
with assisted reproductive technologies such as day of
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Table 4. Reported clinical outcomes from ART in women over 40.
Reference
Our clincal data
Cabry et al., 2014 [17]
Gleicher et al., 2014 [18]
Niinimaki et al., 2012 [19]
Nikolaou & Marinakis 2011 [20]
Tsafrir et al., 2009 [21]
Hourvitz et al., 2009 [22]
Maternal Age (years)
Clinical Pregnancy (%)
Miscarriage Rate (%)
Live Births (%)
40-42
12.52
43-45
5.04
41.5*
8.9
16.85
7.4
40-41
29.15
0
29.15
42-45
11.56
77.51
2.6
46-53
0
0
0
(SET) 19.5
43.59
11.0
(DET) 23.5
42.13
13.6
40-44
40-42
7.7
43-44
5.4
>44
1.9
40
13.9
34.53
9.1
45
2.8
75
0.7
42
7.7
43
5.4
85.3
3.1
44
1.9
Spandorfer et al., 2007 [23]
45.4*
21.2
Van Disseldorp et al., 2007 [24]
40-43
8
42
7.7
43
5.4
Ciray et al., 2006 [25]
Klipstein et al., 2005 [26]
Ron-El et al., 2000 [27]
44
1.9
≥45
0
40
3
41-42
18.8
43
9.6
44
1.6
41
14
50
7
42
9
77.78
2
43
26
50
13
44
0
0
0
* mean maternal age, SET: single embryo transfer, DET: double embryo transfer.
transfer, embryo quality and type of protocol are not taken
into account when analysing results. However, studies such
as these are not without their relevance and the correlation
of similar studies performed worldwide provides a reliable
indication of the effect advanced maternal aging is having
on reproductive potential following ART.
Current strategies
Over the past decade many patient management and
treatment strategies have been developed for women
over 40 years of age. First, patients must be counselled
on both the reduced clinical success rates for ART and
increased pregnancy risks and miscarriage rates associated
with increased maternal age. Here we show a significant
reduction in pregnancy rate and clinical pregnancy rate, in
addition to a substantial increase in the rate of preclincal
pregancy loss (increase from 34.23 % in 40-42 year olds
to 56.23% in 43-45 year olds at 7 week scan). It is known
that a large proportion of miscarriages that occur in the
first trimester of pregnancy are due to chromosomal
abnormalities, a known factor associated with oocytes from
women of increased reproductive age [28].
Due to the low success rates observed following ART,
women of increased reproductive age often undergo the
transfer of multiple embryos in order to obtain a pregnancy.
One previous study reported that the optimum number of
embryos for transfer in order to achieve an appropriate
ARTICLES
pregnancy and live birth rate in women over 40 is five
[29], while many others have suggested 3 embryos to be
sufficient [25, 26, 30]. Due to the increased risks associated
with multiple births, and the increased pregnancy risk
the patient is already subject to, this strategy should be
considered very carefully before being initiated.
Emerging strategies
Preimplantation genetic screening (PGS) is one strategy
which is currently being used by clinics to identify genetic
and chromosomal abnormalities in embryos. This has been
shown to allow for improved embryo selection techniques,
leading to increased pregnancy rates and reduced
implantation rates in women over 40 years of age [31-33].
Another strategy currently in focus for patients of
increased age is the option of oocyte and embryo donation.
Several studies have shown that many infertility issues
associated with increased maternal age can be overcome by
oocyte donation [34, 35]. Nevertheless, patient age also has
an effect on uterine receptivity and implantation; as evident
in the large meta-analysis performed by Vernaeve et al.
[36] which showed that pregnancy rates were significantly
reduced in patients over 45 years of age using donor
oocytes. In such cases surrogacy may also be an option. The
Spanish experience shows cumulative pregnancy rates after
4 cycles of embryo transfer from donor eggs reaching nearly
94%, with clinical pregnancy rates of 53.4% per cycle and
live birth rates of 42.6% [35]. Therefore, oocyte donation is
a very promising option for women over 40 years of age
wishing to get pregnant. In addition, oocyte and embryo
donation eliminates the risk of ovarian stimulation in the
recipient. Before undergoing either oocyte or embryo
donation patients need to be extensively counselled about
the ethical and legal issues associated with such procedures.
Patients need to be aware of the possibility of their child
having full siblings following embryo donation, or half
siblings in the case of oocyte donation. At present in many
countries, including Ireland, there is no legislation in place
to cover the legal issues arising from surrogacy, oocyte or
embryo donation. This leads to ambiguity over who the
legal parents of a child born from such procedures are.
Due to the increased success rates associated with
transferring embryos generated from young oocytes to
women over 40, one option for women is to ‘self-preserve’
their own fertility by banking their eggs when they are in
their 20’s and 30’s. With the development of vitrification,
oocyte cryopreservation has vastly improved, however to
date data are still limited [37]. It has been shown that the
retrieval of 55 metaphase II oocytes is needed for women
aged between 38 and 43 years of age to achieve a pregnancy
following vitrification [38]. The large number of oocytes
needed, in addition to the risks associated with ovarian
stimulation and egg collection may deter women from
availing of this ‘back-up plan’ for their future fertility.
Furthermore, the cryopreservation of oocytes does not
guarantee a pregnancy and the maternal risks associated
with a pregnancy at advanced age are all still present. The
ethics of offering such a service at present, giving the cost
and the low rate of success, need to be questioned.
Ovarian tissue freezing is a novel strategy under
development in order to preserve fertility in women. This
allows storage of a large pool of primordial follicles and
negates the need for hormone treatment at the time of
retrieval [39, 40]. For these reasons it is currently used as
an option for cancer patients with time and/or hormone
sensitive malignancies. Furthermore, this procedure not
only has the potential to restore fertility but has also been
shown to restore endocrine function, and could potentially
radically postpone the onset of menopause [41].
These current and emerging advances in ART techniques
may allow women to achieve pregnancy much later in life.
However, when considering ART at an advanced age, it is
necessary to take into account the severe risks associated
with such a pregnancy [4]. One study observed that 63%
women over 50 who achieved a pregnancy through embryo
donation needed hospitalization, compared to 22% in
women aged between 45 and 49 [34]. In such women,
surrogacy may be the safer option.
Conclusions
Women of age greater than 42 have a significantly
reduced reproductive potential, compared to women who
are between 40 and 42 years of age. This corresponds to
a reduced fertilization rate, pregnancy rate and clinical
pregnancy rate, in addition to an increased miscarriage
rate. With the life expectancy of children being born
today expanding above 100 years of age, the demand for
ART strategies to prolong reproductive potential and/or
postpone the onset of menopause will continue to rise.
Acknowledgements
I wish to thank the embryology staff, nurses, patient
support team and physicians at Human Assisted
Reproduction Ireland (HARI), Rotunda Hospital, Dublin,
Ireland who have contributed to the care of the patients.
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ARTICLES
High-magnification selection of spermatozoa
prior to oocyte injection: confirmed and potential
indications
F Boitrelle*, B Guthauser, L Alter, M Bailly, M Bergere, R Wainer, F Vialard, M Albert, J Selva
Department of Reproductive Biology, Cytogenetics and Gynaecology, Poissy General
Hospital, F-78303 Poissy, rance; EA
2493, Versailles University of Medicine and Science, F-78000 Versailles, France
*Corresponding author address: Department of Reproductive Biology, Cytogenetics
and Gynaecology, Poissy General Hospital, F-78303 Poissy, France.
E-mail address: [email protected] (F Boitrelle).
Florence Boitrelle, MD
Florence Boitrelle, MD is working as an embryologist at Poissy General Hospital’s assisted reproduction unit since 2009.
She is currently working on her PhD thesis on the links between sperm morphology, nuclear quality, acrosome quality,
genetics and epigenetics at the University of Versailles Saint-Quentin-en-Yvelines.
Abstract Intracytoplasmic morphologically selected sperm injection (IMSI) involves the use of differential interference
contrast microscopy at high magnification (at least ·6300) to improve the observation of live human spermatozoa
(particularly by showing sperm head vacuoles that are not necessarily seen at lower magnifications) prior to
intracytoplasmic sperm injection (ICSI) into the oocyte. However, a decade after IMSI’s introduction, the technique’s
indications and ability to increase pregnancy and/or birth rates (relative to conventional ICSI) are subject to debate. In
an attempt to clarify this debate, this work performed a systematic literature review according to the PRISMA guidelines.
The PubMed database was searched from 2001 onwards with the terms ‘IMSI’, ‘MSOME’ and ‘high-magnification, sperm’.
Out of 168 search results, 22 relevant studies reporting IMSI outcomes in terms of blastocyst, pregnancy, delivery and/
or birth rates were selected and reviewed. The studies’ methodologies and results are described and discussed herein.
In view of the scarcity of head-to-head IMSI versus ICSI studies, the only confirmed indication for IMSI is recurrent
implantation failure following ICSI. All other potential indications of IMSI require further investigation.
KEYWORDS: human spermatozoa, IMSI, MSOME, outcome, pregnancy rate, vacuole
This article was published in Reproductive BioMedicine Online, Vol 28, 2014, p6-13, High-magnification selection of
spermatozoa prior to oocyte injection: confirmed and potential indications. Copyright Elsevier. It is reprinted here with
permission.
Introduction
Since its first use in the early 1990s (Palermo et al.,
1992), intracytoplasmic sperm injection (ICSI) has become
a powerful tool for infertile couples – particular in cases of
severe male infertility and low sperm counts. In ICSI, the
‘best-looking’ live spermatozoon is chosen for its motility,
viability and gross morphology, using Hoffman contrast
microscopy and a magnification of x200 or x400. Although
the fertilization and clinical pregnancy rates associated
with ICSI are high (Palermo et al., 2009), it has been shown
that ejaculate characteristics (e.g. normal spermatozoa or
mild or severe oligoasthenoteratozoospermia) (Loutradi
et al., 2006) and the morphology of the individually
selected spermatozoon may affect post-ICSI fertilization,
implantation and pregnancy rates (De Vos et al., 2003).
These results can be explained (at least in part) by the
fact that, even though a spermatozoon’s morphology is
slightly correlated with its chromatin condensation or
DNA integrity, the selection of normal spermatozoa during
ICSI does not enable spermatozoa with nuclear defects to
be excluded (Abu Hassan Abu et al., 2012; Avendaño et al.,
2009).
Hence, over the last decade, some researchers have
tried to improve sperm observation with higher-resolution
microscopy
techniques. Their objective has been to establish
correlations between the morphology of a viable (and
subsequently injectable) spermatozoon and its inherent
quality (in terms of chromosomal content, degree of
chromatin condensation and/or DNA integrity). The most
studied of these novel techniques is motile sperm organelle
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morphology examination (MSOME), which uses differential
interferential contrast microscopy and high magnification
(>x6300), first described by Bartoov et al. (2001). This
observation technique reportedly enables better assessment
of a spermatozoon’s morphology and the visualization of
sperm head vacuoles. The latter structures are not visible
(particularly when they are small) at a conventional ICSI-like
magnification (using Hoffman contrast and a magnification
of x200–x400) (Bartoov et al., 2001). Nevertheless, since the
introduction of IMSI, more attention has been given to the
pre-ICSI detection of spermatozoa that contain vacuoles.
Over the last decade, many researchers have evaluated
IMSI (i.e. the MSOME-based selection of a spermatozoon
and then its injection into the oocyte) and compared it
with the gold-standard technique, ICSI. However, IMSI’s
superiority over ICSI (in terms of pregnancy or delivery
rates) is still subject to debate. The only meta-analysis of
this topic was performed 3 years ago (Setti et al., 2010). It
included three studies and, by pooling all the IMSI results,
did not take account of the specific indication. In fact, the
studies in this field differ significantly in terms of: (i) their
design (e.g. randomized versus nonrandomized studies, or
the comparison of IMSI results with previous ICSI results for
the same couples versus other couples matched according
to various criteria); (ii) the ICSI magnification used; (iii)
the sprm morphology designated as ‘normal’ at an IMSIlike magnification; (iv) the sperm classification; and (v) the
criteria used to assess the outcome (e.g. clinical pregnancy
and delivery rates per couple, per transfer or per cycle).
Hence, the objective of the present literature review
was to assess the outcomes for IMSI vs. ICSI and determine
the clinical situations in which the use of this assisted
reproduction technology is likely to be of greatest value.
Materials and methods
This work performed a systematic review of the relevant
literature, according to the PRISMA guidelines (Moher et
al., 2009). The PubMed database was searched for work
published between 2001 and March 2013 with the following
search terms: ‘IMSI’, ‘MSOME’ and ‘high-magnification,
sperm’. The publications’ titles, abstracts and reference
lists were viewed and only relevant publications (i.e.
those reporting on IMSI outcomes in terms of blastocyst,
pregnancy, delivery and/or live birth rates) in English were
selected and included. This review examined, compared
and discussed study methodologies and results, including
patient characteristics, the magnifications used for IMSI
and ICSI (when stated) and the pregnancy and/or delivery
rates associated with IMSI and ICSI. The results were
subdivided into currently accepted indications of IMSI (i.e.
clinically relevant indications confirmed by several studies,
including at least two randomized clinical trials with a large
sample size) and potential indications (i.e. those requiring
additional research).
Results and discussion
Literature retrieved
The PubMed search identified a total of 168 publications
(58 using the term ‘IMSI’, 28 using the term ‘MSOME’
and 82 using the terms ‘high-magnification, sperm’)
indexed between 2001 and March 2013. After viewing the
publications’ titles, abstracts and reference lists, 24 studies
which directly compared IMSI and ICSI were retrieved.
Following the exclusion of two publications not written in
English, a total of 22 studies were included in this review.
Indication for IMSI
In most studies, ICSI was indicated because of the
presence of at least one male factor for infertility (oligoand/or asthenoand/or teratozoospermia) (see, for example,
Table 1). The indication of ICSI was not specified in three
studies and varied in one other study. The only confirmed
indication of IMSI is recurrent implantation failure
following ICSI.
Outcomes after IMSI
The outcomes of IMSI following ICSI failure are
summarized in Table 1.
In two studies, IMSI was directly compared with
ICSI in couples matched for the number of previous ICSI
failures (Bartoov et al., 2003; Oliveira et al., 2011). Bartoov
et al. studied a total of 100 couples with an mean (range)
of 4.1 (2–8) previous ICSI failures. When compared with
ICSI (performed at a magnification of x200 or x400, n =
50 couples), IMSI (with selection of normal spermatozoa
with no more than one small vacuole occupying <4% of
the sperm head area, n = 50 couples) yielded a significantly
higher clinical pregnancy rate per couple (30% versus 66%,
respectively, P < 0.01), a lower miscarriage rate (33% versus
9%, P < 0.01) and a higher delivery rate per couple (20%
versus 60%, P < 0.01). Furthermore, IMSI yielded even higher
clinical pregnancy and delivery rates per couple (50%, in
both cases) in a group of 12 additional, unmatched couples
with more than eight ICSI failures (although the technique
was not compared directly with ICSI; Bartoov et al., 2003).
Oliveira et al. studied 200 couples having undergone IMSI
(with selection of normal spermatozoa with no more than
one small vacuole occupying <4% of the sperm head area)
and found that clinical pregnancy and live birth rates per
cycle tended to be higher and miscarriage rates tended to
be lower than those of the 100 couples having undergone
ICSI (performed at a magnification of x400), although
these differences were not statistically significant (Oliveira
et al., 2011). The comparison of these two studies suggest
that sperm abnormalities that are not visible at x200 might
indeed be detected at magnification x400. Hence, IMSI’s
superiority over ICSI at ·400 might be less obvious than
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IMSI’s superiority over ICSI at x200.
Hazout et al. (2006) compared outcomes for IMSI and
ICSI (x200) in a total of 125 couples acting as their own
controls. After at least two previous failed ICSI attempts,
each couple underwent two further attempts: an additional
round of ICSI (x200) and then IMSI (with selection of
vacuole-free spermatozoa). When compared with the ICSI
cycle, IMSI was associated with a significantly higher clinical
pregnancy rate per transfer (2% versus 38%, respectively,
P < 0.001) and a significantly lower miscarriage rate (data
not reported). Furthermore, IMSI led to a significantly
higher delivery rate per transfer (34% for IMSI and 0% for
ICSI, P < 0.001) and a significantly higher live birth rate per
embryo transferred (18% versus 0%, respectively, P < 0.001)
than ICSI did (Hazout et al., 2006).
Very recently, a prospective but non-randomized study
compared IMSI and ICSI outcomes in patients with more
than two ICSI failures (El Khattabi et al., 2013). This was the
only study to report that IMSI (with selection of the ‘best
available’ spermatozoon, according to Vanderzwalmen et
al., 2008) yielded much the same results (in terms of clinical
pregnancy and live birth rates per cycle) as ICSI performed
at x200 (24% versus 26% and 21% versus 22%, respectively).
However, the fact that patients were not matched for the
number of previous ICSI failures may have been a source
of bias. The mean number of attempts for the IMSI patients
(4.8) was indeed significantly greater than that for the ICSI
patients (4.1; P = 0.0001).
Randomized studies (and particularly those with large
sample sizes) provide the most robust evidence when
comparing IMSI and ICSI outcomes. Knez et al. (2011)
performed a randomized trial in patients with no blastocyst
ARTICLES
formation in previous ICSI failures. The researchers
compared IMSI (with selection of the best spermatozoon,
according to Cassuto et al. (2009), n = 37) and ICSI (performed
at magnification of both x200 and x400, n = 20). There was
a nonsignificant trend towards a higher number of cycles
with at least one blastocyst in the IMSI group than in the
ICSI group (50% versus 35%, respectively). Likewise, a nonsignificant trend towards a higher clinical pregnancy rate
per cycle was achieved in the IMSI group, when compared
with the ICSI group (25% versus 8%, respectively). Given
that several factors (e.g. low oocyte quality) could be
responsible for absence of blastocyst formation and that the
study’s sample size was small, it remains to be determined
whether IMSI is indeed better than ICSI in this precise
indication of no blastocyst formation.
Another randomized study (Antinori et al., 2008) of
a larger number of couples (n = 446) compared 227 IMSI
attempts (with selection of normal spermatozoa with no
more than one small vacuole with a borderline diameter of
0.78 ± 0.18 lm) with 219 ICSI attempts (at an unspecified
magnification). Overall, IMSI yielded a significantly higher
clinical pregnancy rate per couple than ICSI (39% versus
27%, respectively; P = 0.004). For the 139 couples with
at least two ICSI failures (Table 1), IMSI in 77 couples
was associated with a 2-fold higher clinical pregnancy
rate per couple than ICSI in 62 couples (30% versus 13%,
respectively) and a 2-fold lower miscarriage rate (17%
versus 38%). For couples with no previous ICSI failures (n =
123) or only one previous ICSI failure (n = 184), the clinical
pregnancy rates per couple for IMSI and ICSI did not differ
significantly (Antinori et al., 2008).
Very recently, it was suggested that IMSI may be of value
after just one previous ICSI failure (Klement et al., 2013). In
fact, this group led by Berkovitz performed a randomized
study of a very large number (449) of couples with one
previous ICSI failure. The clinical pregnancy and delivery
rates per cycle were significantly higher for the 127 couples
randomized to IMSI (with selection of normal spermatozoa
with no more than one small vacuole occupying <4% of the
sperm head area) than for the 322 couples randomized to
further ICSI (at a magnification of x200 or x400): 56% versus
38% (P = 0.002) and 28% versus 18% (P = 0.04), respectively.
A multivariate analysis prompted these researchers to state
that the ICSI-to-IMSI switch after the initial failure was
associated with a 3-fold greater chance of clinical pregnancy
and delivery. Furthermore, the study results also showed
that IMSI was no better than ICSI when used in the first
round of treatment.
Other researchers have shown that IMSI is no more
efficient than ICSI in unselected patients (i.e. regardless of
the number of treatment attempts) (Balaban et al., 2011; De
Vos et al., 2013). De Vos et al. (2013) recently analysed the
outcomes of 350 attempts (including 125 IMSI cycles with
the transfer of IMSI-only embryos and 139 ICSI cycles with
the transfer of ICSI-only embryos) in a non-randomized
study of 340 couples. The researchers reported that
IMSI (with selection of vacuole-free spermatozoa, when
available) yielded much the same results (in terms of clinical
pregnancy rates per embryo transferred) as ICSI performed
at a magnification of x400 (34% versus 37%, respectively).
However, most of the patients included in this study were
undergoing their first ICSI/IMSI attempt (188/350, 54%) or
second attempt (72/350, 21%). Hence, this study provided
additional evidence for the lack of superiority of IMSI in
patients with no previous ICSI failures. Similarly, Balaban
et al. (2011) compared the outcomes of IMSI (n = 87) and ICSI
(n = 81) in a randomized study of 168 unselected couples
(i.e. regardless of any previous ICSI or IVF failures). Clinical
pregnancy rates per cycle (54% versus 44%) and live birth
rates per cycle (44% versus 38%) did not significantly differ
when comparing the IMSI group (with selection of normal
spermatozoa with no more than one small vacuole with a
borderline diameter of 0.78 ± 0.18 lm) and the ICSI group
(for which the magnification was not stated in the report).
In summary, the literature review results suggest
that IMSI is only of value (in terms of higher clinical
pregnancy and live birth rates) for patients with one or
more previous ICSI failure and not for unselected patients
or those undergoing their first treatment attempt. Given
that (i) vacuoles were shown to be linked to chromatin
condensation failure (Boitrelle et al., 2011; Boitrelle et al.,
2013; Franco et al., 2012; Garolla et al., 2008; Perdrix et al.,
2011), (ii) chromatin condensation failure is associated
with recurrent abortions (Kazerooni et al., 2009; Talebi et
al., 2012) and (iii) a growing body of evidence suggests
that the degree of sperm chromatin condensation at the
time of fertilization can influence early and late embryo
development (Hammoud et al., 2011), the current work
postulates that the higher pregnancy and delivery rates
and lower miscarriage rates observed for IMSI after ICSI
failure can be explained (at least in part) by the exclusion
of spermatozoa containing sperm head vacuoles of nuclear
origin.
Potential indications of IMSI
Teratozoospermia
Teratozoospermia may be an indication for IMSI. In
all but one of the studies reviewed below, ICSI and IMSI
were indicated because of the presence of at least one
male factor for infertility (oligo- and/or astheno- and/or
teratozoospermia). The article by Berkovitz et al. (2006) did
not specify an indication.
It has already been shown that individual spermatozoa
differ in their ability to produce an embryo capable of
implanting. Indeed, the use of morphometrically normal
spermatozoa with no vacuoles or less than two small
vacuoles has been associated with significantly higher
blastocyst rates than all other types of spermatozoa (i.e.
those with more than two small vacuoles, those with one
large vacuole and those with morphometric abnormalities)
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(Knez et al., 2012; Vanderzwalmen et al., 2008). In
contrast, one study has reported lower blastocyst rates
when vacuole-free spermatozoa were used for injection
(relative to spermatozoa with vacuoles) (Tanaka et al.,
2012); however, the sample size was small and the study
population was not homogeneous because it included both
patients with azoospermia and patients with normal sperm
characteristics. Furthermore, concerning the ability of
individual spermatozoa to lead to a pregnancy, the shape of
the sperm head and the presence of vacuoles were reported
as being significantly and positively correlated with
the chance of achieving a pregnancy with IMSI (r = 0.38;
P < 0.01; Bartoov et al., 2002). Berkovitz et al. (2006) reported
on IMSI in 80 patients: when compared with spermatozoa
with vacuoles or an abnormal morphology (so-called
‘second-choice’ spermatozoa), normal, vacuole-free
spermatozoa yielded significantly higher clinical pregnancy
and delivery rates per cycle (58% versus 26% and 53%
versus 17%, respectively, both P < 0.01) and significantly
lower miscarriage rates (10% versus 33%, respectively;
P = 0.02) (Berkovitz et al., 2006). Hence, the morphology of
individually selected spermatozoa seems to have an impact
on pregnancy and delivery rates.
This is why some researchers have tried to determine
a threshold for the proportion of normal spermatozoa
below which ICSI might be inefficient or, conversely, IMSI
might be of value. Two studies have evaluated ICSI results
as a function of the proportion of normal spermatozoa in
the ejaculate, as assessed by MSOME (Bartoov et al., 2002;
Falagario et al., 2012). It was shown that normalcy of the
sperm nucleus (i.e. a normal shape and with less than one
small vacuole occupying <4% of the sperm head area) was
predictive of the clinical pregnancy rate after ICSI (Bartoov
et al., 2002). Even though <20% of normal spermatozoa
were found in the ejaculate with MSOME, no pregnancies
were obtained after ICSI performed at a magnification of
x200–x400 (Bartoov et al., 2002). Another study contributed
additional data on this matter by reporting that the lower
the proportion of normal spermatozoa in the ejaculate (as
assessed by MSOME, with a threshold of 20%), the higher
the risk of choosing a vacuolated spermatozoon with
conventional ICSI and the lower the clinical pregnancy
rate with conventional ICSI (Falagario et al., 2012). Hence,
the proportion of normal spermatozoa (as assessed by
MSOME) and the quality of the ICSI outcomes seem to
decrease in parallel. Similarly, a group of researchers
compared IMSI outcomes as function of the proportion
of normal spermatozoa (as assessed by MSOME) in
the ejaculate (Berkovitz et al., 2005). They reported that
IMSI in which normal, vacuole-free spermatozoa were
available for injection (n = 126 IMSI cycles) yielded a
significantly higher clinical pregnancy rate per transfer and
a significantly lower miscarriage rate relative to IMSI (n =
38 cycles) in which no normal spermatozoa were available
(respectively 53% versus 18%, P < 0.01; 10% versus 57%; P
= 0.02) (Berkovitz et al., 2005). This finding suggested that
even in the absence of normal spermatozoa (according to
MSOME), pregnancy rates were low but not null with IMSI.
Hence, IMSI might be preferable to ICSI when few normal
spermatozoa (as assessed by MSOME) are present in the
ejaculate. Very recently, a prospective but non-randomized
study (El Khattabi et al., 2013) compared IMSI and ICSI
results in patients with teratozoospermia (defined as <10%
of normal spermatozoa in a spermocytogram, according
to David’s criteria (Auger et al., 2001)). In this study,
IMSI (with selection of the ‘best available’ spermatozoon,
according to Vanderzwalmen’s criteria (Vanderzwalmen et
al., 2008)) yielded higher clinical pregnancy and live birth
rates per cycle than ICSI performed at a magnification
of ·200 (46% versus 26%, P = 0.001 and 38% versus 20%,
P = 0.002, respectively). However, randomized ICSI versus
IMSI studies constitute the only way of robustly testing the
value of IMSI in cases of teratozoospermia.
The only study of this type to date was performed
recently in patients with isolated teratozoospermia (defined
as <14% of normal spermatozoa in a spermocytogram,
according to Kruger’s strict criteria; Knez et al., 2012). In
this randomized study, 52 couples underwent IMSI (with
selection of the ‘best available’ spermatozoon, according
to Vanderzwalmen’s criteria) and 70 underwent ICSI
(performed at a magnification of x200–x400). The clinical
pregnancy rates per couple were significantly higher for
IMSI than for ICSI (48% and 24%, respectively; P < 0.05).
However, Knez et al. did not state threshold values for the
number or proportion of normal spermatozoa (as assessed
by MSOME) below which IMSI (rather than ICSI) could
be indicated because the couples were not matched by the
degree of teratozoospermia (Knez et al., 2012).
In contrast, one can legitimately question whether
IMSI is indicated in some types of teratozoospermia. It
appears that IMSI was ineffective (or no more efficient
than ICSI, at least) in patients with a high proportion of
spermatozoa with enlarged heads (Chelli et al., 2010), since
normal spermatozoa selected at both ICSI-like and IMSIlike magnifications were potentially aneuploid. In cases of
globozoospermia, however, IMSI might enable the selection
of spermatozoa with a small acrosomal bud. Indeed, IMSI
enabled a successful pregnancy for a couple in which the
male displayed almost total globozoospermia (99% of the
spermatozoa were round-headed), in the absence of assisted
oocyte activation (Sermondade et al., 2011).
In summary, IMSI might be indicated in some cases
of teratozoospermia. However, given that only one
randomized study observed higher clinical pregnancy rates
for IMSI than for ICSI in patients with teratozoospermia and
the threshold for the number of morphometrically normal
spermatozoa (as assessed by MSOME) below which IMSI
might produce higher clinical pregnancy and delivery rates
than ICSI remains to be determined, further studies of the
potential value of IMSI in patients with teratozoospermia
are required.
ARTICLES
IMSI and spermatozoa with nuclear abnormalities
IMSI for older women
The use of IMSI might help to avoid the selection of
spermatozoa with nuclear abnormalities such as chromatin
condensation failure, DNA fragmentation and an abnormal
chromosomal content.
First, given that the nuclear origin of sperm head
vacuoles has been linked to chromatin condensation failure,
a high proportion of non-condensed chromatin could be
considered as an indication for IMSI. No data are available
but this question deserves to be evaluated in large-scale,
randomized trials.
A second potential indication of interest is sperm DNA
fragmentation. Indeed, it has been shown that spermatozoa
judged to be normal at ICSI-like magnifications can
present DNA fragmentation and that normal vacuole-free
spermatozoa selected at an IMSI-like magnification are less
DNA fragmented than normal spermatozoa selected at an
ICSI-like magnification (Hammoud et al., 2013). However,
only one study has compared IMSI and ICSI outcomes in
patients with high levels of sperm DNA fragmentation
(Hazout et al., 2006), in which 72 couples with two or more
previous ICSI failures were subdivided according to the
proportion of spermatozoa with DNA fragmentation in the
male’s whole semen: normal (i.e. <30% of spermatozoa with
DNA fragmentation, n = 51), moderately elevated (30–40%,
n = 11) and greatly elevated (>40%, n = 10). Overall, IMSI
yielded significantly higher clinical pregnancy, delivery
and birth rates per transfer than ICSI (performed at a
magnification of x200) did, with values of 38% versus 2%,
34% versus 0% and 18% versus 0%, respectively (P < 0.001
for all comparisons). For patients with normal proportions
of DNA-fragmented spermatozoa, the birth rate was 19%
for IMSI and 0% for ICSI (P < 0.001). The superiority of
IMSI over ICSI (in terms of birth rates) was particularly
obvious in the group with the highest percentage of DNAfragmented spermatozoa (29% versus 0%, respectively;
P < 0.01), although the small sample size reduced the
statistical significance (Hazout et al., 2006). Given that the
sample size was small and the study was not randomized,
it remains to be seen whether IMSI is indicated in cases
of sperm DNA fragmentation. This potential indication
deserves to be evaluated in large-scale randomized trials.
Thirdly, sperm aneuploidy may be considered.
Although two studies have reported that spermatozoa
with large vacuoles are more likely to be aneuploid than
normal, vacuole-free spermatozoa (Garolla et al., 2008) or
spermatozoa from whole semen (Perdrix et al., 2011), IMSI
was found to be no more efficient than ICSI for selecting
euploid spermatozoa in patients with a high proportion
of aneuploid spermatozoa (e.g. patients with a high
proportion of spermatozoa with enlarged heads (Chelli et
al., 2010) or translocations (Cassuto et al., 2011; Chelli et al.,
2013)). Hence, it has not been proved that IMSI can be used
to efficiently select euploid spermatozoa. Indeed, one can
even consider that the opposite is true (i.e. a demonstrated
lack of efficiency).
Interestingly, only one research group has evaluated
IMSI in older women. In a randomized study of patients
with a mean age of 37, preimplantation genetic diagnosis
showed that ICSI (n = 60) was associated with significantly
higher sex chromosome aneuploidy in the embryo and a
significantly greater proportion of chaotic embryos (i.e.
with two or more chromosomal number abnormalities)
relative to IMSI (n = 60). The researchers postulated that, in
older women, oocytes were less able to repair the injected
spermatozoon’s DNA and hence that use of IMSI to select
spermatozoa with fewer nuclear abnormalities could be of
value for aged oocytes and older women (Figueira et al.,
2011). This indication also deserves to be evaluated in largescale, randomized trials.
IMSI for everyone
Some authors go as far as to suggest that IMSI can be
used for all couples in assisted reproduction programmes.
They argue that, relative to ICSI, IMSI increases the
likelihood of obtaining a healthy, normal child (Berkovitz
et al., 2007). In the latter study, children (n = 176) born after
ICSI had a significantly greater risk of major congenital
malformations than those born after IMSI (n = 181; 8%
versus 3%, respectively; P = 0.02). However, the value of 8%
is the highest post-ICSI malformation rate ever reported and
casts doubt on the reliability of the study data (for a metaanalysis, see Wen et al., 2012). Hence, one cannot conclude
as to the potential value of IMSI for reducing congenital
malformations; only randomized studies in a large number
of patients are capable of providing robust information.
Conclusion
A decade after the introduction of IMSI, this technique
continues to divide assisted reproduction professionals.
There are few confirmed indications of IMSI, partly
because few randomized, head-to-head studies have been
performed. According to this systematic literature review,
the only currently and consistently acknowledged indication
of IMSI is recurrent implantation failure following ICSI. All
other potential indications of IMSI must be further assessed.
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ARTICLES
The spatial arrangement of blastomeres at the
4-cell stage and IVF outcome
Goedele Paternot*, Sophie Debrock, Diane De Neubourg, Thomas M D’Hooghe, Carl
Spiessens
Leuven University Fertility Center, UZ Leuven, Gasthuisberg, Campus Gasthuisberg,
Herestraat 49, 3000 Leuven, Belgium
*Corresponding author. E-mail addresses: [email protected] (G Paternot),
[email protected] (S Debrock), [email protected] (D De
Neubourg), [email protected] (TM D’Hooghe), [email protected]
(C Spiessens).
Goedele Paternot, PhD
Goedele Paternot obtained her Masters in biomedical sciences in 2007 at the Catholic University of Leuven, Belgium. In
2011, she finished her PhD in medical sciences entitled ‘‘Selecting the embryo with the highest implantation potential
regarding the morphology and developmental potential in a clinical setting’’ at the Leuven University Fertility Centre (ISO
9001:2008 certified). The main research topics of the centre are embryo scoring and the optimization of in-vitro culture
conditions, quality of patient care and endometriosis.
Abstract This study compared the developmental and implantation potential of tetrahedrally arranged versus nontetrahedrally arranged 4-cell-stage embryos. If the cleavage planes of a 4-cell-stage embryo were perpendicularly
orientated, blastomeres were defined as tetrahedrally arranged, while embryos with parallel-orientated cleavage axes
were considered as non-tetrahedral embryos. The 4-cell-stage embryos (n = 862) examined in this study were obtained
from 299 patients aged <36 years. A total of 299 embryos were transferred as a single-embryo transfer on day 3. This
study showed that tetrahedral embryos developed into a 8-cell-stage embryo on day 3 more frequently (307, 45%
versus 42, 24%; P < 0.0001) and also developed more frequently into good-quality embryos (461, 67% versus 67,
38%; P < 0.0001) and into excellent-quality embryos (290, 42% versus 34, 19%; P < 0.0001). Tetrahedral embryos had
a significantly higher implantation potential (98, 38% versus 9, 21%; P = 0.038), ongoing pregnancy rate (84, 33%
versus 7, 16%; P = 0.032) and live birth rate (84, 33% versus 7, 16%; P = 0.032). In conclusion, tetrahedral 4-cell-stage
embryos on day 2 developed into embryos of better quality on day 3 with a higher implantation potential and live birth
rate compared with non-tetrahedral 4-cell-stage embryos.
KEYWORDS: 4-cell-stage embryos, embryo quality, embryo selection, IVF, live birth rate, spatial arrangement
This article was published in Reproductive BioMedicine Online, Vol 28, 2014, p198-203, The spatial arrangement of
blastomeres at the 4-cell stage and IVF outcome. Copyright Elsevier. It is reprinted here with permission.
Introduction
The mechanisms behind the development of
mammalian embryos from a single cell, the fertilized
oocyte, into a complex structure of a blastocyst consisting of
different cell lineages has been debated for the last few years
(Zernicka-Goetz, 2006). The mouse model has been used to
understand the mechanisms underlying the earliest stages
of development by different research groups. As mentioned
by Cooke et al. (2003), clear definitions are needed for terms
like polarity and poles when assessing development poles
and planes. While polarity defines an ongoing axis of
symmetry throughout pre- and post-implantation embryo
development, the term ‘‘pole’’ (animal or vegetal) defines
two distinguishing parts of the oocyte from which the A–V
plane is formed by a meridional cleavage (Cooke et al.,
2003).
It has been stated that cell fate decisions in the embryo
are crucial for the further development (Edwards and
Beard, 1997; Zernicka-Goetz et al., 2009). The first cell fate
decisions in the embryo can be explained by different points
of views. A recent review focused on the different opinions
(Johnson, 2009) and concluded that a combination of the
early asymmetric hypothesis (Johnson and Ziomek, 1981),
the inside–outside hypothesis (Tarkowski and Wroblewska,
2006) and the polarization hypothesis (Plusa et al., 2005) can
explain the differentiation processes and the formation of
the axis of symmetry in early development. In addition,
studies on transcription factors confirmed the complexity of
the process indicating the presence of different mechanisms
(Plachta et al., 2011; Vermilyea et al., 2011; Zernicka-Goetz,
2011).
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Studies on basic embryology of embryos revealed that
the oocyte consists of an animal and vegetal pole (Antczak
and Van Blerkom, 1997, 1999; Edwards and Beard, 1997;
Edwards, 2002). Although not morphologically visible, this
polarization was found based on the differential expression
of molecular factors (Antczak and Van Blerkom, 1997, 1999;
Edwards and Beard, 1997; Van Blerkom, 2007). Maternal
proteins like leptin and STAT3 are distributed differentially
and are mainly expressed in the cortex of the animal pole
(Antczak and Van Blerkom, 1997, 1999). Differences in
distribution in human embryos was confirmed by the
detection of high concentrations of animal-distributed
leptin/STAT3 in one blastomere of a 4-cell embryo, in two
blastomeres of the 8-cell embryo and finally to trophectoderm
only (Antczak and Van Blerkom, 1999). Controlled cleavage
planes leading to correct distribution of proteins might
have an influence on important developmental aspects of
the embryo (Hansis and Edwards, 2002). Indeed, the pattern
of inheritance of polarized domains in daughter cells
seems to be determined by how successive equatorial or
meridional planes of cell division are oriented with respect
to the domains (Antczak and Van Blerkom, 1999). The first
meridionally orientated cleavage results in two daughter
cells with the same polarity, meaning that both cells contain
animal and vegetal cytoplasm. One cell divides meridionally
orientated while the other cell divides equatorially (or after
the second cleavage plane rotated through 90 degrees)
which result in four cells with different polarity (Edwards
and Hansis, 2005; Gulyas, 1975). The two daughter cells
resulting from the meridionally orientated cleavage have
again the full polarity (animal and vegetal cytoplasm). The
daughter cells resulting from equatorially cleavage differ in
polarity since one cell will contain mostly animal cytoplasm
while the other contains mostly vegetal cytoplasm. These
specific cleavage planes result in a tetrahedral arrangement
of the blastomeres (Edwards, 2002). On the other hand,
other researchers have claimed that the pattern of the
second cleavage is random (Louvet-Valleé et al., 2005)
and, as a consequence, other topologies were found in
4-cell-stage embryos. However, as mentioned by Gardner
(2006) the validity of the latter study is questionable due
to the method used. The only clear experimental evidence
for the second cleavage was published by Gardner, who
described tetrahedral and non-regular tetrahedral 4-cellstage embryos in mice (Gardner, 2002). The last group was
subdivided into two groups based on the positioning of the
polar body (either in contact with all the blastomeres or lay
between only two) (Gardner, 2002) which might result in
blastulation failure (Gardner, 2002; Ebner et al., 2012).
The current manuscript focuses on the arrangement
of blastomeres at the four cell stage (tetrahedral versus
non-tetrahedral arrangement). In turn, this characteristic
can be useful in embryo selection in a non-invasive way.
Tetrahedral 4-cell-stage embryos have been described
with a meridional axis and an equatorial axis during the
second cleavage (Cauffman et al., 2010). Where the two
cleavage planes are oriented otherwise, a non-tetrahedral
arrangement of blastomeres may occur (Cauffman et
al., 2010; Ebner et al., 2012). This can result in a different
distribution of cytoplasm in the daughter cells (for example
different distribution of membrane associated or cortical
factors (Edwards, 2005) or mitochondria (Van Blerkom,
2007). The aim of this study was to compare in a large set
of embryos the developmental and implantation potential
of tetrahedral versus non-tetrahedral 4-cell-stage embryos.
Materials and methods
Patients
The 4-cell-stage embryos examined in this study
resulted from 299 patients aged <36 years, who received a
single-embryo transfer on day 3 at the Leuven University
Fertility Centre. Patients entered their first (n = 254) or
second (n = 45) IVF/intracytoplasmic sperm injection
(ICSI) cycle and were only included once. The stimulation
protocol used in this study has been published (Debrock et
al., 2010). Patients were excluded when the cycle included
biopsy for preimplantation genetic diagnosis or if donor
spermatozoa/donor oocytes were used. In total, the dataset
contained 862 4-cell-stage embryos of which 299 embryos
were transferred on day 3. The study was approved by
Institutional Review Board of the University Hospitals
Leuven (ML4564, 16 November 2007).
Assisted reproduction treatment
After oocyte retrieval, the oocytes were washed
through four wells and placed in a 4-well dish containing
500 μl fertilization medium (Cook medium; Sydney IVF,
Queensland, Australia; or GM501 medium; Gynemed
Lensahn Germany) at 37°C, pH 7.25–7.35 per well, under
mineral oil. Spermatozoa used for the IVF procedure were
prepared using standard density gradient procedures
(Isolate; Irvine Scientific, USA). Sperm samples used for
ICSI were diluted and were centrifuged twice for 10 min at
300 g. Standard IVF/ICSI procedures were performed 2–6
h after oocyte retrieval. During the IVF procedure, oocytes
were inseminated with 300,000 progressively motile
spermatozoa per well (5 oocytes in 0.5 ml). In the case of
ICSI cycles, injected oocytes were incubated together in a
20 ll culture medium droplet under oil. On day 1 (16–20
h after insemination/injection), fertilization was evaluated.
Only normal fertilized oocytes (2 pronuclei) were cultured
individually in a 20 ll droplet of culture medium covered
with mineral oil.
Embryo evaluation
Image sequences were recorded of each embryo on
day 1 (16–20 h after insemination/injection), day 2 (41–44
h after insemination/injection) and day 3 (66–71 h after
ARTICLES
insemination/injection) using a computer-assisted scoring
system (CellCura FertiMORPH; CellCura Software
Solutions, Copenhagen, Denmark). The semi-automatic
embryo quality assessment using this system has been
recently described by Paternot et al. (2011). Based on the 26
sequential images of the same embryo, the system calculates
the total cytoplasmic reduction (which can be interpreted as
the degree of fragmentation) and the size of blastomeres.
The criteria for distinguishing between a blastomere and
a fragment were based on the findings by Hnida et al.
(2005) and Johansson et al. (2003). For this study, embryos
evaluated by this system with 4 cells on day 2 (44% of
the day 2 embryos in the study period) were included
and additionally evaluated based on the blastomere
arrangement within the zona pellucida. If the cleavage
planes were perpendicularly orientated, blastomeres were
defined as tetrahedrally arranged (tetrahedral embryos),
while embryos with rather parallelly orientated cleavage
axes were considered as non-tetrahedral embryos. In cases
where the cleavage axes were not exactly perpendicular,
the embryos were considered as non-tetrahedral embryos
(Figure 1).
In addition, the embryos were evaluated using the
standard scoring system of the Leuven University Fertility
Centre. This embryo evaluation is based on the assessment
by an embryologist who evaluated the number and size
of blastomeres and the degree of fragmentation. On
day 3, embryos with 7, 8 or 9 equally or approximately
equally sized blastomeres (<50% difference) and a degree
of fragmentation <25% were defined as good-quality
embryos. Embryos with 8 equally or slightly unequally
sized blastomeres and <10% fragmentation were defined as
excellent-quality embryos. On day 3 of development, the
best embryo was chosen for transfer based on the standard
scoring system. Embryos with at least 6 equally or slightly
unequally sized blastomeres and <25% of fragmentation on
day 3 were cryopreserved.
Utilization rate was defined as the number of embryos
available for cryopreservation and embryo transfer over
the total number of embryos. A positive b-human chorionic
gonadotrophin result was defined as <25 IU/l 14 days after
embryo transfer. Implantation rate was defined as the
number of gestational sacs observed divided by the number
of embryos transferred. Live birth rate was defined as the
number of deliveries divided by the number of embryos
transferred (Zegers-Hochschild et al., 2009).
Statistical analysis
The developmental and implantation potential of
tetrahedral versus non-tetrahedral embryos was compared
using a Fisher Exact chi-squared test with a significance
level of 0.05.
Results
The characteristics of the patients and cycles are listed
in Table 1.
ARTICLES
Developmental
capacity
nontetrahedral embryos
of
tetrahedral
versus
A total of 862 4-cell-stage embryos were evaluated
based on the blastomere arrangement including 684 (79%)
tetrahedral embryos 178 (21%) non-tetrahedral embryos
(Table 2).
The percentage of tetrahedral and non-tetrahedral
embryos was similar with respect to the type of insemination
and the type of culture medium. A total of 459 (80%) and
225 (78%) tetrahedral and 114 (20%) and 64 (22%) nontetrahedral embryos were found in the IVF and ICSI groups,
respectively. In the Cook medium, a total number of 436
(79%) embryos were tetrahedral and 113 embryos (21%)
were non-tetrahedral embryos which was comparable to
the GM501 medium, in which 248 (79%) embryos were
tetrahedral and 65 (21%) were non-tetrahedral embryos.
When compared with non-tetrahedral embryos,
tetrahedral embryos developed more frequently into the
8-cell-stage embryo on day 3 (307, 45% versus 42, 24%, P
< 0.0001) and also developed more frequently into goodquality embryos (461, 67% versus 67, 38%; P < 0.0001) and
excellent-quality embryos (290, 42% versus 34, 19%; P <
0.0001; Table 2).
On day 3, 299 embryos were transferred (256 tetrahedral
versus 43 non tetrahedral embryos), 370 embryos were
cryopreserved (309 tetrahedral and 61 non-tetrahedral
embryos) and 193 embryos were discarded (119 tetrahedral
and 74 non-tetrahedral embryos). The utilization rate was
significantly higher for the tetrahedral embryos (565, 83%)
compared with the non-tetrahedral embryos (104, 58%; P <
0.0001; Table 2).
Implantation potential of tetrahedral versus nontetrahedral
embryos
Since no significant differences were found in the
live birth rate of embryos of patients entering their first
(77/254, 30%) or second (14/45, 31%) IVF/ICSI cycle, the
transferred embryos were analysed as one group (Table 3).
A total number of 256 (86%) patients had a single-embryo
transfer with tetrahedral blastomere arrangement on day
2 while 43 (14%) patients received an embryo that had a
non-tetrahedral blastomere arrangement on day 2. When
compared with non-tetrahedral embryos, tetrahedral
embryos had a significantly higher implantation rate (98,
38% versus 9, 21%; P = 0.038), pregnancy rate (84, 33% versus
7 16%; P = 0.032) and live birth rate (84, 33% versus 7 16%;
P = 0.032; Table 3). The number of biochemical pregnancies
(18, 5% versus 2, 7%) and miscarriages (13, 5% versus 2, 5%)
were comparable between both groups (Table 3).
Discussion
The aim of this study was to compare in a large set of
embryos the developmental and implantation potential of
tetrahedral versus non-tetrahedral 4-cell-stage embryos. An
advantage of this study is the use of single-embryo transfers
on day 3, which makes it possible to link the orientation
of the cleavage planes to the implantation potential of the
transferred embryo. In addition, this study contained a
large set of 4-cell-stage embryos, which make the results of
the developmental potential analysis reliable.
The study of Ebner et al. (2012) found fewer embryos
with a suboptimal blastomere configuration in IVF cycles
compared with ICSI cycles or ICSI cycles including testicular
spermatozoa and assumed that the sperm centrosome
composition or function might be affected and as a
consequence lead to an abnormal blastomere configuration.
This could not be confirmed in the current study since no
differences were found between IVF and ICSI cycles nor
between cycles with or without male infertility. A full
comparison between both studies is, however, difficult
since Ebner et al. (2012) focused on a specific type of nontetrahedral embryos (planar embryos) while this study did
not define subgroups.
A significantly higher embryo quality (good and
excellent quality) was found for the tetrahedral embryos. In
contrast, Ebner et al. (2012) reported only a positive impact
on blastocyst development and not on embryo quality on
days 2 and 3. Cauffman et al. (2010) also found a higher
proportion of good- and top-quality blastocysts when
embryos had a tetrahedral shape on day 2 compared with
the non-tetrahedral group of embryos.
In line with the results reported by Ebner et al. (2012),
this study found a higher implantation and live birth rate
when a tetrahedral embryo was transferred. This is in
contrast with the results described by Cauffman et al. (2010),
with the same study design as this study, who found no
differences in implantation potential in the group of single-
ARTICLES
embryo transfers on day 3 (n = 60). The same was found
for the single-blastocyst transfers on day 5 (n = 53). This
difference could be due to a low number of nontetrahedral
transferred embryos (19 on day 3 and 9 on day 5) in the
study of Cauffman et al. (2010). This latter finding could be
due to the fact that these embryos had a lower quality on
day 3 and were therefore not chosen for transfer.
The question remains, however, as to why the
nontetrahedral group had a lower developmental potential.
One hypothesis, as mentioned by Louvet-Valleé et al.
(2005), is that the zona pellucida exerts a constraining effect
on the blastomeres. This would indicate that blastomeres
of non-tetrahedral embryos are more compressed within
the zona pellucida than in tetrahedral embryos, which
can in turn affect cell fate or vitality. Based on the finding
that the distribution of maternal proteins and regulatory
domains are important for embryo development (Antczak
and Van Blerkom, 1999; Hansis and Edwards, 2002),
another hypothesis is that the distribution of maternal
proteins is disturbed in this type of embryos. A nontetrahedral embryo can result from two cleavage patterns.
When the 2-cell-stage embryo cleaves with both cleavage
axes meridionally orientated, blastomeres retain the
polarity of the mother cells and the embryo may recover
from the disturbed distribution. However, when a 2-cellstage embryo cleaves with equatorially orientated axes,
this can be more detrimental to embryo development
since fundamental studies on cell differentiation and
cell replacement have found that cells with predominant
animal or vegetal cytoplasm have a lower developmental
competence compared with fully potent cells (Edwards and
Hansis, 2005). This is, however, hypothetical and has to be
confirmed in molecular studies. In addition, it is based on
the findings of Antczak and Van Blerkom (1999), which are
so far not confirmed by others. Ebner et al. (2012) postulated
that the mitotic spindle might have been affected which in
turn might have led to the observed cleavage anomaly.
Another hypothesis is that the contact between blastomeres
in non-tetrahedral embryo is lower compared with
tetrahedral embryos (Ebner et al. 2008), which can influence
the developmental potential. Indeed, it has been stated that
an increase in the number of contact points will facilitate
compaction because of a larger number of tight junctions
available (Ding et al., 1999). The aim of the current study was
not to give an answer on the general molecular mechanisms
of the blastomere arrangement in human embryos since it
focused on the use of the spatial arrangement of a 4-cellstage embryo as a non-invasive morphological characteristic
to improve the embryo selection.
In conclusion, this study shows that tetrahedral
4-cell-stage embryos have a higher developmental and
implantation potential. This characteristic should be
included in the embryo evaluation. In the case of selecting
a single embryo for transfer, one should choose an embryo
with a tetrahedral arrangement over a non-tetrahedrally
arranged embryo.
References
Antczak, M., Van Blerkom, J., 1997. Oocyte influence on early
development: the regulatory proteins leptin and STAT 3 are
polarized in mouse and human oocytes and differentially
distributed within the cells of the preimplantation stage
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of fragmentation in early human embryos: possible effects
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domains. Hum. Reprod. 14, 429–447.
Cauffman, G., Verheyen, G., Haentjens, P., Devroey, P., Liebaers,
I., Van de Velde, H., 2010. Developmental Capacity and
Pregnancy Rate of Tetrahedral Versus Non-tetrahedral 4-Cell
Stage Human Embryos. Oral Presentation, Annual Meeting
ESHRE Rome, (O-153). Cooke, S., Tyler, J.P.P., Driscoll, G.L.,
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on embryonic cleavage plane and early development. Hum.
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Ding, T., Rana, N., Dmowski, W.P., 1999. Intracytoplasmic sperm
injection into zona-free human oocytes results in normal
fertilization and blastocyst development. Hum. Reprod. 14,
476–478.
Debrock, S., Melotte, C., Spiessens, C., Peeraer, K., Vanneste, E.,
Meeuwis, L., Meuleman, C., Frijns, J.P., Vermeersch, J.R.,
D’Hooghe, T.M., 2010. Pre-implantation genetic screening for
aneuploidy of embryos after in vitro fertilization in women
aged at least 35 years: a prospective randomized trial. Fertil.
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Ebner, T., Shebl, O., Moser, M., Sommergruber, M., Tews, G., 2008.
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Ebner, T., Maurer, M., Shebl, O., Moser, M., Mayer, R.B., Duba,
H.C., Tews, G., 2012. Planar embryos have poor prognosis
in terms of blastocyst formation and implantation. Reprod.
Edwards, R.G., 2002. Aspects of the molecular regulation of early
mammalian development. Reprod. Biomed. Online 6, 97–113.
Edwards, R.G., 2005. Genetics in polarity in mammalian embryos.
Edwards, R.G., Beard, H.K., 1997. Oocyte polarity and cell
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Edwards, R.G., Hansis, C., 2005. Initial differentiation of
blastomeres in 4-cell human embryos and its significance
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Gardner, R.L., 2002. Experimental analysis of second cleavage in
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Gardner, R.L., 2006. Weaknesses in the case against prepatterning
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Hansis, C., Edwards, R.G., 2002. Cell differentiation in the
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Hnida, C., Agerholm, I., Ziebe, S., 2005. Traditional detection
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Johansson, M., Hardarson, T., Lundin, K., 2003. There is a cutoff
limit in diameter between a blastomere and a small anucleate
fragment. J. Assist. Reprod. Genet. 20, 309–313.
Johnson, M.H., 2009. From mouse egg to mouse embryo: Polarities,
axes and tissues. Annu. Rev. Cell Dev. 25, 483–512.
Johnson, M.H., Ziomek, C.A., 1981. The foundation of two distinct
cell lineages within the mouse morula. Cell 24, 71–80.
Louvet-Valleé, S., Vinot, S., Maro, B., 2005. Mitotic spindles and
cleavage planes are oriented randomly in the two-cell mouse
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Paternot, G., Debrock, S., D’Hooghe, T.M., Spiessens, C., 2011.
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Embryol. Exp. Morphol. 18, 155–180.
Van Blerkom, J., 2007. Translocation of the subplasmalemmal
cytoplasm in human blastomeres: possible effects on the
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VerMilyea, M.D., Maneck, M., Yoshida, N., Blochberger, I.,
Suzuki, E., Suzuki, T., Spang, R., Klein, C.A., Perry, A.C.F.,
2011. Transcriptome asymmetry within mouse zygotes but
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Zegers-Hochschild, F., Adamson, G.D., de Mouzon, J., Ishihara,
O., Mansour, R., Nygren, K., Sullivan, E., van der Poel, S.,
2009. The international committee for monitoring assisted
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Organization (WHO) revised glossary on ART technology.
Hum. Reprod. 24, 2683–2687.
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Zernicka-Goetz, M., 2011. Proclaiming fate in the early mouse
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PATIENT’S CORNER
by Gloria Calderón, PhD & Nuno Costa Borges, PhD
Embryotools SL, Barcelona, Spain
You can contact Gloria Calderón at [email protected]
and Nuno Borges at [email protected].
D
esigning a new human IVF laboratory is not an easy
task. Several important factors must be taken into
account in order to construct a high standard IVF
laboratory, which we know to be the key to success of any
ART center.
One of the most important factors to be considered is
the location of the site. Specialists working in human IVF,
are aware of the importance of air quality when working
with human gametes and embryos. Air quality can vary
enormously depending on the geographic location of the
lab. Commonly, a clinic located in a small village with
little traffic, and surrounded by parks or a forest, will
give the site one of the best external air qualities. On the
contrary, if the site is located in a crowded city, the air
quality might not be optimal. Therefore, when the lab
is designed, an appropriate ventilation system must be
considered, in order to protect and isolate human gametes
and embryos from external pollution. In this sense, the first
recommended step would be to measure the concentrations
of volatile organic compounds (VOC) in the external air.
After performing a deep examination of the results, we
should be able to design, with the help of engineers, an
appropriate ventilation system that covers the
needs of the chosen site. It is also important to
emphasize that IVF labs and operating rooms
should not share ventilation systems or air
flow. The embryology and cryopreservation
laboratories, should have the highest positive
pressure with airflow towards the operating
rooms and sperm laboratory.
Another important aspect to take into account is the
size and shape of the building, because this will define the
final layout of the center, including the size and location of
the IVF Laboratories.
The number of IVF cycles that the center is expected to
carry out within its first 10 years is also valuable information
during the design phase. This allows us to better determine
how to dimension the IVF laboratories, avoiding the need
for disruption of renovation and extension of the clean
areas due to the lack of space in the following years.
Another very important design aspect to consider is
the materials used for construction. We must always avoid
the use of plastics, glue, wood, paint, and in general, all
components that could potentially generate long-duration
VOC emissions. Taking care of these details will help us to
preserve the quality of the air.
In recent years, the use of prefabricated modules for the
construction of IVF laboratories has become very common
Gloria Calderón, PhD
Nuno Borges PhD
and convenient. These modules offer several advantages
over classic construction, including antibacterial finishes,
layout and size high flexibility, and maximum asepsis. They
also provide active support for the air treatment system, as
their structure has been designed for compartmentalization,
allowing for perfect integration of any kind of equipment.
Moreover, it is highly advantageous to use prefabricated
SMS® (solid mineral surfaces), panels that are bacteriostatic,
uniform, non-porous and very easy to clean or disinfect.
As mentioned before, in order to maintain stable
air
quality
conditions
inside
the
lab, operating
rooms
and
laboratories
must be sealed
and positively
pressurized, in order to protect these spaces from the other
areas of the building. Therefore, it is recommended the
installation of an independent ventilation system, together
with VOC and HEPA filters, as well as the conventional
filters types with biggest porous size at the beginning of the
ventilation system. Additionally, a UV light system should
be included at the end of the ventilation system for a more
efficient removal of VOCs and better protection against
possible bacterial, viruses or fungus contamination.
The ideal layout plan of an IVF lab should include at
least 3 different rooms: one for cryopreservation, a second
one for sperm preparation, and a third one for embryology
work. The embryology lab should be connected through a
door with the cryopreservation lab. The cryopreservation
lab should be wall to wall with the liquid nitrogen tank
room. This will not only save liquid nitrogen, but will also
provide a direct connection of the liquid nitrogen to the
tank storage of gametes and embryos. The sperm lab can
One of the most important
factors to be considered is
the location of the site.
PATIENT’S CORNER
be located inside the clean area, but not necessarily next
door to the embryology or cryopreservation lab. It is also
advisable to design a storage room close to the IVF area,
with enough space to keep all material and disposables
needed in all labs.
In all laboratories it is convenient to avoid the use of
excessive furniture inside the IVF area, and all surfaces
should be made of stainless steel and glass.
The location of the equipment inside the laboratory
is also another key point that must be considered during
the design of a new lab. We recommend organizing the lab
in individual working units/workstations. Each of these
should include an anti-vibration table with the inverted
microscope and adapted micromanipulation system; a
vertical laminar flow hood with heated surface and a
working incubator. Regarding incubators, we should tend
to use bench top incubators for gamete and embryo culture,
because they are more stable than regular cell culture
incubators. They can be located on one of the laboratory
walls on shelves, stacked at different heights to save space.
Our last recommendation would be to install an
alarm system to continuously monitor the equipment, in
particular, all incubators, storage tanks and refrigerators
with all media.
Last, but certainly not least, the budget available
for the project needs to be sufficient to provide for fully
equipped laboratories with high quality and state-of-the art
equipment.
We believe that by following these simple but crucial
rules, the performance of your IVF lab can improve.
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PATIENT’S CORNER
Cassandra’s prophecy: why we need to tell the women of the future
about age-related fertility decline and ‘delayed’ childbearing
Jane Everywoman1
C/O Duck End Farm, Park Lane, Dry Drayton, Cambridge CB23 8DB, UK
E-mail address: [email protected]
Abstract This anonymized paper describes the author’s experience of age-related infertility and unintended childlessness.
It outlines her journey from diagnosis to treatment success and clinical pregnancy through assisted reproduction using
oocyte donation, followed by subsequent early miscarriage. It makes subjective observations about treatment she
received and presents her impressions of how discourses of knowledge dissemination, communication and care were
constructed in the organizations she encountered. It sets her own reflections alongside broader observations on the
challenges facing women today when planning a family and draws attention to what she perceives to be the misleading
myths and misunderstandings concerning reproduction that these women are now subject to. In the light of this, it offers
some suggestions for modified public health messages and new approaches to sex education and health screening
that may consequently help to empower tomorrow’s women (and men) to take full control over their reproductive lives in
the 21st century. The paper takes as its mascot the figure of Cassandra, daughter of King Priam and Queen Hecuba. She
was loved by Apollo, but resisted him. In consequence, he rendered useless the gift of prophecy that he had bestowed
on her by causing her predictions never to be believed.
KEYWORDS: abortion, assisted reproduction, infertility, IVF, oocyte donation, premenopause
1Pseudonym
This article was published in Reproductive BioMedicine Online, Vol 27, 2013, p4-10, Cassandra’s prophecy: why we
need to tell the women of the future about age-related fertility decline and ‘delayed’ childbearing. Copyright Elsevier.
It is reprinted here with permission.
A personal story
At the age of 32, I suddenly began to suffer considerable,
cyclical gynaecological discomfort. By age 37, my weight
was inexorably increasing and my menstrual flow became
lighter and would begin unpredictably between day 21 and
35. I had the occasional night sweat and hot flush. Trips to
the doctor where these symptoms were discussed alongside
other things elicited no further comments.
During this time, I had started trying to conceive
because, even though married 5 years before, at age 27, I
had earlier lacked the emotional and financial confidence to
have a baby. As a school teacher and academic researcher,
I had made many sacrifices to gain several higher degrees
and professional certifications. By 32, I had a full-time job,
a husband and a comfortable home – things that were hard
and time consuming to attain – and I felt the conditions
were exactly right to begin the family that was always
firmly on my agenda, if not for a long time at the top of
it. After the first time of unprotected sex on cycle day 14, I
looked forward to my forthcoming offspring.
To my surprise, no baby was conceived. I had assumed,
like many of my contemporaries (Edwards, 2011), that sex
without contraception always resulted in pregnancy. This
was because since childhood I had been educated to believe
that ‘all’ acts of unprotected intercourse would inevitably
result in a live birth – which, if it was unplanned, was
naturally morally suspect and to be avoided until ‘the right
time’, which was always left undefined. Months passed
and no baby materialized. Time and time again, pregnancy
announcements came from family, friends and colleagues
and irritated my profound and unrelenting pain. I forgot
what it was like not to be constantly and secretly sad, and
carrying the burden of loss of control blanched the colour
from every aspect of my life all the time.
At around age 34/35, I consulted a doctor, who told me
that the hormonal imbalances that I informed her I suspected
I had (but could not make sense of), would be easily
‘corrected’ by a pregnancy. Another GP told me to ‘wait 2
years’, because women are only assessed as infertile if they
have been trying unsuccessfully for that long. Respectful
around doctors, I went away, misconstrued his advice and
waited for 24 attempts at trying to conceive, which actually
turned into three chronological years, possibly because of
my unacknowledged reactive depression and busy work
schedule.
During this time, my husband and I also attended
a family planning and women’s health clinic because,
we assumed, our issues were surely relevant to their
mission. Yet we were turned away. At smear tests, nurses
in whom I confided told me to ‘have patience’. Another
GP consultation at age 37 where I again talked of my
PATIENT’S CORNER
lack of pregnancy alongside other issues simply elicited
the response ‘go home and relax’. Still no baby resulted.
I purchased ovulation sticks which apparently showed I
was ovulating. What I was unaware of at the time is that
these devices detect merely hormonal surges. They do not
prove an egg has been released nor measure its potential
to develop into a live birth and therefore they are, in some
contexts, misleading.
I felt ashamed, vaguely and constantly unwell and
very confused, especially since the clinicians in whom I
placed my trust seemed unconcerned. I disclosed nothing
to close family or friends because of the very private nature
of my struggles. I assumed it was something I was doing
wrong, and given that women are so urgently told to use
contraception at the very earliest opportunity to prevent
pregnancy, I believed that it must have been my inadequacy
alone.
The reason I felt I had plenty of time for childbirth
was also everywhere around me. No one in my wide and
international social and professional circle had given birth
before about 33. In media reports, numerous celebrities
were also getting pregnant for the first time at 40+, often
with twins (Freedman, 2007; Mamamia, 2007; Morrell,
2008; Sager, 2010). Some were admitting to the use of IVF
treatment, but rarely did they (understandably) disclose
how many pregnancies were lost in the process, whether
donor eggs from a much younger woman were used, the
toll taken on their own or their baby’s bodies, what the total
cost was, or the number of attempts needed for a live birth
(Mail Foreign Service, 2010). Therefore I ‘knew’ that IVF
would easily ‘cure’ me – if only I had the courage to go to
a clinic – and, since no GP had formally referred me, I still
assumed all I had to do was wait, and I was also terrified
at the prospect of the cost. No one had explained to me
how the process would work, how long it might take, how
it would be paid for, what tests might be done and what
criteria would be used to make me eligible for treatment.
As I still menstruated – to me an obvious sign of fertility – I
firmly believed my pregnancy was just around the corner.
Having never been given a diagnosis, I wonder, with
hindsight, if my symptoms were those of perimenopause,
the transitional period of hormonal shifts that can, in some
women, occur years before final menopause (Corio, 2000).
Tragically, this had been suggested to me by a herbalist I
consulted only in desperation. However, because for me
she had less authority than a formally-qualified clinician,
I doubted her. Aged 42 now, I expect my menopause
to happen around age 43–45 – just within the ‘normal’
spectrum. I know this now because late in 2008 at the age
of 39 I finally attended a private IVF clinic through selfrecommendation. Two quick but expensive blood tests told
me I had a raised FSH and low anti-Mu¨llerian hormone.
They revealed my ovaries were almost ‘closed down’ and
my egg reserve was virtually completely empty. I had
nothing to make a baby with, no matter how regularly I
had sex. The clinic doctor informed me my menopause was
imminent and only the use of donor eggs would lead to
pregnancy. He said, ‘Treatment here costs £8000 per cycle
including drugs and you’ll probably need three cycles for
it to work. Our waiting list is about 2+ years and it costs
£1500, refundable against future treatment, to put your
name on the list now.’
Since then, my personal IVF experience means that
my husband and I have haemorrhaged money as well as
blood. Our life savings are around £18,000 lighter, for the
NHS would not treat me because at age 39/40 I was by
then ineligible for funding (although no one ever pointed
this prospect out to me when I had previously sought
advice). I have had two gynaecological operations, endless
vaginal scans and blood tests whose results were essentially
meaningless as there is currently no formal consensus on
what they reveal (Rai et al., 2005). I managed unexpectedly
quickly to get a first attempt at donor egg IVF through
egg sharing at a second clinic (where it also cost a nonrefundable £450 simply to place my name on the waiting
list), but it failed. A second attempt a year later gave a
positive pregnancy test. Two weeks afterwards, a missed
miscarriage was diagnosed. After this, I was told by a nodoubt well-meaning clinician that all I needed to do was
simply ‘try harder’ to get pregnant. This puzzled me, since
I had exhausted my life savings to get that far and taken the
most powerful drugs I had ever used in order to conceive. I
wondered what more I could possibly have done.
Making sense of my experience was made all the more
difficult because staff, perhaps desperate to convince me
(and reassure themselves) that the process does work
sometimes, bombarded me with anecdotes of unexpected
IVF ‘miracle’ babies, who had been born to patients with
very poor prognoses. This merely intensified my sense of
personal failure. As I passed out during the operation to
remove the products of conception, the anaesthetist told me
that it was better that the embryo had miscarried because
it would not have been viable. I was crying as I drifted
into unconsciousness and struggled to tell him that it was
a donor egg from some anonymous woman, and I had no
more money to pay for another. Becoming conscious one
hour later, the nurse by my side told me that miracles do
happen as her relative at age 44 gave birth to her first child
that year. I was crying even before I had opened my eyes.
Once recovered from the miscarriage, I was bound to try
a third cycle with embryos cryopreserved from the previous
attempt (if only to rule out a pregnancy if I subsequently
wanted to adopt). Mere ‘shadows of possibility’, treatment
with these resulted in an initial negative result, which was
subsequently reinterpreted as a ‘weak positive’. I was then
instructed to continue medications until another blood test
could determine whether there was an ectopic pregnancy.
To pay for all this, my husband and I took on two jobs
each, and for 2 years juggled these with appointments for
time-consuming treatments and tests in several different
places. Although infertility consumed every element of our
lives for what has ultimately amounted to 10 years, I did
PATIENT’S CORNER
not confess to a soul outside of my clinics that I was unable
to do what I perceived everyone could do effortlessly – get
pregnant – and my sense of shame cut into my heart. This
was even more so since I personally felt that clinical staff
in IVF centres tended to hint the lack of success was my
failure rather than that of the science and technology they
used. This is probably because from their perspective the
treatments work (as they sometimes do), and it is only a
deficiency in women’s stamina that makes them withdraw
before their goal is achieved (Cross, 2010). Ultimately I
had to resign from work in order to fully recover from my
experiences and this merely exacerbated my sense of loss
of control.
This writing describes only my personal truth – a
narrative that could be told in many ways. Some may
dismiss me as naïve for, as one journalist has said, ‘it
would be impossible . . . to exist in society and not know
that having children became problematic in one’s mid30s’ (Williams, 2005). Whilst initially I would have agreed
with this, I have reflected at length on my experiences
and assumptions and have wondered about just how
flawed or unrepresentative they might be and what can
be learned from them. Although admitting to ignorance
about reproduction in our information-saturated, highly
sexualized culture is embarrassing, I am also beginning to
suspect that I am definitely not alone and that assuming
‘everybody knows’ may be far too simplistic an explanation
(Birrittieri, 2005, 2006; Bretherick et al., 2010; Bunting and
Boivin, 2008; Cooke et al., 2010; Daly, 2011; Peterson et al.,
2012). Indeed, my private tragedy transcends the personal
and embraces the political domain, as it speaks for the asyet unidentified community of women in the developed
world who inevitably and sadly will, in future years, trudge
the arduous path I have traversed.
I would argue that what women know and understand
and what they relate to and use in their own lives may be
subtly different things (Elkind, 1981). I did know about
fertility ‘decline’ with age; but I did not really understand
it in an adequate way, perceiving it merely as ‘risk’ easily
managed through excellent prenatal care. I had no idea
that late-age pregnancy is ultimately difficult because of a
decline in egg quantity and quality – the key building blocks
of life. Nor did I really believe the risk especially in the light
of media reporting of first babies at age 40+ and the fact that
no clinician outside my fertility clinic communicated to me
that age was a relevant issue (Cohen, 2010). For me, highly
educated and ‘well informed’, it was extremely difficult
to relate to my own life all the competing, confused and
fragmented threads of information that I had gleaned since
childhood about pregnancy and fertility from a wide variety
of sources and then to step back in order to see that in fact
I really was ageing and moving fast towards chronological
middle age and reproductive old age. For I still felt young,
looked young and perceived myself to be in relatively good
health.
Thus, I feel it is reasonable to claim that I am as much
a victim of a deficiency of information as I was solely
responsible for my miserable plight. I lacked key information
to make appropriate choices about reproduction as a result
of the sex education I had received and, despite great effort,
I failed to access it from public health agencies. I was also
falsely given hope by the oblique reporting of ‘miracle’
babies born through IVF (Jenny, 2010). I therefore write
because, as a teacher, I have a duty of care to ensure that
the next generation of adults, who could be struggling with
many of the issues I had to make sense of on my own, are
well informed. For, statistics show that there is a global
trend for delaying childbearing beyond 30 years of age and
by 2007, 20% of British women had waited until after 35
to begin their family (Botting, 1992; BUPA, 2009; Campbell,
2010; Cooke et al., 2010). Moreover, infertility issues affect
around one in six of the population (Templeton, 1992).
Gustafsson (2001) estimates that as many as half of women
in their thirties will have some sort of fertility problem
and she also comments on the ‘spectacular’ increase in the
percentage of women who have not yet given birth by 34
in Europe, of whom many, as a result, will be ‘ultimately
childless’.
Myths and misunderstandings
A number of misunderstandings and myths may need
to be addressed. First, it may be that teens and young
adults are genuinely confused about what are, in the
developed world, stark dissonances between chronological,
reproductive and cultural age. A well-groomed celebrity
can be 47, look 32 and be perceived as ‘young’ especially
since life expectancy is around 70–80 years. As her egg
reserve was fixed at birth and from that point began a slow
decline, reproductively she is almost certainly unable to
conceive her own genetic child, or indeed any child unless
donor eggs from a much younger woman are used. When
the journalist Kasey Edwards was told by her gynaecologist
that she was approaching infertility, she responded ‘But I’m
only 32 . . . Surely that’s not old’ (Edwards, 2011, p. 17). For
the girls in schools now, 40 really is the new 20, a phrase
increasingly in common parlance (Cougar Town, 2010).
Second, such is the entrenched potency of the ‘family
planning’ rhetoric and so extensive is contraceptive use
in developed societies that anything to do with baby
making carries with it a strong sense of personal control,
as if implicit in every act of contraception is a guaranteed
conception and live birth that we simply choose to defer
(Marks, 2001). This may drive men and women to ‘delay’
families precisely because they partly assume that starting
them is their decision alone. Posters in GP surgeries talk
of ‘planning’ (not ‘hoping for’) a baby; ovulation monitors
indicate when sex should occur to conceive; the habit of
The Pill allows you to control when you will menstruate.
As Kasey Edwards has written, ‘in sex-education classes in
school, it’s implied that there is a one-to-one relationship
between unprotected [intercourse] and getting knocked up’
PATIENT’S CORNER
(Edwards, 2011, p. 105) and similar assumptions are not
uncommon (Kardashian, 2011). Yet, in clinical reality, if 100
ova are exposed to spermatozoa at ovulation, only around
31% will become a live birth (Leridon, 1977, p. 79; Macklon
et al., 2002). As one author has pointed out, these discourses
mean that for some women reproduction is viewed as so
inevitable it is relegated to the lowest point on their ‘to do’
list. She admitted ‘I didn’t consider motherhood as any sort
of accomplishment, because . . . anybody could do it . . .
that’s hardly an achievement’ (Edwards, 2011, p. 10).
Third, it seems that there exist key misunderstandings of
what IVF treatment can actually achieve (Adashi et al., 2000;
Hashiloni-Dolev et al., 2011). It may be that many perceive
it (before they try it) as a failsafe ‘cure’ for infertility (Parkin,
2007). As one journalist admitted, ‘I honestly thought that
you start to worry at 38 and then you go to doctors at 39 and
then you would have treatment to help you get pregnant’
(Bewley et al., 2009, p. 348). Thus, many do not view it as
a treatment that must work in harmony with one’s own
body and that relies on viable eggs or spermatozoa being
available. Furthermore, it is in reality a treatment with a
rate of ‘success’, which, overall, currently hovers around
the 20–30% mark worldwide (Bewley, 2005; Throsby, 2001).
For the 39,879 women who had IVF in the UK in 2008,
there were only 12,211 successful live births resulting from
50,687 cycles of treatment (HFEA, 2011). Therefore, it is also
reasonable to describe it as an unreliable and unpredictable
process which does not work for the majority trying it and
when it does, its success is often the result of multiple, timeconsuming, expensive attempts. It is a stark ‘truth’ that, if
around 4 million IVF babies were born since treatment first
began, many more millions of couples who were treated
were left empty handed (Brown, 1979; Groskop, 2010;
Nobel Prize, 2010). Indeed, as one clinician has admitted,
‘We would probably have said 20 years ago that by 2008 we
will be able to pick the best embryo to transfer but it is still
beyond our grasp’ (Bewley et al., 2009, p. 330).
Moreover, IVF is regularly perceived by the lay person
as a treatment specifically for older women. As one woman
has reported ‘. . . with all these medical breakthroughs
women can have children later and later . . . it means I can
put off having children until my forties. It’s such a relief’
(Hewlett, 2002a, p. 184). As the Princeton-educated actress
Brooke Shields exclaimed when told she would need IVF,
‘Isn’t that for older women? I’m only thirty-six’ (Shields,
2005, pp. 8–9). Hewlett (2001) and Hewlett (2002b) reported
that 90% of women aged 28–40 who participated in her
2001 study stated that they believed that reproductive
technologies would allow them to get pregnant into their
forties. IVF does tend to be used more by older women
because it is precisely that group that struggles most to
conceive. Yet after around 36/37, ‘success rates’ plummet
and by the age of 40 they are almost negligible for patients
using their own eggs (HFEA, 2011). Thus, it is possible to
argue that IVF is in reality a treatment that usually works
best on younger people and it cannot be the guaranteed,
failsafe back-up plan for older women as two recent movies
imply (The Back-up Plan, 2010; The Switch, 2011).
Similarly, it is quite possible that miscarriage rates are
also greatly misunderstood. Unfortunately, these can occur
in one in four or five pregnancies overall and in one in two
women over 40 years old (Holman et al., 2000; KlugerBell, 2000; Leridon, 1977, pp. 54, 63–65, 75–79; Miscarriage
Association, 2010). Although seldom discussed, upon
disclosure a woman can often find that they are far more
common than she originally anticipated. Such an experience
can delay pregnancy plans and, whilst waiting for recovery
from them, fertility still continues its inexorable decline.
Moreover, many women may be uncertain of the
wide individual variation in levels of fertility across the
population and the fact that it is not something fixed and
unchanging, but a characteristic that briefly blossoms and
then declines in females and males (although comparatively
‘slowly’ in the latter) (Faddy et al., 1992). For example,
analysis of school curriculum materials reveals that it is
often unhelpfully characterized as something ‘without an
end’ and universally guaranteed, rather than something
that is, for all women, finite (Kisby Littleton, 2012). Neither
are women aware that some members of the medical
community have suggested that subfertility and infertility
can occur at least a decade or more before menopause,
which can vary widely between about 40 and 60 years
(Lobo, 2003; te Velde, 1997). Whilst some women do give
birth easily in their late thirties and beyond, what many of
them (or those ‘planning’ to emulate them) will probably
not really understand is that it is ultimately biological
serendipity that permitted this and that personal volition
only plays a secondary role.
New directions for education
In Greek mythology, the beauty of Cassandra (daughter
of Queen Hecuba and King Priam) caused Apollo to grant
her the gift of prophecy. When his love was not returned,
Apollo placed a curse on her so that no one would ever
believe her predictions until they had come to pass (Harvey,
1986, p. 92). She thus possessed a combination of deep
understanding and powerlessness and I know how she
must have felt. Although positive reproduction experiences
are thankfully common, similar stories to mine have been,
and will continue to be, replayed around the developed
world as women are educated to expect fulfilling careers,
may have to work for economic reasons, anticipate meeting
the perfect partner and continue to struggle with work–life
balance and the decision to have a baby whilst they are still
enmeshed in career structures suffused with patriarchal
values (Barker, 2009; Harris, 2005; Hewlett, 1988). Although
in the short term we cannot change many of the very
complex issues that lead to this situation, we can modify
the subsequent public health message that the women of
the future experience at the school and adult level – if we
have the will to do it.
PATIENT’S CORNER
Twenty-first century culture tells women that they
can ‘outsmart Mother Nature’ (Tampax, 2011a, 2011b). At
present, a ‘two-dimensional’ version of reproduction is also
disseminated in schools – man + woman + unprotected sex
= guaranteed baby – and, until some discover otherwise,
most carry this notion into adulthood (Skoog Svanberg et
al., 2006). Most adults know the options for dealing with
unplanned parenthood; most know how to nourish a
developing baby in utero and we mostly have complete
faith in the medical profession to care for our baby and
young child. Most dangerously, many feel a strong moral
sense that, if a family should be ‘delayed’, it is simply
personal choice, a lack of a suitable partner and is no-one
else’s business. Most harbour a vague notion that, if they
are so unlucky as to experience a problem conceiving, an
IVF clinic can provide a ‘cure’ (Dickson, 1992).
These beliefs have validity, but together they may not
make up the whole story that couples need to know now.
Thus although my account does not have the epistemological
authority of a formal study, it is still possible to make some
suggestions on the basis of it (DasGupta and Charon, 2004).
While it should not be necessary to cause anxiety around
reproduction, it may be that now more balanced and honest
information needs to be disseminated, first in schools and
then later also supported by official public health messages
disseminated at the grassroots level and directed at young
adults – not through media headlines, ill-informed TV
documentaries or websites of dubious quality (Marriott et
al., 2008).
This information should be designed to empower the
women who will grow up in cultures very different from
those in which ‘developed’ society’s original messages about
sex education were first formulated (Hall, 2009). It may be
that these women should be encouraged to take adequate
notice of their natural menstrual patterns if they have used
chemical contraception for a long time (Grigoriadis, 2010),
and also be sensitively warned of the relative frequency
and potential disruption of early miscarriage (Tough et al.,
2007). They need to be briefed about the wide spectrum
of ages of menopause and the fact that a woman’s egg
supply is fixed at birth and the implications of the decline
in quantity and quality of eggs should be made crystal
clear to them in a timely fashion. The telling changes that
may occur in the transitional phase before a woman’s final
period should be clearly outlined, lest women walk into
perimenopause with their eyes closed before their family
is complete (Feinmann, 2008). It may be that, at the same
time, honest, more balanced and easily accessible accounts
(beyond statistics or abridged stories of straightforward
patient success) of the current limitations of the fertility
industry (including egg-freezing technology) would be
helpful so that these women do not assume they can fully
rely on it to conquer Nature’s plan if the need should arise
(Extend Fertility, 2011; Parkin, 2007).
Most crucially, it may be that for these women the
debate about ‘delayed childbearing’ needs to be moved
from the moral sphere where it usually sits (Frostrup,
2006) and be repositioned within the biological one too.
Whether it is desirable or not for older women to bear
children, because fertility potential varies and ultimately
ceases in women, and to a lesser extent men, not all couples
can have the choice. Indeed, it may be that women cannot
avoid ‘delayed’ childbearing until they are made more fully
aware of the true concept of it and are (re)introduced to
the basic notion of ‘optimum time for birth’ (van NoordZaadstra et al., 1991; Maheshwari et al., 2008). These basic
notions could also be set forth alongside the traditional
warnings about teenage pregnancy currently disseminated
at the school level (Allen, 2007; Boseley, 2008; Dickson,
1992; Kisby Littleton, 2012; Peterson et al., 2012). They could
also be reinforced carefully in nurse-led fertility support
sessions offered to women who have not conceived by age
29/30, where current fertility myths are also challenged
(Asthana and Hill, 2009). These information sessions
would be a modified form of the ‘fertility MOTs’ already
suggested, for they would avoid the potential complacency,
expense and possible inaccuracies that could result from the
formal assessment of ovarian reserve through blood testing
(Hussell, 2010; Macrae, 2009; Visser et al., 2006).
How can people be expected to properly ‘know’ what
appears to be currently formally untold? Clearly, what are
considered to be ‘our’ reproductive choices are to some
extent cultural illusions divorced from the reality that
human reproduction is ultimately dependent on biology’s
plans. In the light of all this, it is important this paper is
not interpreted as yet another piece telling women at what
age to have a baby, nor a judgement on those that delay
childbirth, or even a criticism of the fertility industry. It is
none of these. Rather, it is a call for appropriate education so
that people become fully empowered to make reproductive
decisions for themselves. It is also a hint that perhaps
infertility clinics could further integrate medical, social and
psychological models of care and learn from palliative care.
Relentless positivity and descriptions of others’ success
does not always assuage emotional pain; but sensitive
validation of that pain is usually reassuring to its sufferer.
Perhaps, at the beginning of the third millennium, women
who consider themselves ‘emancipated’ should be helped
to re-engage in a more respectful dialogue with Mother
Nature alongside the one they have with Science, and all of
us should be made to listen more carefully to Cassandra as
well. For, reproductive health and rights are not only about
the choice not to have a baby, but they are also about the
choice to have one too.
Acknowledgements
I am grateful to Professor Susan Bewley, Professor Bill
Ledger, Antonia Rodriguez, Irenee Daly and Kate Bentley
for their sensitivity during preparation of this article.
PATIENT’S CORNER
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Fertility Magazine • Volume 18

Transcripción

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